Stories of Success, Volume 46: Personal Recollections XI (Comprehensive Biochemistry)

COMPREHENSIVE BIOCHEMISTRY Series Editor: GIORGIO SEMENZA Swiss Federal Institute of Technology, Department of Biochemis...

Author: Giorgio Semenza

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COMPREHENSIVE BIOCHEMISTRY Series Editor: GIORGIO SEMENZA Swiss Federal Institute of Technology, Department of Biochemistry, ETH-Zentrum, CH-8092 Zurich (Switzerland) and University of Milan, Department of Chemistry, Biochemistry, and Biotechnologies for Medicine, I-20133 Milan (Italy)

VOLUME 46 STORIES OF SUCCESS – PERSONAL RECOLLECTIONS. XI Volume Editors: VLADMIR P. SKULACHEV Moscow State University, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia

GIORGIO SEMENZA Swiss Federal Institute of Technology, Department of Biochemistry, ETH-Zentrum, CH-8092 Zurich (Switzerland) and University of Milan, Department of Chemistry, Biochemistry, and Biotechnologies for Medicine, I-20133 Milan (Italy)

AMSTERDAM BOSTON LONDON NEW YORK OXFORD PARIS SAN DIEGO SAN FRANCISCO SINGAPORE SYDNEY TOKYO 2008

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK First edition 2008 Copyright r 2008 Elsevier B.V. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (þ44) (0) 1865 843830; fax (þ44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://www.elsevier.com/ locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-444-53225-1 ISSN: 0069-8032

For information on all Elsevier publications visit our website at elsevierdirect.com

Printed and bound in Hungary 08 09 10 11 12

10 9 8 7 6 5 4 3 2 1

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PREFACE TO VOLUME 46 (Vol. XI of the ‘‘Stories of Success. Personal Recollections)$ ‘‘Luck’’ does not come to a scientist the way it does to a winner of a lottery. Luck in science is man-made, mostly scientist-made, through his or her own ingenuity, hard work, endurance (or, stubbornness, as others might say). Fruitful ideas come to the ‘‘prepared mind’’ only, as Pasteur [1] and the mentor of one of us, Tiselius used to say [2]. ‘‘Luck’’ can be a helpful ingredient among the components, which lead to a discovery. However, the unprecedented explosion of molecular biological sciences began during the ‘‘age of extremes’’ of the so-called ‘‘short 20th century’’ [3]. Many scientists, their families and friends, like other citizens, were hit by the avalanche of horrors which swept through Europe and much of the rest of the world before, during and after World War II. ‘‘Luck in science’’ was often nullified by these catastrophic events. Many were drafted and sent to the front; those who returned would find their countries in ruins and shambles. Even after the aftermath of World War II, emigration (which is always traumatic even in the most favorable of circumstances) was often a required condition to be scientifically active at all. The Jews – always prominent in the molecular biological scientific community – were discriminated against and persecuted well before World War II began, and even more so during the war, not only in Nazi-occupied Europe; but also often in Soviet-block countries too. Emigration had become for many a disguised sort of good luck. It is well known that Jews could not regard themselves as safe in the Soviet Union. Often they were silenced for years, or

$

Parts of this Preface are taken from that of vol. 45. Note: Vol. 40 (1997, vol. V of the ‘‘Personal Recollections’’) and subsequent ones are online at http://www.sciencedirect.com/science/bookseries/00698032

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PREFACE

even in some cases forever, for whatever whimsical political [4] or pseudo-scientific [5] reasons (to name but two examples). To better illustrate this, let us comment briefly on the first chapter in this volume, that of Prof. Lev L. Kisselev. He, then a boy, his brother, his mother and his aunt were evacuated during World War II west of Moscow (whereas his father stayed to work in Moscow). They were taken by the Germans, and had to work and follow them also when they retired west. What saved their life was that Lev’s father was incredibly far-sighted: he never wanted that his children and family should carry his own name – Silber. Such an obviously Jewish name would have immediately given them away as half-Jews to the Nazi – tantamount to a death sentence. At the end of the war the Kisselevs were as far west as Dresden. Prof. Silber had no idea where they were, and they had no news about him, either. Their worry of perhaps never finding Prof. Silber was justified: he was notoriously anti-Stalin, for which he had been arrested various times. By shear luck, however, the family was united again. Miracles do happen – but not always. Prof. Michel Goldberg’s family, originally from Poland, went through the ‘‘French version’’, so to say, of Kisselev’s teen-age life; they were often in jeopardy in occupied France. Prof. W. Tanner and Prof. R. Timpl (see Prof. J. Engel’s chapter) were born in Sudetenland and shared as children the destiny of the Germans in that region. Prof. Viktor Mutt (see Prof. H. Jo¨rnvall’s chapter) was born in Estonia and came as a refugee to Sweden via Finland during World War II. Prof. H. Feldmann and Profs. B. and H. Jockusch experienced World War II and its aftermath as kids or teen-agers. Yet, they succeeded to enrich the scientific community with significant contributions. This takes us to the second, very important goal of this volume and of this sub-series. The ghosts of the past, which our generation thought had been killed for ever, are lurking and, occasionally, spring back to life. Beware. Those who forget their past are prone to repeat past errors – and suffer past horrors anew. Our children and children’s children must not be tempted to repeat the mistakes, which our fathers and fathers’ fathers tragically made. Not just molecular biosciences, but indeed any intellectual and decent human initiative can thrive only in freedom and peace. We hope that these volumes will convey this message to our present and – importantly – to our future colleagues.

PREFACE

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It is unfortunate, we think, that this subseries ends with this vol. 46 (XI of the ‘‘Stories of Success: Personal Recollections’’), in spite of its good success on the market also. Whatever the reasons, we, series editor and volume editors, find difficult to understand this publisher’s decision. Still – spes ultima dea. ACKNOWLEDGMENTS

The editors wish to thank the authors for having written such excellent (auto)biographic chapters, and the staff of Elsevier BV for their help and co-operative attitude. REFERENCES [1] [2] [3] [4] [5]

See, e.g., in Dubos, R. (1960) Pasteur and Modern Science, republished by the Amer. Soc. For Microbiology, in 1998. Tiselius, A. (1968) Annu. Rev. Biochem. 37, 1–24. Hobsbawm, E. (1995) Age of Extremes. The Short Twentieth Century, 1914–1991. London, UK, Abacus (Little, Brown and Company). Bayev, A.A. (1995) The paths of my life. In Comprehensive Biochemistry Vol. 38 (IV of the ‘‘Personal Recollections’’ Sub-series) (Slater, E.C., Jaenicke, R. and Semenza, G., eds.), pp. 439–479. Amsterdam, Elsevier. Levina, E.S., Yesakov, V.D. and Kisselev, L.L. (2005) Nikolai Vavilov: Life in the cause of science or science at the cost of life. In Comprehensive Biochemistry Vol. 44 (IX of the ‘‘Personal Recollections’’ Sub-series) (Semenza, G. and Turner, A.J., eds.), pp. 345–410. Amsterdam, Elsevier.

Swiss Institute of Technology, Zu¨rich, Switzerland, and University of Milan, 2008 Moscow State University, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia, 2008

Giorgio Semenza

Vladimir P. Skulachev

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CONTRIBUTORS TO THIS VOLUME BIRGITTA AGERBETH Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden

¨ RGEN ENGEL JU Biozentrum University of Basel, Switzerland

HORST FELDMANN Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Molecular Biology. Schillerstrasse 44, D-80336 Mu¨nchen, Germany

MICHEL E. GOLDBERG Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France

HARALD JOCKUSCH Developmental Biology and Molecular Pathology, University of Bielefeld, D-33501 Bielefeld, Germany

BRIGITTE M. JOCKUSCH Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092 Braunschweig, Germany

¨ RNVALL HANS JO Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden

LEV. L. KISSELEV Engelhardt Institute of Molecular Biology, the Russian Academy of Sciences, 32 Vavilova, Moscow 119991, Russia

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CONTRIBUTORS TO THIS VOLUME

WIDMAR TANNER Cell Biology and Plant Physiology, University of Regensburg, D-93040, Regensburg, Germany

MICHAEL ZASLOFF Department of Surgery & Pediatrics, Georgetown University School of Medicine, Washington, DC 20007, USA

V.P. Skulachev and G. Semenza (Eds.) Stories of Success – Personal Recollections. XI (Comprehensive Biochemistry Vol. 46) r 2008 Elsevier B.V. All rights reserved. DOI: 10.1016/S0069-8032(08)00001-6

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Chapter 1

Half a Century Later and, Still, I’m Not Disenchanted with Science LEV L. KISSELEV Engelhardt Institute of Molecular Biology, the Russian Academy of Sciences, 32 Vavilova, Moscow 119991, Russia E-mail: [email protected]

Abstract I became a scientist in 1959 and continue to pursue that profession to this day. All these years I have worked at the same institute of the Academy of Sciences. Today the Institute of Molecular Biology is named after the great biochemist who was my teacher, Wladimir Engelhardt. The key words in my areas of research in biochemistry and molecular biology are, I would say, transfer RNAs, aminoacyl-tRNA synthetases, termination of translation in eukaryotes, reverse transcription, and tumor suppressor genes. The reader may not read every page in this chapter. He or she will gain an impression, nevertheless, of how biochemistry and molecular biology have developed and evolved during the last half century in the USSR and then in the Russian Federation. The brief description of a lifetime of scientific activities is supplemented by a quite extensive list of my published works. Keywords: Transfer RNAs; Aminoacyl-tRNA synthetases; Termination of translation; Eukaryotes; Tumor suppressor genes; Reverse transcription

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Instead of a Preface I was born in Moscow on 14 August 1936. There I remained until war broke out in June 1941, when my mother took me and my 8-month-old brother to a dacha outside the city. In late autumn the invading German forces overtook us and when they began to retreat we were forced to move with them. So it was in the town of Chemnitz, 60 kilometers west of Dresden, that we were liberated by the Red Army on 6 May 1945. For most of the war my father Lev Zilber was imprisoned by the Soviet authorities. Arrested and released twice during the 1930s, he was again arrested by the NKVD, the Soviet secret police, in 1940 and would not be freed until March 1944. All the time we spent in German captivity we knew nothing of my father’s fate – and he had no idea what had happened to us. In July 1945 the family was re-united and thereafter we lived together in Moscow. I entered Moscow University in 1954. In 1955–1956 the Communist Party organization within the biological faculty persecuted the students in my year for ‘‘Mendelism-Morganism’’ and for our hostility to the ‘‘teachings’’ of Lysenko. An attempt was made to expel us from the university but it failed. The rector of Moscow University was Ivan Petrovsky, a mathematician and member of the USSR Academy of Sciences. He did not approve our expulsion. His decision became possible since in February 1956 the Party held its 20th congress and there Nikita Khrushchev made his ‘‘secret’’ speech about ‘‘cult of personality’’ under Stalin. Since then my whole life, as a biochemist and molecular biologist, has been linked with the Academy’s Institute of Molecular Biology. It is named after one of my mentors Wladimir Engelhardt, who is a classic figure in the discipline of biochemistry [1]. My other two teachers, I consider, were my father Lev Zilber, whom I describe in this chapter (for more detail see [2]), and Vladimir Skulachev, who supervised my undergraduate studies at the university’s department of biochemistry [3]. My first work performed at the Institute is dated 1961 [4]. The main subjects of my scientific interests have been the transfer ribonucleic acids (tRNAs), the aminoacyl-tRNA synthetases (aaRSases) and the translation termination factors eRF1 and eRF3. These important components of the protein-synthesizing system of cells are all linked because they participate in the specific RNA–protein interactions.

I’M NOT DISENCHANTED WITH SCIENCE

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My first trip abroad was in 1964. Since then contacts with foreign colleagues have constantly expanded, especially since the Gorbachev’s ‘‘perestroika’’ (1985) and the final fall of the Communist regime (1991). As a result, a great many of my studies have been carried out in tight collaboration with scientists from France, Denmark, the USA, Japan, Sweden, the Czech Republic, China, Austria, and Britain. Extended periods of work in France and Denmark have proved interesting, productive, and pleasant. As a full member (academician) of the Russian Academy of Sciences I am entitled to continue my scientific investigations for as long as I wish; and though I recently turned 70 I do not yet feel the desire to cease my studies.

My Parents When I was born, my parents, Valeria Petrovna Kisseleva and Lev Alexandrovich Zilber, were not officially registered as man and wife. As was then common (and indeed is the case today in Russia), theirs was a ‘‘civil marriage.’’ Therefore, in accordance with Soviet laws, I was entered in my mother Valeria Kisseleva’s identity document and given her surname. My parents were only legally married in 1946, due to the pressure of external circumstances (on a trip to the south the hotel management refused them a double room). Mama then suggested that when I and my brother reached 16 and received our own identity documents, we take Father’s surname. He was categorically opposed. It will become clear from what follows, why he took that decision. My mother graduated in art studies from the history faculty at Moscow State University. Her special field was medieval Russian painting. Since there was no secular painting in that epoch, she studied Russian icons. This was not a very safe occupation under Stalin – one might be accused of promoting religion, which was a kind of ‘‘anti-Soviet activity.’’ Mama was a most energetic and active woman. The prospect of becoming a modest guide at the Tretyakov Gallery would not have suited her at all. Abandoning her training, she studied to become an assistant in a microbiology laboratory: in the USSR, once you had graduated, it was not permitted to become an undergraduate in a different discipline. Successfully completing the course, Mama got a job at the Institute of Microbiology and it was there that she met Father

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already a well-known microbiologist and in charge of one of the laboratories. Lev Zilber was 9 years her senior and he had no children although married 3 times before. Two previous wives remained warmly attached to him until he died. Maria Shevlyagina was a highly thought of general practitioner and professor of medicine. Zinaida Yermolyeva was a well-known microbiologist and would subsequently become a member of the USSR Academy of Medical Sciences. It was Yermolyeva, with Father’s younger brother, the famous Soviet writer Venyamin Kaverin, who several times waged a desperately bold and exhausting struggle to free Zilber from imprisonment. It was thanks to their efforts, and to the support of many outstanding scholars and of Father’s own pupils, that he was 3 times plucked from Stalin’s dungeons. For those times it was a unique achievement. Lev Zilber attended Universities both in Petrograd (St. Petersburg) and in Moscow, graduating from the natural sciences and medical faculties, respectively. The superb biological and medical education he received between 1912 and 1919 formed the basis of his scientific career. Today the field is called biomedicine. In 1923–1927 Father discovered the serological transformation in bacteria, several years before the renowned experiments conducted by Griffiths. In 1930–1931, at risk to his own life, he suppressed an outbreak of plague in the Caucasus, in the mountain region of Nagorno-Karabakh. There followed his first arrest. The absurd accusation was that Father considered the outbreak to be natural in origin: the NKVD demanded his admission that spies had introduced the infection from neighboring Iran. Fortunately, intervention from Moscow secured his comparatively rapid release. He immediately returned to the capital from Baku when he was a director of the Microbiological Institute. In 1937 he led an expedition to the Soviet Far East which culminated in a triumphant success. After three and a half months of intense, wearisome labor in the forests of the untamed taiga, Father and his young co-workers, identified the pathogen of the spring-summer encephalitis that afflicted the region. It proved to be a new virus and he demonstrated that it was transmitted by ticks. Throughout the world this virus is today referred to as the tick-borne encephalitis virus. The outcome of these efforts, in autumn 1937 after Zilber had returned to Moscow, was his second arrest on the monstrous accusation of


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being a Japanese spy. According to NKVD version, the virus Father discovered had been brought from Japan and was the Japanese encephalitis virus. Thousands who lived in the Far East and worked out in the taiga, and the soldiers and officers of the Red Army who were based there, were saved by the discovery made by Zilber and his colleagues. That did not halt the secret police. It took enormous efforts to secure his release in 1939. Yet in 1940 he was arrested, once again, on the very same ludicrous charges and this time Father was sentenced to 10 years in the camps for being ‘‘a Traitor to the Motherland’’ (the sadly notorious Article 58 of the Soviet Criminal Code) (Figure 1).

Fig. 1. A portrait of Lev Zilber (1943) drown by anonymous author in GULAG at far-north of the European part of Russia near Pechora river (archive of Zilber’s family).

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Only in March 1944 did a direct appeal to Stalin, passed to the dictator’s immediate entourage and signed by two dozen of the country’s leading scientists and scholars, resulted in Father’s immediate release, though the charges against him were not withdrawn. Lev Zilber was a man of iron will and great gifts and he endured a fate in which tragedy mingled with joy. I shall not dwell further on his life here. For those who read Russian, however, there exists a large 700-page biography that I wrote with the biologist and historian of science Elena Levina [5]. While in prison Father created in 1943–1944 the viro-genetic concept of the origin of malignant tumors. His work was published in the USSR in 1945 when the Iron Curtain rendered it quite unknown to the outside world [6,7]. Only in the late 1950s when Zilber was able, once again, to travel to other countries did foreign colleagues learn of his theories and investigations. His unexpected and sudden death on 10 November 1966 at the age of 72 was a great grief to his family, his students, and colleagues. I am proud that he entrusted me with the task of writing a chapter in his last book, which he finished a day before his death [8]. Undoubtedly his qualities and character influenced me greatly. He set an example as a scientist, displaying inexhaustible optimism, vitality and love of life, an enormous capacity for work, limitless erudition and possessed a rare combination of qualities, being both a deep thinker and a virtuoso experimenter. Despite that, Father never directly intervened in my education or upbringing. He did not ask how I was doing at school or university; he never ticked me off or interfered in my private life. I lost my father when I was 30 and to this day I feel his absence, and a void that nothing, and no one else, can fill. At the request of a family friend, Professor George Klein of the Karolinska Institutet, Stockholm, I wrote a scientific biography of my father. My co-authors were his closest pupil and scientific heir Professor Garry Abelev and my younger brother Fyodor [2]. After the biography was published I received a letter from the Nobel laureate Howard Temin. It is included here in the form of a photocopy without commentary (Figure 2). Cancer Research journal illustrated on one of its covers Abelev’s discovery of the alpha-phetoprotein, which was made in Zilber’s department. What follows are merely a few of the English-language references to his investigations [9–19].


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Fig. 2. The letter addressed to myself from Howard Temin as a commentary to the publication about L. Zilber [2].

On our return from German captivity, Mama resolved to devote her life wholly to her husband and family. She quickly learned to use a typewriter and typed up Father’s manuscripts – between 1945 and 1966 he wrote six major monographs; she became

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particularly adept with lists of Russian- and English-language references. Mama was dearly loved by her numerous friends, many of whom she had known from their schooldays together at the pre-Revolutionary gymnasium. They became friends of the family and I have heart-warming recollections of these kind, loyal and genuine members of the intelligentsia. My mother liked to entertain and invite guests to eat with us; she was exceptionally good in the kitchen. When Professor O. Muhlbock, a friend of my Father, was about to return to Europe he insisted that Frau Valeria make some of her pies for the journey. Enchanted, she fulfilled this request. Our home was open to visitors and friends and on New Year’s Eve or my parents’ birthdays a great many gathered there. The merrymaking was endless. We put on theatrical performances, composed humorous verse and epigrams or birthday and New Year greetings, and we sang romantic ballads of our own making. This, moreover, was in the period from 1946 to 1966 when there was still a long way to go before the Communist regime was abandoned. Father’s sudden and untimely death was due, in part, to 7 years and 6 months spent in prison and in the camps. In part, it was the result of his ceaseless, intense work as a scientist. After he died, Mama devoted all her efforts to bringing up two granddaughters and a grandson. She outlived her husband by 15 years but could never reconcile herself to his demise and slowly faded away. Mama’s own father, Pyotr Kisselev was a very well-known Moscow surgeon who died early, before I was born. From her tales I heard of his difficult character and this, probably, to some extent passed down to his daughter. When both parents are very strong characters, the family life cannot be a simple, straightforward matter. Yet it was thanks to this strength and staunch endurance that my parents survived, preserving their family and raising children during the most terrible years in the 20th century history of Russia (Figure 3).

Fate of the Family During the War, 1941–1945 When Hitler invaded the USSR early on the morning of 22 June 1941, Mama was working at the Central Institute for Epidemiology and Microbiology. There, before his arrest, Father had headed a laboratory. We lived in an apartment building owned by the


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Fig. 3. Lev Zilber and Valeria Kisseleva at the celebration of Zilber’s 70th anniversary birthday at Gamaleya Institute in Moscow (March 28, 1964).

institute and my family continues to live there today, almost 70 years later. In the Soviet terminology of the time Mama was an FMTM, namely a Family Member of a Traitor to the Motherland. It was courageous of Lev Yankelevich, director of the institute, to give her a job and our family is eternally grateful to him. Had mother not found employment, her two under-age children, aged 6 months and 5 years, would have simply starved to death. Rumors quickly spread: the Germans were about to carry out a terrible bombing campaign against Moscow (as it turned out that was not the case). Mama decided to send us away with our nanny to friends living at a dacha. It was approximately 60 kilometers west of Moscow, not far from the small town of New Jerusalem. On Saturday evenings she brought us food from the city and on Sunday she returned to work, taking the local train. We had not the slightest idea where the fighting was. Black loudspeakers hung in public places – it was strictly forbidden to possess your own wireless receiver – and they spoke of battles near Brest, on the Soviet Union’s western border. In fact, the Germans were moving rapidly towards Moscow. By the middle of October, it became impossible to conceal that the Germans were not far away and panic broke out in the Soviet capital, with mass flight to the east and the south. That Saturday Mama, as usual, brought food from Moscow but she could not

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return. Local trains and buses were canceled. The radio endlessly reported that all approaches to Moscow had been mined and called on the population to stay put. Quite by chance, on the Sunday when they closed all routes back to Moscow, Mama’s younger sister Anastasia came to the dacha. (At home we called her Naka, a short name I invented since her full name was too hard for a little boy to pronounce.) Naka wanted to help mother with the children and had also brought some necessities. My aunt worked as a maintenance engineer at the electric lamp factory in Moscow. Her failure to turn up for work could be regarded, under wartime conditions, as desertion and lead to her arrest. Luckily, she managed to ring through to the factory administration and explained what had happened. The Germans appeared but did not do us any harm. We were saved by two fortunate coincidences. During their childhood Mama and her younger brother Andrei had spent the years 1910–1912 in Zurich. There they lived in a pension and learned to speak German fluently. They were addressed only in German, naturally, and were not allowed to talk Russian, even among themselves. (Both retained knowledge of the language until they died.) When Mama answered the officer in a ‘‘most superior’’ German he was astounded. ‘‘Is madam German?’’ he immediately asked. ‘‘No,’’ Mama calmly replied, ‘‘I am Russian.’’ On hearing perfect German from a Russian woman the soldiers behaved in an entirely civilized fashion and quickly left. This gave Mama time to recover and gather her wits about her. More terrifying was that many who lived nearby knew Mother’s husband to be Jewish. Had anyone dropped a hint to the Germans, my brother and I – and, probably, Mama and Naka as well – would have received one-way tickets to a certain death. No one said a thing. No informers were to be found there; we escaped that fate. It helped, of course, that we bore a profoundly Russian surname. (It derives from an old and popular dish, formerly served as a dessert in Russian villages: kisel is made of starch, boiled with berries or fruit juice, and is a favorite with tiny children.) My brother and I were entered in Mama’s identity document and there was no mention of Father’s name there. There were no photographs of Father at home though I knew that Mama hid a very small photo of him somewhere and kept it with her throughout the war.


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The frosts were bitterly cold and the oil in the German tanks froze. The dacha was heated with logs and with firewood that we gathered in the surrounding woods. The German soldiers felt the cold terribly since their leather boots and clothing were not intended for daytime temperatures of about minus 30–35 1C and they went from one house to another, confiscating sheepskin coats and warm felt boots from the inhabitants. Round their heads they wrapped knitted hoods so that only their noses and eyes were visible. I well remember that they presented quite a sorry spectacle. The German forces had come to a halt although they could already see Moscow through their powerful artillery binoculars. They had been stopped by the winter cold, Russia’s main ally, and by the army divisions from Siberia that had managed, at the very last moment, to place themselves between the invaders and the city. When the counter-attack began, a German officer appeared and told Mama that we must be ready within 2 hours. We should take the minimum necessary and would be evacuated with the retreating German forces. Little Fyodor, Fedya, was a baby in arms, not yet 11 months old. We took bags of meal with us (Mother’s milk had dried up long before), and put on all our warm garments. I insisted we take my beloved cat and he was placed in a basket, tied up with a rag: with one hand I clutched the basket, with the other I held on to my Aunt Naka. We were loaded onto an enormous truck, like a Studebaker with a canvas top. The vehicle was crammed with empty tank-fuel barrels. The Germans did not abandon these containers, even in retreat, and they sat us on top of them. It was awful. The frost was so severe that if you touched the barrel your skin immediately froze to it. ‘‘Leave the metal alone,’’ Mama kept warning us: ‘‘don’t touch it.’’ When the truck set off the barrels began to rattle. We were mercilessly tossed about on the cratered roads that had been of poor quality before the bombs started falling. On one of these potholes the cat jumped from the basket and out of the truck. He did not want to remain in captivity. We were driven westward with those barrels, all the way to Smolensk. There they put us up in the railway station, which had been bombed earlier by the Germans. No roof was left and we sat, squeezing together, on the oak benches in the former waiting room, trying to preserve what little warmth we could. Fedya, starved and chilled through, had stopped howling. He only made a

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quiet whimpering. A slender, blond-headed German lieutenant passed by. Mama said something quietly to him in German. He stopped, looked at us, said something in reply and went away. After a while Mama left, leaving Naka to look after me and Fedya. Some time later she reappeared, concealing a loaf of bread wrapped in cellophane. Later I learned that the officer told Mama where he would hide us some bread from his own ration. Mama had gone to fetch it. Once again her excellent knowledge of the language had saved us. At the station the Germans were handing out boiling water and that also helped. It was frightfully cold, about –40 1C. Above our heads the pure stars burned brightly, shining through the shattered roof. When we had been driven as far as Polotsk (today it’s in Belarus) Mama was taken on at the army hospital. As well as knowing German she had received a medical education. Though it did not qualify her as a doctor, it was more than sufficient for her employment as a nurse. The German military doctors were welldisposed towards her and would call her Frau Valeria. There was no call for an electrical engineer at the hospital, my Aunt Naka’s occupation, so she was given the job of stoking the furnace and washing the dishes. They were both very fortunate to get such work. Mama had access to the doctors and to medicine while Naka in the kitchen kept warm and well-fed and could bring some food home for my brother and me. We lived in a one-storey wooden house on the outskirts of Polotsk. Its previous inhabitants had died or had gone into hiding or, perhaps, they had fled from the Germans. All day long Fedya was in my care and so, at the age of 6–7, I found myself playing the role of a very juvenile father. Naka was then 27, unmarried and a most attractive woman: she was a classic Russian beauty, with large eyes, rosy cheeks, and regular features. Mama was terrified that the soldiers, as they got better, would begin casting eyes her way. Rubbing soot on Naka’s cheeks, spoiling her hair-do and dressing her badly, Mama did all she could to make her sister unattractive. She ordered her not to leave the kitchen and let the soldiers see her. It all helped. Let me recall two episodes that remain fixed in my mind from our time in Polotsk. Fedya hardly grew. He was ‘‘pint-sized,’’ very small and thin, and due to a lack of any vitamins and to undernourishment he acquired impetigo. It is a little-known illness these days. The face becomes covered with a crust, peeling in scabs and only the eyes remain unaffected. An unbearable itching


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opens the way for secondary infection. Especially for the mother, the child’s appearance is terribly distressing. ‘‘Frau Valeria’’ described Fedya’s condition at the hospital. A German doctor came and examined my brother. He wrote out a prescription for medicines that Mama was then issued at the hospital. She was given vitamins and allowed to take certain foods from the kitchen. Fedya quickly began to recover. The traces of impetigo would remain on his face, however, for years to come. There were many partisan units around Polotsk and at night, from our house, we could see the flickering of fires in the forest. After dark the Germans did not walk about the town and most certainly did not risk venturing beyond its limits. Then Polotsk would be plunged into utter darkness so as not to offer any target for attack by Soviet planes. During such a pitch-black night two people came to our house. They knew where Mama worked. They said they needed medicaments for wounded partisans and asked her to get them from the hospital. It was a very dangerous situation. German motorcycle patrols rode around the outskirts of the town and might notice the appearance of these outsiders. We would all be shot for having links with the partisans. If it was noticed at the hospital that medicaments had gone missing, the Germans would know who was responsible and we would also suffer the ultimate punishment. Even if Mama managed to take what was needed undetected, a second visit from the partisans would be no less dangerous than the first. The visitors were unshaven, dirty and thin, as mother later described. How she could see this I do not know. The partisans asked her not to light a candle and their conversation took place in the dark. I could only hear quiet male voices and Mama’s yet quieter replies. After the war I learned that Mama had somehow managed to take much that the partisans requested and pass it to them: gauze, bandages, iodine, streptocide, and more besides. She was told that the partisans’ commander had ordered, in gratitude, that the whole family be transferred to the partisan camp and then to the ‘‘mainland.’’ (The partisans had quite regular links by air to the military command in the unoccupied territory or ‘‘mainland’’.) After agonizing uncertainty, Mama turned this offer down. She never gave me the reasons for her decision. However, I think, as the wife of a ‘‘Traitor to the Motherland,’’ a woman who was now working for the Germans – it did not matter

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that this was not voluntary – she and her family would have little chance of survival once they fell into the hands of the NKVD. She could not say this to the partisans. She would be of more use to her country here, she said, since medicaments might again be needed and then, once more, she would try to help. Her words would be passed on to their commander, they replied, and, if they were in need, they would come back. They did not return. I do not know if the Germans discovered that anything was missing. By then they were preparing to retreat further. The front was approaching Polotsk. I shall not recount all that happened to us then. That would form a lengthy story of one family’s experiences during World War Two while the present work is a single chapter in a series of personal recollections of scientists involved in molecular biological sciences. Let me recall the last stage of all. We were in Germany at a labor camp in the industrial town of Chemnitz. Mama and Naka were set to work, in 12-hour shifts with a 30-minute lunch break. Their job was to turn out basic metal parts, the purpose of which was unknown. To maintain some contact with the children, Mama and Naka took different shifts: one would work nights from 6 pm to 6 am, the other worked days from 6 am to 6 pm. The factory was some way from the camp and they were herded there on foot in any weather. The hardest part was to keep standing at work. After the war Mama developed serious varicose veins in her legs, which were a great trial to her. Naka, who is today over 92, has suffered for many years from varicose ulcers of the legs. The camp was located in a former toy factory. The building had thick brick walls and large workshop floors where the machines had been torn from their bases to make room for the plank beds. The beds were wooden, two-tier constructions, with the head against the wall. To increase stability they were screwed to one another at the head and this created a wooden runway for the rats. At night the rats ran literally over the heads of the tormented people, and brazenly bit away parts of their ears, lips, or noses. A terrible cry would ring out, the light was turned on and any object near to hand was hurled at the rat. It was little help. There was no rest at nights, and the shrieks were repeated many times. Finally, the prisoners could stand no more. A ‘‘delegation’’ was sent to the camp commandant and Mama acted as interpreter. The Germans responded and workers came with


15

liquid cement, mixed with broken glass, and filled the numerous holes in the floor, the ceiling and the walls of that huge workshop (I well remember that 60 people slept there). It helped. The rats declined notably in numbers but they did not completely vanish. It was there I learned that a hungry rodent, with its unbelievably sharp teeth, could gnaw through cement – that was why broken glass had to be added. They attempted to introduce cats but the rats quickly ate them or the cats turned tail and fled. When a student biochemist and, later, a young research assistant, I came to work with white rats. Outwardly they have nothing in common with those common grey rats, either in size, form or color. Yet I would mercilessly behead them. They were quite innocent but a hatred of their kind, born of the night-time horrors of our life in that camp, proved firmly embedded and impervious to reason. For the most part, the guards did not treat the prisoners badly. There was one short, fat guard, however, with a luxurious moustache, who would come (at 4.30 am) to wake the morning shift. People were worn out by the work and sometimes, due to the rats, had hardly slept at night. By morning, as a result, their sleep was particularly deep. This bewhiskered sadist would bend over their ear and blow into a special, very shrill whistle. His victim leapt about the plank bed, usually crying out, unable in their drowsy state to understand a thing. ‘‘Aufstehen!’’ yelled the guard, and was extremely pleased with the result of his efforts. From great to small, he was hated by everyone. The other guards, it was said, considered him a degenerate. While the adults worked at the factory, the children, aged from 2 to 14, remained locked in the camp. Anyone older than 14 worked alongside the adults but there were few of that age among us. Surprising as it seems, the Germans paid the prisoners for their work. The cynicism lay in our inability to spend this money, since we could not leave the camp. It was considered that those who worked could buy something at the factory: food of some kind could be bought there, it seems. Since they were not free to move around at work, this was a fictional opportunity. A way round was found. Nights were passed in utter darkness and if any town-dwellers infringed the blackout the Gestapo could simply arrest them. To avoid attacking one another, the Germans fastened a patch of luminescent material to their clothes, visible from a distance of 1–2 meters. It was a rather phantasmagoric

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spectacle. No one could be seen, merely pale white patches that moved towards one another and then parted. The Germans were desperately afraid of aerial bombardment by the allies and an extremely thorough blackout was their only defense. This worked in our favor. We devised the following strategy. Several families put together their modest earnings and entrusted them to a thin child, like myself. I was hoisted onto the shoulders of the tallest person and was then able to climb on top of the tall brick wall surrounding the camp. The guards did not walk about the camp – evidently, they were afraid – neither did they venture outside. Instead they kept together in the guard room next to the massive metal gates (these opened only twice a day, to let the workers leave and return). No one prevented us climbing over the wall. In order to scramble down the other side the absconder had a rope with which he cautiously lowered himself to the pavement. It all had to be done very quietly so as not to draw anyone’s attention. There were few passers-by after dark so the risk of landing on someone’s head was not great. The patrols moving about the town did present a danger: they were looking for narrow beams of light from doors and windows, an infringement of the blackout. They rode on motorcycles with dimmed and shielded lights or walked about in groups of two or three. German punctuality meant that their visits to our street always occurred at roughly the same time. This enabled us to avoid encountering them. Once I found myself outside, the money tucked inside my shirt, I made for the nearest shop. It was very close, about 100 meters away. Caution was needed when entering. I had to make sure, by listening outside, that there were no customers within. Then I entered in such a way as not to disrupt the total blackout. As a rule, there was a large, dense black curtain hanging behind the door. Once I had opened the door I checked that the curtain was in place. Closing the door behind me, I drew back the hanging and entered the shop. They chose to send me because, like another two of the boys, I was able to talk to the shop owner in German. Very politely I greeted her, wiping my feet at the entrance and respectfully enquired, imitating a grown-up, how she was. Then I would proceed with the purchases, telling her what I wished to buy and the sum I was carrying. The shop owner told me how much I could buy and then poured the meal (pearl barley, semolina, millet – the cheapest there was), the sugar and, sometimes, other items into rough-woven bags. There was no


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chance to haggle, naturally. But we knew the prices and she did not cheat us. Sometimes she gave me a handful of the cheapest sweets, ‘‘For the little ones.’’ Of course, the shop owner knew very well whence I came in the surrounding darkness. Business is business, however, and no one prohibited selling things to children. She was not breaking the law. I bade her farewell, expressed my thanks and quietly, carefully disappeared. All the purchases I tied behind my back on a special belt: my hands must be free to climb and we did not have a rucksack. Then, feeling my way back along the wall that bounded the camp, I would give quiet signals which were answered from the other side. A rope was let down, I grasped it with all my might, while three lads pulled it back. I returned to the camp. All I brought with me was then divided between the ‘‘contributors.’’ Making use of that food was a problem in itself. A porridge was made of the meal in kettles in the back yard where the guards feared to go. As fuel we used the peat and coal briquettes with which the old toy factory, that is our camp habitation, was heated. An enormous truck would empty the briquettes in the yard and they were then shifted to the basement where the furnace was located. Theft of the briquettes was allotted to the smallest among us. Their job was to sidle up to the pile unnoticed (Fedya was among this group), shove a briquette under their shirt, and quickly make their getaway. It was a dirty task – their bodies, especially their tummies, turned a brownish-black – but it worked very well. One briquette was enough to boil a kettle full of runny porridge. Few were entrusted to do the cooking. It was important neither to waste a single calorie of heat nor to disrupt the blackout. The flame had to fit the bottom of the kettle exactly or be of slightly narrower radius. In the basement, children under 6 years old were given a mug of milk every day. Fedya stood in line, received his portion and drank the milk, then passed the mug to me. After a little while I went up to the woman distributing milk and said, in German: ‘‘For my little brother, please.’’ She filled the mug although the deception was obvious. All the workers had to wear a stitched OST (Easterner) on their clothing but sometimes it could be turned about so that it was not visible. Mornings adults were given ersatz coffee, 200 grams of bread, a small dab of ersatz butter, and some jam (one teaspoonful). Work continued 7 days a week but on Sunday they

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worked until the mealtime and in the evening they were allowed to move outside the camp. On 6 March 1945 American and British planes bombed Chemnitz all night long. The town was destroyed. We were all in the basement under one of the camp buildings. When the explosions of the bombs and aerial mines had died away we clambered out. Dawn was not yet breaking but it was as bright as day from the fires. The buildings of the camp were still standing. One bomb had plunged into the earth nearby without exploding. Another had fallen between the supporting walls and penetrated two stories without reaching the ground floor and basement. Not a single pane of glass remained in the windows, however. The rooms were covered with fragments of broken glass and brick dust. The engineering plant had been hit. Work halted there. Living in the camp became impossible without electricity, water, and sewage disposal. The walls and roof had been partially damaged. The administration announced that it was transferring everyone to a stadium beyond the city limits. I very clearly remember how we moved from the camp to the stadium across the burning city. We went on foot, carrying all we needed. The smoke stung our eyes, there was a rasping in our throats and the streets were blocked with rubble. All the time, we had to find a way round the obstructions. We walked for ages. I began to feel a pain in my lower stomach and it was very hard for me to keep going. We took it in turns to carry Fedya. At the stadium we were given places beneath the seats and conditions were better than in the city: there were no fires burning round. A week later when the fires had died out and some of the streets had been cleared we were taken back to the camp. Electricity and water had been restored. The walk back was much easier. I shall never forget the bombs falling, and the road through the burning city. On 6 May 1945 Soviet tanks burst into Chemnitz. Before their arrival an SS motorcycle unit swept through the town, retreating from east to west. They were in a hurry to surrender to American troops, which were positioned outside the city but had not attempted to capture it. It was a frightening moment. The fleeing motorcycle riders fired at the windows of the houses where white flags, sheets, pillow cases, or anything white was hung. The war was nearly over and the civilian population, naturally, wanted to avoid bloodshed. The crazed SS men tried to break into the camp


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and attacked the gates, demanding that the guards open up. All the adults among us rushed towards the guards to stop this happening – but it turned out that they were just as afraid of the SS men. They defended the gates, which were of such thick iron that they easily resisted sub-machine gunfire. When the motorcycle riders understood the futility of their efforts, they flew off, further westwards, and in a few hours we ourselves entered the town. The guards had unlocked the gates and fled. After liberation, we were transferred to Dresden, and then to Breslau (today Wroclaw in Poland) where there was a camp for displaced persons in the former German military cantonment. Twenty seven thousand of us waited there for repatriation to the Motherland. Mama dictated to Naka the text of a postcard addressed to their mother, my granny Sofya Pervushina: We were alive, it said, and waiting to be taken back home. It contained the phrase: ‘‘If Lyova [i.e. Father] is alive, pass this postcard on to him.’’ Since November 1941 we knew nothing of his fate, just as he knew nothing of ours. He had not even seen his younger son, who was born when he was already in prison. It took 30 days for the postcard to reach Moscow. When my grandmother received it, she took it straight to Father who had been trying, by every conceivable means, to learn what had happened to his family ever since his release in March 1944, over a year before. Everywhere the reply was a discouraging ‘‘Missing, fate unknown.’’ When he saw the postcard and read its message, his heart must have leaped in his breast. Father was acquainted with the then People’s Commissar (Minister) of Health G.A. Miterev. The minister knew about the disappearance of Professor Zilber’s family and about Father’s tribulations (3 times arrested and thrice released), and he helped Father take a military plane from Moscow to Berlin. Then Father was driven by chauffeur in a medical vehicle to Breslau. On board they had two sub-machine guns for protection: in July 1945 there were still isolated SS units that had not surrendered. Father found me and Fedya sick with stomach typhoid fever. I shall not describe our reunion. Even today, more than 60 years later, I find myself short of breath at the recollection and my heart begins to beat frantically. We drove back to Berlin in the same vehicle, taking every possible medical precaution: the doctors feared I might suffer an intestinal haemorrhage since I was very weak (Fedya had already

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begun to recover). In Berlin our military doctors dissuaded Father from flying us straight back to Moscow and insisted that I be treated first. Father gave way and, at the request of the Soviet military command, immediately began to study the German medical and biological institutions in Berlin and the surrounding district. On 30 July we boarded a Douglas military plane at Schoenefeld aerodrome but when the plane pulled onto the flight strip a signal was given to stop. A Willis jeep drove up and we were asked to descend. An officer told Father that we would take the next plane. This one was urgently needed to transport captured German generals to Moscow. We and our belongings were loaded into the Willis and driven through the pouring rain to a small building on the aerodrome. Forty minutes later we were put aboard the next Douglas and this time flew all the way to Moscow. The trip took 6 hours. Mama and Naka were unwell during the flight. The plane shook and shuddered, it dived up and down but, despite my feeble state and illness, I looked out the window with enormous interest. It was my first time in a plane and an international flight, at that: there I was, flying from Berlin to Moscow. We returned to Moscow and to Father’s pre-war apartment. Thanks to the noble behavior and selfless support of his friend Tatyana Dvoretskaya, the flat, by some miracle, had not been confiscated. Anxious weeks followed. In turn, Mama and Naka were taken to the NKVD, and separately cross-examined about everything that had happened to them, literally day-by-day, weekby-week, from November 1941 to May 1945. When they left each time for a new interrogation Father was beside himself with worry: he had no confidence that they would return home again. We knew that many who survived German captivity were sent straight to Soviet camps and never came back. To our great relief these regular summonses finally ended. Mama and Naka were given back their identity documents, their residence permits to live in Moscow and were left in peace. After the war we all became fatalists. Had we not known the truth our story would have seemed incredible. Father had survived and was still able to work though he had spent more than 7 years in Stalin’s camps and prisons. Two women with two under-age children had all returned, alive and well, from Germany after the war and Mama and Naka had not then been herded off to Siberia. By an extraordinary coincidence, the whole


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family had narrowly escaped death on 30 July 1945. Half a minute before the plane’s departure we were taken off a flight that never reached Moscow but crashed en route y War strips everything down to essentials. It renders all black or white. During those almost 4 years we came to know many noble, brave and humane people, both among our fellow citizens and among our enemies, the Germans. We also encountered cruelty, base behavior and heartlessness and, again, it was to be found among both Russians and Germans. I learned that wartime lesson once and for all. It’s the reason I do not distinguish people according to their language, skin color, religion, or ethnicity. I divide them into the good and the bad. My Father and Mama had exactly the same attitude.

After the War. The ‘‘Doctor’s Plot.’’ The Death of Stalin We returned to Moscow on 30 July 1945. School re-opened on 1 September. I was 9 years old but I did not know how to write and I read with difficulty. At that time my grandmother Anya, Father’s mother, was living with us in Moscow and she gave me a book Hari and Kari about Indian elephants, which contained wonderful pictures. A day or two later Grandma asked me how I liked her gift. I answered honestly: the pictures were nice. ‘‘But what about the story?’’ enquired Grandma Anya. ‘‘I find it difficult to read,’’ I answered, diplomatically. She went straight to Father: ‘‘Your boy can’t read!’’ Papa remained imperturbable, ‘‘Never mind, he’ll learn.’’ Mama was deeply offended: ‘‘You should be thankful we’re alive! We had no time for books there.’’ I was old enough to join the third class at school but it was pointless. The family discussed what to do. They decided I would study at home with the help of Naka (my aunt was eminently well-suited to be a teacher) and take lessons, 2 times a week, from a schoolmistress at her apartment. In a single school year I absorbed the program of study for the first two classes and on 1 September 1946 I entered the 3rd class. The majority of my classmates were a year younger but our excellent teacher was Anna Prosvirnina, the same who had privately prepared me to go to school. I found the work easy although I missed many lessons in the 5th and 7th classes due to two serious illnesses, polyneuritis and viral jaundice. On graduation from the school

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I won the gold medal, which entitled me to go to Moscow Lomonosov State University without sitting an entrance exam. The years from 1945 to 1953 were very hard in the Soviet Union. Cities had been bombed and people were disfigured by the war. There were vast numbers of widows and orphans and of devastated and burnt-out villages. There was an acute food shortage and criminal gangs roamed the large cities. Against such a background ideological campaigns were waged, against ‘‘fawning on the West,’’ ‘‘cosmopolitanism,’’ and ‘‘formalism in the arts.’’ The great poet Anna Akhmatova, the wonderful writer Mikhail Zoshchenko, and the outstanding composer Dmitry Shostakovich were subjected to harassment and persecution. In 1948 the August session of the Academy of Agricultural Sciences completed the rout of genetics in the USSR: it was a campaign that had begun before the war and led to the death in prison of the remarkable biologist Nikolai Vavilov (see [20]). In chemistry it was the theory of resonance that came under attack. Physicists were left alone: they were busy making the atomic bomb and, later, the hydrogen bomb. In addition to Trofim Lysenko pseudoscientists of a lesser calibre also appeared. One was Olga Lepeshinskaya, who is not to be confused with her namesake, the famous ballerina. Another was Georgy Boshyan, a veterinary doctor, who proclaimed that bacteria might be transformed into viruses and vice versa. They all exploited the support of the Party and the state apparatus. In 1952 the ‘‘Doctors’ Plot’’ was fabricated. The best doctors in the country, who treated members of the government and the Politburo, were accused of being ‘‘saboteurs,’’ and were put in prison. Most were Jewish and their arrest formed part of a statesponsored campaign of anti-Semitism. Father was not a medical doctor but he was a member of the Academy of Medical Sciences. A very well-known figure, he had publicly argued with Boshyan and Lepeshinskaya and, most important of all, he was a Jew who had been imprisoned 3 times before. Expecting arrest or, at best, deportation from Moscow, Father burned notebooks containing the telephone numbers of friends, acquaintances, and colleagues. (He also tore out dedications inscribed in books from his extensive library.) Indeed, he stopped using the telephone altogether. Phone conversations were being tapped and, after his death in 1966, the device used for this purpose was, before our eyes, torn out of the wall next to our


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flat. At the laboratory, Father met with each of the research staff and discussed in detail his plans for the next 1–2 years: previously he made arrangements no more than 1–2 months into the future. Father asked them not to phone him. Some agreed only at his insistence. Others, a small minority, decided not to phone him in the interests of self-preservation. No fourth arrest followed. Two circumstances worked in Father’s favor. With his staff Father discovered specific tumor antigens, thereby opening a new chapter in oncology and immunology (see [11]). This discovery enabled the lab to begin developing vaccines against cancer and his superiors were informed of this. The work was recognized to be of special importance and classified as a state secret. The staffing of the laboratory was even expanded and a vehicle was provided so that tumorous material might be collected from medical clinics. In March 1953 Stalin died. The imprisoned doctors were soon released; two of them had died in prison. How many grey hairs these dreadful years added to the heads of my parents, I do not know. Both did all they could to prevent their children being affected. We knew little of what was going on. We guessed more from snatches of conversation, hints and the expression on people’s faces or in their eyes. Dozens of books have now appeared in Russia about the history of that period and it appears yet more frightening than it seemed to us at the time. At home there was never any talk about politics and the names of Soviet leaders were not spoken aloud. Our parents set an example. The children followed their lead. We lived next to the institute where Father worked. It was, literally, a 2-minute walk and I could run off to see him there at any free moment. He was relaxed about my visits: he neither encouraged nor hindered them. There, for the first time, I saw a centrifuge. It was a manually operated model, bolted to the table, with two test tubes. I was allowed to ensure their weight balanced on the nearby scales. It was there I saw my first microscope. They let me look at a slide of a blood sample after it had been dyed with haematoxylin. I watched Father skilfully draw blood from a rat’s tail or a vein in a rabbit’s ear. The only condition was that I did not disrupt anyone’s work. Since several of Father’s researchers were not just colleagues but friends of both parents (Mama took an active interest in the laboratory), I was surrounded by a kindly, ‘‘family’’ atmosphere.

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I remember the special rooms (‘‘boxes’’) for sterile work – the snow-white gowns, gloves, and masks. They taught me to count the constituent elements of blood and I would sit at the microscope, wearing a serious expression and noting the number of leukocytes, erythrocytes, and so on. When I reached the final classes at school I had so many lessons that I almost stopped visiting the lab. However, from Father and his conversations with Mama, I still knew what went on there. The institute had an enormous stable, holding up to 600 horses. It was also near our apartment and the laboratory. The institute was engaged in production as well as research: the horses were kept to provide immune sera. I would go and see them. To the stable lads I was ‘‘the professor’s son’’ and they were friendly, permitting me to lead the horses by their bridle, and letting me ride on them. The beasts were neatly groomed, plump and well cared for (otherwise they would not have given good-quality serum!). When I mounted a horse, it walked calmly in a circle, without bucking or trying to throw me, casting an eye my way. These visits gave me great pleasure but, just in case, I did not tell my parents. I did not know how they might react.

School Life The secondary school I attended was almost 3 kilometers from home. No public transport went that way so I made the journey on foot. It took about half an hour to get there. Part of the way took me through a very pretty park. Times were hungry and in the first years after the war they did not feed children at school. I took sandwiches, which Mama prepared in large quantities so my hungry schoolmates could share them. Once I was stopped by three unfamiliar lads (they were not from our school), who tried to rummage through my knapsack in search of food. They seized all the sandwiches and off they ran. It was no use resisting. It was three to one and two of them were much older and stronger than me. When my schoolmates found that the knapsack lacked its usual sustenance I had to explain. They were indignant. The head boy in our class was a fine fellow, who enjoyed undisputed authority among the teachers as well as his classmates. Half Ukrainian, half Tatar, he was 3 years older than me: he spent 2 years in each class since he lived in appalling conditions and could


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barely do his homework there. He demanded that I describe the robbers in detail. ‘‘Don’t be frightened, Kissel (my nickname at school that means Fruity),’’ he said: ‘‘Tell your mother that we’ll look after you.’’ I didn’t say a thing to her and, as usual, she made a large number of sandwiches. At the very same place, the same three lads appeared and surrounded me like an old acquaintance. They expected I would open up my knapsack but at that moment our head boy and three more of my schoolmates loomed behind them. Their revenge was brutal. The unlucky robbers fled. I walked the rest of the way to school without interference and handed out the sandwiches. A few minutes later my defenders returned and there was rejoicing and merriment. Next day my three tormentors again appeared, bearing clear traces of yesterday’s battle. ‘‘Don’t be scared,’’ they called to me, from some distance: ‘‘We’ll see you to school so nobody bothers you.’’ I never expected such a noble response. Wary of a trap, I was cautious but we walked to the school together, where they shook my hand and started to go. Opening my knapsack, I gave them each a sandwich. And that was the end of the sandwich wars. The school was located on what were then the outskirts of Moscow. It was mostly a working-class district and the school itself, as Russians put it these days, was ‘‘not prestigious.’’ The teachers and the students, however, were decent people, despite the difficult times we lived in or, perhaps, because of them. Many of the teachers were Jews, driven out of schools in central Moscow as part of the rabidly anti-Semitic campaign. Some were superb pedagogues and, like many others, I am obliged to them for the fine education I received. All my life I have remained friendly with certain teachers and classmates from those years and it’s a great pity that many have not been with us for some while now. At school I was regarded as a student of the humanities. I was the best when it came to literature and the Russian language. Others led the field in maths and physics. When we sat our leaving exams things went seriously wrong. The maths teacher had returned from the war, suffering from concussion. This affected his memory and concentration but we knew he had fought at the front and forgave his sometimes strange behavior. At our final algebra exam he set the wrong equation. As a consequence the final result looked very odd. Not trusting my own judgement I took the advice of our best mathematician. I had given the correct answer, he said, but Nik Nik (that’s what we

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called Nikolai Nikolaevich, our teacher) had probably made a mistake when he copied out the equation. We handed in our work and because of that fateful error not one of us was awarded the highest grade. The examiners had looked at our answers, not the equation. The class was strong and expected to take several gold and silver medals. It was a fiasco. We went to the director of the school and explained what had happened. She was a tough lady and immediately drove to the district education department to put things straight. With great anxiety we awaited her return. Next day, we were informed that the entire class would sit a new exam with quite different examples, supervised by two unfamiliar teachers sent by the education department. We were overjoyed but the director should not to punish Nik Nik, we told her. The effect on his shattered nervous system might be too much. She gave her word. This time the exam went brilliantly. Half of the class received the highest grade, which was even better than we had expected. The director pinned up the list of those awarded gold and silver medals and we went off to the nearest bar and downed more than a few beers to celebrate. With a gold medal one could go to university without sitting the entrance exams. At that time, there was very stiff competition for entry to almost all the faculties at Moscow University: the widespread corruption that flourishes today in Russian higher education was unheard of. Had I not won a medal, in other words, I would have needed to prepare very seriously. When they heard at the school that I had applied to enter the faculty of biology and soil sciences most of the teachers were totally bemused. One of them rang our apartment and began seeking my mother’s support. Could the family not exert some pressure on me, the teacher enquired and explained, at some length, that I was a natural student of literature or, at the very least, a humanities man. Mama reassured the caller that it was not the custom in our family to pressurize someone when they were making such an important decision. I had made up my own mind and my parents approved my choice. ‘‘But his uncle is Kaverin, a famous writer!’’ exclaimed the teacher: ‘‘What if Lev is also gifted as a writer?’’ Mama burst out laughing. She didn’t believe that literary talents could be transmitted from uncle to nephew, she said. ‘‘But if, as you assure me, Lev speaks and writes well, then that will be an advantage to him, whatever his future profession.’’


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From talking to Uncle Venyamin, I knew that writers led an extremely difficult life in the USSR. He had been forced, he told me, to write new episodes and replace others in a manuscript novel that then became known as An Open Book. It was impossible to grasp what was bad about the way he depicted the life of Soviet microbiologists in that major work but the censors were implacable. There was then no chance, Father told me, of pursuing the arts or humanities in the Soviet Union. ‘‘You’ll write what they tell you, not what you think,’’ he said. ‘‘Believe me, such a life will not be to your liking.’’ I was very attached to Vera Vasilyevna, our chemistry teacher. A plump, ironic lady with large pale blue-green eyes, she was passionate about her subject and demanded a great deal more from me than was in the school textbook. That rather pleased me. She sent me to participate in chemistry Olympiads, that is competitions between pupils and schools, and she set me to work, helping younger pupils who were lagging behind. Vera Vasilyevna thereby played, most cunningly, on my indubitable self-respect and ambition. As a result, during my first year at Moscow University there was nothing in chemistry for me to do – I had already covered it all in the last two classes at school. Pure chemistry, it struck me, was closer to industry than science. A discipline in which so much had already been done left little room for development. If, on the other hand, one were to apply chemical knowledge to living things then a vast unexplored territory stretched ahead. Probably there was more intuition than genuine understanding in this response. Nevertheless, as life would show, the thoughts of a pupil in his last years at school were close to reality. Those who left school with gold medals still had to face a spoken examination. Professors and lecturers from the biology and soil faculty asked me various questions. Some concerned knowledge of particular subjects, mainly aspects of biology and chemistry. Some were of a different type: ‘‘Why do you want to join this faculty and not any other institute?’’ Thanks to Vera Vasilyevna, I gave well-supported answers to certain of the chemistry questions and could see, by the reactions of the commission, that they appreciated this. When asked how to plant an apple tree, I could also reply. Two years earlier Papa had bought an old wooden dacha and we set about planting fruit trees in the garden. That was knowledge won outside school. Next there was an

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episode, which became a family legend and the subject of endless teasing. It was an innocent question. ‘‘Which birds do you know?’’ After a certain pause I replied: ‘‘A hen and a sparrow.’’ An ornithologist had asked and, doubtless, he was expecting something rather exalted: a nightingale, for instance, a golden oriole and a wagtail. It clearly cost certain members of the commission considerable effort to keep a straight face. One of the professors came to my rescue. ‘‘You’ve answered correctly,’’ he said in a calm voice. ‘‘But perhaps you can name some other birds?’’ For some reason the first to enter my head was the parrot. I was reluctant to say that aloud: it would have been too much, after the hen and the sparrow. If I remember rightly, I said something like ‘‘eagle, swallow and swift.’’ With that the grilling ended and I became a student of Moscow University.

University: Student Community and Lysenko I entered the university in summer 1954. Stalin was no more but Lysenko was still very much alive. The biological faculty had been purged and was now named as the faculty of biology and soil sciences. The departments of genetics and of Darwinism were each dominated by people who called themselves followers of Michurin. Actually, they were typical supporters of Lysenko: among them were such odious figures as Faina Kuperman, Noi Feiginson, and Fyodor Dvoryankin. For the most part they had been drafted in from far away, using Party connections, in order to ‘‘strengthen’’ the biological faculty. Intellectually and in cultural terms they were no match for the established university staff. The notable geneticists A. Serebrovsky and V. Sakharov were hounded out of the faculty. So were the histologist and cytologist G. Roskin and the plant physiologist D. Sabinin. Fortunately, certain outstanding scientists escaped this purge: Lev Zenkevich and Jacob Birshtein, invertebrate zoologists, Andrei Belozersky who was professor of the plant biochemistry department, the zoologists B. Matveyev and G. Dementyev and a few others. Before I became a student, the dean of the faculty was I. Prezent, one of the most rabid supporters of Lysenko and his right-hand man. He had pretensions to philosophy and wanted to prove to his Party superiors that ‘Michurinist biology’ (i.e. Lysenko’s doctrines) was in complete agreement with dialectical


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materialism, the official philosophy of the Soviet Communist Party. When he was caught in certain extremely dubious activities it proved possible to remove him. The physiologist Leonid Voronin took his place but since the Party bureau at the faculty was almost entirely made up of Lysenko’s followers, Voronin had a very difficult time. I joined at a most fortunate moment. Many fellow students in that first year had intelligentsia parents who were professors or researchers at the university or other institutes. We quickly got to know one another and made friends, no matter what subjects we later selected. Our student friendship proved very strong and has lasted a lifetime. Two sisters, Elena and Natalya Lyapunova, were in the same year as me. Committed biologists, they had studied the subject at school. Their father, Alexei Lyapunov, was a major Russian mathematician from an extensive family of mathematicians bearing that name. (The institute where I work is next to Lyapunov Street, which was named after one of Lyapunov’s kin.) A man of passionate emotions and striking appearance, Lyapunov was among the founders of cybernetics in the USSR and worked with the mathematicians Kolmogorov, Sobolev, and Berg, who themselves established major schools in the discipline. As well as mathematics and cybernetics, Lyapunov had a professional understanding of certain fields in mineralogy and biology, above all genetics – the real kind, not the Michurin-Lysenko variety. On the bookshelves at his apartment in Khavsko-Shabolovsky Street lay a wealth of rare and beautiful minerals: many he had found on expeditions, others were gifts from friends who knew of his pastime. From his daughters Lyapunov learned what was going on at the biological faculty and decided to remedy the situation. Courses of lectures were organized and held in the evenings at the family apartment. Lyapunov himself taught us the correct use of statistics in biology. He invited professors who had been driven out of the university or research institutes to address us: they were either unemployed or had been forced to seek work in quite different fields. (For instance, a well-known professor of biology, Evgeny Vermel, a histologist and cytologist, had to give up his work and moved to the All-Union Institute for Scientific and Technical Information. This was good for the reference journal Biology but meant that Vermel could no longer do research.) So many started coming to these lectures at the Lyapunov apartment

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that people had to sit on the floor. There were not enough chairs. Special arrangements were made when the outstanding biologist Nikolai Timofeyev-Resovsky spoke. After the war he spent time in prison and the camps and had gone almost totally blind: a diagonal space was cleared for him in the largest room so he could walk up and down as he delivered his lecture. Among the speakers were famous geneticists, such as Sakharov, Zhebrak, and Dubinin. Thanks to these lectures and the free discussion afterwards with the speakers we gained access to the findings of genuine contemporary science and were able to fill the yawning gaps in the biology we were being taught. The contrast with all the poppycock about ‘‘creative Darwinism’’ and ‘‘Michurinist biology’’ at the university was striking. I can recall one of the experiments we were asked to perform at the department of Darwinism. We took two eggs, one from a Leghorn, the other from a speckled hen. We made an opening in both shells and exchanged the liquid white without touching the yolk. Next the opening was closed and the eggs were placed in an incubator. We were then supposed to observe that the white introduced from the speckled hen’s egg caused the Leghorn to lose its white color, and vice versa. This would demonstrate that acquired characteristics were inherited since it was known that the chicken embryo was attached to the yolk, surrounded by the egg white. Any biologist, any educated person, knows that the result would be negative. The coloring of the hen is not influenced by the white of the egg, but wholly depends on the embryo. What could Lysenko’s followers be up to? Why did they make us do experiments that could only prove them wrong? Two weeks later we found out. The department offered different explanations to different groups of students. Someone had turned off the incubator, the eggs had cooled and the embryos had died. That was the first explanation. Others were told that we had not resealed the shells properly. The eggs had become infected and gone off. The final explanation was that someone had thrown out the eggs or, perhaps, had simply eaten them. Not one of the 15 experimental eggs produced a chick. The lecturer said that we were not to blame for this failure. There was no time to repeat the experiment, however, so he asked us to write down what should have been the result, that is a change in color following the exchange of egg whites. To add conviction to his words he drew pretty pictures of white and speckled young chickens. ‘‘That’s


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what you should have seen,’’ he said, pointing to the picture. ‘‘These are results obtained by other students where everything went properly.’’ That was how the followers of Lysenko taught us to falsify scientific results. It was an unforgettable lesson. The alchemists of the Middle Ages appeared highly trained men of science by comparison. Quite soon the biological faculty became aware of the home study group at the Lyapunov apartment. None of us made a secret of its existence. A great many people attended. Some came to every lecture, others were occasional visitors, when they had the time or a lecture of particular interest to them was being given. Many shared their impressions with other students, urging them to come and listen, or they boasted about learning things that were not taught at the university. One student told his sister and she quickly informed the Party bureau at the faculty that Professor Lyapunov was hosting an ideologically flawed (antiLysenko) and privately run study group (i.e. it was unofficial and not subject to the Party’s obligatory supervision). The persecution began. The Party committee at the university was asked to deal with the communist Lyapunov. The professor had joined the Party during the war, when fighting at the front. One of his daughters, Elena, was forced to move to Leningrad (today St. Petersburg) to continue her studies. She was joined there by a fellow Moscow University student N. Vorontsov, who was her fiance´ and soon to be her husband. Later he would become one of Russia’s leading zoologists and authorities on evolution. We were then in our second year of studies and I was secretary of the Komsomol. The faculty’s Party bureau decided it must rapidly remove me from that post and summoned a meeting of all Komsomol members in our year. Almost every student had to belong to the Komsomol. It was extremely difficult, otherwise, to get into the university although I did know of two students who managed to enter the biological faculty without such a membership card in their pocket. The meeting was noisy. Party members among the students demanded, on the instructions of the Party bureau, that I should be removed. (Students who had come to university straight from the army belonged, in the main, to the Party.) Furthermore, they demanded that I be issued with a severe reprimand, to be entered in my Komsomol records, for ‘‘promoting Mendelism-Morganism,’’ ‘‘fighting against the Party line in biology,’’ ‘‘ideologically

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incorrect views,’’ and so on. Two or three of those who spoke were rabid in their hatred of native Muscovites (they came from the provinces) and of ‘‘the favored sons of professors’’ and the ‘‘rotten intelligentsia.’’ They spoke with conviction and passion but others were quite lukewarm and unenthusiastic in their demands for our punishment. Many students spoke in our defence. I had prepared well for these attacks and would not let discussion be diverted from scientific disagreements to issues of ideology. When it came to a vote the great majority voted for me to remain secretary of the Komsomol for our year. About a dozen of Party members were against. After the winter examinations in January 1956 the student record books of the 12 most active attendants of the Lyapunov study circle (his two daughters, myself, and several more capable students) were taken to the rector’s office. The suggestion was that we be expelled from the university. The Party bureau of the biological faculty had adopted such a resolution and the dean could not block the proposal, or was afraid to do so. All now depended on Ivan Petrovsky, the rector of Moscow University. A member of the USSR Academy of Sciences, he was an outstanding mathematician and a decent man. He knew Lyapunov very well, incidentally, since they worked in the same faculty and, even, I think, the same department. Petrovsky knew perfectly well what was going on in the biological faculty and clearly saw who Lysenko and his followers were. At the same time, Petrovsky had to take the Party’s view into account. He did not sign the expulsion order. We were reprieved and though a ‘‘severe reprimand’’ was entered in the files the Komsomol kept on each of us it did not prove fatal. After the Party’s 20th Congress and Khrushchev’s ‘‘secret speech’’ about Stalin and the cult of personality the Party activists in the university faculties displayed markedly less enthusiasm. The following year I ceased to be a leading Komsomol member and the reprimand vanished from sight. It would have been naı¨ve, however, to imagine that was the end of the story. The World Youth Festival, which took place in Moscow in summer 1957, was a dazzling event. One focus of its activities was Moscow University since the new ‘‘skyscraper’’ on the hills above the Moskva River had recently opened and many of the delegates were accommodated there. The rooms for students looked very attractive and, of course, the building itself made quite an


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impression. To make it easier for the Soviet students and the visitors, many from Africa, Latin America and Asia, to communicate there was a need for translators. A competitive examination in spoken English was held in all the faculties. I passed and was allocated to a group that underwent intensive study. Three months with good teachers greatly improved my English and I received a kind of diploma as a guide and translator. My job was to show the guests who were interested around the university. For instance, I would take them up to the top floor of the new building with its panoramic view of Moscow and show them the geological museum located beneath the spire of the central tower. Then I would show them the laboratories, lecture halls, sports facilities, and so on. Naturally, this made a great impression on young visitors from poor countries. It was a most interesting experience. Every day I conducted 4–5 tours, each of 2 hours duration, so the language practice was highly intensive. I had to talk all the time and the questions were many and varied. Some of the visitors’ enquiries were so naı¨ve that it was clear they had never heard anything about the USSR. Other visitors, in all probability, had been specially prepared before the trip and tried to catch us out, by asking pointed questions. Before the festival opened we had been called to a meeting where anonymous individuals explained how we should behave. They did not introduce themselves (we were told they represented the organizing committee). In particular they instructed us how to avoid ‘‘provocation.’’ By this they meant ‘‘awkward’’ questions. The variety of human types at the festival was fascinating. Nowhere had I yet come across such a range of skin color, language, temperament, educational background, and interests. The cultural program was very full. The translator’s badge, which I wore on my jacket, gained me admission to different events, including film shows. It was there I first saw Andrzej Wajda’s Canal. I was utterly staggered by the film. Certain scenes would come to mind, unbidden, for decades after. There were meetings with writers and poets, whom I would not have otherwise seen ‘‘in the flesh.’’ I particularly recall Konstantin Simonov, Nazim Hikmet, and Ilya Erenburg. Of course, our guests could not appreciate the realities of Soviet life from the few days they spent in Moscow. The festival was a well-prepared propaganda exercise and thoroughly professional in

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its execution. The aim was to promote Communist ideology. Yet less than 5 years since the decades of Stalinist terror had ended, it was undoubtedly step forward, another hole in the Iron Curtain. Only a year after the 20th Party Congress had rehabilitated millions of innocent victims, who had spent many years and in some cases, decades in prisons and camps, the festival offered a rare opportunity to catch a glimpse of the outside world through our young visitors. That year also saw the release of film The Cranes are Flying. I first saw it at the Udarnik Cinema, one of our best cinemas, nearby the Kremlin. The film went on to win the Palme d’Or at the Cannes Film Festival and I have watched it a half-dozen times more, at the cinema or on television. It is an inspired work, in my view. The gifted acting of Tatyana Samoilova, Alexei Batalov, and Vasily Merkuryev is combined with the extraordinary camerawork of Urusevsky, under the profound direction of Kalatozov, in a film that deals with eternal themes: love, treachery, fidelity, betrayal, honor and disgrace, duty, and base behavior. A year ago I watched the film again, almost 50 years since it was made. That is an unthinkably long time for a cinematic work. Comparing the reactions of a young man who was barely more than 20 with the feelings of a 70 year old, I was amazed. The passion, sincerity and authentic emotions of the film had just the same irresistible effect. I very much want my grandsons to watch The Cranes are Flying when they grow up. I now moved to the biochemistry department. There were two brilliant professors there. Today Wladimir Engelhardt is a worldfamous scientist who first discovered oxidative (respiratory) phosphorylation and the ATPase activity of the myosin (for details, see Ref. [1]). Engelhardt gave a special lecture course on enzymology. Profound, logical and systematic, his lectures were devoid of special effects and elaborate phraseology. The prompt cards, to which he occasionally referred, were a sign of the thoroughness with which he prepared each lecture. The professor, who headed the department, was Sergei Severin. A brilliant speaker, he was most erudite: an excellent teacher, he knew just how to attract the interest of students, especially female students. His blue eyes flashed and he made expressive gestures. The chalk flew across the board as Severin exploited an inexhaustibly rich variety of intonation. He seemed the complete opposite of his friend Engelhardt, who was a far more restrained man and


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outwardly rather phlegmatic. (This was a mistaken impression, as I would later realize.) They complemented one another perfectly. We received a well-grounded education in biochemistry. The department teaching staff was strong and evenly matched and they were strict with their students but indubitably well-disposed. We valued that. The department trained a number of outstanding biochemists who later acquired a worldwide fame. The supervisor of my undergraduate diploma work was Vladimir (Volodya) Skulachev, a second-year postgraduate. It was an extraordinary piece of good fortune. Volodya was only 18 months my senior and there was no age barrier between us. Tremendously enthusiastic about his own studies, his enthusiasm was infectious. He knew a great deal more than many of his age and so I gained unlimited access to a greater range of knowledge than I myself possessed. We carried out experiments together, sat in the laboratory until nightfall, and visited each other’s homes. Volodya’s mother Nadezhda enchanted me. She was an endlessly kind, gentle and domesticated woman, and most hospitable. Professor Severin approved of our comradeship. Volodya was his favorite postgraduate although Skulachev’s scientific interests were nearer, in fact, to the area in biochemistry being studied by Engelhardt. Volodya described his scientific carrier in an excellent essay published in this series [3]. My first publication was based on my undergraduate work and appeared in 1959, the year I graduated from university [21]. There followed more publications [22,23]. My contribution to these articles was, of course, secondary and purely experimental. The ideas came from Volodya and it was he that wrote the articles, from beginning to end. It was a wonderful education for me, both the ideas and the experiments, and I have remained indebted to Volodya for that instruction all my life. He was, moreover, an amusing teacher, neither tedious nor keen to show off his superior knowledge. In the long line of postgraduates and last-year students who passed through Volodya’s hands I am proud that I was Number 1. In the Soviet period all students had to be ‘‘allocated’’ after finishing their studies. This meant they were directed to a particular job that had received the approval of the faculty administration (and above all the Party bureau). I had to think where I was going to work. The first invitation came from Roman Khesin. A geneticist by training, he had managed to complete his

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education at Moscow University before the genetics was destroyed. After the notorious 1948 session of the Academy of Agricultural Sciences he was forced to retrain as a biochemist. The founder of biochemical genetics in the USSR, Khesin would discover the early and late messenger RNAs in collaboration with M. Shemyakin (a fellow student from my year). In 1958 a start was made in setting up the radio-biological section (today the Academy’s Institute of Molecular Genetics) at the Institute of Atomic Energy. Khesin was, effectively, the scientific leader and ideologist of the new section. He needed young biochemists and invited myself and Misha Shemyakin. Khesin did not know me but had a high opinion of my father’s work and, so far as I can judge, held him in great respect. In choosing me Khesin was hoping, I think, that the father’s genes had been passed to his son (if only partially). It also helped that Khesin knew about the ‘‘Lyapunov Affair’’: in scientific circles it was widely known. My ‘‘Mendelism-Morganism’’ was, for him, the highest recommendation. Misha Shemyakin was reputed to be a brilliant experimenter, a fame that was quite justified. Khesin had probably heard of his reputation and his choice of this assistant was entirely understandable. Khesin handed me the questionnaires I had to complete before I could be taken on at the Institute. The Institute of Atomic Energy was engaged in highly classified research (e.g. the atom bomb) and although the radio-biological section had nothing to do with such work all employees had to be strictly checked by the ‘‘competent bodies.’’ There were several reasons why I accepted Khesin’s invitation. One, he had great personal charm and a charisma that all found irresistible. The subject he was proposing to explore was most enticing: the actions of genes and the mechanisms of expression of genetic information. Two, I had not then received other invitations and feared to find myself in the clutches of the Party bureau. They might make me go and work in the back of beyond where no one needed biochemistry. Three, I lived near Khesin’s institute. It was only 15 minutes on foot compared to the 90 minutes I traveled each way to the university. I would save a great deal of time and energy! Three months later Khesin invited me to the pre-Revolutionary professor’s apartment where he lived, behind the old university building in the center of Moscow. ‘‘Lyova!’’ he said, in his typically calm and ironic way, ‘‘Why didn’t you warn me you are a


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German spy?’’ I was stunned. Then I laughed and replied: ‘‘How could I possibly warn you if I myself didn’t know!’’ In the questionnaire I had answered that during the Great Patriotic War (1941–1945), from the age of 5–8, I had been in a part of the USSR temporarily occupied by the fascists and was liberated by the Red Army in the town of Chemnitz. This proved more than enough for the ‘‘competent bodies’’ to classify me as insufficiently reliable, if not actually a German spy. Khesin smiled but his eyes were sad. He had discussed the absurdity of the situation, he added, with Kurchatov and Alexandrov, both powerful figures in the scientific establishment and directors of the Institute. They told Khesin the objection was ‘‘insuperable.’’ Until his dying day Khesin remained my friend and I will always be thankful to him for that. All who knew him regard Khesin as a brilliant scientist and an outstanding personality. I did not join the Institute of Atomic Energy but Volodya learned that I had been invited there. Without delay he went to Severin to organize a postgraduate place for me. Severin’s attitude was very positive and the application was forwarded to the dean’s office (with my agreement, naturally). The prospect of keeping in close contact with Volodya was very attractive, especially because he considered that in the year I spent on my undergraduate dissertation I had completed half of my Ph.D. thesis. Volodya was keen to keep an already trained and capable in his opinion postgraduate near at hand. In early 1959 Engelhardt was at last able to break the resistance of Lysenko and began Institute of Radiation and Physicochemical Biology (IRPB) at the USSR Academy of Sciences. The struggle had required the active support of physicists, chemists, and mathematicians: Kurchatov, Alexandrov, Tamm, Artzimovich, Knunyantz, and others. The institute would need young research staff and in April 1959 Engelhardt called me to his office at the biochemistry department and invited me to join him at the IRPB. I was, of course, flattered. A new institute at the Academy, new equipment and Engelhardt, a world-class scientist and a classic figure in the discipline! I told him I had already received an invitation from Severin and agreed to become a postgraduate in the biochemistry department. Engelhardt thought for a moment. He looked at me attentively and said: ‘‘Yes, Professor Severin told me about it. But you may encounter difficulties staying on at Moscow University and then the invitation from my institute might come in handy. I’ll send it

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all the same, if you’re agreed.’’ I thanked him and gave my consent but I felt disconcerted. What difficulties? I had the highest-rated graduation diploma, publications based on my diploma work and an excellent reference from the department. Why did Engelhardt invite me to join his new institute? When I submitted my answers to the examination he set in enzymology I did not just rely on his lectures. I also drew on biochemistry books I had by then read, in particular Baldwin’s Dynamic Aspects of Biochemistry. In my reply to one of the questions I made use of such additional information and this caught Engelhardt’s eye. ‘‘How do you know that?’’ he enquired. ‘‘I didn’t mention it during my lectures.’’ ‘‘I picked it up from Baldwin’’ was my reply. Engelhardt smiled and his eyes twinkled. Evidently, he had not forgotten. There was another, no less important consideration. Engelhardt and Lev Zilber had studied in the same year at Moscow University’s medical faculty. Later he was among those who signed a letter in Father’s defence which saved him from prison. Engelhardt regarded my father with enormous respect and, like Khesin, he probably hoped I had inherited some of Father’s scientific ability. When the Party bureau met to decide where the faculty’s graduates would work a row broke out. They would not allow Severin to take on the ‘‘best student’’ as a postgraduate: that was how Severin described me when he spoke in my support (so I was later told by a member of the bureau). Postgraduate Kisselev was a threat to the faculty. In their words, I would teach ‘‘MendelismMorganism’’ to the students and oppose the ‘‘doctrines of Lysenko.’’ Severin was indignant. The Party bureau had not forgotten the ‘‘Lyapunov Affair,’’ however, and the shameful defeat the students had inflicted at the Komsomol general meeting. The revenge was belated and ineffective since I joined Engelhardt’s institute instead. Much as they wanted, they could do nothing about that. Volodya Skulachev was very disappointed. Our friendship, however, withstood this fateful blow (or, rather, intervention by the Party bureau) and we remained lifelong friends.

Linus Pauling Visits Moscow University Knowledge of spoken English was then rare in the USSR: why study a language when you have no hope of using it? My newly


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acquired ability served me well, both in later life, of course, and in my student years. The visit of Linus Pauling to the Soviet Union was a major event. He gave a public lecture at Moscow University in which the main subject was ascorbic acid, Vitamin C. The vast assembly hall was full and I was selected to be his translator. Pauling spoke a few sentences which I then translated into Russian. (This was much easier than synchronic translation, something that I did quite frequently later on.) After the first minutes of anxiety I calmed down, got into the swing of things, and ceased trembling. Pauling was not a young man and spoke refined English behind which one could sense an experienced lecturer and public speaker. Soon I forgot that he had twice won the Nobel Prize and was a great scientist. On two occasions I did not understand what Linus was saying – he himself said I should call him that, though in company I addressed him as Professor Pauling – and I told him so. Quite calmly he found other words to explain his meaning that I could translate. When we were talking off-stage before his lecture he drank tea and took a box of large tablets out of his pocket. It was, of course, Vitamin C and he took one before speaking. At that time the dose was considered enormous: Soviet doctors usually prescribed 50–100 mg/day but Pauling took 1 gram at a time. As he proved, people need much larger doses and he himself set an example of the benefits. After swallowing the tablet he offered me one. It was very useful before work, he said. With a certain apprehension I agreed and managed to swallow the large tablet with some warm tea. It proved not to be acidic: it had been neutralized beforehand. After the lecture I translated many questions from the audience and Pauling’s precise replies. As we parted he thanked me for my good translation, which was kind (he did not know Russian and could not judge), and he wished me success as a biochemist. The true stature of the scientist, I understood, lay in his simplicity and approachability: it was demonstrated by his ability to explain complex things in a simple fashion. I regret it was our one and only meeting.

The Engelhardt’s Institute: First Years It took 2 years before the IRPB could be established. In early 1957 a decision was taken to set in frame of the Academy of Sciences,

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but the proposal met with frantic opposition from Lysenko and his entourage. They operated through the Central Committee’s agricultural department where their influence remained strong. Lysenko promised Khrushchev that he could restore Soviet agricultural output within 2–3 years and the new leader, like Stalin, was taken in. Engelhardt made use of the enormous authority he enjoyed in scientific circles to gain the support of our great and influential physicists. With their help he was successful and in April 1959 the institute began its official existence. Engelhardt became its first director and remained in that post until his death in 1984. The premises of the Institute of Mining were handed over to the new institute. Designed by the well-known Soviet architect Zheltovsky, it was built in neo-classical style with a large number of powerful columns (no less than 18, I believe) decorating its facade. In the early 1950s such buildings were in vogue and evidently reflected Stalin’s taste. On coming to power Khrushchev decided that the Institute of Mining should be moved nearer the mining industry and out of a city where no coal was extracted. It ended up somewhere in the surrounding Moscow region though so far as I know there are no coalmines there either. When we took over the building we inherited enormous machines, which the mining engineers had probably found difficult to dismantle and were now quite obsolete. Major repair work was necessary and it soon began. Temporary electricity cables lay along the corridors. There were no laboratory tables, no biochemical equipment, and no reagents or instruments. There was a great deal of enthusiasm and much talk about the new science of molecular biology – although there was not a single molecular biologist at the institute then. Our work would be supported, said Engelhardt, by the three great pillars of biology, physics, and chemistry, hence the title of the institute. Among those invited to work there were: the physicist L.A. Tumerman, followed later by the physicist M.V. Volkenstein, the physical chemist Ya.M. Varshavsky, the virologist V.I. Tovarnitsky, the cytologist I.A. Utkin, the microbiologist M.N. Meisel, the outstanding biochemist A.E. Braunstein, who brought with him a large group of organic chemists (R.M. Khomutov, M.Ya. Karpeisky, E.S. Severin, and B.P. Gottikh). Engelhardt transferred some of his laboratory colleagues from the Bach Institute of Biochemistry: A.A. Bayev, T.V. Venkstern,


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R.I. Tatarskaya, and others. To these notable scientists were added inexperienced youngsters like myself. Later Alexandra Prokofyeva-Belgovskaya, a leading cytogenetisist, joined the institute, as did G.P. Georgiev, who had just completed his doctorate but was already an experienced molecular geneticist. The first year of the institute’s existence, from summer 1959 to summer 1960, was spent on fundamental repair work, acquisition of the essentials, and reading of the scientific literature that gradually began to reach the institute. I did not enjoy such activities. At the department of biochemistry I was used to intensive study but here it was not yet possible to conduct experiments. Without permission I would sneak back to Volodya Skulachev and continue my work on oxidative phosphorylation and respiration. The experiments were carried out on pigeons. They were a classic subject of research. It was in the nuclear erythrocytes of pigeons that Engelhardt, then working in Kazan, discovered the phenomenon of respiratory (oxidative) phosphorylation in the early 1930s. (Read more about Engelhardt in [1].) The birds were everywhere in Moscow and they cost little to buy. After conducting the experiments, for which we required their livers, we ate them. We didn’t eat the whole bird, just the breast. Roasted on a pan we kept for the purpose they were delicious – especially at midnight when the experiment finished, the lab was empty and we were both famished. At that time we were interested (above all, Volodya, of course) in how to uncouple respiration from phosphorylation, that is in the possibility of diminishing the P/O ratio. (For more detail, please read Volodya’s recollections [3].) This coefficient was brought into scientific usage by Vladimir Belitzer, a wonderful biochemist who was working in Kiev. We wanted the energy of oxidation to be expended not on the formation of ATP but directly transformed into heat, into calories. To attain this Volodya thought up a way of despatching the pigeons, which involved plucking them almost naked and then putting them in a large refrigerator at temperatures below 0 1C. There were no ‘‘greens’’ then, or animal rights activists. Had they existed they would not have approved of our experiments. After the pigeons had been sufficiently ‘‘frosted’’ we quickly separated out the mitochondria from liver and measured the absorption of oxygen in a Warburg vessel while the amount of organic phosphate in the incubation mixture was measured according to the Fiske-Subbarow method.

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A great many Warburg vessels were needed for these experiments (I won’t describe the equipment since they are familiar to every biochemist of the older generation). Many lay around the department but they were not in working order: inexperienced students tended to break the glass. Late in the evening when no one else was around we would visit all the laboratories in the department looking for the broken Warburg vessels and confiscate any we found. If the owners left them lying about we assumed that they did not need them. We were free to take them and advance the cause of science. The cracked and damaged vessels were passed to a glass-blower of our acquaintance and we received dozens of restored, re-usable vessels, in return for money we ourselves provided from our postgraduate fellowship and apprenticeship stipends. Our productivity greatly increased but we had not foreseen the consequences, however. Severin’s right hand in the department, Nina Meshkova, discovered that the broken vessels had gone missing. When she learned of their fate she demanded that we be severely punished: we were ‘‘thieving’’ from the department, she considered, out of narrow self-interest. Severin did not pass judgement. We kept the repaired vessels but magnanimously declared that anyone who had need of them could borrow them from us. When it became possible to undertake research at IRPB, I concluded my university experiments under Skulachev’s supervision and devoted myself entirely to a new subject. From autumn 1960 onwards there were ‘‘brain-storming’’ sessions at the institute where all present could propose any scientific problem within the framework of molecular biology. Most scientists date the birth of the new science from the publication in April 1953 of Watson and Crick’s famous article about the DNA double helix. Other researchers place its beginnings earlier, in 1944, when Avery, MacLeod, and McCarty established that genetic information is stored in DNA, which constitutes the chemical foundation of heredity. There can be no doubt that these were the two discoveries that laid the foundations of molecular biology. By the end of the 1950s ribosomes, tRNA, the aminoacyl-tRNA synthetases had all been discovered. By then Francis Crick had created his ‘‘adaptor hypothesis,’’ the existence of messenger RNA had been predicted and then confirmed and the unraveling of the genetic code was becoming a reality. Our institute had to find its own place and profile in the new science. From among the


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numerous possibilities it had to choose those lines of research that would establish its position in the world and forge its researchers into a team. The search for such themes and major scientific tasks was one of the purposes of the ‘‘brain-storming’’ sessions at IRPB. Many who joined the institute, of course, brought with them previous experience and accumulated knowledge and, not seeing a place for themselves in the new science, they wanted to continue their former studies. L.A. Tumerman, for instance, wanted to go on studying photosynthesis as a physicist. That did not suit Engelhardt. A.E. Braunstein had discovered the transamination reaction, one of the key reactions of nitrogen metabolism within the cell. He wanted to deepen the study of these reactions, using chemical and physical methods. We termed this area of investigation molecular enzymology and it was, of course, closer to biochemistry than molecular biology. Since Braunstein’s work was being conducted at a very high theoretical and methodological level, however, Engelhardt supported its continuation at the institute. Engelhardt’s own laboratory offered an example of a radical shift in direction. Until A.A. Bayev joined the institute he studied the nucleotide exchange as part of Engelhardt’s former research in the energy metabolism (see [24]). Now Bayev decided to work on decoding the primary structure of tRNA, a subject he had never studied before. Meanwhile I decided that, under the direct supervision of Engelhardt, I would simultaneously take up the study of tRNA and the aminoacyl-tRNA synthetases. It was an area of research that the Nobel laureate De Duve later designated ‘‘the second genetic code’’ [25]. In summer 1961 the 5th International Congress of Biochemistry was held in Moscow. This was, without doubt, a major event for Soviet biochemistry and molecular biology. The congress was held at Moscow University on the Lenin (now Vorobyovy) Hills and the plenary sessions took place in the assembly hall. Engelhardt led the section on ‘‘Biological Macromolecules’’ and Bayev made great efforts to ensure that the abstracts of the congress were published. All the leading lights in world biochemistry and molecular biology had gathered in Moscow. The Iron Curtain had collapsed and the Thaw, under Khrushchev, had begun. Scientists abroad wanted to find out what Soviet biology was like, after decades of isolation and the depredations of Lysenko’s pseudo-science. Many who came were

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subsequently awarded the Nobel Prize and became classic figures, whose works would be standard texts. Of that enormous list let me name just a few: Francis Crick, James Watson, Marshall Nirenberg, Matthew Meselson, Paul Doty, Jacques Fresco, and Marianne Grunberg-Manago. The general knowledge of foreign languages among our scientists was still very low. Decades of isolation, the futility of studying languages one could not use, and the extreme shortage of foreign scientific literature outside the major centers of study in the Soviet Union, had all taken their toll. The organizers decided that the reports read out at the congress must have synchronic translation. At first they turned to professional interpreters with an impeccable knowledge of English, the most needed language, who had worked for the UN, UNESCO, and other international bodies. However, almost all of them proved incapable of translating the reports since they did not know the subject and the specialist terminology. When biochemists such as Braunstein, Orekhovich, and Engelhardt listened to the results they could hardly contain their laughter. Afterwards numerous stories circulated privately about those comical errors and misunderstandings. It was decided, instead, to invite young scientists to help out. An examination was organized at which prominent biochemists, not language specialists, tested the candidates. If I recall rightly about dozen people were selected of whom I was one. Among the others were my friends and fellow students from the biological faculty A. Antonov, G. Gauze, M. Verkhovtseva, M. Kritsky, and A. Rubin. The Academy of Sciences paid for an excellent teacher V. Karzinkin to bolster our preparation. He was a wonderful person with an excellent grasp of the language and we greatly enjoyed our studies with him. During the early years of IRPB’s existence it was visited by many outstanding scientists. This also made it an interesting place to be. The visitors were attracted, I think, by the personality of Engelhardt. He was widely known abroad, not just for his discoveries in biochemistry but for his charisma, mastery of several European languages, welcoming disposition and the pleasure he took in contact and conversation with others. A second obvious reason for this interest was that the young institute had a positive reputation. It was opposed to Lysenko, forward-looking, it was developing new fields in science and had


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many talented young researchers: this was not the case with many of the Academy’s older institutes. I was almost the only young member of staff who had mastered both the fundamentals of molecular biology and conversational English. Engelhardt therefore made me interpret when famous visitors spoke at the institute. Not only did my own education greatly benefit from listening to some of the most outstanding scientists of the time. Through translating the lectures of Doty, Meselson, Chapeville, Rich, Zachau, and many others I also made their personal acquaintance. With some horror I recall the visit of the American scientist James Bonner. He had just discovered the conservation of histone structures in animals and plants and Engelhardt asked him to give a lecture on the subject. I was introduced to the speaker in the director’s office and quickly realized that it would be a disaster if I attempted to interpret his words. Bonner was a Southerner. As many will know, the dialect he spoke is characterized by a lack of clear diction and it seemed as though he was mumbling without opening his mouth. Leaning towards me, away from Bonner, Engelhardt said quietly in Russian: ‘‘I don’t understand what he’s saying.’’ Engelhardt’s hearing was partly impaired and to him Bonner’s drawl was not only incomprehensible, it was inaudible. What was I to do? Politely, I asked Bonner if he had some off-prints relating to the theme of his lecture. If he would give them to me then I could prepare better. He liked the idea and, digging in his briefcase, he pulled out an off-print and a manuscript. For the most part this was what he was going to say during the lecture, he replied. Grabbing the texts I immediately took my leave. Bonner was taken out to lunch and I rushed off to the laboratory to read. There was only an hour before the talk. Danger sharpens our perceptions. I quickly read both papers, in places skimming the contents, and then ran to the conference hall to translate. Speaking in public Bonner was no easier to understand than he had been in Engelhardt’s office. It was the same mumbling through gritted teeth. At that moment, I thought to myself, if Engelhardt and I can barely understand what he’s saying, then the audience will be even more in the dark: I can say anything I like and no one can complain that I’ve translated incorrectly! Emboldened by that thought, I began to paraphrase what I had read in Bonner’s articles, keeping an eye on the slides he was displaying on the screen and adjusting my ‘‘translation’’

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accordingly. The crisis came later, when certain curious listeners began to ask questions. As I translated these enquiries into English I faced two dangers. The question might be addressed not to Bonner but to me, if I had added something the speaker had not referred to. On the other hand, the question might be appropriate – but could I translate Bonner’s reply? A mixture of impudence and intuition (it’s hard to say in what proportions) came to my aid. Fortunately, there were relatively few questions. Following the lecture I had a frightful headache and kept well clear of Engelhardt and his guest speaker. The next day the director called me to his office. Bonner had said he was pleased with the questions he’d been asked. They showed, Bonner remarked, that his lecture had been well translated. ‘‘I simply can’t imagine how you interpreted what he said,’’ Engelhardt added. ‘‘It’s a long time since I’ve heard someone speak so appallingly!’’ I did not try explaining the little tricks of my trade. From that moment on, my reputation as a translator was unimpeachable. Thus myths are born. Alex Rich was, on the contrary, a model lecturer. It was extremely easy, even pleasurable, to interpret for him. The astonishing clarity with which Rich composed his phrases, his irreproachable diction, and the moderate and comfortable tempo of his delivery reflected the clarity of his thought and the irrefutable logic of his presentation. I envied American students who could listen to an entire course of his lectures. His articles were written with the same transparent simplicity and logic. I understood the enormous gulf separating Massachusets Institute of Technology (MIT) from the southern states. Later I became acquainted with many other professors from leading East Coast universities (Harvard, Yale, or MIT) and in the mid-West (Wisconsin) and the great majority were excellent lecturers. For me, however, Alex Rich remains the ideal. Engelhardt had his own ideas about running the institute. He did not pay great attention to degrees, titles, and official posts. For him people were judged by what they did, not by any formal categories. So he did not care whether, where or when I would submit a thesis, and on what subject. He was very keen, on the other hand, to discuss ‘‘pure’’ science, how to set up experiments and interpret the results. By 1963 I had completed the first cycle of studies to determine the macromolecular structure of tRNA in solution. I was ready to defend my Ph.D. thesis but the institute


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did not have its own ‘‘thesis’’ council, approved by the All-Union Accreditation Commission that could examine theses and award scholarly degrees. Engelhardt was adamantly opposed to its introduction. Such a body was a waste of time, in his view. ‘‘Why should we pay attention to the bad work done by outsiders?’’ was his main argument. ‘‘We would do better to tell others about our good work.’’ I had to find my own way out of this predicament and went to see Alexander Oparin, director of the nearby Bach Institute of Biochemistry. A member of the Academy of Sciences, Oparin headed the department of plant biochemistry at Moscow University and created a theory of the origin of life that gained worldwide fame. His institute did have its own ‘‘thesis’’ council. The situation was tricky, however, because relations between Engelhardt and Oparin were not that good. Oparin was close to the ‘‘followers of Michurin/Lysenko’’ and as a pupil of Engelhardt I might be refused outright. Fortunately, Oparin was very understanding and magnanimously agreed to accept my thesis for submission. In March 1963 I was awarded an Ph.D. degree in biochemistry. When it came to the vote I was rather surprised that two people opposed the award. I had no scientific competitors in Oparin’s institute. On the contrary, I was on good terms with many of its staff so I asked Nikolai Dyatchkov, the long-standing scholarly secretary of both the institute and the ‘‘thesis’’ councils, what might be the explanation. He smiled. ‘‘One black ball was for Father, Lyova,’’ he said, ‘‘and the other was for you.’’ This commentary was intriguing and I asked him to explain. A member of the council ‘‘had it in for Lev Zilber,’’ he said, probably for his anti-Lysenko views and ‘‘everything else.’’ Another council member believed I persuaded someone to feed me questions I had prepared for. When I was getting ready I indeed tried to imagine what questions might come up. My preparations not only involved thinking about the answers but preparing slides to illustrate my replies. When one of the expected questions was asked I showed a ready-made slide not shown during my talk. No one had actually fed me previously agreed questions but my conscientious preparation earned me a black ball. Truth to tell, it did not influence the overall positive result. Not only my family and close friends were in the audience when I defended my dissertation. There were also friends of the family. One was the wonderful soprano and People’s Artist Natalya Schpiller. ‘‘Of course, I didn’t understand a word you were

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saying,’’ she commented afterwards, ‘‘but you did mention ‘naked chains’. I’m a little curious.’’ (There was a reference in my report to the single stranded short tRNA chains, linking double-stranded helical regions.) Nothing improper was intended, I had to explain. She was reassured and this added to the general hilarity. Under Engelhardt’s directorship there was a very lively social life at the institute although the dominant atmosphere in the country, under Brezhnev, hardly encouraged people to mix freely. Evening events were quite regularly held in the institute’s dining hall, with the most varied performers or speakers. They were always of interest to the audience. The Nobel laureate Igor Tamm, for instance, the outstanding theoretical physicist and friend of Engelhardt was one speaker. The legendary Vladimir Vysotsky, who composed and performed his own songs and was an incomparable Hamlet at Lyubimov’s Taganka theatre, came and sang for us. I recall a brilliant lecture by Natan Eidelman, who spoke with great erudition and incisive wit about Russian history. One evening was hosted by Alexander Shirvindt and Mikhail Derzhavin from the Satire Theatre. Very young then, they were already famed throughout Moscow for their sharp repartee and, then as now, were idolized by the intelligentsia. Tatyana and Sergei Nikitin also performed for us. Entertainments organized by the young staff at the institute were also immensely popular. We call them kapustniks, and there is no direct translation in English (kapusta literally means cabbage!). The institute’s grand and famous scientists were depicted in various situations – taking part in a meeting of the scholarly council, say. It was comparatively easy to write and act out such a scenario since the council of the day contained many striking personalities, starting with Engelhardt himself. Taking part in a kapustnik was a risky venture. It was remembered long after and if a character was portrayed with sufficient irony or even, sometimes, satire the consequence might be far worse than any formal and official reprimand. In most cases the irony tended more to humor than satire. I well remember an episode in which I acted the part of Alexander Braunstein, who had two well-known characteristics. A love of foreign words and recondite scientific terms often took the place of simple Russian expressions in his speech. Poor hearing, from childhood years, gave his lectures a most distinctive phonetic range and they were easily and immediately


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recognizable. As Braunstein I delivered a speech from the stage while the scientist, listening attentively, sat in the front row. The audience clearly liked my performance. After the kapustnik, however, when we were leaving the hall, Braunstein walked straight in front of me (I leaped out of his way), without turning his head or saying a word. By chance, we met in the corridor the next day. Braunstein did not respond to my greeting although he was a well-mannered person and his gaze was cold. I was in serious trouble. I had not yet defended my thesis and the attitude of a leading light such as Braunstein could strongly influence my future as a scientist. I went to Engelhardt and told him everything. His reaction was very lively. To begin with, he congratulated me on the success of the kapustnik, for which I had been not only a performer but one of the script-writers. The portrayal of Engelhardt at our entertainment, I should add, was quite ‘‘biting’’ but kindly meant, just like that of all the other characters. My tale greatly amused Engelhardt and he laughed until he cried, wiping the tears from his eyes with a handkerchief. It did not seem at all funny to me. Finally, his light blue eyes begun to sparkle. Evidently, he had a plan. Pensively, he quoted Trollope: ‘‘Disrespect is one way of showing affection.’’ When we parted he promised to save me from the wrath of Alexander Braunstein. However, he did not say when or how. The conclusion of this tragicomic story was a triumph for Engelhardt. About a month later, the scholarly council met as usual in the same conference hall – the real council, that is, not our comic version. Usually I attended if scientific reports were to be presented. Judging by the agenda on the notice board nothing of the kind would happen and I did not intend to go. Fifteen minutes before the meeting began, however, I received a call at the lab from Vera Belenitskaya, Engelhardt’s secretary. The director wanted to know whether I would attend. I was not planning to go, I replied. After a pause she said: ‘‘Perhaps, nonetheless, you might go?’’ I always found Vera a most considerate person, and I decided not to ignore her advice. The meeting was dull and I sat at the back, outlining the next experiment in my notebook. At the very end, before Engelhardt closed the meeting, he rose slowly, as always, from his chair and unexpectedly began to discuss things that were nowhere on the agenda. A new science, molecular biology, was taking shape, he said. The institute must become the leader of the field in the

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USSR and that required the presence of many striking personalities, of scientists with a pronounced individuality. So far we were doing very badly. At the institute’s last kapustnik, for instance, only 6 of the 18 members of the scholarly council were portrayed. The rest must be faceless individuals if they could not even be caricatured at such an event. I was exultant. When the meeting closed I tried to catch Braunstein’s eye. He spotted me, gave a paternal smile and, stretching out his hand, congratulated me on a very successful kapustnik. ‘‘You gave a most sympathetic portrayal of me,’’ he added. I was saved. I never again depicted Braunstein at a kapustnik, however. I passed that role to David Beritashvili, who made a very good job of it. We did not avoid controversial subjects. At one kapustnik Lysenko was brilliantly depicted by Gena Zavilgelsky. The similarity between his portrayal of Lysenko and Hitler was obvious. A physicist, Gena became a molecular biologist. Later, unfortunately, he left the institute due to his successful work, he got a good position at the other institute. Our Institute lost excellent artist and scientist. Kapustniks were possible, of course, thanks to the support and protection of our director. Engelhardt detested bureaucratic attitudes and did a great deal to encourage signs of talent not just in science but also in other areas. Our kapustniks were organized by a group that included Luda Minchenkova, Rita Timofeyeva, Valery Ivanov, Yury Morozov, Veta Grechko, Robert Beabealashvili, David Beritashvili, Gena Zavilgelsky, and others, such as myself. After Engelhardt’s death in 1984 our new director Andrei Mirzabekov tried to support these admirable traditions. Quite soon, however, they died out. The times had changed, we were older and Engelhardt was no longer with us. All good things come to an end, sooner or later. Another expression of our public-spirited outlook was our wall newspaper. During the Soviet period such publications had to appear regularly in each organization. Ours differed markedly from those in other institutes. It was more witty, ironic, and sharply worded and it tackled more controversial subjects. People would crowd round the stand when the latest issue appeared. I was quite an active participant, both as author and editor. Among other contributors were Sam Tulkes, an artist of near professional standard, and Olga Epifanova who eventually became a writer after a career as a cellular biologist.


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One episode concerning our newspaper springs to mind. It was displayed in a hall on the third floor opposite the director’s office. When a new issue appeared Engelhardt would always examine it attentively. On one such occasion I was walking across the hall when he called me over and, pointing to a particular column, clearly indicated his disapproval. Feverishly, my eyes scoured the headlines but I could see nothing wrong and decided to ask Engelhardt the cause of his displeasure. ‘‘Lyova! You know when it became clear that Macmillan should retire? When they stopped publishing cartoons of the prime minister in English newspapers. There’s no mention of the director here. Time for me to retire, is it?’’ I gave him my word that he need not retire: the ‘‘mistake’’ would be corrected in the next issue. Engelhardt withdrew to his study, pleased with my dismay and the impression he had made. The newspaper’s editors were quickly gathered and I told them of this exchange. Sam Tulkes drew an excellent full-length caricature of Engelhardt in a typical pose with an ironic expression on his face. Probably it took up a quarter of the entire space allocated to the issue. A little while later the newspaper appeared in its usual location and I rang Engelhardt’s secretary Vera, asking her to ‘‘casually’’ walk our director past the stand. The mission appealed to her. Within a couple of hours I learned that Engelhardt had laughed so hard he had cried: ‘‘Those rascals. They even make fun of the director. I shall sack the lot of them!’’ There could be no higher words of approval.

A Digression from Biochemistry into the Physics of Biopolymers The early 1960s in molecular biology were distinguished by a rapid parallel development of both structural and functional investigations. ‘‘If you do not understand function, then study structure,’’ ‘‘The way to understand function lies in the structure’’: these were very popular slogans after Watson and Crick’s double helix had uncovered the principle whereby DNA was replicated. It was very difficult to study the function of biopolymers at the institute. We lacked the necessary equipment, substantial internal reconstruction of the building was required, and the Soviet Union did not produce the necessary reagents (their purchase abroad required hard currency, which was strictly rationed).

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There was no system of grants. Instead, all depended on the plan, which decreed that we might only buy reagents once a year. As a consequence, we had to foresee what we would need in a year’s time and in science that is extremely difficult and sometimes simply impossible. In such conditions I decided to begin with research not into the functions but the structure of biopolymers, since this was simpler at that time from both a methodological and technical point of view. My decision was, of course, influenced by the work of Doty, Rich, Fresco, and Spirin. They were then having great success with the macromolecular structure of natural nucleic acids and synthetic polynucleotides. Naturally, I did not intend to duplicate their excellent research but apply their methods and certain others they had not adopted in order to analyse the spatial structure of the molecules of tRNA in solution. These then attracted great interest thanks to the adaptor concept of Francis Crick and the studies of M. Hoagland in P. Zamecnik’s laboratory. The adaptor function of tRNA in protein synthesis had been proved experimentally by F. Chapeville, working in the laboratory of F. Lipmann [26]. I decided I would use physical methods that had already been tested on viral and ribosomal RNAs and were accessible in the USSR: we had both the highly qualified specialists and necessary equipment. It would have been ideal to work with tRNA in a crystalline form, considering their tiny dimensions. I did not attempt that approach. I myself had no experience of crystallizing proteins, no one had then attempted to crystallize nucleic acid, and the X-ray structural analysis of biopolymers was only in an embryonic form in the USSR. Our group was represented by Elena Rebinder, Ludmila Frolova, and myself [27]. The chemistry and physics of polymers was developing in the USSR very intensively in the early 1960s. A leading center was the Academy’s Institute of High Molecular Compounds in Leningrad and I was eager to go there, since I had thoroughly purified preparations of yeast tRNA. Joining forces with the V. Tsvetkov’s laboratory at that institute, we were able to carry out a cycle of investigations [28–30]. Another series of studies was carried out within the institute. We worked with staff from Tumerman’s laboratory [31–34], although he himself took no part in these investigations. The outside world, in particular the English-speaking scientific community, had almost no knowledge of these works since all the papers were published in


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Russian. There were major reasons for this. In order to publish abroad one had to undergo a complicated procedure during which one was obliged to give a written assurance that there was nothing new (!) in the work. I was young and impetuous. It would be impossible, a disgrace, for me to describe my efforts in such terms. Nor did we then know how to write scientific articles in English. Entrusting the task to a professional translator was risky since he would not know the terminology. It was also expensive and the institute did not cover such expenses.

Macromolecular Structure of Transfer RNA in Solution The difficulty consisted of the small sizes of tRNA molecules and hence their weak polymer properties. However, it was possible to apply a combination of physical methods for tRNA studies, which included: sedimentation, viscosity [27–29], flow birefringence [30], circular dichroism and optical rotatory dispersion, spectrophotometry, fluorescence (including polarization of fluorescence) [31–34], microcalorimetry [35], and small angle X-ray scattering [36]. The physical methods were supplemented by chemical modification of tRNA bases [37–40]. These studies were carried out in tight contact with physical Russian laboratories, and provided experimental evidence for the following conclusions [27–43]: (1) in spite of rather small sizes, secondary structure of tRNAs behaved in the same manner as high molecular weight viral or ribosomal RNAs investigated by other groups (P. Doty, J. Fresco, A. Spirin, etc.) and the ratio of double-helical and single-stranded regions was elucidated in tRNA structure; (2) tRNA molecule consists of two domains positioned at some angle to each other (this conclusion was later supported by X-ray analysis of phenylalanine tRNA by A. Rich, A. Klug, and others); (3) tRNA molecule contains three exposed sites located outside the double-helical regions; these are the anticodon loop, the CCA-end, and a site between two domains; this conclusion based on experiments with valine tRNA was confirmed by other authors for tRNAs of different amino acid specificity; (4) studies of folding and unfolding of tRNA in solution revealed a role of Mg2þ in this process.

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Using phenylalanine tRNA and poly(U), specific codon–anticodon interaction was demonstrated in ribosome-free solution; these experiments [44] provided experimental evidence for the existence of exposed anticodon in the spatial structure of tRNA in solution and its ability for specific duplex formation with mRNA codon; 5 years later X-ray analysis of tRNAPhe confirmed the correctness of this conclusion. All these results together with data on primary structures of tRNAs obtained in many laboratories in the 1960s and 1970s created the structural background for investigation of functional properties of tRNAs during their interactions with aaRSases and functioning within the ribosomes.

Aminoacyl-tRNA Synthetases Our laboratory paid special attention to eukaryotic aaRSases (see for review [45]), as they were less studied compared with the bacterial enzymes. Using classical methods of molecular enzymology (isolation of individual enzymes, kinetic analysis, use of substrate analogs, etc.) and modern gene engineering, the following results were obtained. The amino acid sequences of human [46] and rabbit [47] tryptophanyl-RSases were elucidated. Our team has characterized exon–intron organization of the human tryptophanyl-RSase gene and demonstrated that this is an interferon-dependent gene and the gene transcript undergoes alternative splicing [48–51]. We also elucidated the spatial organization of mammalian tryptophanyl-RSase: this enzyme is a homodimer (a2) and each subunit consists of two non-equal domains. The N-terminal domain (representing about 1/3 of the subunit size) is not essential for its ability to catalyze synthesis of tryptophanyl-tRNA [52,53]. Using monoclonal antibodies, we demonstrated the presence of aaRSases in nuclei of animal cells [54,55]; later these results were confirmed in other laboratories. They explained early observations by V.A. Gvozdev, who found amino acid activation in mammalian cell nuclei. Binding of substrates (amino acid, ATP, and tRNA) and products (AMP, pyrophosphate, and aminoacyl-tRNA) to aaRSases may follow two kinetic mechanisms [56–60]: ordered (for tryptophanyl-RSase) and random (for phenylalanyl-RSase). Later


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both kinetic mechanisms were also demonstrated for other aaRSases. Tryptophanyl-RSase can form covalent intermediates. A tryptophanyl residue can be transferred from the enzyme onto tRNATrp in the absence of ATP; the latter suggests high-energy bond formation between Trp and tryptophanyl-RSase [61–67]. Molecular mechanism of tryptophanyl-RSase functioning involves negative cooperativity between subunits [68–70], ordered substrate binding, and covalent enzyme–substrate complex formation (see for review [71,72]). We provided experimental evidence that in contrast to most mammalian aaRSases, tryptophanyl-RSase is a Zn2þ-dependent enzyme; Zn2þ ion is ultimately required for enzyme functioning and its absence alters catalytic properties of this enzyme [73,74]. In the absence of tRNA, tryptophanyl-RSase can form Ap3A dinucleotides [75,76], an important regulator of cell processes [77]; this appears to be a characteristic feature of this enzyme as other aaRSases lack such property. Mammalian glycyl-RSase and Escherichia coli lysyl-RSase demonstrate molecular polymorphism [78,79]. Using immobilized substrate, our group obtained the enzyme– substrate complex between aaRSase and tRNA, and this approach allowed the development a new method for affinity purification of aaRSases and study of conditions required for complex formation [80]. Development of polyclonal and monoclonal antibodies to various domains of mammalian tryptophanyl-RSase and their subsequent use for enzymological and immunocytochemical studies provided important insight into the highly conservative structure of this enzyme in all kingdoms of living organisms; antigenic determinants are unequally distributed between domains, and abnormally high tryptophanyl-RSase content has been found in the ruminant pancreas [81–84]. Functionally essential histidine residues have been found in mammalian tryptophanyl-RSase [85], which also contains covalently bound carbohydrate [86]. We also synthesized covalent inhibitors of aaRSases and used them for selective inactivation of one (or two) active sites of tryptophanyl-RSase [87–91]. Our team selected cell lines resistant to tryptophan analogs and characterized properties of tryptophanyl-RSase in these cells [92,93].

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The gene encoding tryptophanyl-RSase has been localized on the human and bovine chromosomes [94]. The interaction between aaRSase and its cognate tRNA is accompanied by mutual conformational changes [95]. Conditions of in vitro aminoacylation influence the behavior of isoacceptor forms and transcripts of tRNA genes [96–98]. Our team employed computer analysis for comparative investigation of interaction of aaRSases with tRNA in prokaryotic and eukaryotic systems [99]. We also investigated the structure of two-dimensional crystals formed by tryptophandtryptophanyl-RSase complex [100]. Studies of aminoacyl-RSases have been accompanied by development of new methods for investigation of these enzymes, including analysis of catalytic activity and enzyme–substrate interactions [101–104]. Through high long-term research activity of our laboratory and the use of wide ranges of strategies and approaches mammalian tryptophanyl-RSase became at that time the most studied eukaryotic aaRSase (see reviews [105–107]). Many of the abovementioned results were obtained in collaboration with other laboratories, in particular, with the labs headed by D. Knorre (Novosibirsk, Russia) and J.-P. Ebel (Strasbourg, France).

The Role of Anticodon in the Second Genetic Code In the process of genetic information decoding, the stage of amino acid attachment to cognate tRNA is especially important. It is clear that the specificity of this reaction is determined by mutual recognition of certain sites of tRNA and the cognate aaRSase [107]. In 1964, a hypothesis explaining specificity of such interaction was suggested. We proposed dual function of tRNA anticodon: (i) it is involved in tRNA–mRNA interaction in the ribosome by forming anticodon–codon duplex and (ii) the anticodon specifically interacts with aaRSase of the same amino acid specificity as tRNA [108,109]. The principal advantage of this hypothesis consisted in strict coupling between acceptor and adaptor functions of tRNAs, which is required for correct decoding of genetic information. This hypothesis that aaRSase recognizes tRNA anticodon as a specific element has experimental background [108–111]. Chemical modification of tRNAs revealed


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correlation between nucleotide composition of tRNA anticodon and sensitivity of acceptor function of tRNA to chemical modification. In particular, the acceptor function of tRNAVal, having C as the third anticodon letter, was highly sensitive to modification of this base by hydroxylamine or O-methylhydroxylamine. The tRNA lacking C in their anticodons were weakly sensitive to this modification. Later it was demonstrated that anticodon C is indeed responsible for loss of tRNAVal activity after modification. In A.A. Bayev’s laboratory at our Institute use of the ‘‘dissected molecule’’ method demonstrated similar phenomenon: anticodon removal of two ‘‘root’’ nucleotides (AC) from tRNAVal 1 was accompanied by total loss of acceptor activity [24]. Later our laboratory also demonstrated the crucial role of tRNATrp anticodon for recognition by tryptophanyl-RSase [112,113]. It should be noted that in the very beginning of the appearance of this ‘‘anticodon hypothesis’’ we emphasized that in the tRNAs encoding amino acids with six codons (serine, leucine, arginine) anticodon plays secondary role (if any) and in these cases another recognition mechanism is involved. Experiments with synthetic polynucleotides inhibiting aminoacylation of tRNAs, containing anticodons, which had the same nucleotide composition and sequence as the added polynucleotides provided independent evidence supporting the role of tRNA anticodons in recognition by aaRSases; this suggested competition between anticodons and polynucleotides for the same site on the enzyme molecule [114]. In the end of the last century, the hypothesis of the role of anticodon in determining acceptor function of tRNA was directly confirmed by means of classical enzymological methods and also X-ray analysis of tRNA–aaRSase complexes (reviewed in [115,116]). This was strictly proven for at least half of all aaRSases; for some others this hypothesis was confirmed by various indirect methods. Nevertheless, at least three of 20 aaRSases employ other site(s) of tRNA molecules (acceptor stem, variable loop, etc.). It was also shown that together with anticodon, many aaRSases contain a second recognition site; this increases specificity of tRNA–enzyme interaction. Accumulation of convincing evidence validating the correctness of the ‘‘anticodon hypothesis’’ took more than 30 years (1964–1995). Now the role of tRNA anticodon as the key element determining specific recognition by cognate aaRSases is firmly established. This

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dogma has been included into textbooks on biochemistry and molecular biology.

IUB Congress in New York: The First Trip Abroad Here let me digress and describe my participation in the International Union Biochemical (IUB) congress held in New York in summer 1964 (Figure 4). Engelhardt was pleased with our

Fig. 4. Wladimir Engelhardt, Boris Gottikh, and Lev Kisselev (IUB Congress, 1964, USA).


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findings about the spatial structure of tRNA and decided that I should go to the congress, not least because I could describe our results in English. Such journeys outside the Soviet Union were a major event at that time and permission depended on a great many circumstances. Those running the Academy of Sciences and the ‘‘competent bodies’’ both considered me a wholly unsuitable candidate. I did not belong to the Party and was already too old for the Komsomol. I was unmarried and so might defect. Only recently had I defended my Ph.D. thesis whereas the delegation was composed mainly of professors and members of the Academy. I was frank with Engelhardt: I was not a suitable choice. I had not even been to Bulgaria before. (‘‘A chicken isn’t a bird, and Bulgaria isn’t abroad,’’ as we used to joke.) Yet he was proposing that I be sent to the USA, straight into the ‘‘lair of imperialism.’’ Engelhardt came up with an inspired suggestion. The delegation was to be headed by Oparin, the president of the USSR biochemical society. He knew no English and was rather deaf. In Moscow these shortcomings were fully compensated by his wife Nina who was an English teacher and would whisper a translation in his ear, even when the subject was biochemistry. For the congress, Engelhardt suggested, Oparin would need a young translator. The head of external relations at the Academy had a great respect for both scientists and asked Engelhardt to find a suitable candidate. Our director kindly agreed but when he named me the department of external relations objected, for the reasons I have mentioned. Engelhardt displayed great determination, bringing the matter before various important officials, until he had his way. For his part, he guaranteed that I would not remain in the USA or give in to ‘‘provocation.’’ The trip was well organized and as well as participating in the congress we also managed to visit major centers of biochemical research, such as Harvard and Bethesda, where we met leading American biochemists (Figure 5). I accompanied Oparin at the congress but very quickly he released me, saying that his wife, who was always with him, could help with translation. I was free. It was a kind act on his part since our scientific interests did not coincide and I would have been forced to listen to reports that did not interest me and translate them in public, thereby disturbing others in the audience. I was very thankful to Oparin and, naturally, made good use of my freedom.

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Fig. 5.

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In the laboratory of Alex Rich (1964).

Jacques Fresco had been given a laboratory in Princeton, after working for some time with Doty and together publishing classic works on the denaturing and renaturing DNA. He invited me to give a seminar at Princeton since he was very interested in tRNA and intended to study it himself. What was I to do? It was impossible to go to Princeton by myself, without asking permission from our ‘‘authorities.’’ They would discover I was missing, report the misdemeanor and it would prove my first and last trip abroad. If I asked to go, on the other hand, they would certainly not permit me. If someone went off on their own it was clear they did not intend to come back. I knew Fresco’s work and very much wanted to go. His field of interest was close to mine at that time and I was flattered by the invitation to one of the best centres for biology in the USA. I went to Oparin, the head of our delegation, and requested permission to make a trip to Princeton to give a seminar. An experienced diplomat, he did not turn me down but asked me to discuss the matter with Professor Severin, the deputy head of the delegation. No less diplomatically, Severin neither refused nor agreed, hinting that it did not actually depend on him. I went to see the man who had introduced himself as someone who would protect


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us from ‘‘provocation.’’ I told him of my conversation with the two Academicians. He smiled. ‘‘And why’s it so important to you?’’ he asked. I explained. ‘‘How and when are you coming back?’’ I would take the local train, I said, which stopped near the hotel where we were staying. They would make sure I caught the return train in Princeton. ‘‘When you get back, give a knock at my door, okay?’’ I said it would be no earlier than 10 pm, perhaps 10.30. ‘‘Never mind, I won’t be asleep. Don’t worry, knock loudly.’’ I thought he would offer to accompany me, which would have been hard to explain to Jacques Fresco. I’m thankful to our ‘‘guardian,’’ who did not fear to take such a decision. It would have been much easier and much less worry for him to refuse. The seminar proved a very tough experience. It lasted about 2 hours since I was interrupted by questions after almost every phrase. My questioners, moreover, did not bother to speak clearly and sometimes I didn’t understand them but had to guess what they might be asking. It was a typical American working seminar. It was thoroughly business-like and scientific but I was unused to a form of scientific discussion where you had to answer immediately and then continue from the point at which you had been interrupted. I stood the test but afterwards I was desperately tired. They had quite worn me out. Jacques took me to dinner where we continued to discuss science and topics of more general interest. After a substantial meal, washed down with beer, he drove me to the train. Buying my ticket to New York, he told me how to recognize my station and saw me off. About 10.30 I was back in the hotel and knocked firmly at the door where our ‘‘guardian’’ was staying. ‘‘Lyova? Is that you?’’ ‘‘Yes, it’s me. Everything’s fine, don’t worry.’’ ‘‘That’s good. Get some sleep and tomorrow you can tell me all about it.’’ I went to bed. The consequences of my seminar were a little unexpected. About 2 months later an official letter on Princeton University note-paper reached Moscow. It was addressed to Wladimir Engelhardt. Dr. Lev Kisselev was invited for 2 years to the USA, it said, to work at Professor Fresco’s laboratory on the macromolecular structure of tRNA. The director called me to his office. Engelhardt knew about the seminar, of course. I had given him a detailed account when we were still in New York. ‘‘What are we going to do?’’ he asked. I was in no doubt that I wanted to go. Fresco’s laboratory had seemed magnificently well-equipped, compared to our institute. There were many other young

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researchers there and I had been given a warm welcome. On the other hand, 2 years seemed a very long time and I was sure that I could do a great deal in a year if I prepared well. My reply was diplomatic. I would like to know, I said, what Engelhardt himself thought. What exactly would I be able to do in Princeton, he asked, that I could not do here? I explained. He thought for a moment. Then he enquired: ‘‘Will the family let you go?’’ There was a hidden meaning to this question. Not yet married, I was already attached. Engelhardt, I realized, did not want me to go for such a long time. I therefore suggested we discuss the possibility of a year’s absence. He decided to discuss this with the Academy of Sciences and went to the Presidium with the letter. I don’t know what he said or with whom he talked. The outcome was plainly negative. The Academy had no wish to let me go to the USA for 2 years or for one. I was not a Party member, I was unmarried, I’d just defended my Ph.D. thesis and I knew English. I would defect. Such a visit would benefit Soviet science, Engelhardt argued, taking advantage of foreign experience, the invitation was in itself an honor, and so on. Clearly these were unconvincing arguments when set against the aforementioned ‘‘shortcomings.’’ Engelhardt was asked, he complained, ‘‘Why do they invite some young postdoc instead of our renowned professors?’’ Many of the renowned professors did not speak English and had long ceased doing experiments themselves. All Fresco needed was a young and capable post-doctoral student. The president of the Academy of Sciences was M.V. Keldysh, a famous mathematician and one of those in charge of the Soviet space program. Keldysh was very well-disposed towards Engelhardt and his institute but in this case he was powerless. Engelhardt simply did not reply to Fresco’s letter. Offended that his invitation had not been acknowledged, Fresco then wrote to Keldysh, insisting that I be allowed to join him and complaining about our director’s silence. The whole situation distressed Engelhardt. I took things calmly since I was quite sure, from the very first, that I would not be allowed to go. I don’t know what Keldysh wrote to Fresco or what reply was written on his behalf. Whether Fresco wrote to Engelhardt again I did not enquire, so as not to rub salt in the wound. Thus, ended my first, but not last, invitation to work abroad. Since I’ve told the story of the Fresco seminar let me also mention the seminar I gave at Harvard during that same trip.


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I was invited by Alex Rich, with whom I was already acquainted, to talk about experiments in the chemical modification of tRNA as a method of studying its interaction with aaRSases. The seminar was attended by Watson, Rich, Meselson, and certain other key figures of the time in molecular biology. I was terribly nervous. The work was still not complete and though the first results had been published the conclusions were very radical and directly contradicted the then dominant view on the specific interaction of tRNAs with aaRSases. The atmosphere at the seminar was perfectly friendly and the questions were fair and tactful, without any attempt to catch me out. At the end they thanked me and wished me success but I felt that my audience had not been very convinced by my conclusions although none of them expressed doubts as to the value of the experiments. The problem was that the primary structure of tRNA had not yet been deciphered. It therefore seemed rather ‘‘dashing’’ to prove the role of the anticodon of tRNA in recognizing cognate aaRSase when the very existence of the anticodon had not been directly proved. At the same time, no one at the seminar suggested that my whole argument was incorrect (though that was also a possibility). The leading lights of molecular biology had given me a hearing, which in itself was a great boost to morale. I must return to the 1964 biochemical congress in New York. At that time one of my idols was the Nobel laureate and great biochemist Fritz Lipmann. He had made an enormous contribution to the study of energy metabolism and classic enzymology (read more about Lipmann in [117]). A refugee from Nazi Germany, Lipmann was no longer a young man when he found the strength to become a molecular biologist and make an outstanding contribution to that branch of science as well. Knowing that Lipmann worked at the Rockefeller Institute in the very center of New York I set out, without telling anyone where I was going, to pay him a visit. I was afraid not to find Lipmann at his laboratory. Perhaps the secretary would say I must give at least a month’s notice of my visit and agree a time and day? Entering the building I easily found a giant board that listed (without titles, posts, or academic distinctions) the full-time employees and the rooms where they might be found. Making my way upstairs I found the door inscribed with the name Fritz Lipmann and, holding my breath, knocked. An elderly voice with a strong German accent said, ‘‘Come in.’’

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I entered and with an anxious breathlessness gave my explanation: I was a post-doctoral student at Engelhardt’s lab in Moscow, I was studying tRNA and aaRSases and I asked the professor if he would give me a little of his time. Engelhardt had a deep respect and admiration for Lipmann. I knew that they had met on numerous occasions and that Lipmann also valued Engelhardt’s ability to speak German, which evidently gave him more pleasure than talking in English. My calculated reference to Engelhardt proved well-founded. At his name Lipmann came to life and immediately demanded, ‘‘Wladimir! How is he? What’s he doing now?’’ We began to talk and Lipmann, seated in an armchair behind the desk, turned to face me with benign smile. His office was small and contained a great many books in English and German. To one side stood a typewriter (it was the precomputer era!). Gaining confidence I gave a brief account of the institute, Engelhardt, the general situation and mentioned Lysenko. The reaction to that name was swift: ‘‘He killed Vavilov!’’ (learn about Vavilov more in [20]). Then Lipmann asked, was it true that anti-Semitism was less widespread in the USSR since Stalin died. I confirmed that it was. I gave a short account of our method for isolating individual tRNA [118]. His response was very prompt. He liked it, he said, and asked me to send a protocol in English so that they might use it for isolating other tRNAs in his laboratory. I felt happy and proud. No longer intimidated, I asked his advice. With which aaRSase should I work? Which, in his view, were the most important? Lipmann’s response was lively. Some time before, two post-doctoral students (one was Hans Zachau [119], later my friend) isolated partially purified tryptophanyl-RSase from the bovine pancreas. When however, they left the lab, the work was discontinued and no one took charge of the enzyme. I should take it over, was Lipmann’s vivid advice, complete its purification and make use of it. ‘‘The bovine pancreas contains a lot of the enzyme,’’ he said, adding, ‘‘I don’t know why. Furthermore, it’s very stable and the raw material, the bovine pancreas of cows, is very cheap and easily available. I hope they didn’t despatch cows to the Gulag?’’ I was presented with an offprint describing the procedure for obtaining partially purified tryptophanyl-RSase. I made good use of Lipmann’s kind advice. In our group and then in the laboratory the enzyme was purified to a homogenous state and used in dozens of studies (see section ‘‘Aminoacyl-tRNA


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Synthetases’’). Many years later the results were summarized and published in a survey [106] that I wrote for the journal Biochimie at the request of its chief editor Marianne GrunbergManago. When I went to see Lipmann I took with me two long-playing records – large, heavy, black discs that, probably, you can only see today in museums or the private collections of fanatic musiclovers. Engelhardt had told me that Lipmann loved classical music but I did not know his tastes. My favorite Russian composers were and are Shostakovich and Prokofiev, so I took a record of each to New York though I was not at all certain I would see Lipmann. As we made our farewells, I held out the records and said that these composers had been persecuted under Stalin but I considered them brilliant. Not knowing whether they were available in the USA I had brought them as a gift for the professor. The choice was well made! A smile lit up Lipmann’s face and he said it was a wonderful gift. That very evening he would listen to the records at home. He asked me what works were being performed. I dictated the titles and he wrote them down. I could see he was genuinely pleased by this modest gift. He placed the records in a box from his cupboard and then put the box in the briefcase next to his table. As I left Lipmann thanked me once again not just for the gifts but for my visit. It was pleasant to make the acquaintance of the younger generation of Russian biochemists, he said. Sending warm greetings to Engelhardt, he wished me every success. I had spent a little under an hour with the professor: I checked my watch after leaving his office. It was unbelievable! Twice I had asked Lipmann if I should leave – I had turned up without invitation, spontaneously – but both times he replied that if I was not in a hurry (!) then he was happy to go on talking. Our meeting left an enormous and quite indelible impression. I could not forget the charm of the man, the kindness that shone in his eyes, his uninhibited reactions and a certain special ease of communication between people of different generations: I was not yet 30 while Lipmann was almost 70. The interest he showed in what a wholly unacquainted post-doctoral student from the distant and incomprehensible USSR had to tell him was entirely genuine. There was not the slightest hint of his own importance or unique stature or, I have no qualms in saying so, his genius: I was astounded. Of course, I did not carry a business card but at

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Lipmann’s request I wrote down my surname and address in English. There was no point in leaving him off-prints of my articles since not a single one had appeared in English in 1964. Lipmann had a good memory. Next year Engelhardt received a letter from Professors Kennedy and Kaplan. Academic Press was preparing a festschrift, a volume celebrating Fritz Lipmann’s 70th birthday, and Professor Engelhardt was invited to contribute. Their letter ended with the comment that Lipmann had met a student of Engelhardt’s in New York, Lev Kisselev, and he might also be invited to make a contribution. The director quickly called me in and showed me the letter. He suggested to write an article together. I had worked in Engelhardt’s laboratory under his supervision for more than 5 years but we had co-authored only one work [110]. He was exceptionally fastidious and categorically refused to add his name to our articles because, he would say, he no longer conducted experiments himself and that was the important part of the work. Yet every article we wrote underwent his thorough ‘‘censorship.’’ Not only did he edit the text, eliminating cliche´s and officialese (which he detested), and clarifying the formulations. Often he also suggested interesting ideas that we then used in discussing the findings at the end of such articles. Naturally, I was flattered and overjoyed. Together we wrote our article [114] and after the book appeared a letter of thanks arrived from Lipmann. I believe that Lipmann’s kindly genius and irresistible charisma illuminated and accompanied the success of the research using this enzyme, bovine tryptophanyl-RSase, already described above (section ‘‘Aminoacyl-tRNA Synthetases’’).

Visits to France, 1967 and 1974 In 1967 I was in a deeply distressed condition. On November 10, 1966 my father suddenly died of a massive heart attack in his office at the Institute of Epidemiology and Microbiology. For me it was a vast tragedy and an irreplaceable loss. My father meant far more to me than is usually the case with grown children. Father was, for me, an unattainable ideal as a scientist and as a person. I was totally unprepared, psychologically, for his death and long felt an enormous void, an emptiness that nothing and no one could fill. As a close friend of my father, Engelhardt was most upset by


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his loss. I knew this and he, in turn, understood my feelings and sympathized deeply and sincerely. A wise and humane man, Engelhardt decided that if I went abroad it would help me recover. I had already attended the international biochemical congress in New York as Oparin’s translator as described above but then I was part of a delegation and under supervision. This time I was to go by myself and for 3 months, a much longer period. Engelhardt was always in favor of wide international contacts among scientists and made constant and effective efforts to support such ties. He decided to expand contacts with France. It was a good choice. French molecular biology was then, with Britain, the undisputed leader in this discipline in Europe thanks to the work of Jacob, Lwoff, Monod, Grunberg-Manago, Gros, Chapeville, Monier, Chambon, and others. For his part, Charles de Gaulle, who respected the USSR for its part in the victory over fascism, ensured that French attitudes to Soviet scientists were, on the whole, favorable. The traditional French interest in Russian culture also played its part. It was decided to send a ‘‘micro-delegation’’ of two. Boris Gottikh, an organic chemist who had studied under the outstanding Russian scientist N.K. Kochetkov, was now a deputy director (responsible for research) at our Institute and a member of the Communist Party. Married, with a daughter, he counterbalanced the major shortcomings of Lev Kisselev, the other half of the ‘‘delegation,’’ who did not belong to the Party, was still unmarried and held no administrative post. It was our good fortune that Boris and I had been friends from the first moment we joined the institute. The proposal suited me perfectly. Boris was incomparably better informed about advances in chemistry and could assess the work the French had done, which I found difficult. The aim of our visit was very precisely defined by Engelhardt. We were to go to all the major centres of molecular biology in France and gain a detailed knowledge of new methods, ideas and equipment. While there we would use lectures, seminars, and meetings to promote and publicize the attainments of the young discipline of molecular biology in the USSR. The underlying purpose of our trip was to establish personal contacts with the leading French scientists in the field. Then we would be able to organize productive, two-way scientific cooperation. Engelhardt ordered us to rapidly learn French. Knowledge of the language, he rightly hinted, however imperfect, would still be

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far more use in our dealings with the French, who took a passionate pride in their native tongue, than our more advanced English. I only managed to take 15 lessons but even that minimal achievement was to prove extremely useful. Boris made greater progress and after we arrived in France began making active use of his modest vocabulary and knowledge of French grammar. When the time came to fly to France, Boris met no hindrance but my departure was blocked by the Academy’s department of external relations. This wrecked the whole idea of a trip that combined a biochemist and molecular biologist with an organic chemist. Everything was already agreed with the French, however, so it was quite impossible to postpone or cancel the trip. Boris flew alone. Engelhardt was furious. A plan on which he spent so much time and energy had been wrecked by the Russian Academy’s bureaucrats. I took things calmly and decided to get my kayak ready for a summer vacation. By then I greatly enjoyed such river expeditions and regularly went on them with Uncle Venyamin’s son, my cousin Nikolai Kaverin (today a member of the Academy of Medical Sciences). I had underestimated Engelhardt’s strength of character, however. He took what had happened personally, as an expression of distrust in him and his recommendation, and used every available channel to obtain agreement to my trip abroad. Engelhardt contacted the highest ranking officials in the ‘‘competent bodies’’ and, personally vouching that I would come back, he secured permission for me to travel. The possibility that I might remain in France simply had not entered my head though the ‘‘competent bodies’’ did not suspect that. I was leaving behind my mother and aunt to whom I was passionately attached, the woman I loved, the work I loved and numerous friends and relations. I flew to France a month late. Engelhardt had phoned me at home, something he never did (though he had often called Father) and said that I should go to the Academy’s department of external relations the next morning to pick up my passport with visa and ticket. The next day I would fly to Paris. I had already assembled and tested the kayak and put France out of mind so the call was totally unexpected. I could detect joy in Engelhardt’s voice, that he had achieved his goal, and also pride that he had overcome resistance and mistrust. His efforts on my behalf had been kept a secret. I knew nothing and, probably, he did not want me to be disappointed a second time if he was unsuccessful.


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It was hard for me to imagine that I would be in France for 2 months. I had no experience of such extended visits and feverishly finished preparing the slides and transparencies for overhead projection. (There were no electronic slides then, after all, and though computers had just begun to appear they were not to be found in biology laboratories.) All night Mama packed my case, first putting one set of clothes into the suitcase and then taking them out and replacing them with others. The trip was financed by the USSR Academy of Sciences and so the funds were insufficient to buy anything for one’s personal needs. In Paris I was met at the airport by Professor Francois Chapeville. He knew me from a visit he had made to Moscow some 2 years before. By chance (or on purpose?) he drove me past all the world-famous sites in Paris and I recognized them in an instant: the Louvre, the Eiffel Tower, the Champs Elysees, the Arc de Triomphe, Sacre Coeur, the Palais de Justice and Sante Chapelle, the bridges over the Seine y It was a dream, I thought, it wasn’t happening to me. My mother dreamt of seeing Paris. Her dream would never come true; mine had. I was euphoric. Finally, we arrived. Franek, as his close friends called him, managed to park the car – despite the difference in age Boris and I were soon calling him that as well – although even then it was difficult to find a parking space. When on the corner building I saw rue Jacob I asked automatically in French: ‘‘Ou est la rue Monod?’’ Franek stopped, gazed at me for a second and then roared with laughter. Everyone had then heard of Jacob and Monod, who worked together though they were quite different in character. The street name, in fact, had no relation to Francois Jacob. Unlike the Soviet Union, streets in France were not named after the living. My spontaneous and elementary joke so appealed to Chapeville that he told it everyone he met. As I discovered when I first visited a French laboratory. ‘‘Are you the Russian who was looking for the rue Monod in Paris?’’ I was asked. I thus acquired fame among molecular biologists that had no connection with my scientific achievements. We faced a very full program of seminars and meetings and were eager to encompass as many scientists and laboratories as we could. This was made easier by the groundwork Boris had done by the time of my delayed arrival in Paris. Our starting point in the French capital was the laboratory of Marianne Grunberg-Manago at the Institute of Physico-Chemical

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Biology. There were several good reasons. Marianne knew Engelhardt and spoke Russian fluently. Her parents had left Russia in 1922, taking Marianne with them. She loved the country and very much wanted Russian biology to become a part of world science after decades of isolation under Stalin’s tyranny. Marianne was an exceptionally sociable person who knew everyone in Paris. Incredibly well-disposed and energetic, her recommendations opened every door and this was very important since at that time few people knew us. Above all else, Marianne was an outstanding scientist, a classic figure in biochemistry and molecular biology, and contact with her was immensely enriching. As it happened, a friend of mine was already working in Marianne’s laboratory. Belcho Belchev had studied in the same group as me at the Moscow University department of animal biochemistry and he spoke French freely, though with a strong Bulgarian accent. Belcho had lived for some while in Paris and was a great help to us in many situations, concerning scientific matters or in everyday life. Also then working in Marianne’s laboratory was the outstanding chemist Michelson, one of the founders of the chemistry of nucleotides and nucleosides, he was a striking and unconventional man. It was interesting and useful for Boris to make his acquaintance. Another colleague was M. Tang, who was from China but had long settled in France. One of the laboratories at the institute was then headed by Francois Gros, a friend of Marianne. One of those who discovered messenger RNA, Gros had previously worked at James Watson’s lab. A postgraduate student in Gros’ lab, Moshe Yaniv, was working on the aaRSases of bacteria, a subject of great interest to me since I was studying the same enzymes but in the higher eukaryotes. Moshe proved a charming person and a major scientist, as was shown by his subsequent brilliant career at the Pasteur Institute. Our scientific interests later diverged greatly but my liking and respect for Moshe, and for his wife Josette Rouviere-Yaniv, only grew with the passing years. Acquaintance with Gros left a great mark on my life. It is now 40 years since I first met that wise and most educated man, one of the most striking figures in French and world molecular biology. I am proud that this accessible and ever helpful man and his wife Danielle have honored my wife and myself with their friendship.


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Another laboratory where Boris and I received a wonderful welcome was that of Professor Francois Chapeville, Franek, which was at the center for nuclear studies in Sacle, nearby Paris. There we got to know A.-L. Haenni. Before she became Franek’s right hand, this remarkable woman had worked with Lipmann in New York. Anne-Lise had grown up speaking French, German, and English and she also knew Spanish. Over the years our friendship was reinforced by a close scientific collaboration that is revealed in a long list of joint publications. When we were later working together I and my wife often stayed in Anne-Lise’s little apartment on tiny Christine Street near the Odeon metro station. Also then working in Chapeville’s laboratory was the jolly, redheaded Suzanne Chousterman who was so full of the joys of life. She and her husband Michel also became firm friends. Nearby, in a laboratory headed by Professor Pierre Fromageot, worked Willi Guschlbauer, an Austrian by birth who was a connoisseur both of classical music and the physical chemistry of nucleic acids. He was married to Chapeville’s secretary MariePierre and they also joined the long list of our French friends. Visits to other parts of France were an essential part of our program. I rank our acquaintance with Strasbourg and JeanPierre Ebel as the most important. Ebel, a former inmate of the Nazi concentration camp and a member of the Resistance, was the father of five. Now heading the institute of biochemistry, he attracted young researchers who were inflamed by his energy, enthusiasm, and devotion to science. His great personal charm and open, genuine character helped to create a domestic atmosphere at the institute. The staff called him Papa, made a joke of his most healthy appetite, and often sought his advice on matters that bore no relation to their work. There is a very good account of him by his pupil, colleague and friend Guy Dirheimer [120]. Ebel’s wife Jacqueline, a soft feminine woman was the perfect foil to her tumultuous husband’s stormy character. A brilliant organizer and teacher, Ebel created one of the best schools of molecular biology in France, which won worldwide renown in its study of the early stages in the biosynthesis of proteins (aaRSases and tRNAs). There was good reason why a great many Soviet and, after 1991, Russian scientists studied alongside Ebel’s pupils in Strasbourg. In Marseilles we were in the kind and attentive hands of Professors Lissitsky, Monier and Denoe¨l during a visit that expanded our conception of French biology beyond biochemical

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and molecular biology. Many more names might be added but I must mention other visits and friends. The main aim of our trip was to take a closer look at our scientific neighbors and let them examine us. Indubitably, that aim was achieved. We gained faithful friends, both in science and in life, and laid solid foundations for cooperation in molecular biology. The evidence is to be found in many dozens of joint publications by Soviet (later Russian) and French scientists in the years that followed. On our return to Moscow Engelhardt received a detailed report, which emphasized how timely and valuable our visit had been. He gave it his full approval and passed it on to the Presidium of the Academy. Forty years have elapsed since, but those two unforgettable months in France remain one of the dearest memories of my life. I have since visited France many times but the impression it first made on a 30-yearold visitor will always be the most vivid. There is no need to explain how much I wished to visit France again. The next time, however, I wanted not to travel about, meeting people and seeing the country, but to engage in specific experimental work. It would require prolonged effort and the continued support of Engelhardt before, in 1974, I was able to achieve my goal. For a month I worked at the Jacques Monod institute in Paris in the laboratory of Francois Chapeville who, by then, was director of the entire institute and then I went to Strasbourg. There Ebel had become director of the Institute of Molecular and Cellular Biology and for another month I worked in his laboratory. In Paris I joined the group of Anne-Lise Haenni, which was then working on the Turnip Yellow Mosaic Virus (TYMV). I proposed to obtain a DNA copy for the viral RNA by means of reverse transcription, a procedure I had then begun to employ. A lab assistant or one of Anne-Lise’s students would isolate the RNA of that virus while from Moscow I would bring the purified revertase. (The latter was a term invented by Engelhardt to denote the RNA-directed DNA polymerase and is still widely used in Russian scientific literature.) To obtain DNA complementary to viral RNA I used a homopolimer primer to the homopolimer sequence present in this viral RNA. After prolonged incubation with revertase a cDNA product was synthesized; the size of this product was as we anticipated. During last 2 days before departure we run polyacrilamide gel electrophoresis in four hands


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with Anne-Lise and were able to complete the work which was published [121]. In Strasbourg I worked in quite a different area. Together with Franco Fasiolo and Gabi Nussbaum we conducted kinetic experiments on aminoacylation of yeast tRNAPhe by phenylalanyl-RSase. Soon after we began Franco went skiing in the Alps, leaving Gabi and myself to continue the work. I was quite content since Gabi was a wonderfully competent and intelligent assistant. Over 28 days we performed about 100 kinetic curves, each with 10 individual measurements (‘‘points’’). I then took back the long reels of data to Moscow before forwarding them to Ernest Malygin who was working under the supervision of Dmitry Knorre in Novosibirsk. We selected six most probable kinetic mechanisms of substrates binding and compared them with experimental results obtained in Strasbourg. To do that, a special computer program had been designed by Novosibirsk colleagues. It turned out that only one mechanism fits nicely the experimental results [60]. To my knowledge, it was the first case when the order of substrate binding to aaRSases had been established by this way later on widely used in molecular enzymology. This work made use of one of the first Soviet computers built at Akademgorodok in Novosibirsk. That was how the Strasbourg-Moscow-Novosibirsk axis came into being. It lasted in various forms for several decades and gave rise to a great many joint publications. On the last day of work in Strasbourg, on the eve of my departure, Ebel gathered all the institute staff. Bringing bottles of champagne, a cake and sweets, he spoke very warmly and said I could regard his institute as my home. He would be glad to see me. All clinked their glasses and wished me success. I was very touched and did not want to go, I was leaving so many friends behind.

The Revertase Project: Deviation from the Mainstream The 1970 discovery by David Baltimore and Howard Temin of reverse transcription, the RNA-directed synthesis of DNA staggered Engelhardt. He immediately appreciated the scale and significance of this advance. With a youthful energy that belied his 75 years, he set about organizing international cooperation so as stimulate Soviet work in this area. He quite rightly foresaw that this discovery would have not just theoretical but enormous

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practical consequences. Engelhardt decided that an international project should be set up in the Soviet Union, for the first time in biology that involved scientists from Czechoslovakia, East Germany, Bulgaria, and Poland. The project did not have separately allocated funding and each participant could count only on his or her own capacity and resources. There would be the widest association between participants, which should speed up the work. Engelhardt appointed me as executive director of the project while he remained in charge of research program. We carried out a number of meetings in the participating countries and the program was entitled the ‘‘Revertase Project’’ (as I’ve said, revertase was a term that Engelhardt invented). In collaboration with other participants our laboratory successfully obtained a series of results that were quite on a par with world achievements in this field. For example, we found that short heteropolymeric synthetic oligodeoxyribonucleotide could serve as a specific primer for cDNA synthesis directed by revertase in the presence of RNA template [122,123]. Later those primers became widespread and were used by many labs. Apart of that, we demonstrated the ability of very short primers to initiate cDNA synthesis at low temperature [124]. Finally, using revertase we were able to synthesize double-stranded cDNA, that is, intronless genes. At that time many labs were engaged in such type of experiments. We believe that probably the most interesting result we obtained in framework of the Project was a demonstration of the ability of revertase to utilize for cDNA synthesis a covalently interrupted template RNA [125]. This observation was very important because it explained how revertase functioned in propagating oncornaviruses, namely, how proviral DNA was formed. This result was confirmed by American virologists. Our active partners in the investigations on oncoviral RNA reverse ˇ iman and transcription were our colleagues from Prague J. R M. Travnicˇek [126,127]. It was natural that from revertase we moved towards its inhibitors [128]. The main tasks of the project were completed by 1978. The following year the USSR State Prize was awarded to a group of the most active participants, among them Engelhardt, Ludmila Frolova, and myself (Figure 6). After completion of the Project, we decided to go further and focused on search of proviral DNA sequences in human genome, products of the action of revertase during


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Fig. 6. The Revertase Project in action (1975). From left to right: W. Engelhardt, L. Frolova, and L. Kisselev.

evolution of humans. The summary of this work could be found in my review presented at Moscow FEBS Congress in 1984 [129]. Russian Version of the Human Genome Project In 1988 Alexander Bayev (see also [24]), by then widely known for his work on yeast tRNAVal, and the primary structures of ribosomal RNA and DNA, proposed that the Soviet Union set up a Human Genome Project. This would form part of the international project, which had James Watson, one of the fathers of the double helix, as one of its chief proponents. As our laboratory worked very actively on problems tightly related to Human genome [130–133] we also decided to participate. After the death of Engelhardt in 1984 and the changes that began in the USSR there was a marked deterioration in the funding of science as a whole. In its early stages the new project received financial support from the state and this played a not insignificant part in our decision. We joined in the work but at the end of 1994 Alexander Bayev suddenly died. Without a director the program faced the

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Fig. 7. Lev L. Kisselev at the time when he was in charge of the Russian Human Genome project (2002).

threat of closure. Andrei Mirzabekov, Engelhardt’s successor at the institute, proposed to the Ministry of Science that I be appointed to head the program. It was a great honor to follow Bayev but the work took much of my time and energy that would otherwise have been devoted to purely scientific activities (Figure 7). In frame of the project we concentrated mostly on analysis of repeats very widespread in human genome [134–136] and on chromosomal regions and genes associated with cancer origin and progression [137–139]. Apart of that, we paid attention for development of some new techniques applicable in genome research. Results of these efforts are described [139a–139d]. In 2002 the Ministry of Industry and Science decided to close Russian Human Genome Project. In my view, this was clearly premature. The work was progressing very actively, as can be seen from the published scientific report on the program [140]. The ministry’s expenditure was very modest but the results fully matched international standards, especially in the fields of bioinformatics and medical genomics. As part of the project’s activities the scientific council of which I was head organized a series of conferences and symposia, which were very lively and interesting. Despite the closure of the program we continue to work in the area, above all on the oncogenomics.


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I handed over the direction of this research to my very gifted pupil Eugene Zabarovsky: today he is a professor at the Karolinska Institute in Stockholm. As the impressive series of our publications [141–149] about the structural-functional mapping of chromosome 3 shows, Zabarovsky coped successfully with this role. This led to the discovery of a series of new tumor suppressor and onco-significant genes and the development of new methods. Like much other work performed in our laboratory, these studies required wide cooperation, within Russia and internationally, and this is vividly reflected in the lists of publications.

Paris Once More y The early 1990s were a tragic time for Russian science. The collapse of the Soviet Union, the transition to a ‘‘wild’’ capitalism, administrative chaos and the rampages of the so-called democracy in place of elementary order led to an almost total cessation in the funding of fundamental science. It held no interest and even hindered those impatient to take control of the oil, gas, aluminium, diamonds, and timber in which Russia is so rich. It became almost impossible to carry on working. I understood, however, that to leave science, even temporarily, in these conditions would mean to abandon science forever. A scientist has certain things in common with a footballer or a pianist. If you do not practice for a week, you notice it yourself. If you stop practicing for a month then other specialists can tell. If you give up for 3 months the spectators begin to notice and they stop coming to your concerts or to watch your play. Science demands daily exercise. Fortunately, our French friends came to the rescue. Franek Chapeville invited me for a year as a visiting professor at the Paris-7 University and the CNRS supported this with a grant for international cooperation under the PICS program. This allowed me to return to Paris but on this occasion I was no longer alone. I was accompanied by Luda Frolova, who by then had long been not only my close colleague but also my wife (Figure 8). We worked at the Jacques Monod institute with Anne-Lise Haenni, returning once in a while to Moscow so as to ensure that the laboratory there did not collapse. Work continued there as well, despite our somewhat nomadic way of life. Other researchers still firmly associated our laboratory with tRNA and the aaRSases (and

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Fig. 8. Chaire Internationale Blaise Pascal awarded. L. Frolova and L. Kisselev. Paris (1996).

to this day I receive invitations to conferences about tRNA and aaRSases although I have long moved into other areas of research). Gradually, however, we began to win an ‘‘ecological niche’’ for ourselves in ribosomology though there was already a settled and rather inward-looking scientific community in that field. Some time after our return to Moscow the municipal administration of the Ile-de-France, which also embraces the towns adjoining the French capital, announced a competition for ‘‘highranking foreign scientists’’ who would be able to work for a year in Paris and the surrounding area. The grant, moreover, was of such proportions that it could be stretched to cover a second year and include not just salaries of the 2–3 participants but the costs of inexpensive equipment and the essential reagents. Our friend Dick D’Ari saw the announcement in Nature, I think, and wrote suggesting that we apply. On reading the details I could see that only three grants were being offered for all the natural sciences. Paris was an enticing prospect for anyone. There would be a great many contenders, I thought, and decided not to get involved. Seeing that the application was very short and simple, Luda persuaded me to take the time to fill in the forms, nevertheless, adding that our chances of success were minimal. I quickly completed all the documentation (thankfully the papers required


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were few in number and all relevant!) and despatched them by e-mail to Paris. Notification arrived that they had been received and admitted to the competition. Months passed and I forgot about the Chaire Internationale Blaise Pascal, as the competition was called. Luda and I were making one of our regular visits to Paris when an e-mail arrived from my secretary in Moscow that I had won one of the three grants: I was the only biologist and the only successful candidate from Russia (the other two were scientists from the USA and Britain). Much later I learned that a member of the judges panel, one of the most outstanding contemporary biologists Changeau had singled my project out for particular praise (a total of 22 biologists had entered the competition). We were not personally acquainted since his work was in an area remote from my scientific interests. By then we had already published a series of joint articles with French researchers, in such journals as Nature, TIBS, and EMBO J, and this played its part in our success. We were based at the Curie Institute where, thanks to the efforts of our colleague and close friend Professor Armand Tavitian, excellent working conditions were provided. A room was set aside for our experimental work and specially redecorated for the purpose. We were given a small office with telephone and Internet connection and all essentials. An apartment in the grounds of the institute was made available so that we could forget about public transport and thus save a great deal of time and effort: since the flat belonged to the institute we paid half the rent a similar apartment in Paris would have cost us (Figure 9). At that moment the French contract of Tatyana Merkulova, a gifted and favorite postdoc of mine, came to an end. On my recommendation she had been working under Professor Gros with Dominique Lazar at the College de France. This was another happy coincidence. We invited her to join us and she proved a brilliant co-worker. In a few months she mastered a dual-hybrid yeast system that was entirely new to us and obtained very promising results on the interaction between translation termination factors eRF1 and eRF3 (see below). Her achievements in this respect were noted and appreciated and today she herself is a mature scientist in her own right, and directs her own research group. Tatyana gained her Ph.D. in my lab in Moscow and then, having learned French, did new work and defended her doctoral thesis in Paris, delivering her report in good French.

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Fig. 9.

L. L. KISSELEV

Paris. Nearby the Luxembourg garden (1998).

The completion of our work at the Curie Institute was marked with great festivities. A reception was given at which several leading French molecular biologists spoke and I gave a report on our work that was received with much approval. Throughout our stay we were looked after by Vladimir Mercouroff, professor at the Ecole Normale Superieure, which administered our grant. An admirable person and scientist, his concern for us extended beyond financial cares to create an exceptionally friendly and welcoming atmosphere. He and his wife Olga joined our many friends in France.


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Termination Translation in Eukaryotes: Last Love? Scientists may be classified by the most varied characteristics. One such distinction is between ‘‘romantics’’ and ‘‘systemizers.’’ A romantic takes up the subject that interests him at a particular moment and is not concerned with how it relates to his previous research. Examples of the romantic in biology are Pasteur, Mechnikov, Lipmann, and undoubtedly my father. The systemizers, at the very beginning of their scientific career, ‘‘saddle up their favourite hobby-horse’’ and ride it all their years in science: in other words, they develop a single scientific line of research. Such biochemists in Russia were Alexander Braunstein, who devoted his long life as a scientist to nitrogen metabolism or Sergei Severin [150] who studied the muscle components, carnosine, and anserine. There also exist scientists, I think, who represent a ‘‘hybrid’’ stage between these two extremes: they work on a few problems but, in each case, do so fruitfully and for some length of time. For me Frederick Sanger, twice awarded the Nobel Prize for achievements in different fields of biochemistry, could be considered an example of such a ‘‘hybrid.’’ As I am approaching the end of my scientific career I think that I also belong to this ‘‘mixed’’ category. It is shown by what I have written thus far, and is confirmed by what follows. When Ludmila Frolova, with her young colleagues, decoded the primary structure of human tryptophanyl-RSase in our laboratory [46], we were astonished by the almost total identity of this sequence with the description of the amino acid sequence of the rabbit translation termination factor established at the same time in Tom Casky’s laboratory [151]. At that very moment the primary structure of bovine tryptofanyl-RSase was established in the laboratory of Bernard and Julie Labouesse [152]. This proved practically identical to our human enzyme and Casky’s termination factor. The similarity of the tryptophanylRSases was expected since these enzymes in eukaryotes are highly conservative but the similarity to the termination factor was totally inexplicable since these proteins fulfilled quite different functions in the cell. French researchers [152] and our group [153] gave different interpretations of this unexpected similarity. We proposed in our article that the clone of cDNA used in sequencing did not belong to the termination factor but to the rabbit tryptophanyl-RSase. French colleagues attempted to

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provide a different explanation. We carried out a special investigation with Anne-Lise Haenni’s group at the Jacques Monod institute in Paris and Warren Tate from New Zealand: Tate had already done a great deal of work on the translation termination in both eukaryotes and prokaryotes. Together we provided convincing evidence that Caskey’s team used preparation of the translation termination factor containing traces of rabbit tryptophanyl-RSase; they wrongly attributed cloned rabbit cDNA to that factor resulting in serious error [153,154]. These studies raised a question about the actual structure of eukaryotic translation termination factor. This problem was solved in collaboration with researchers from other laboratories. Their efforts culminated in reliable determination of primary structure of family of translation termination factors from higher and lower eukaryotes [155], and that report was accompanied by special comments in Nature [156]. The authors demonstrated unique structure of eRF1 (as it was termed in [155]), which did not share similarity with either tryptophanyl-RSases, or bacterial termination factors RF1 and RF2 known at the time. We suggested that prokaryotic and eukaryotic termination factors have a different evolutionary origin and lack a common ancestor [155]. Although this viewpoint had been questioned [157], subsequent analysis employing a representative number of amino acid sequences followed by their correct alignment confirmed the hypothesis of independent origin of eRF1 and RF1/RF2 [158]. Subsequent X-ray analysis of eRF1 [159], RF2 [160], and RF1 [161] also demonstrated principal differences of spatial structure of these factors. Identification of eRF1 and decoding of its primary structure helped understanding of functions of some proteins with known primary structure (but with unknown functions at that time). These include yeast, X. laevis, and human proteins. Comparison of these proteins revealed high similarity of their primary structures; this suggested that amino acid sequences of the eRF1 family are highly conserved [155,158,159]. In collaboration with colleagues from Novosibirsk, our team identified tripeptide Gly-Gly-Gln (GGQ) as a common structural motif of all class-1 translation termination factors [162]; this includes eRF1, RF1, RF2, and mtRF1 families and also aRF1 family (archaeal factors are close to eRF1). This motif is located at the tip of the M-domain of the human eRF1 molecule [159].


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Modification of its glycine residues is accompanied by loss of RFactivity in vitro and in vivo. This was reported for human [162], yeast [159], and E. coli [163] class-1 factors. According to our hypothesis [162], GGQ motif is involved in peptidyl-tRNA hydrolysis in the ribosomal peptidyl transferase centre. Biochemical [164] and X-ray [165] data obtained for prokaryotic ribosomes support this hypothesis. However, it should be noted that a suggestion on direct involvement of the glutamine residue of this motif in orientation of water molecules near the ester bond [159] was not experimentally confirmed [163,166]. Based on our experiments on point mutagenesis of the M-domain of human eRF1 in conjunction with the Nuclear Magnetic Resonance (NMR) data on the spacial structure of this domain obtained in collaboration with two NMR groups (one in Russia and the other in Britain) [167] we arrived to a hypothesis according to which the indispensable GG dipeptide of the GGQ motif coordinates the water molecule nearby the hydrolysable ester bond whereas the hydrolytic reaction is catalyzed by conservative and invariant amino acid moieties located in space nearby the GGQ motif (Alkalaeva, Ivanova, and Kisselev, in preparation). Experiments revealed methylation of glutamine residue of GGQ motif both in eukaryotes and prokaryotes; this reaction requires a special methyltransferase, and methylation increases activity of eRF1 ([168] and ref. therein). Interestingly, methylation of eRF1 required its complex formation with class-2 termination factor, eRF3 (see below). We (partly in collaboration with other labs) obtained the biochemical evidence that the N-domain of eRF1 is responsible for decoding of mRNA stop codons in the ribosome [169–172], as suggested earlier [173], from indirect genetic data. This function is mediated through direct contact between the nucleotide bases and amino acid residues [174,175]. In parallel with elucidation of the functional role of the M- and N-domains, our team in collaboration with Russian, French, and Danish groups found that the C-domain of human eRF1 did bind to the other termination factor eRF3 both in vitro and in vivo [176–179]. This interaction also involved the C-terminus of eRF3 [178]. Similar results were obtained in Y. Nakamura’s and M. Tuite’s laboratories for yeast factors. Interestingly, after

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depletion from the C-domain eRF1 was able to induce ribosomal peptidyl-tRNA hydrolysis in in vitro experiments (eRF1 was used in excess) [179]. Consequently, C-domain is not involved in decoding and hydrolysis of peptidyl-tRNA, but it is responsible for physical contact with eRF3 and this significantly influences the activity of both factors as described below. During recent years, our laboratory has focused attention on a search for sites of eRF1 molecules responsible for specific decoding of mRNA stop codons at the ribosome. This problem is a mirror reflection compared to that solved when studying tRNA and aaRSases (see section ‘‘The Role of Anticodon in the Second Genetic Code’’): in that case we were looking for a tRNA site (anticodon) responsible for protein recognition, whereas in the case of eRF1 and mRNA we were looking for a codon-recognizing a protein site. Solution of a new problem required use of a different strategy. The results were quite interesting. First, it was demonstrated that class-1 factor but not the ribosome is responsible for decoding specificity [180]. This was silently suggested earlier, but the first experimental evidence was obtained for eRF1. Experiments also revealed that archaea class-1 factors are characterized by the same spectrum of decoding stop codons as eRF1; this followed from experiments in which aRF1 also decoded all three stop codons in the mammalian ribosome [181]. After localization of decoding specificity in the eRF1 N-domain (see above), a principally important fact was determined: two different sites of the N-domain are responsible for decoding of the first (U) nucleotide of stop codon and of the second and third nucleotides. In 3D structure of the protein, these sites are closely positioned, but within the primary structure they are distantly located (positions 61–64 and 125–131 in human eRF1). This result has shown that in contrast to tRNA anticodon, which recognizes mRNA codon by forming a 2D codon–anticodon duplex, in the case of eRF1, this is 3D-recognition involving various fragments of the polypeptide chain ([182] and ref. therein). So, it is irrelevant to apply the term ‘‘protein anticodon’’ for eRF1; it is more accurate to employ a 3D-protein discriminator for characterization of this interaction. Identification of two key conservative motifs of eRF1 (NIKS and YxCxxxF) involved in decoding made possible artificial in vitro simulation of the process, which happened a long time ago: point substitutions of two amino acids in the N-domain converted


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omnipotent human eRF1 into the unipotent factor recognizing only UGA stop codon [170]. Such type of stop codon recognition is typical for some ciliate infusoria (Stylonychia, Tetrahymena, etc.) possessing a variant genetic code [183]. This experiment confirmed the hypothesis of molecular evolutionists suggesting that uni- and bi-potent factors originated from omnipotent ones [180]. Based on results of these experiments, we proposed a new hypothesis that transition from omnipotent into unipotent or bipotent states is achieved due to negative elements, which block some side chains of amino acids and therefore prevent their involvement in recognition of one or two stop codons [170]. Indirect support for this hypothesis came from joint experiments with Nakamura’s team [171]. This study demonstrated conversion of unipotent factor into omnipotent one achieved by simple temperature increase. This effect may be attributed to the increased mobility of side chains of amino acids attenuating or abolishing their negative effect on recognition. The concept of ‘‘negative elements’’ [170] responsible for loss of omnipotence in variant-code organisms has recently been confirmed in direct experiments. Using the method of inter- and intra-domain molecular chimeras developed in our laboratory, we identified negative element in the N-domain of Stylonychia eRF1 [184]. In this infusorian eRF1 recognized only one stop codon, UGA. This negative element is positioned at 122–124 (using human eRF1 numeration) near conservative, functionally important motif YxCxxxF. It probably prevents contact of the second nucleotide of stop codon, A, with eRF1 recognition site (the YxCxxxF loop). In this position of human eRF1 there is tripeptide threonineserine-lysine (TSL), lacking properties of negative element. Interestingly, in another ciliate Euplotes, recognizing stop codons UAA and UAG, there is another mechanism limiting omnipotence and eRF1 lacks negative element positioned at 122–124 and fragments of the negative element are located in completely different sites of the eRF1 molecule. The third type of stop codon restriction was discovered for UGA-only eRF1 from Paramecium which differed both from that in Stylonychia and Euplotes [184]. Since the shape of prokaryotic RF1 and RF2 molecules in crystals significantly differs from that of eRF1 crystal structure, a reasonable question on eRF1 behavior in solution has arisen. The shape and size of eRF1 (Figure 10) does not differ significantly

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Fig. 10. Low-resolution shape reconstruction from SAXs data (a) and the crystal structure of human eRF1 (b) in two projections (901 turn about the vertical axis) (courtesy of Artem Kononenko).

from that of the crystal structure [185]. This emphasizes big differences between eukaryotic and prokaryotic class-1 RFs. However, this 3D structure is non-adjustable to the dimensions of the ribosomal A-site where it has to be placed within the ribosome. It implies that eRF1 should undergo a conformational adaptation and change the angles between domains, in particular, diminishing the distance from the NIKS motif to the GGQ motif ˚ to no more than 76 A ˚ . Molecular dynamics analysis from W100 A has shown that it could be achieved due to flexible linkers connecting domains with one another [186]. Molecular modeling shows that the shape of eRF1 inside the ribosome could fit the A-site cavity. More importantly, in two models generated by computer, the functionally essential NIKS and YxCxxxF loops appeared to be close to the nucleotide bases of a stop codon. Potentially, the domains of eRF1 exhibit an ability to interact with one another as shown by differential scanning calorimetry [187], isothermal titration calorimetry [188], and circular dichroism [187]. To reconcile all these data [185–188], apparently controversial, one may assume that in solution and in the ribosome the domains interact with one another by their proximal parts located at the center of the protein globule, forming a ‘‘core’’ which melts as a single unit.


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Our team made substantial contributions to discovery and study of class-2 termination factor eRF3. This factor was identified [176a,176b] soon after discovery of prokaryotic RF3. In collaboration with French scientists and colleagues from St. Petersburg, we demonstrated that eRF3 belongs to a class of GTPases [189]. GTPase activity of human eRF3 was experimentally demonstrated before demonstration of such activity in E. coli RF3. In contrast to RF3 and other GTPases, manifestation of this activity in eRF3 requires the presence of both ribosomes and eRF1 [189]. eRF1 and eRF3 exhibit mutual influences: GTPase activity of eRF3 requires the presence of eRF1, whereas manifestation of eRF1 activity in vitro at low concentrations of template oligonucleotides requires the presence of eRF3 [189]. After it was discovered that human eRF1 and eRF3 physically interact with one another [176,178,179], no quantitative analysis of the eRF1deRF3 complex formation was performed for a long time. Recently, we applied two different methods to measure this binding using isothermal titration calorimetry (ITC) [189] and fixation of the complex on the nitrocellulose membranes [190]. In the first case this work was done in collaboration with A. Makarov’s laboratory in our Institute, in the second case with M. Ehrenberg’s laboratory at Uppsala University. The most exciting result of these works was a discovery that eRF3 despite possessing a GTP binding site is unable to bind to GTP, while being complexed with eRF1 it acquired this ability. Furthermore, the MC-domain of eRF1 is sufficient to induce GTP binding to eRF3 in the absence of the N-domain of eRF1 [188]. We assume that a quaternary eRF1deRF3dGTPdMg2þ complex is formed in solution and probably in cytoplasm that enters the ribosome in which GTP hydrolysis could proceed in the absence of stop-codon binding to eRF1 [188,189]. The thermodynamic constants of interactions between eRF1, eRF3, Mg2þ, guanine nucleotides, and individual domains of eRF1 are presented in Table 1. Various hypotheses were suggested with regard to possible biological role of eRF3 in translation termination in eukaryotes [191,192]. It was emphasized that from genetic, biochemical and structural reasons it follows that RF3 and eRF3 are neither structural nor functional homologs in contrary to what was thought by others earlier. Very recently the major function of eRF3 has been elucidated (see below).

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Thermodynamic constants of interactions between human eRF1, eRF3, Mg2þ, guanine nucleotides, and individual domains of eRF1 in solutiona (courtesy of A. Kononenko and V. Mitkevich)

Sample

eRF3 eRF3 eRF3 eRF3C eRF3M eRF3MC eRF1eRF3 eRF3 eRF3 eRF3GDP eRF3 eRF3 eRF3 eRF3 eRF3M MþC eRF3 eRF1eRF3 eRF1eRF3 eRF3C eRF3M eRF3MC eRF3(MþC) eRF1eRF3GDPh eRF1eRF3GTPh

Ligand

GDP GDP GDP GDP GDP GDP GDP eRF1 eRF1 eRF1 N-domain M-domain C-domain MC-domain C-domain eRF3 NM-domain GTP GTP GTP GTP GTP GTP GTP GDP

Kdc DHd TDSe DGf MgCl2 Kab 1 (mM) (M ) (mM) (kcal/ (kcal/ (kcal/ mol) mol) mol) 0 2 10 2 2 2 2 0 2 2 2 2 2 2 2 2 2 0 2 2 2 2 2 2 2

9.1 105 5.6 105 3.6 105 4.8 105 7.7 105 6.4 105 5.1 105 6.0 105 1.4 106 4.9 106

1.1 1.9 2.8 2.1 1.3 1.6 2.0 1.7 0.7 0.2

6.7 105 1.0 106 3.5 106 1.3 106 2.7 106

1.5 1 0.3 0.8 0.4

3.6 105 2.0 106

2.8 0.5

2.6 106 7.5 105 3.1 106 4.6 105

0.4 1.3 0.3 2.2

9.8 9.2 2.1 12.3 12.8 11.2 11.8 7.4g 7.2g 3.0g ND 3.7g 16.2 8.8g 13.5 7.7 ND 1.1 2.2 ND ND 2.8 0.5

1.7 1.4 5.5 4.5 4.8 3.3 4.0 0.5 1.2 6.0

8.1 7.8 7.6 7.8 8.0 7.9 7.8 7.9 8.4 9.1

11.6 8.0 0.1 5.2 1.1

7.9 8.2 8.9 8.3 8.8

8.7 6.4

7.6 8.6

5.9 7.5

8.7 8.0 8.9 7.7

ND, Not detected; N, M, and C, individual domains of eRF1; NM and MC, two-domain eRF1; MþC, equimolar mixture of two individual domains of eRF1. a All measurements were performed 3 or 4 times in 25 mM K2HPO4, pH 7.5, 10% glycerol, 1 mM DTT, 0.1 M KCl. b Ka – association constant; standard deviation was 720%. c Kd – dissociation constant; calculated as 1/Ka. d DH – enthalpy variation; standard deviation was 78%. e DS – entropy variation; calculated from the equation DG ¼ DHTDS. f DG – Gibbs energy; calculated from the equation DG ¼ RTlnKa. g DH was calculated taking into account the effect of protonation as described [189]. h The model of competitive ligand binding was used [189].

Some more facts elucidated in our group about eRF1 and eRF3. In joint study with M. Yarus’s laboratory (USA) we obtained RNA aptamers, which selectively bind to eRF1 and to eRF3 and inhibit their activity [193]. In collaboration with A. Favre et al. and using


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the method of selective covalent cross-links between short mRNA and eRF1 [194], we found that eRF1 binding with the ribosome occurs in two steps, and the first one does not depend on stop codon, whereas the second step depends on stop codon [194]. In collaboration with Tatyana Pestova and her group we created a completely reconstituted eukaryotic translation system (RETS) from individual components [195]. Thanks to this system we established three fundamental facts concerning the translation termination mechanism in eukaryotes. First, we showed that eRF3 is an analog of prokaryotic RF3 neither in terms of its functioning nor the structure. eRF3 strongly accelerates peptidyltRNA hydrolysis within the ribosome via binding to eRF1. This interaction is mediated by the C-domain of eRF1. In contrast, RF3 is not involved in this reaction but facilitates release of RF1/RF2 from the ribosome as shown in Ehrenberg’s laboratory. Second formation of pretermination complex is accompanied by a large conformational change revealed by toe-print technique. Third, GTP hydrolysis takes place before not after peptidyl-tRNA cleavage as opposed to what is known for prokaryotic system. All that is summarized on a scheme (Figure 11). I believe that all new data obtained in our lab and in collaboration with other groups demonstrate a profound difference in molecular mechanism of translation termination between eukaryotes and prokaryotes. We also employed methods of bioinformatics for analysis of stop codon contexts. These results [196, 197] have been subsequently confirmed by other laboratories. For example, in prokaryotes and eukaryotes nonrandom distribution of some nucleotides at –1 and þ4 to the stop codon was observed. Bioinformatics also assisted

Fig. 11. A model for translation termination in eukaryotes. Step 1: Binding of eRF1eRF3GTP complex to the pretermination complex. Step 2: Translocation/ structural rearrangement of the pre-TC. Step 3: GTP hydrolysis by eRF3. Step 4: Peptidyl-tRNA hydrolysis (courtesy of Elena Alkalaeva).

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in identification of a new site specific for bacterial termination factors RF1 and RF2 [198]. Summarizing results obtained in our laboratory, we came to the following main conclusions. In the eRF1 protein family, there is a clear correlation between domain organization and functions [199]: the N-domain is faced to the mRNA side; it is partially positioned on the small ribosomal subunit and provides direct interaction of eRF1 with one of three stop codons. The M-domain is faced to the large ribosomal subunit; using GGQ motif it interacts with peptidyl-tRNA in the peptidyltransferase center and induces hydrolytic reaction of peptidyl-tRNA cleavage into polypeptide and free tRNA. Apart of that, the M-domain binds to eRF3 [188]. The C-domain binds to class-2 termination factor eRF3 and jointly with the M-domain induces its GTPase activity within the ribosome; this interaction does not require the N-domain, and the MC-domain is fully competent for realization of this function (Figure 12).

Fig. 12. Functional anatomy of eRF1. Locations of the functional sites are boxed. The N-terminal domain is in charge of stop-codon decoding; the M (middle) domain interacts with the eRF3 protein and peptidyl transferase center of the ribosome; the C-terminal domain binds to the C-terminal domain of eRF3. NIKS, a conservative functionally important motif; GGQ, invariant functionally important motif; BS, binding site; RBS(s), ribosome binding site(s); PRIS, peptidyl-tRNA interaction site; TCRS, termination codon recognition site.


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The second conclusion is related to resemblance between eRF1 and tRNA. Y. Nakamura et al. underlined the structural similarity between tRNA and class-1 termination factors [157]. Based on detailed analysis, I proposed a concept of functional similarity of tRNA and class-1 termination factors and provided convincing arguments that these are true functional homologs [200]. I referred the proteins of class-1 termination factors to a special type of proteins defined as ‘‘protein nucleic acids’’; being proteins in nature, these are functionally equivalent to nucleic acids. This idea may have intriguing consequences because besides class-1 termination factors other proteins functioning as nucleic acids may also exist. Thus, in spite of basic chemical differences proteins and nucleic acids do not have a functional gap.

Human Frontiers Science Program: Intercontinental Cooperation The Human Frontiers Science Program was set up by the international scientific community, drawing largely on funds from the USA and Japan. Its main goal is to encourage international or, to be more precise, intercontinental scientific collaboration, in the most currently important fields of biology of an inter-disciplinary nature. The prestige of this competition is very great though the grants it offers are not large and sufficient only for three researchers, at most, from each side. The Tokyo University geneticist and molecular biologist Yoshikazu Nakamura, who has successfully studied the termination of protein synthesis in bacteria, and then in yeast, suggested to me to prepare a joint grant application. Warren Tate from the University of Otago in New Zealand and Michel Philippe of Rennes University in France were invited to join the project. The result was a four-sided group in which each could act independently within the framework of the common program and in cooperation with the others. In parallel we studied the termination of translation in bacteria and eukaryotes, exchanging information, and plasmids, and, when necessary, working in the laboratories of our program partners. I was entrusted to write the draft of basic text of our application, in Rennes and then in Moscow, following which it was expanded and edited by our

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partners. To our great joy, the project was given a high assessment and was successful. For 3 years we enjoyed financial support and though modest in scale it was quite sufficient for the work of our small group. The laboratories working actively in the same field at that time were involved in our project. Instead of competing we interacted and helped one another. In a number of our publications it is indicated that the work receives the support of the Human Frontiers Science Program.

Editorial Activities Laboratory work occupied much of my time, naturally, but I wanted somehow to expand my scientific horizons. I did not want to know only what was essential to carry out my own experiments and supervise those of my students and postgraduates. During my first years as a scientist my income, like that of other Soviet researchers, was extremely modest. A desire to improve my finances led me to prepare abstracts for the Biochemistry section in Biology, a reference journal published by the All-Union Institute of Scientific and Technical Information (AISTI). It added little to my salary: the rates of pay were extremely low. More importantly, I considerably widened my knowledge of biochemistry and significantly enriched my English vocabulary. Usually I did the work at home in the late evenings, immediately writing up the abstract in Russian on a typewriter. This taught me to express myself clearly since there was no time or desire to retype the texts. After I defended my doctoral thesis in 1972 I decided to found a new reference journal Molecular Biology that, until then, had not been among AISTI’s publications. The first issue was published in 1975 and it continues to appear today. A great role in its creation and successful existence was played by Valentin Milgram. He had a great experience of working at AISTI and invested much energy and knowledge in the new publication’s organization. The journal undoubtedly played a considerable role in the development of Soviet molecular biology. In the 1970s and 1980s access to foreign scientific journals was extremely limited, especially outside the major research centers, and so the reference


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journal Molecular Biology was for many the sole source of scientific information from abroad. Not to mention the very mediocre knowledge of English that was then typical of our research staff. Alongside the journal Milgram and myself also managed collections of articles (there were nine in the series) that gave an overview of molecular biology and discussed both issues of general topics and of methods. Undergraduates and graduate students, I was very pleased to learn, made active use of these publications when preparing for exams: this served, at least in part, to make up for the severe shortage at that time of study aids in molecular biology. I was chief editor of the reference journal Molecular Biology, from 1975 to 1986. The volume of my work at the institute steadily grew. The situation at AISTI was ever less conducive to work and so I decided to end my activities there. The acquired experience would prove very useful, however. At the request of Andrei Mirzabekov, who succeeded Engelhardt as director of the institute in 1985, I began to help him run the journal Engelhardt had founded in 1967 and edited until his death. He became the chief editor of Molecular Biology (MB) and I was his deputy editor. Mirzabekov began to spend more and more time in the USA, where he developed biochip technology, and in 1996 he handed over the running of the journal to me. My many years experience of working with AISTI and the broad scientific perspective I acquired there now proved of great value. I have now been the journal’s chief editor for more than 10 years and it gives me satisfaction. The work we do as editors with our young authors indubitably encourages their growth as scientists: it teaches them to express their thoughts clearly and to avoid the long-winded texts and unproven assertions that some are prone to. A number of articles published in the journal have been singled out for prizes by the publisher, Nauka/Interperiodika. Unlike many international journals that levy a charge for publication on their authors and do not pay reviewers for their work, we offer small fees to both authors and reviewers. Since Molecular Biology is published in both Russian and English it has, of late, begun to attract authors from other countries, above all China and India, where interest in fundamental biology has been on the increase.

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Probably, my activities as publisher and editor did not pass unnoticed and I was invited to become one of the editors of two international journals: FEBS Letters (Amsterdam) and Biochimie (Paris). In the first case, I was recommended by Vladimir Skulachev who was by then already working for FEBS Letters; in the second instance, I received an invitation to participate from Marianne Grunberg-Manago. In both cases I considered I had no right to refuse. I did not then appreciate how much work would be involved. ‘‘It’s dangerous work, Lev!’’ said my friend Giorgio Semenza, for many years chief editor of FEBS Letters. Indeed, when you have to turn down an article by someone you have known for many years because your pitiless reviewers have spotted defects in his work, your duties as an editor and a friend come into conflict. At such moments there’s a strong temptation to leave the job and take up more peaceful work. Since I began working with FEBS Letters there has been a notable shift in the geographical distribution of our authors. In the 1990s articles from Europe dominated. Today a great proportion of the articles come from Japan, Korea, and the USA. This genuinely reflects the rapid growth of biochemistry in Asia and the fierce competition in the USA that makes certain scientists keen to ‘‘take refuge’’ in Europe where there are fewer American editors and reviewers. From time to time the editors of FEBS Letters gather to discuss the pressing issues that arise in our demanding and very responsible work. I find these meetings most interesting. They give us an opportunity for direct contacts and sharing of experience, and a chance to compare our criteria for accepting or declining particular manuscripts.

Scientific Research as a Source of Optimism The life of my generation (Figure 13), for most of the time, was overshadowed by the totalitarian, Communist system. That existence embodied a paradox, however. In the humanities and in literature, music, and art creative freedom hardly existed. There was undoubtedly creative freedom in the natural sciences, on the other hand. This was despite the destruction of Soviet biology by the dominance of Lysenko, the distortion of Pavlov’s ‘‘dogma,’’ and the efforts of Lepeshinskaya and Boshyan in the


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Fig. 13. Among friends: With Elena Lyapunova (a biologist) and Vladimir Arnold (a mathematician).

1950s. Science formed an ‘‘island of liberty.’’ It attracted the young and especially the most gifted among them, those who had no wish to serve the regime as Party functionaries or State officials. In the decades following Stalin’s death Soviet science thus gathered around it the best representatives of the younger generation: emigration was not an option and their striving for self-realization pushed them into research. As a result, new scientific institutions emerged in the late 1950s and early 1960s, the years of the post-Stalin ‘‘Thaw’’ under Nikita Khrushchev, that would become centers for modern biological research of European and even world stature: the Radio-Biological department at the Institute of Atomic Energy, the Institute of Molecular Biology, the Institute of Protein Research, the Institute of Physico-Chemical Biology at Moscow State University, the Institute of Bioorganic Chemistry, and the Novosibirsk Institute of Cytology and Genetics. Among those who played a major role in their establishment and development were A.N. Belozersky, V.W.A. Engelhardt, M.M. Shemyakin, A.S. Spirin, D.G. Knorre, Yu. A. Ovchinnikov, R.B. Khesin, M.A. Prokofiev, and A.A. Bayev. The Engelhardt Institute was our own ‘‘island of liberty’’ and enabled us to pursue the scientific research that was of interest to

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us. To our great good fortune the Party and the KGB did not tell us which molecules were good and which were bad; they did not dictate what we should and should not do. Nobody tells us what research we must do today either. The ideological oppression of the past has now been replaced, however, by economic pressures. Funds are mainly channeled towards purely practical goals and fundamental science suffers as a result. A system of funding through the allocation of grants, which is generally accepted in the western scientific world, has been adopted in Russia but the grants are not distributed after objective assessment by experienced and qualified specialists. Russian democratic institutions are weak, there is universal corruption among state officials and clan loyalties prevail in science. In these conditions, lacking a genuine and effective community that can exercise peer group review, the allocation of sums, which are often very substantial, is made according to the desires or instructions of functionaries who divide scientists between those whom they recognize and those they do not. (It is not hard to guess how such distinctions are made.) Nowadays the struggle to obtain such funding takes more and more of the time and efforts of the best scientists. This, in turn, reduces their opportunities for leisure. Yet such leisure moments, such ‘‘idle’’ time, form one of the most important elements of scientific creativity – periods when unexpected ideas and profound thoughts may visit the scientist. The strength of the Russian approach to science, it seems to me, has always lain in its preference for tackling problems on the grand scale, the originality of its methodology, its fresh perception of established facts and their inventive interpretation. These strengths remain today and are valued in those Russians who work abroad by the majority of western laboratories. The conditions under which Soviet science formerly existed, and especially biology, which, thanks to Lysenko and his cronies, was set back dozens of years, might have led to its total degradation. Isolated from the world scientific community, it had meagre access to scientific publications and the quality of its equipment and reagents was poor. Yet it did not succumb. The totalitarian regime, I believe, led to the formation of an ‘‘iron’’ generation that was able, despite all obstacles and restrictions, to practice science and attain results of world calibre. In such conditions it was, of course, important not to follow the fashion in any discipline or to


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trail in the wake of others ideas and achievements. You had to set off in your own direction, experimentally confirming your ideas, not those of others. Imagine what would happen if contemporary post-doctoral US students, who are accustomed to ready-made protocols, an easily available range of reagents and to following the mainstream of research, were subjected to the conditions under which we labored in the 1960s to 1980s. The great majority, I believe, would find the task impossible and quickly give up science. During the past 15 years, when there has been a mass exodus of our young (and not so young) scientists, the West has come to appreciate the advantages of the Soviet educational system, which are partially preserved in today’s Russia. The Russian schools of mathematics and physics have enriched American and European science with numerous outstanding works. Against this background Russian biologists make a less striking impression but they also are in no way inferior to the best researchers at work in Europe today. Contemporary science, including biology, constantly encounters two problems. One, the accumulation of great volumes of new information requires an ‘‘industrialization’’ of the process. Large teams of researchers, most often international in complexion, are assembled under the leadership of a powerful figure who is sometimes more of a manager than a true scientific supervisor. It is often difficult for individuals to express their own creative potential in such large groups, since they are only one among many and work according to a division of labor laid down in a single plan. Two, research is becoming ever more expensive. The equipment is more complex and, as a result, costs increase, while individual components are replaced by sets of components that are many times dearer. And there is a third problem. Society today exerts its own pressure on scientists. This mainly takes the form of a demand for immediate practical benefit from any research and a striving to obtain some return or income. As a consequence, fundamental research is under-financed and, worse still, the prestige of scientists who engage in such pure study is diminished. Undoubtedly, the explanation lies in public ignorance. The philistine has no idea that any application of science depends entirely on prior achievements in fundamental research. When no such scientific research is being conducted in a country

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its progress is entirely at the mercy of imported technology and knowledge and that comes at a high price. Science in Russia is no exception. It suffers from the problems I have just outlined. There are aspects, however, that are specific to our country. Russian biologists, unlike many of their physicist colleagues (those working on the ‘‘atom project,’’ for instance), have not had a tradition of industrial-scale activity. The Russian genome project, to take one example, did not extinguish the creative individuality of its participants. Fundamental science, undoubtedly, will benefit from the fruits of such collective labor but, I hope, will not be transformed into something similar. I have never wanted to run a large laboratory and have always preferred quality to quantity. Three weak researchers can never replace a single strong researcher. The under-funding of Russian science is not entirely negative. It has led to a quest for original solutions, unusual subject matter and a greater creative freedom. When I observed at close hand the work of certain European and American laboratories I was amazed how wasteful they could be. For the same money it would be possible to obtain 2–3 times as much information in Russia. In one laboratory, I remember, each researcher who was working with radioactive GTP would open the tube and use no more than 10–20% of the contents. No one thought of using all there was y Stalinism and the legacy of totalitarianism burden Russian science to this day. An attitude of suspicion and lack of trust towards our scientists may still be found in the West. As we in Russia have long noted, certain international scientific periodicals avoid publishing our articles if we do not have American or European co-authors. Recently I was due to speak at an international scientific conference in the USA. I was given an entry visa to the USA on the last day of the event, although I had submitted my documents almost 2 months earlier. It remained unclear what threat I presented to the United States if I had been invited by the conference organizers to participate. It is behavior reminiscent of our Communist leaders in times gone by, when they did not permit certain Western scientists to visit the USSR. Looking back, I see two major peaks in my scientific career. The first covers the years from the early 1960s to the early 1970s. The second started in 1991 and hopefully is not yet completed. They were separated by a very long period. During that time I wrote a


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great many articles (by mid-2007 I had over 450 publications to my name, including 3 books and more than 30 reviews); I received the USSR State Prize (1979) and the Gregor Mendel Gold Medal (1982); and in 1990 I was elected a corresponding member of the USSR Academy of Sciences. Despite all these outward signs of success, however, I have a sorrowful feeling that I might have produced so much more and achieved more significant advances in knowledge. During those years, perhaps, quantity prevailed over quality. In any case, it cannot now be changed. The only advice I can offer the younger generation is very simple though probably not easy to follow. Sooner or later, you will attain the state of scientific independence. At that point, stop working every day and for a short while reflect on how you want to advance further. Then set yourself goals for the next 7–10 years. It is my impression that 7 (or, at most, 10) years is a sufficient span within which to reach significant results. Two, don’t be afraid to profoundly change the topic of your studies. In many cases (but, of course, not always) this can be most stimulating and productive. Finally, don’t try to do everything within the confines of your lab. Collaborate with strong groups inside and outside your country, otherwise you may grow stale and unproductive. Never forget: science is the most enjoyable and interesting thing you can do with your life on Earth.

ACKNOWLEDGMENTS

I am deeply thankful to Giorgio Semenza, who invited me to prepare this chapter, provided me with good advice and made very helpful comments on the first draft of the manuscript, to Vladimir Skulachev, who kindly corrected some parts of the text and shared with me some good ideas, to John Crowfoot who contributed enormously to the English version of this chapter, to Svetlana Tvardovskaya, who typed and attentively corrected this text many times, to Luda Frolova who is in science and life with me for more than 45 years. I warmly and sincerely thank all my students, co-workers, collaborators and colleagues mentioned in my publications for their significant, valuable, and numerous contributions to what was done by myself in science.

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REFERENCES Before the Second World War and soon after my father transcribed his name as Silber because most of the publications at that time appeared in German. Later, when he published mostly in English, he transcribed his surname as Zilber. Russian journals Biokhimiya and Molekularnaya Biologiya are available in English. Although the volumes and years coincide in both versions, the page numbers differ. Here they are given for Russian edition if not specified otherwise. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]

Kisselev, L.L. (1990) Wladimir Engelhardt: The man and the scientist. In Comprehensive Biochemistry (‘‘Personal Recollections’’. III) (Semenza, G. and Jaenicke, R., eds.), Vol. 37, pp. 66–99. Amsterdam, Elsevier. Kisselev, L.L., Abelev, G.I. and Kisseljov, F. (1992) Lev Zilber, the personality and the scientist. Adv. Cancer Res. 59, 1–40. Skulachev, V.P. (2002) A risky job: In search of noncanonical pathways. In Comprehensive Biochemistry (‘‘Personal Recollections’’. VII) (Semenza, G. and Turner, R., eds.), Vol. 42, pp. 319–410. Amsterdam, Elsevier. Kisselev, N. and Kisselev, L. (1961) Electron microscopy of the soluble ribonucleic acids. Dokl. Akad. Nauk SSSR 141, 980–983. In Russian. Kisselev L.L. and Levina E.S. (2005) Lev Aleksandrovich Zilber (1894–1966): Life in Science, p. 699. Moscow, Nauka. Silber, L. (1945) Viruses and tumours. Clin. Med. (Klinicheskaya Medizina) 23(12), 79. In Russian. Silber, L. (1945) On the origin of malignant tumours. J. Microbiol. Epidemiol. Immunol. 9(43), 4–5, 16. In Russian. Zilber L. (1968) Viro-Genetic Theory of Tumour Origin, p. 299. In Russian, Nauka Publishing House. Silber, L. and Soloviev, W. (1946) Far-Eastern tick-borne spring-summer (spring) encephalitis. Am. Rev. Sov. Med. (Special suppl.), 1–80. Silber, L. (1946) On the filtrability of tumors induced by 1,2,5,6dibenzanthracene. Am. Rev. Sov. Med. 4, 100. Zilber, L. (1957) Studies on tumor antigens. J. Nat. Cancer Inst. 18, 341. Zilber, L. and Kriukova, I. (1957) Haemorrhagic disease of rats due to the virus of chick sarcoma. Acta Virol. 1, 156. Zilber, L. (1958) Specific tumor antigens. Adv. Cancer Res. 5, 291. Zilber, L. (1960) Progress in experimental virology of cancer. Progr. Exp. Tumor Res. 1, 1. Zilber, L. (1961) On the interaction between tumor viruses and cells: A virogenetic concept of tumorigenesis. J. Nat. Cancer Inst. 26(6), 1311. Zilber, L. (1962) Interaction between tumor viruses and cells in cysts and tumors induced by these viruses in various animal species. Cold Spring Harb. Symp. Quant. Biol. 27, 513. Zilber, L. and Shevljaghyn, V. (1964) Transformation of embryonic human cells by Rous sarcoma virus. Nature 203, 194. Zilber, L., Lapin, B. and Adgighytov, F. (1965) Pathogenicity of Rous sarcoma virus for monkeys. Nature 205, 1123.

I’M NOT DISENCHANTED WITH SCIENCE [19] [20]

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[23] [24] [25] [26] [27]

[28]

[29]

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[32]

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A FAREWELL Prof. Lev L. Kisselev passed away April 12th, 2008. You reader, who have read his chapter, have certainly sensed that in writing it, while recounting his life through WW2, and his remarkable scientific achievements in Soviet and in post-Soviet Russia, he was, in fact, writing his moral and scientific testament. We, the editors of this volume (see our Preface to it) and many colleagues and friends are close to his family in mourning him. His unflagging uprightness, his profound and self-effacing scientific curiosity we will miss forever. We, both in the so-called ‘‘West’’ and in the so-called ‘‘East’’ knew that he would always be ready to help, to co-operate across both real and fictitious frontiers. We do have a hope: that the young generation of scientists will remember Lev and follow his example, in terms of both personal and scientific honesty, reliability and search of the truth. G. Semenza, Zurich

V.P. Skulachev, Moscow


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Chapter 2

Jacques Monod, Protein Folding and Me MICHEL E. GOLDBERG Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France Email: [email protected]

Abstract Jacques Monod, a prominent figure of the golden age of Molecular Biology, is well known for his contributions to understanding the regulation of gene expression (the ‘‘operon’’ model) and the regulation of enzyme activities (the ‘‘allostery’’ model). Few people are aware of his deep interest in the mechanisms of protein folding, which he foresaw would become a problem of major importance. The author of the present chapter describes the meandring pathway which led him to join the laboratory of Jacques Monod and the way in which Jacques Monod triggered his interest for protein folding, inspired his first discoveries and influenced many of his contributions to an emerging area of research which now stands at the forefront of biological research. Keywords: Protein folding; Protein aggregation; Physical biochemistry; Institut Pasteur

The Pathway to the Institut Pasteur: Chance or Destiny September 28, 1938. Sure! I was born to be a happy one! I was told that my mother emerged from the pains of childbed in an outburst of relief and joy upon hearing that Hitler, Mussolini, Chamberlain, and Daladier were meeting in Munich to solve peacefully the Sudete crisis. And indeed, 2 days later, the Munich Treaty was signed. There would be no war y

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September 28, 1938. The 43rd anniversary of Louis Pasteur’s death. Was I born under the sign of this monumental figure of science? Was I coined by destiny to become one of his many admirers and followers? Thinking it over retrospectively, one could have thought so: my father was a tanner, like Louis Pasteur’s. The Nanny who took care of my older brother and of myself was Mademoiselle Meister, a niece of Joseph Meister. Her uncle had been the first person treated by Pasteur with antirabies vaccine. The first human being to escape the thus far inescapable death that followed bites from an enraged dog. I was told years later that Mademoiselle Meister used to talk frequently about Louis Pasteur, with much admiration and gratitude. Yet, I do not believe that Mademoiselle Meister had a significant influence on my future career, since she left us when I still was a baby. I should rather say that we left her y September 1, 1939. The hopes for a peaceful future, briefly raised by the Munich Treaty, had for long been forgotten. World War II broke out. A war which would have a tremendous impact on our and Mademoiselle Meister’s families. Until the war, Joseph Meister worked at the Institut Pasteur. He took care of the horses used for producing therapeutic immune sera. In 1940, soon after Paris was taken over by the Germans, officers from the Wehrmacht came to the Institut Pasteur and asked to enter the crypt where Louis Pasteur is buried. Joseph Meister, a proud Alsacian, refused to open them the crypt. He returned to his room and, unable to overcome the sorrow of France’s defeat, shot himself dead with the army pistol that he had kept after World War I. We did not witness Mademoiselle Meister’s reaction to this tragedy. We had left her behind when the German army invaded France. My parents, like many others, fled to escape the fighting’s. I have been told that, during our exodus, in the endless line of cars, trucks, buses, horse carts, bicycles, pedestrians that slowly moved southwards, under the bombing and shooting of the German fighter planes, I spoke my first words: ‘‘Tank’’ and ‘‘Airplane.’’ Not ‘‘Maman’’ or ‘‘Papa.’’ After a few days, the fighting’s were over. My father, a confirmed optimist, decided to bring us back to Paris and see how one could manage with life in the occupied city. Rather than settling back into the villa, which my parents owned in Montmorency, a charming residential suburb some 10 miles north of Paris, we moved into a

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flat they had been renting since their wedding. It was located near the Buttes-Chaumont Park, on the second floor of a rather elegant building. It had a balcony overlooking the avenue Laumie`re. This feature enabled me, at the age of one and a half, to perform one of the first acts of resistance against the Nazi occupation: while playing on the balcony with a set of wood skittles, I threw one over the handrail. Skill, chance, or destiny? The skittle hit a German officer on his head and knocked him down for a couple of minutes. We luckily escaped severe retaliation through the fearless intervention of a French policeman, on ward in front of the nearby police station, who had witnessed the ‘‘aggression’’ and certified that it was nothing but an accident caused by a careless baby. Shortly after this incident, worried by the anti-Jewish measures taken by the Vichy government, my parents decided to leave Paris and take refuge in the ‘‘free zone’’ (i.e. not occupied by the Germans) of southern France. A wise move because of my parents’ visibly Jewish origins. Both my parents were born in Poland, in orthodox Jewish families. My mother’s maiden name, Rottenberg, reflected her direct descent from an uninterrupted lineage of prestigious rabbis that started with the Ma’aram of Rotenburg (1220–1293), a prominent scholar who lived in Rotenburg auf der Taube (Germany) and was buried in Worms years after he died in prison for forbidding the Jewish community to pay the enormous ransom requested by Emperor Rudolf I of Habsburg for his release. My father’s ascent was no less prestigious. His mother was a descendant of Rashi (1040–1105), one of the (it not ‘‘the’’) most famous Talmud commentators, who was born and spent most of his life in Troyes (France) except for about 5 years spent studying in the most famous rabbinic schools of Mainz and Worms in Germany. Both my parents were impregnated with this cultural heritage, but none of them was really religious. They stuck to tradition much more than to religious belief. My father had immigrated to France in 1924 for business reasons. He met my mother in Warsaw during one of his frequent visits to his parents. In spite of the numerus clausus that severely restricted the number of Jewish students that could register at the universities in Poland, and though she was a girl and therefore expected to stay at home rather than study, she had graduated in Roman Philology from the Warsaw University, and was studying

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at the school of journalism in Warsaw. They got married in 1932 and my mother enthusiastically left Poland behind and settled in France. Both loved France. They did not regret Poland a single minute. They had suffered so much, since their childhood, from violent, constant, omnipresent anti-Semitism. And here they were in France, foreigners but free to be themselves. With equal rights. Protected by law. These were happy years. Which ended in 1940 with the Nazis arriving in Paris and the Vichy regime sneakingly but steadily restricting the rights of Jews until the adoption of the anti-Jewish laws in 1941. With the obviously Jewish name of Goldberg, and with their heavy Yiddish/polish accents in French, my parents could hardly hide their belonging to the hated race. They felt the danger and, by mid-1941, decided to leave for the ‘‘free zone.’’ Before we left, and because Jews were no longer allowed by the Vichy regime to own a business or a house, my father sold his business to the foreman in his tanning-trade, Mr. Pelletier, who was a gentile and whom my father trusted with no restriction for having known him and worked in close collaboration with him for years. The sale was a fake. In a secret written contract it was mutually agreed that as long as my father would be away and the anti-Jewish laws valid, Mr. Pelletier would run the business and send my parents some money to allow for their daily expenses during our exile. It was also agreed that my father could claim the restitution of his belongings when better times would come. In a similar fake transaction, my father sold the building of his leather factory, his retail shop and his villa in Montmorency to Mrs Jacquelin, a business partner and friend of his, also a gentile. In October 1941, after crossing nightly the line between the occupied and free zones, walking through creeks and forests with the help of the underground, we ended up in Nice. My parents managed to get false identity papers, with the name of Colbert. A typical French name, sounding similar enough to Goldberg to allow for my brother or I to mistakenly use our old, rather than our new, name. We stayed there for a few relatively calm months, but hunger drove us away. The food restrictions were such that, even with the money sent by Mr. Pelletier, my parents were hardly able to feed us. In July 1942, we moved to Luchon, a charming thermal resort in the Pyrenees, close to the border with Spain. There, we lived in the Hotel d’Etignies, owned by Jean Lafon, a tall, strong, joyful, generous man, closely associated with


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the underground. He risked his life hiding Jewish families, and helping several of them escape illegally to Spain. We were about to leave for Spain with his help when my father developed a severe sciatica which prevented him from walking even a few steps out of his bed. Jean Lafon offered to carry him on his shoulders to Spain across the mountain. But this was clearly too risky. The flight was postponed. When my father recovered, the border had become much more hermetic, and the chances of success much smaller. We gave up the idea of taking refuge in Spain. Under the growing pressure of the militia who had found our track and was actively searching for us, we had to leave Luchon in a hurry. In January 1943, we moved to Albi, a small, quiet town in the southwest of France. Though the Germans had invaded the free zone in 1942, we lived in Albi a few relatively quiet months. We lived in a rented apartment, together with its owner Madame Donadieu, an old miserly lady with whom we had to share our frugal meals. According to my parents, she would always help herself first, taking the biggest and best parts of the food that my mother managed to prepare in spite of the severe shortage. But not all were as miserly. I remember the day when I got ill, with a very bad, painful sore throat, lots of fever, and no appetite. My mother wanted me to eat something, but I stubbornly refused everything she offered me. All I wanted were canned sardines. My mother burst into tears and shouted on me ‘But where do you want me to find sardines!’. And here comes a knock on our window, on the third floor. And the knock comes from a can of sardines, hanging from a string. I have not the faintest memory about who was living on the floor above us. But sure enough, his generosity, and even more so the miraculous way in which he expressed it, is one of the numerous ties which link me to the French people y An other one is the unknown French policeman working in the headquarters of the militia who leaked out to us, via the underground, an information accordingly to which the Gestapo had traced us and was about to arrest us. I shall not dwell on the queer scenario organized by our family doctor, Dr. Lapeyre, to save my father, stuck again in bed by uncontrollable sciatica. To cut a long story short, he declared that my father was dead, showed his corpse to the men of the Gestapo and milita and signed a death certificate. Thus, my father was officially dead. My parents managed to get new false papers. Our name became Lafon, a name I would carry until the end of the occupation. We thus could stay in Albi for a

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few more months until, on a bright Sunday afternoon, upon returning from a walk with my parents, we are approached at the corner of our street by a neighbor who insistently prompted us no to go home: four Gestapo and militia men were waiting for us. My father took me in his arms, my mother took my brother’s hand, and we hurried away. Not knowing to where. But urgently away. This is one of my first memories. I do remember to rush, the anxiety, the fear. I do remember this unknown man who near the Saint-Salvy Cloister, asked my father in a whisper it we were in trouble. Father hesitatingly nodded. And we were Jews? Father nodded again. ‘Just follow me’ said the man. And he took us into the house of Jules and Rose Cavalille´s, active members of the underground, who hid us for a few days until they organized our escape: my parents hid in a cache in an apartment in Albi. Only Mr. Pelletier new the address so that he could bring in the money my parents needed for surviving. They stayed there about three months, until the Gestapo came to their apartment. The Gestapo knew my parents false name and address. Fortunately, they could not find the cache. And this was the last time my parents were harassed by the Gestapo. From then on, we were no longer bothered because my parents realized why the Gestapo had been able to trace us so efficiently and rapidly. Each time we changed name and moved to a new place, my father informed a few trustworthy persons of our new identity and address, including Mr. Pelletier so that he can bring us the money. This time, only Mr. Pelletier, and nobody else, had been informed. From that, they inferred that nobody else than he could have given the information to the Gestapo. Therefore they no longer contacted him until the end of the war. They immediately left Albi and took refuge in a tiny little hamlet in the Tarn region were they stayed until the liberation of France in August 1944. My brother and I did not stay in Albi, nor did we follow my parents in their rural exile. The situation had become so critical in France, the risk of being caught by the French militia or by the Gestapo had become so high, that my parents decided to split the family. Because they had a strong foreign accent, they could easily be identified as originating from Central Europe, and thus suspected of being Jews. While my brother and I spoke French with a local accent and sounded like ‘authentic’ French kids. To increase our chances of survival, my parents sent us to one of their former employees who had rented an isolated farm, in an isolated hamlet near an isolated village in


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the Landes (South–West part of France). There, we spent the last year or so of the German occupation. There, with absolutely no contact with my parents, I literally forgot them. I lived the happy life of a countryside little boy. I grew up among ducks and hen, playing in the fields, with no school to attend, riding the ox hooked to a plough, climbing the high pine trees, scratching the melting tar of the road on hot summer days to build the only toys I could play with. I keep only warm, sunny, gay memories of this period in Batz. My obviously selective memory has erased any rainy day, any sad thought, any longing after my family. I lived happy. I do remember the days when I saw German trucks hurrying north, loaded with gaunt-faced soldiers. I remember that exhausted, elderly German soldier, scarlet from riding on old bicycle under the stifling sun, who stopped at our farm begging for some water. I remember my insisting request to my host ‘Max, give him to drink!’. Was it fear? Was it pity? I can’t say. I also remember the couple of black Matford cars, decorated with French flags, rushing northbound with their loads of armed underground people y I remember my trip to Paris by train, crossing the river Loire by foot on a hastily constructed wooden bridge. And I remember that lady, waiting for us in the subway station ‘Gare du Nord’. When she saw us, she rushed towards us, and crushed me in her arms. I hated her kisses. I did not understand them. I did not know who she was y my mother. My mother took us to the villa in Montmorency. Indeed, by the end of August 1944, as soon as Paris was liberated, my parents showed up there. Mr. and Mrs Pelletier were having lunch in our villa that they rented from Mrs Jacquelin. When they saw my parents, they stood still, petrified. With no difficulty, they handed the factory back to my parents, so that our family could immediately move back into it. My mother wanted to retaliate against the Pelletiers. My father, for whom justice was not a vain word, refused. He claimed that he did not have the formal proof that the Pelletiers had given us away to the Gestapo. He therefore did not act against them. He even did not fire Mr. Pelletier and kept him in his factory until he himself decided to retire. A few years later, we heard that Mr. Pelletier had died. His wife passed away shortly after him. On the following Yom Kippur (the Day of Atonement, that is the most sacred holyday in the Jewish religion), while we were having diner after the celebration, someone knocked at the window of the dining room. My mother

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went to the window and opened it. A man in his forties told us that he was the son of the Pelletiers, that he knew we were celebrating Yom Kippur, and that he had purposedly chosen to come on that day to ask my parents for forgiveness. Indeed, after his parents’ death, while cleaning up their house before selling it, he found letters showing that, on several occasions, his parents had given our fake identity and address to the Gestapo, thinking it a good way to ascertain that they would keep ‘‘their’’ factory. After this discovery, the son of Mr. and Mrs Pelletier could not find rest. He needed to confess his parents’ deeds and ask for forgiveness y Nothing in this period prepared me to my future academic career. Nothing seemed to relate me anymore to Louis Pasteur. My parents were the last ones to think, or hope, that I might become a respectable scientist. All they expected from me was to turn the young little uneducated scoundrel I had become into decently well-behaved townsman. Not an easy task! Neither for them, nor for me. They had become total strangers for me. The mere notion of parental authority was something unknown and unacceptable to me, after the freedom I had experienced in Batz. What really changed my relation to them, as well as to society, was the sudden appearance, during the years 1945–1946, of holocaust survivors. Because my parents were alive (an extremely rare privilege in a family that had been devastated in the ghettos and extermination camps), because they had recovered their house in Montmorency, and because of their immense generosity, a flow of human wrecks drifted into our home, bringing in relatives, friends, friends of friends, who had been released from the camps after the German defeat. This flow of desperate, hardly human-looking, grey, skinny, silent figures brought me a terrifying feeling of the sufferings they had undergone. Not from what they said: they spoke very little, and mostly in foreign languages that I could not understand. Rather it was their physical weakness, the slowness of their gestures, their silence, their constant fear, the absence of any smile on their face when they felt unobserved, the sadness of their empty eyes that elicited an impression of extreme anxiety, of unutterable sorrow, of unspeakable physical and moral pain. I was, and still am, fully aware that this perception I had of what the victims of Nazism had undergone was only a pale, subdued, superficial representation of reality. It was nevertheless enough to drag me out of my


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unconscious happy childhood and thrust me into a drastically different vision of life and humanity. From then on, I questioned my parents about why did we survive, why did we not share the fate of so many of our family, friends and people, and learnt from them what evil and what good men can achieve, depending on their choices. From then on I knew that I would do my best, under any circumstance, to be among those who would work for, not against, humanity. The underground people were my heroes, the Germans and their collaborators my foes. I developed a strong feeling of solidarity, of quasi-identification, with the victims of the holocaust, those who were exterminated, as well as those who survived but would bear with them the indelible traces of their lives in the camps. I watched with passion, vibrant hopes, and ultimately immense joy, the fight that led to the creation of the State of Israel. I started school in October 1944. I took it upon myself to be a model pupil. As opposed to my behavior at home, I was very polite, calm, and submissive. My parents heard only compliments from my schoolteachers. I did my best to be the best in my class. I had two good reasons for that. One was that I had to do as well as my brother, who was 3 years older than me (and 4 years ahead of me at school) and had left in all classes he attended the image of the perfect pupil y The second reason was that, as a member of a community who had undergone so much hatred and contempt during the war, and who was experiencing mainly pity after the war, I felt the profound need to assert myself as someone deserving respect and admiration. This attitude at school stuck onto me throughout my studies, from elementary school to high school. Without much effort, I always did enough to be the first of my class. I felt being the second at the end of a school year (which did, after all, happen once) as a personal failure that had to be immediately corrected, which resulted during the next year in a bit more time and effort devoted to learning. I thus succeeded in doing as well as my brother (a major incentive!) and to graduate from high school with brilliant marks and a reasonably solid background, particularly in my two ‘‘majors,’’ mathematics and physics. This however did not prevent me from devoting much of my time playing with my electric train, building complicated machines with my Meccano, riding my bicycle dauntlessly up and down the abrupt slopes of our hilly environment, collecting stones and Dinky Toys miniature cars, running, escalating the front wall

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of our house till the third floor and calling my mother to scare her when she saw me hanging from the highest cornice. I thought little about my future. But when I did, I considered either becoming an electrical engineer (an idea clearly generated by my interest in complex circuitry and tinkering with my electric train) or working with my father in his tannery. Yet, two events marked my last year at high school and decided on my future. During the summer of 1955, getting close to my 17th birthday, I spent my vacations in Israel on a Zelidja Fellowship. These competitive fellowships were granted, each year, to a few high school students on the basis of their projects for study visits to foreign countries. The project I had submitted dealt with trying to find some relations between the economy of modern Israel and indications in the Old Testament on the natural resources of the Holy Land. The financial support provided by the Zelidja Foundation was minor. But it was accompanied by a letter, written in some 25 different languages and signed by the French Ministry of Education, who described the laureate and his project in highly laudative terms and requested that full support should be given to him. This letter opened many doors. Including that of the Weizmann Institute of Sciences in Rehovot. Oil had just been found in the Neguev Desert, in southern Israel, and samples were being analyzed at the Weizmann Institute to assess its commercial value. I was introduced in the laboratory of Physical Chemistry where the tests were being done. Though not absolutely certain of it, I think I remember that the man running the laboratory was Gerhard Schmidt, one of the pioneers of X-ray crystallography, and founder of a brilliant school of crystallographers at the Weizmann Institute. There, I got some indications, unfortunately not very optimistic, about the interest of the recently discovered oil field. But more important to me, my host also introduced me to several of his colleagues who took me to their laboratories and showed me around. This was a determining experience! I was fascinated by what I saw. Like one of the very first computers in the world (remember, this was in 1955!) that was working on diode bulbs and electrical relays (reminiscent of the gadgets I used to build for my electric train). Or like a ‘‘deaf chamber’’ (a room with total phonic isolation) where I could hear the beats of my heart and the blood-flow in my veins. But above all, the scientists who entertained me struck me: these old men (probably in their late


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30s or mid-40s y) were all enthusiastic, happy, and seemed to spend their time playing rather than working. On the spot, the dazzling idea came to my mind that I too would spend my life playing the game of scientific discovery: I would become a researcher. The image of research I got from my visit at the Weizmann Institute thus determined my professional life. Back in France, I started my last year of high school in the strongest scientific section, ‘‘Elementary Mathematics,’’ with a program centered on mathematics and physics. Natural Sciences was also part of our program, but it was looked at with some contempt by the fans of ‘‘hard sciences.’’ When Mrs Giraudeau, our Natural Sciences teacher, started her first course in October 1955, she made it clear to us that she more or less shared our view on the limited interest of what she had to teach us. But she pointed out that a minor part of our program dealt with heredity, and that a major discovery had just been made which made it possible to understand the mysteries of the transmission of genetic traits in terms of molecular properties. She obviously was referring to DNA, whose structure had been understood only about 2 years earlier by Watson and Crick. Mrs Giraudeau was aware of this discovery, and, 7 years before the Nobel Prize recognized its importance, she pointed out to us its foreseeable tremendous impact. She decided that she would spend the two last trimesters of the academic year teaching us heredity and ‘‘molecular heredity.’’ Her course fascinated me: she taught a course, but also made us rediscover the laws of Mendel by growing peas and crossing flies, showed us chromosomes under the microscope, told us how the random distribution of chromosomes between the next generation offered a straightforward interpretation of the laws of Mendel, explained us the chemical structure of DNA and the base pairing between complementary strands and showed us how maintaining chain complementarity during replication could account for the transmission of an intact genetic heritage from cell to cell. I was swept away by the logics underlying the molecular mechanisms of heredity. I imagined that, in a similar way, simple molecular mechanisms should underlie other fundamental aspects of life. By the end of the school year, I decided that my life as a scientist would focus on molecular aspects of life. I would become a ‘‘molecular biologist.’’ For that purpose, I envisaged undertaking medical studies. When I told my mother about my project, she bluntly opposed it.

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Her point of view was that she had always raised my brother and me in exactly the same way, that my brother had entered the Ecole Polytechnique, and that it was obvious that I had to follow the same track. Let me point out that the Ecole Polytechnique is a very prestigious school that belongs to the peculiar French system of Grandes Ecoles (let us translate it as Schools of Higher Education). This system parallels, and competes with, the Universities. In France, any student who graduates from high school with the baccalaureate (end of school examination) is entitled to enter any French university without additional selection. As opposed to this very permissive system, the Grandes Ecoles rely on a highly discriminating process for selecting the (supposedly y) best students: after getting their baccalaureate, the students who choose this hard way enter ‘‘preparatory classes,’’ that is 2 years of hard training in a limited number of subjects with the unique aim of obtaining the best possible grades at the competitive examinations organized by each Grande Ecole. For entering the Ecole Polytechnique, mathematics was by far the major topic on which the selection was based, though several other ‘‘minor’’ subjects (none related to biology however) were also included in the competition. Moreover, the Ecole Polytechnique was considered by public opinion as the very top, most prestigious, Grande Ecole and consequently, it was the most competitive one, together with the Ecole Normale Supe´rieure. Entering 2 years of convict’s work focused essentially on mathematics (which I did not like particularly and did not consider as my strongest talent) did not appeal to me at all. Moreover, the chances of ending up ranked better than the 300th among several thousands of the very best students in France seemed to me very weak. I therefore firmly rejected my mother’s suggestion, arguing that the Ecole Polytechnique was not the proper track for becoming a biologist. Knowing how stubborn I could be, she did not insist. But she informed my father. So many years after the end of the war, we had rebuilt familial relations. I had come to respect and admire my father immensely for his honesty, courage, deep sense of justice, and generosity. When he came to me and asked me to reconsider my decision, I listened carefully at his reasons. His financial situation was very difficult. His leather factory had burnt a few years before, and the insurance covered only the building, not the goods in it. As a consequence, his company was facing terrible debts that my


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father had decided to pay off rather than go on bankruptcy. And indeed, he spent the rest of his active life refunding these debts. He pointed out to me that medical studies were long, costly, and that I would not be earning my first franc for 7 years, while if I succeeded at the Ecole Polytechnique I would gain financial independance after only 2 years. Indeed, studies (including housing, food, uniforms, etc.) at the Ecole Polytechnique are free of charge, and students even get a small money allocation. Considering my father’s struggle for closing the family’s monthly budget, and the efforts my mother was putting forth to keep a comfortable household in spite of the money shortage, I immediately surrendered to my father’s reasons and promised him I would do my best to help him. To be honest, I was convinced that my limited gifts in mathematics would result in my eviction from the preparatory class after the first year and that I might then turn back to biology. In October 1956, I thus entered the class of ‘‘Mathe´matiques Supe´rieures’’ at the Lyceé Louis le Grand, one of the three best places in France to prepare the competition for the Ecole Polytechnique. The location was superb, just in the heart of the Quartier Latin, facing La Sorbonne, close to the student-crowded boulevard Saint-Michel and the gardens of the Luxembourg. What a change from the bourgeois, quiet, dull residential suburb in which I had been living for the last 12 years. But what a change too when the courses started. I felt lost. Things were going much too fast for me. Most of my classmates seemed to follow the pace rather easily. During the courses, all I succeeded in doing was to take notes. And I had to work for hours at home to understand what I had noted. After the first test it clearly appeared that, from my position as the permanent class leader in my suburban high school, I had come to being in the weakest quarter of the class at Louis le Grand. This was what I had expected from my talents in mathematics. Nevertheless, I studied. And studied. And studied. I worked from 7 in the morning till 1–2 am. There was no weekend. Just 1 week of skiing during Christmas vacations. I worked hard, first ploughing into the courses to understand every detail, then learning all I could by heart. I spent all my free time trying to solve math problems. And to my surprise my rank in the class kept improving. To such an extent that I was easily admitted to the second year. I must admit that, in spite of this divergence from my initial plan, I felt happy, and encouraged by this partial

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success. My second year at Louis le Grand, in ‘‘Mathe´matiques Spećiales,’’ very much looked like the first one. Same feeling of having to swim hard in order not to get drowned. Same dense days fully devoted to mathematics, except for the courses in physics, chemistry, philosophy, French, English, drawing, which we considered as entertainment but represented only a minor part of our schedule. Same regret to be so close to all these university students happily hanging around in the Quartier Latin, so close to us, and not being able to share with them the banks of the Luxembourg garden, the sunshine over the roofs of Notre-Dame, the blooming trees when spring came back, the seats at the open cafe´s on the boulevard Saint-Michel. Like an ox painfully dragging a plough in a dry field, I kept studying. Then came the competition exam. As could be predicted from my status in my class, I ended up with a decent but embarrassing rank: not good enough to enter the Ecole Polytechnique, but good enough to give hopes of success if I would spend one more year in Mathe´matiques Spećiales and try again. Though this was a grim prospect, I felt I owed my parents this additional effort. I also found it more and more challenging, in spite of my limited selfconfidence, to test my own limits and find out whether or not I might succeed in entering the Ecole Polytechnique. This is why I set off for a third year of hard labor. In the same mood as the two previous years, except for the fact that I felt more and more at ease with mathematics. And when on a warm summer afternoon the results of the competition exam finally came out, I found my name in the list of those admitted. For the rest of the summer, I felt extremely excited and relieved. For 3 years I had been in a tunnel, not knowing where I would emerge. And here I was, with the goal I had been aiming at ultimately reached. I was happy. Happy for my parents. With a feeling of self-satisfaction I had never sensed before. In early September 1959, I went through the wide-open gates of the Ecole Polytechnique, signed my incorporation in the army (since its creation by Napoleón, the Ecole Polytechnique is a military school ruled by the Ministry of Defense) and stood in military order with my 299 schoolmates in the impressive courtyard of the school. We listened at a welcome speech delivered by the Colonel, commander in second of the school. He praised us as being ‘‘the elite of the nation,’’ which at this stage I was ready to accept. I felt proud, glorious, intelligent y But after a couple of


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months, I had already spotted among the students a few members of this so-called ‘‘elite’’ who seemed to me rather mediocre people, and many others who appeared to be no more than ordinary young men. Very few showed the features of supermen. The more I knew my mates, the more my respect for our ‘‘e´lite’’ declined. At the same time, the courses had started. Except for the thermodynamics course, where we were introduced to ‘‘statistical thermodynamics,’’ none of the subjects interested me. The teaching was exclusively theoretical. No practical course. No laboratory work. Little by little, I sunk into despair. By the end of December, the euphoria of success had completely vanished, my lack of interest for the teaching we were receiving had become obvious to me, and I wondered what orientation I should take. I thought of Economy, but was discouraged by the lack of reliability of this discipline. I thought of Astrophysics as a substitute for Biology. After all, the intellectual approach to the infinitely distant is somehow similar to that of the infinitely small. But the impossibility of performing direct experiments on the stars and planets deterred me from entering that field. So, rather than studying subjects that did not appeal to me, I undertook catching up on all I failed to do during my years in the preparatory classes. I devoured books from the school library, mainly novels. I discovered American writers, which were never mentioned in French high schools and read their books first in French translations and then in the original version. I attended practically all theater plays that were performed in Paris, became a devoted admirer of Maurice Be´jard after having attended, merely petrified, his performance of Stravinsky’s ‘‘Coronation of Spring,’’, visited all painting exhibits, heard the most outstanding soloists that performed in the best concert halls in Paris. The intense artistic life of this city, which used to be one of the world’s main centers of culture fascinated me, and distracted me from my studies. I held the main role in a theater play written, directed and performed by students for the ‘‘Point Gamma,’’ the great celebration organized each year by the Ecole Polytechnique. After the performance, the godmother of the celebration, Romy Schneider, came to congratulate the actors. She asked me where I had studied acting. I told her that I never did. She seemed quite impressed and tried hard to convince me to become a professional actor. I was extremely flattered: Romy Schneider was world famous for her beauty, charm and sensitivity in the movie

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Sissi (1956). She asked me to accompany her during the evening at the Point Gamma. Which I did with much pleasure and pride. We were both 21 years old. Until dawn, I took her around the various attractions, bars, restaurants improvised in the Ecole, and spent hours waltzing with her. An unforgettable night; yet not enough to convince me of becoming an actor. Some months later, I played a very modest role, and attended the shooting of a movie by Paul Vecchiali, a famous film director and producer of the ‘‘Nouvelle Vague,’’ with three superb actors, Danielle Darrieux, Michel Piccoli, and Nicole Courcel. I learnt horse riding, was an active member of the rowing team, learnt skiing. I also received a military training: marching, shooting with all kinds of weapons ranging from the pistol to the bazooka, driving a Jeep, detecting and neutralizing mines, automobile repair, tactics, and telecommunications. All these activities filled the vacuum created by my lack of motivation for the scientific cursus (Figure 1). In spite of my lack of interest, I attended all the lectures: some sleeping under the seats (an officer would check on the attendance at the beginning of each lecture), others listening eagerly at some outstanding professors. Two of them had a great impact on my life. One was Laurent Schwartz, our teacher in Analysis, a world famous mathematician, winner of the Field Medal (equivalent to the Nobel Prize which does not exist for mathematics). His incredible pedagogic talents rendered his teaching so lively, so clear, and so easily understandable, that this difficult matter seemed straightforward. Going out of his lectures, I thought I had understood everything. But when we approached the end of term exams and I tried to reach a minimum level of knowledge, I realized how far I was from having grasped the essence of his teaching. In addition to being an outstanding mathematician and pedagogue, Laurent Schwartz was also a courageous, inflexible, and just man. Though member of the staff of a military school, he publicly opposed the government’s policy during the war in Algeria and signed in September 1960 the ‘‘Manifeste des 121.’’. This appeal by 121 prominent French intellectuals (among whom one could find personalities like Simone de Beauvoir, Jean-Paul Sartre, Alain Renais) requested the right to default for soldiers opposing the behavior of the French army in Algeria. He was pressed by the authorities to retract, but steadily refused. As a consequence, he was fired from his position at the Ecole Polytechnique. We went on strike to


Fig. 1.

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The author in his parade uniform at the Ecole Polytechnique in 1961.

support him, but he urged us to abandon our action and stay out of this conflict. Laurent Schwartz later had a leading role in planning the reformation of the Universities in France. His charismatic figure, his intense care for his students, his commitment to serve justice and human rights inspired me profoundly when, many years later, I became a professor at the Paris University. For quite different reasons, a second professor profoundly influenced my career. He was Bernard Gregory, one of our physics teachers. This excellent nuclear physicist was involved in building bubble chambers, using them to collect particle collision data at synchrotrons like that at Saclay near Paris or later at the CERN (European Center for Nuclear Research) in Geneva, and analyzing the images to find and characterize new

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elementary nuclear particles. Bernard Gregory ended up being the Director of the CERN. In the 1960s, Bernard Gregory was a coworker with Louis Leprince-Ringuet, also my professor at Polytechnique, a renowned nuclear physicist whose reputation came from his early (1935–1938) determination of the energy spectrum and mass of the mesons. My brother Jacques was working on his PhD thesis in the laboratory of Louis LeprinceRinguet. In early 1961, Jacques was sitting at the ‘‘electric calculator’’ (the single ‘‘computer’’ then available in the whole laboratory) located in Bernard Gregory’s office, when he overheard a conversation between one of my classmates (Jean Thierry) and Bernard Gregory. Jean was asking our professor what areas of research were open to Polytechnique students. This was a rather unusual question since very few students considered research as their possible future profession. Paradoxically, in spite of the intense and deep scientific training we received at Polytechnique, most students would become engineers, bankers, administrators, or technocrats of various sorts, but very few became involved in research. Bernard Gregory gave the classical list of research areas: mathematics, applied mathematics, physics, chemistry, and astronomy. But he added Biophysics to the list, stating that some good biology laboratories were looking for students with a solid background in maths and physics to implement physical techniques, and physical concepts in biological research. My brother immediately thought of me, called me on the phone, reported this conversation, and urged me to contact Bernard Gregory. This phone call came as a ball of fire in my gloomy thoughts about my future. I immediately called Bernard Gregory, met him, got more details about the information he had on Biophysics in France, and went out with the names of two laboratories I should contact. One was that of ˆce, the military Pierre Douzou, then located at the Val-de-Gra hospital and medical school of Paris. Pierre Douzou was lively, full of humor, passionate. Later in his scientific life, he became a pioneering figure in cryoenzymology, the study of enzymatic mechanisms at very low temperatures that enable one to freeze the reaction at various intermediate stages and characterize enzyme-linked intermediates in the catalytic cycle [1]. Pierre Douzou strengthened my enthusiasm, confirmed that biophysics was the future of biology, urged me to persist in my quest for a good laboratory, and y threw me out of his lab. He claimed that


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the army was supporting his research only because of its possible implications in biological warfare and that no interesting research could be done in this environment. Indeed, it did ˆce and join the Institut not take him long to leave the Val-de-Gra de Biologie Physico-Chimique (an institute run by the CNRS) where he stayed for the rest of his active life. He suggested me to meet Rene´ Cohen, who was working in the newly established Department of Biology at the CEA (Center for Nuclear Energy) at Saclay, a southern Parisian suburb. The chairman and founder of this department was Jean Coursaget, both a physicist and a physician, the second person whom Bernard Gregory had advised me to meet. My visit to Rene´ Cohen was most exciting. He was an expert in analytical ultracentrifugation, a method he would cleverly use a few years later in the characterization of active enzymes [2]. He brilliantly and enthusiastically explained to me how centrifugation worked, and how Messelsohn and Stahl had used it to demonstrate the semi-conservative nature of DNA replication [3]. The beauty and simplicity of this experiment, its perfect fit with the teaching of Mme Giraudeau during my last year of high school, and Rene´ Cohen’s enthusiasm sealed my decision: I would join the Saclay group. I therefore met Jean Coursaget who encouraged me to spend my 2 weeks of Easter holidays in the photosynthesis group of his Department. When I came into this laboratory, I had the surprise to find there Jean Thierry and another of my classmates, Maxime Schwartz, who had followed the track suggested by Bernard Gregory and ended up in the same laboratory. The three of us were introduced to ‘‘experimental biology.’’ Our project involved building an optical bench to illuminate an experimental cell with light of a selected wavelength, with the purpose (if I remember correctly) of quantifying the amount of gas produced by photosynthesis as a function of the energy of the incident light. We used a lot of thinking and of manual skill in designing, cutting, and assembling pieces of cardboard to shield the light beam and the cell from external light, and to channel the gas flow to a detector. The three of us enjoyed a lot this introduction to bench work. Apparently, we did well enough. At the end of the 2 weeks, Jean Coursaget expressed his satisfaction about our achievements (!) and offered each of us to join his Department with a fellowship he would provide to work on our PhD. I got ecstatic. The dream was becoming reality: my detour through

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Polytechnique was not a dead-end. I would after all be able to go back to my hobby and become a biologist. But despair soon followed ecstasy. When leaving the Ecole Polytechnique, students must enroll into a government institution that will host and support their activities for the next 10 years. If they fail to do so, they have to refund the costs of their studies, including the housing and living expenses of all kinds. A sum that was obviously prohibitive for my family. The CEA was an approved governmental institution, and receiving a CEA fellowship would have fulfilled all administrative requirements. But just a few days before the deadline for signing our enrolment, Jean Thierry received an unexpected phone call from Jean Coursaget. The CEA had provided only one fellowship and, because Jean Thierry had by far the best grades among the three of us, he would receive the fellowship. Maxime Schwartz and I were thus left jobless, with no alternative to the prospect of accepting some dull position still open in one of the government offices that nobody in the class had wanted to join. I was severely punished for not having been a serious student and not having tried to get better grades. I was comforted only by the idea that Jean Thierry was ranked second out of 300, and that even by working like crazy I would not have been able to reach his level. Maxime’s father, an eminent biostatistician, aware of his son’s difficult situation, noticed a poster in his laboratory. It advertised ` la pre-doctoral fellowships from the De´le´gation Geńe´rale a Recherche Scientifique et Technique (DGRST – a government structure under the direct control of the prime minister aimed at developing new research and technology domains) for students with a good background in mathematics or physics wishing to turn to biology. It gave as a contact Dr. Elie Wollman, one of the founders of Bacterial Genetics and co-inventor with Franc- ois Jacob of interrupted bacterial mating in Andre´ Lwoff’s laboratory [4]. Maxime met Dr. Wollman, who directed him to Jacques Monod. At the end of his meeting with Maxime, Jacques Monod offered him to work for a year, as a test period, in his laboratory at the Institut Pasteur. To my good fortune, Maxime kindly mentioned to Jacques Monod the existence of another Polytechnique student who was looking too for a position in a biology laboratory. Jacques Monod expressed his interest in meeting me. Next morning I called him at the Institut Pasteur and, because I could hardly escape from school during the day, he asked me to come to his


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apartment, any time after 9 pm on the same evening. Fortunately, I did not know who Jacques Monod was exactly, and what incredible privilege it was meeting him. Had I known, I am not sure that my legs would have taken me into his home. For even without knowing, I was trembling with anxiety and shyness when I entered the building on the avenue de La Bourdonnais, a very elegant residential district close to the Eiffel tower. I was introduced into Jacques Monod’s office by a very kind lady, probably his wife Odette, and left there waiting for a very short time, but long enough to let me look around. The office was furnished in a rather old, elegant bourgeois style. It was crowded with books, antiques, and pictures on the wall. A stylish table lamp covered with a parchment-like shade dimly lighted it. A cozy atmosphere of art and intellect haunted the place. As soon as he came in, I relaxed. Jacques Monod was handsome looking, smiling joyfully, and talking in a friendly and charming way. The interview was not at all like what I had expected. Rather than questioning me on my scientific background or my knowledge of biology (which I feared above all!) he asked me about my family, how did we manage during World War II, what were my parents doing, what my hobbies were. We talked at length about my interest for electric trains when I was a teen-ager. About my parents’ painting collection. He tested my abilities to speak English. He wanted to know why I wanted to get involved in biological research. The story about my discovery of molecular genetics at high school clearly made him happy. He asked me how I saw my career as a scientist. I stood baffled. I had never thought of research as a career but as a way of spending my life doing something pleasant and useful. The discussion went on for at least an hour, after which Jacques Monod offered me to join his laboratory with a DGRST fellowship. This was enough to meet the requirements of the Ecole Polytechnique. For the first time since Jean Coursaget’s phone call I felt serene again, though the prospect of entering the Institut Pasteur did impress me. I was however not particularly flurried by the idea of working with Monsieur Monod. I did not realize who he was. In May 1961 (the date of our first meeting) there was no Internet to find someone’s curriculum. And Monsieur Monod’s reputation was not yet established in France: the Nobel Prize was to be awarded to the pasteurian trio (Lwoff, Monod, and Jacob) only 4 years later, and Monsieur Monod was even not a member of the French Academy

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of Sciences. His scientific stature therefore did not inhibit me. On the contrary, our interview had left me with an impression of grandeur, culture, and cheerfulness that made me anticipate happy hours working with him. With extreme kindness and efficiency, Elie Wollman helped me clear the administrative aspects of my enrolment at the DGRST. I thus could complete my last months at school with a clear prospect about my future. I would get my engineer’s degree by the end of the academic year, comply with my military obligations and join Monsieur Monod’s laboratory 2 years later. Indeed, it was compulsory for students finishing the Ecole Polytechnique to serve in the army. The normal length of this service was 1 year. But France was fighting a long, tough war in Algeria and needed more soldiers. All young men were drafted for up to 3 years. Officers freshly graduated from Polytechnique had to serve for 2 years. Because I was good at sports and at military tests, my military ranking was very good. This enabled me to choose to serve in an office of the air force located in Versailles. This was an immense privilege. Rather than fighting a dirty war in Algeria, I was appointed as a statistician in the CEIPAA (Air Force Psycho Technical Investigation and Instruction Center). In contrast with my two previous years, the time I spent in the army was a period of extremely intense work. Indeed, in addition to my duties in the army, I had to prepare my future career in science. My contract with the government, through the DGRST, imposed that I should obtain my PhD within 5 years after the end of my military service. While this was rather easy for my classmates who entered research in mathematics or physics, it was a challenge for me: I had no background in biology whatsoever and would have to make up for that. Moreover, until 1970s, students from Polytechnique had to obtain a BSc before being eligible for a PhD program. I therefore registered at the Sorbonne. My plan for the first year was to obtain four out of the six units required for the BSc. Three would be easy for me in view of my former studies (Thermodynamics and Physical Mechanics, Electricity, Mathematical Techniques for Physics) and the fourth, Genetics, would be my real introduction to academic studies in biology. During my second year, I would work at the two last units required for my BSc, General Biochemistry and Advanced Biochemistry. Running in parallel my activities in the army and my studies at La Sorbonne gave me a hard time. I would get up at


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6.30 am, drive to Versailles, start working at 7.45, stop for a quick lunch in the officer’s mess, sleep for 30 minutes on my desk, work again until 5.45 pm, drive home, have a rapid dinner and then start studying until 1 or 2 am. On Wednesday evenings, I would attend the lab courses in Thermodynamics, on Saturday afternoons those in Electricity. For the practical course in Genetics, the most important for me since I had everything to learn there, there was no session that would not overlap my working hours in Versailles. This would have barred me from the unit of Genetics, were it not for the extreme kindness and availability of Jean Mousseau (then a young assistant professor) who organized special condensed sessions just for me during the winter and spring vacations of the academic year 1961–1962. I easily succeeded in all the exams, except for one oral test in Genetics, which should have been by far the easiest for me. I was asked to solve a statistical genetics problem about the blood groups and Rhesus factor. Having had Laurent Schwartz as a professor in probability, I had not the faintest hesitation about the statistics. But I did not know whether the blood groups and Rhesus factor loci are linked on the same chromosome. I told that to the examiner (a very famous French geneticist) who could not admit my ignorance. I explained him that I had not attended his courses because I was in the army and that his book (which I had studied in detail from A to Z) did not give any indication on the linking between these two loci. He lost his temper. I lost mine too and told him that anyway I did not care a bit about statistical genetics and that my interest was exclusively in molecular genetics. He dismissed me on the spot with a zero (the worst grade) at his test. Fortunately, I had plenty enough points at the written exam and the other oral tests to obtain the Genetics unit in spite of this zero. In the mean time, the Evian Agreements were signed between France and the Algerian rebellion. The war in Algeria was coming to an end. The soldiers went back home y and my service was reduced to 1 year. I immediately notified Monsieur Monod of this unexpected good news. He did not hesitate a second before asking me to join him as soon as the army would release me. This occurred on September 30, 1962. I was just 24. On October 16, I visited Monsieur Monod in the brand new, vast, wood-paneled office he had just fit up when he moved into the new premises of his Cellular Biochemistry Unit. After a brief hearty welcome,

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we discussed the way in which I would simultaneously complete my undergraduate studies and get trained to bench work. He called Ed Lennox, explained him my situation, and laid on him the task of being my mentor for my first steps in research. It was decided that I would start on the next Monday. Was it chance? Was it destiny? Was I born to become a pasteurian? On October 22, 1962 I walked through the gates of the Institut Pasteur as a member of its scientific community.

The Pathway to Protein Folding In 1962, the Unit of Cellular Biochemistry was actively working on two subjects, which corresponded to Monsieur Monod’s two major lines of interest: the lactose operon in Escherichia coli as an experimental system to investigate the mechanisms of gene expression regulation, and allostery as a model to account for the regulation of enzymes involved in key reactions of metabolic pathways. Maxime Schwartz had chosen to work on gene expression. I was left with enzymes. While the concept of the operon had been worked out in details, had already received a wealth of experimental support and was widely accepted in the scientific community, the ‘‘allosteric model’’ was a brand new concept. It had just been conceived by Monsieur Monod based on Jean-Pierre Changeux’s work for his PhD thesis on L-threonine deaminase. When I joined the laboratory, the first paper referring to ‘‘allostery’’ [5] had not yet been published. I was thrilled to get involved in such a new subject. But I quickly found out that it would take a lot of time and learning before I would be able to bring any personal contribution. My first steps in the laboratory were rather discouraging. So were my first contacts with the biochemistry courses I attended at the Paris University. To obtain the BSc degree required for my enrolment in an MSc-PhD program, I had to add two Biochemistry units to my courses. I thus attended the General Biochemistry unit during the first semester, and the Advanced Biochemistry unit during the second semester. So did Maxime Schwartz. There were only about 20 students following these courses, all with a solid background in biological chemistry acquired in college. Some were veterinarians, some others students in agronomy schools. Compared to these students,


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Maxime and I started the year with a heavy handicap. But we attended all classes, always sitting on the front row not to be distracted. We studied all courses, worked hard, exploited all the learning techniques acquired during our years in preparatory classes. Little by little, we filled the gap so that Maxime was ranked first, and I was third at the end of the first term exam. At the end of the year exam, Maxime was first and I was second. Though we had to work long and hard hours, I keep excellent memories of the short time I spent at the university. At last, I was following courses that were of immediate interest to me (Figure 2). In parallel with studying at the university, Maxime and I spent a few hours each day at the Institut Pasteur. I was asked

Fig. 2.

Monsieur Monod in his office – Paris 1967.

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by Monsieur Monod to work on two b-D-galactosidase mutants, U178 and CZ1. Monod, Jacob, and Ullmann had just identified some galactosidase mutants which gave rise to intracistronic complementation in vitro. Mixing inactive extracts of such mutants restored enzymatic activity. The mechanisms by which inactive mutant polypeptide chains could interact to regenerate a functional enzyme seemed to Monsieur Monod of much interest in relation to his views on allosteric enzymes. Indeed he assumed that while wild type galactosidase was a tetramer, individual mutant chains probably failed to undergo homo-oligomerization. Hetero-oligomerization might however occur between complementing chains. Intersubunit constraints within the heterotetramer might then force the polypeptide chain into the native, active conformation. To test this hypothesis, Monsieur Monod gave me as a project to characterize the oligomerization state of two complementing mutants and of the active enzyme they generate. Ed Lennox, my mentor, had to teach me the basic techniques I needed to approach this problem. To start with, Ed was quite reluctant to take care of me. He was at the Institut Pasteur for a sabbatical, waiting for the completion of the Salk Institute in La Jolla where he was to open his own laboratory. He was expecting a quiet, relaxed year in Paris and did not care for the responsibility of training an inexperienced student. Yet, after a short period of observation, he felt deeply involved in my training and looked after me with tenderness and amusement. In fact, Ed had been a physicist before becoming an immunologist. By watching my gaps and my mistakes, he remembered his own first steps in biology. He thus could easily guess what he had to teach me, and what was unnecessary. The first thing he showed me was how to grow bacteria: picking bacteria from an isolated colony on a Petri dish, seeding a tube of broth, following the cell growth by optical density (in fact turbidity). All this was novel to me. He then gave me the composition of the minimal medium into which the inoculum should be injected, and asked me a most puzzling question. What bacterial density did I expect to have in the culture on the morning? Ed knew very well that I would be unable to answer that question. During half an hour, I tried to figure out how I could find the answer, but could not even imagine how to approach the problem. Ed came with a sweet, gentle smile. He did not give me the answer. Rather, question after question, he showed me the way to the solution: when will the bacteria stop


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growing? What is the limiting element in the growth medium? How much of it is there in the medium? How much of it is there per gram of E. coli? And he referred me to the books where I could find the missing data (carbon content of bacteria, correlation between optical density of a culture and the number of bacteria, etc.). Just solving Ed’s original question filled the gap there had been in my mind between inert and living matter. Ed taught me protein purification. How to fill in a purification table: activity, total protein, specific activity, and yield. How to perform ion exchange chromatography. How to use Sephadex for gel filtration. I remember when he came back from a visit to Sweden with a sample of the first batch of Sephadex G-200 ever produced. One had to remove the ‘‘fines’’ by carefully decanting the gel suspension. One had to compromise between thorough decanting which left only a minor fraction of the precious particles, and incomplete decanting which resulted in clogged columns flowing at an unacceptably slow rate. Fraction collectors were unreliable. A hidden devil seemed to interfere with them: they would stop changing tubes just when the peak of the protein of interest would come out of the column. One therefore had to keep an eye on them. Thus, preparing purified U178 and CZ1 took a lot of effort, and taught me care, patience, and obstinacy. Then came their characterization. Sucrose gradient centrifugation was used to determine their sedimentation coefficients and infer from it their oligomerization state. The swinging bucket rotor we used could hold three tubes. One was for U178, the second for CZ1, the third for a mixture of U178 and CZ1. After a full night of centrifugation, the tubes were recovered, their bottom pierced with a needle, and the fractions collected drop by drop in about 35 test tubes per bucket. Each tube was then assayed for protein concentration and enzymatic activity. Testing all the samples (about 100 all together) kept me busy all the day and a good part of the night. But the results were neat. U178 was shown to be exclusively monomeric, CZ1 was essentially dimeric with only traces of tetramers, and the stoichiometric mixture of both showed essentially tetramers. Furthermore, no activity was detected in the samples corresponding to U178, a trace of activity was found for CZ1 at the position of the tetramer, and all the activity of the complemented protein sedimented as the native galactosidase tetramer. The hypothesis of Monsieur Monod was beautifully verified: U178 and CZ1 indeed associate into a

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tetrameric, wild type-like, oligomer and activity indeed arises only in the tetramer. I got excited and happy about my result. I immediately drew the sedimentation profiles (both activity and protein) of the three samples on the same graph, using a different color for each of the six curves, and as soon as Monsieur Monod arrived in his office I went in to show him my graph. To my surprise, he did not react. I had expected one of his warm, encouraging smiles, but nothing on his face! He looked, and looked at the graph as if something were not clear. While he usually was so fast in grasping the essence of what one would tell him, he seemed not to understand what I was trying to explain. David Perrin, one of the co-discoverers of intracistronic complementation in galactosidase had helped me a lot in this experiment, showing me how to run a sucrose gradient experiment. He had come with me in Monsieur Monod’s office and was waving all kinds of signs at me. At first, I could not understand them. But I suddenly realized that David was trying to indicate that Monsieur Monod was color-blind. He could not distinguish the six graphs from one another and therefore could by no means follow my enthusiastic explanations! I went to the blackboard, drew the curves one by one, and then got the smile I had expected y Several years later, Monsieur Monod told me that he had discussed my experiment with Leslie Orgel and Francis Crick when they produced their model of intra-cistronic (or inter-allelic) complementation [6]. This was my main achievement during my year of part-time training with Ed Lennox. It seemed to satisfy Monsieur Monod, as a couple of weeks later he confirmed that he would be happy to have me stay for a second year in his laboratory. Thus, by the end of my first year at the Institut Pasteur, I had obtained my BSc and had done well enough at the bench to obtain a mark of satisfaction from Monsieur Monod. This could have been enough to make me happy. But another event had marked that year and was about to come to a conclusion. On October 13, 1962, a week before starting at the Institut Pasteur, I had met a young, pretty, elegant, bright girl who was living in Roanne (some 400 km south of Paris). Cećile and her father were visiting an old cousin of my father they had come to know during World War II. During my last week of freedom before joining Monsieur Monod, I showed her my favorite places in Paris, took her out for dinner, went twice to the theater. Two weeks later, I spent the weekend


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in Roanne visiting her. Two more weeks, she was back in Paris for a couple of days. We had spent a total of 52 hours together and exchanged two or three letters (there was no e-mail then, and telephone calls between Paris and the province were expensive!) when I asked her whether she would marry me. She said yes without a second of hesitation. She completed her academic year in Lyon where she was studying mathematics for her BSc, and in July of 1963, we got married. Today, 44 years later, neither of us regrets our hurried, somehow immature decision. I thought I had to mention this personal aspect of my life, because it undoubtedly has had a major influence on my scientific life. While I had envisaged spending my life single, entirely devoted to science, I had to share my time and passions between my family and science. It was not always easy. Compromises had to be found. But after all, we managed to build a happy, warm, tightly united family. And, though I shall leave it to others to judge my scientific achievements, I dare say that my scientific and academic activities have brought me immense satisfactions and a better than decent career. In September of 1963, I started my second year at the Institut Pasteur, my first one full time in the laboratory. The Salk Institute had just been completed and Ed Lennox had left for San Diego. Monsieur Monod asked David Perrin to become my mentor. David Perrin was a few years older than me. He was a peculiar, highly lovable, generous person. Grandson of the Nobel Prize winner physicist Jean Perrin, son of the prestigious physicist Francis Perrin (one of the founders of the CEA), David had studied biology. He was a living encyclopedia. He knew all papers that were recently published, was perfectly documented about any information that appeared in the newspapers. He could tell you the number of tons of coal produced in China, the time it takes a sparrow to fly from France to Egypt, the Latin name of any worm or tree, you name it. Moreover, he could tell you where to find any type of equipment, glassware, chemical, gadget you needed for your experiments. Everyone in the Monod-Jacob group would come to him several times a day to get help. Moreover, David was aware of all experiments being run in the lab and was giving precious advice and stimulating suggestions to whoever would ask him. He would also perform experiments by himself. Inspired by Anfinsen’s experiments on the renaturation of ribonuclease, David had observed that heat inactivated E. coli

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b-D-galactosidase could be dissolved in urea and renatured by dialysis [7]. After a few preliminary unfruitful experiments aimed at investigating the kinetics of complementation between U178 and CZ1, I undertook studies on the kinetics of reactivation of urea denatured wild type galactosidase following dilution into urea-free renaturation buffer. Urea denatured galactosidase was diluted at least 100-fold in urea-free buffer containing ONPG (ortho-notrophenyl-beta-D-galactopyranoside, a very convenient chromogenic substrate of galactosidase) and the appearance of active enzyme was monitored by recording the optical density at 420 nm as function of time. Nowadays, such experiments would be straightforward, and modern computerized double beam spectrophotometers would provide precise and rapid determination of the activity as a function of time. In 1963 however, there was no double beam spectrophotometer in the lab. There was no automatic recorder. There was no software in the spectrophotometer to compute the slope of a curve. There was no computer at all in the laboratory to analyze the data. The procedure was time consuming: with a timer in hand every 15 seconds one would reset the zero of the spectrophotometer on the blank cuvette, measure the absorbance of the sample cuvette, and write it down on a paper. At the end of the kinetics (between 5 and 30 minutes usually) one would plot the absorbance as a function of time on a graph paper. Then, at each point, one would estimate the slope of the curve by means of a mirror and a ruler, write down the result for each point, and then plot the slope as a function of time in the appropriate way. One could thus test whether the kinetics were best approximated by a first order, second order, or higher order model. The reactivation kinetics thus obtained after diluting galactosidase out of 8 M urea showed a lag followed by complex reaction with an order somewhere between 2 and 3. Such kinetics indicated that the renaturation is a sequential process, with the folding of an intermediate during the lag phase being required for the oligomerization into active enzyme. With the aim of separating the folding of the intermediate from the association phase, I tried to find an intermediate urea concentration that would not prevent the folding but block the association. Thus, galactosidase denatured in 8 M urea was first diluted into buffer at various urea concentrations ranging from 1 to 8 M, incubated for some time to let the folding proceed, and diluted again into urea-free buffer to let renaturation go to


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completion. The resulting protein was then assayed for enzymatic activity. All samples incubated at intermediate urea concentrations, except 4 M, showed approximately the same final activity as the protein directly diluted into urea-free buffer. After incubation in 4 M urea, the protein failed to refold upon dilution in urea-free buffer. Thus, except for this ‘‘4 M urea trough,’’ incubation at intermediate urea concentrations did not prevent the efficient renaturation of galactosidase. Samples of the protein preincubated at various intermediate urea concentrations were then diluted into the urea-free buffer assay mixture and the kinetics of enzyme activity recovery were recorded and analyzed as indicated earlier. The result was striking and clear. Samples preincubated at urea concentrations higher than 4 M showed the same biphasic kinetics as those observed by direct dilution into urea-free buffer, that is a lag followed by a multimolecular reaction. Conversely, samples preincubated at 2 or 3 M urea showed monophasic kinetics. The lag phase had disappeared and the kinetics reflected the same multimolecular reaction as the association phase observed in the one step renaturation procedure. Thus, preincubation in 2 or 3 M urea had indeed let the protein fold into an association competent intermediate. The sedimentation coefficient of this intermediate was determined by analytical centrifugation in 2 M urea. From its value and from the known molecular weight of the polypeptide chain, the frictional coefficient and axial ratio of the intermediate could be determined. Their values indicated that the intermediate was folded into a condensed globular structure. This set of studies on the renaturation of urea unfolded galactosidase led to several undisputable conclusions. One was that the renaturation of galactosidase is not a ‘‘one step’’ transition. Rather, it is a sequential process involving the formation of a folded monomeric intermediate. A second conclusion arose from the characterization of the inactive protein obtained after preincubation in 4 M urea. The inactive protein was shown to be made of aggregates that could be reactivated by dissolution in 8 M urea followed by one step dilution into ureafree buffer, indicating that they were made of chemically intact, but misfolded polypeptide chains. Moreover, no interconversion was observed between native enzyme and aggregates in the absence of denaturing agent. Thus, depending on the renaturation conditions, two distinct stable states separated by a high energy barrier can be reached. Which of these two states was

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more stable than the other? One could not tell. But one could no longer assert that the native conformation coincides with the thermodynamically most stable state. I did not realize how unorthodox my findings were. I had no idea about how deep rooted in people’s minds the current dogmas were. Indeed, the common belief was that protein folding was a two state process, with no intermediates between the native and denatured states, while I observed a folding intermediate during galactosidase renaturation. The common belief was that the native state of a protein was the thermodynamically most stable one, while I questioned that dogma. The common belief was that aggregates were nothing more than a nuisance resulting from protein damage, while I considered them as a possible alternate outcome of protein folding. Monsieur Monod was extremely interested in my observations and conclusions. We had several intense discussions, particularly about the peculiar behavior of the protein in 4 M urea, before I wrote down and defended my Master’s thesis in June 1964. This marked the end of my studies on galactosidase renaturation. I described in details these experiments, made by a naı¨ve student working for his master’s degree, to show how chance sometimes decides on one’s interests. These extremely simple experiments were not originally meant to solve general problems related to protein folding. Yet, because they went so much against the accepted dogma, they were to influence my whole vision of protein folding and condition most of my research during the rest of my scientific life. The identification of folding intermediates, characterization of folding pathways, discovery of alternate folding pathways, characterization of aggregates and search for procedures to prevent their formation, to which my laboratory devoted most of its efforts, all found their roots in these early experiments on galactosidase.

A Detour Through Molecular Enzymology: The Catalytic Mechanism of Tryptophan Synthase Shortly before I completed my Master’s degree, Monsieur Monod called me to him, one Saturday morning, in a solemn tone. Frowning, without a smile, he stated that his laboratory really needed an expert in protein physical-chemistry. That though I


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seemed to be interested in that subject, I was far from having the required knowledge of the proper experimental methods. That his laboratory had taught me all it could and would not be able to provide the appropriate additional training. I turned livid, guessing that he was delicately firing me out of his laboratory. He saw my disarray (which he clearly had wanted to trigger), burst into a loud laughter, and told me that I might become a permanent member of his group if I would agree to complete my training in the US. He then exposed the plan he had imagined. I would spend 2 years with Robert (Buzz) Baldwin in the Biochemistry Department at Stanford University. The DGRST would provide my fellowship during these 2 years, and would support my travel expenses. The NATO (North Atlantic Treaty Organization) would pay for my wife’s travel and for our moving expenses. Everything was organized. There was only my OK missing to close the file y I told Monsieur Monod that a priori I was very interested, but needed to think it over during the weekend. Indeed on the one hand this offer seemed fantastic: it paved my way to a position at the Institut Pasteur in Monsieur Monod’s group on my return; I would discover the US, where I had never been; it gave me the opportunity of getting an advanced training in a field that fit my interests and where France was trailing way behind the US and UK. On the other hand, my wife had not yet completed her studies at the University; her father and mine were both very sick and leaving them behind was not an easy decision; I knew how reluctant my wife would be to leave Paris after only 1 year of living there. Moreover, I knew very little about my putative future mentor, Buzz Baldwin. Buzz had been spending a sabbatical year at the Institut Pasteur with Franc- ois Jacob, two floors above my laboratory. But he had frequent discussions about allostery with Monsieur Monod, mainly focused on the symmetry within proteins and the energetics of protein–protein and protein–ligand interactions. Monsieur Monod told me how much he appreciated Buzz’s sharp and precise analysis during these discussions. I had seen him only at seminars and at the lunch table, but hardly heard him. He was extremely shy, looked hardly older than me, and would express himself only through short, precise, highly ordered, and documented statements. Only once did I have a personal contact with him. He was writing a paper on some analytical centrifugation experiments and got stuck with the integration of a differential equation. He asked Franc- ois

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Jacob to direct him to a mathematician who might help him solving the equation. Franc- ois sent him to Maxime Schwartz and me. Since Maxime was not available, Buzz came to me, showed me the equation, and in a matter of minutes I could solve it. It had not yet forgotten all my mathematics from Polytechnique! Buzz did not show any sign of surprise, thanked me and went away. I learnt later that this brief encounter had impressed him favorably. This young, shy, not-as-good-as-me-in-mathematics guy who was learning how to grow phages did not look like the charismatic professor of physical-chemistry I imagined as my future mentor. Yet, after discussing Monsieur Monod’s offer over the week-end, my wife and I jointly decided to give it a try. Thus, in July 1964, we took the ‘‘France,’’ one of the nicest transatlantic ships ever built, and sailed off to New York. We spent about a week in New York, bought a second-hand, somehow run down 1960 Valiant and visited Washington DC before leaving for California. A long zigzagging drive took us from New York to the Niagara Falls (an absolute must for French visitors), the Painted Desert, Petrified Forest, Grand Canyon and Brice Canyon National Parks, Las Vegas, and finally California. We arrived in Palo Alto on a Saturday afternoon in late August, parked in front of the President Hotel, stepped out of our car and looked around. Cećile burst into tears. ‘‘I do not want to spend two years in such a desert. I want to be back in Paris. I do not want to live in a provincial town like Roanne again y .’’ Indeed, Palo Alto did not look attractive. There seemed not to be a single living soul in the streets. All shops were closed. But we soon found out that this was not the normal aspect of the city: we had arrived on the Labor Day week-end! It took us no more than a couple of days to rent an apartment and buy a convertible sofa and the minimal cookware for surviving y until the American presidential elections. Indeed, some aspects of the American political life shocked us and the campaign of the republican candidate, Barry Goldwater, scared us. We had decided that if he were to win the elections, we would go back immediately to France. But Johnson made it, and we stayed. When I met Buzz at Stanford, he introduced me to the other group leaders of the Biochemistry Department: Arthur Kornberg (Head of the Department), Bob Lehman, Paul Berg, Dale Kaiser, Lubert Stryer, Dave Hogness, and Georges Stark. Buzz then showed me my lab and the half bench he had allocated to me.


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The other half bench was for Elliot Elson, a brilliant PhD student. Tom Link, a PhD student working under George Stark, used the other bench. Buzz took me to the library, gave me its key and asked me to choose a research subject. He himself was no longer working on proteins. Following his sabbatical at Pasteur, he was studying the ‘‘sticky ends’’ of bacteriophage lambda DNA by means of analytical centrifugation. But, since he had promised Monsieur Monod to train me on proteins, I was free to select whatever subject I wanted. The single advice he gave me was to work on tryptophan synthase since a colleague in a building nearby, by the name of Charles Yanofsky, had just sequenced the tryptophan synthase A subunit and identified several point mutations in its sequence [8]. This, Buzz said, might turn out to be a useful system. I was a bit disoriented. In the French academic system, the mentor usually imposes a subject to his PhD student. There I was, with the complete freedom – but also the responsibility – of choosing my own subject. I thought at first that this reflected Buzz’s lack of interest for proteins, but later found out that this was a common procedure in the American system. I read all I could find in the literature about tryptophan synthase and finally came out with a question I thought might be the backbone of my thesis: since tryptophan synthase was made of two types of subunits, then named A and B, I thought it of interest to find out if the renaturation kinetics and yield of the A subunit are or not influenced by the presence of the B subunit, and whether the renaturation of A precedes or follows its association with B. Answering these questions in experimental terms required several preliminary studies: setting up a convenient, sensitive spectroscopic assay to monitor the kinetics of reactivation; determining the stoichiometry of the A and B subunits in the active complex; setting up an optical test to monitor the association of the A and B subunits; finding a proper set of conditions for obtaining efficient renaturation of both the A and B subunits. Buzz approved this project. He took me to Charles Yanofsky to tell him about my project and ask for his help in teaching me the purification of the two subunits and the preparation of the radioactive substrate (indoleglycerol phosphate) that was routinely used for assaying the A subunit. When I told Charlie about my project and mentioned the need to determine the stoichiometry of the A and B subunits in the complex, he told me that it had just been determined by means of

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gel filtration and that a paper he was about to publish with Irving Crawford showed that the complex contained one A and one B subunit. I told him that this is impossible. Charlie asked why and my answer was abrupt: A is a monomer (it contains one a chain), B is a dimer made of two identical b chains and therefore, because the dimer is symmetric, it must bind two a chains. Thus the complex must be either a2b2 or a multiple thereof. Charlie was taken aback, and asked why must the dimer be symmetric. And my blunt reply was that Monsieur Monod said so in his theory of allostery. Charlie laughed, was probably not convinced by this argument and suggested that I work it out. He very kindly hosted me in his laboratory during a couple of weeks and asked John Hardman to show me the preparation of the substrate, the assay, and the purification of a chains, while Tom Creighton was in charge of teaching me how to purify b2. This short visit to Charles Yanofsky’s laboratory marked the beginning of a long-lasting relation which turned into a solid friendship when the barrier created by our age and status differences progressively vanished with time. Back into Buzz’s lab, I set up a highly sensitive assay for a. The hydrolysis of indoleglycerol phosphate was coupled to a change in NADH concentration, which was monitored by means of a spectrofluorometer just built by Lubert Stryer in the department. The assay turned out to be extremely sensitive, which was my aim. Indeed, because John Hardman and Charles Yanofsky had agreed to provide me with pure a chain, I felt I had to spare it as much as I could. I therefore worked at very low enzyme concentrations, of the order of a couple of micrograms per milliliter. But when I performed the assay, I ran into an unexpected problem. The kinetics were not linear. It looked as if the enzyme became partly inactivated with time. During a couple of minutes, the enzymatic activity decreased, and then reached a steady state. The inactivation phase looked like a first order reaction. I shall not dwell on the various assumptions I made to interpret this observation and to understand why part of the enzyme got inactivated while an other part did not, why the relative amounts of ‘‘stable’’ and ‘‘unstable’’ protein changed with pH, with the concentration of enzyme in the essay tube, with the temperature, with the nature of the buffer. I tested all these assumptions but all of them were ruled out by experimental observations. I had already spent about 2 months trying to solve


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this problem. My time at Stanford was short, and there I was, stumbling on my first experiments. Buzz could not find an interpretation either and was of little help until he came in one morning and, without any explanation, asked me to repeat my experiment in a plastic test tube he gave me. I did it immediately and to my surprise the inactivation phase was no longer there. The kinetics were perfectly linear. I showed this to Buzz. With no comment, he asked me to repeat the experiment in the usual glass tube, but with 1 mg/ml of serum albumin added to the buffer. There again, the kinetics were perfectly linear. This looked so strange to me. I did not understand the underlying mystery. Buzz then told me about the adsorption of proteins onto glass surfaces. The kinetics of ‘‘inactivation’’ I had observed were nothing but the kinetics of protein adsorption on the walls of the test tube. Silicone tubes did not adsorb proteins. Excess serum albumin had coated the glass walls, thus preventing the enzyme to get adsorbed. I was shocked. I had been working for months, spending about 10% of my time at Stanford, on a trivial uninteresting artifact. Yet, I learnt a lot from this experience. Those things may look interesting and be worthless. That one should not get discouraged by failure (as the rest of my time at Stanford was highly productive). And that one can learn a lot from failures. Indeed, not only was I never caught again by protein adsorption on glass, but also 40 years later, my last experimental work dealt with distortions of infrared spectra of proteins recorded by means of an attenuated total reflectance accessory (ATR). These distortions had, for years, prevented spectroscopists from using ATR infrared spectroscopy to study proteins. When in 2004 (i.e. 40 years later) I studied these distortions to find out their cause, I soon realized that they were due to the adsorption of protein on the crystal of the ATR. The evanescent light has to cross a monomolecular layer of adsorbed protein, which absorbs light and brings to the observed IR (infrared) spectrum a significant contribution that adds to the spectrum of the protein in solution. This led me to propose a convenient way to correct the observed spectrum for this spurious contribution. Thus, the time I lost on protein adsorption on glass as a student at Stanford helped me in developing an easy and reliable procedure to obtain undistorted IR protein spectra using the very convenient ATR spectroscopy [9].

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After the disappointment caused by these useless experiments, it took me only a few days to find conditions for efficiently renaturing a and b2. Renaturation of a was so fast that I could not monitor it, but the kinetics of b2 renaturation could be easily followed with the spectrofluorometric assay I had developed, using the ability of native b2 to activate a. I then undertook to determine the stoichiometry of the a and b chains in the tryptophan synthase complex. To do that, Buzz suggested an extremely elegant approach based on sedimentation in the analytical ultracentrifuge. Several samples containing a at a constant concentration and increasing concentrations of b2 were prepared and run in the centrifuge. A rapidly sedimenting boundary (the complex) and a slowly sedimenting one (the excess of a over added b2) were observed as long as there was not enough b2 to saturate a. But above saturation, there was no a left and only a rapidly sedimenting boundary (the sedimentation coefficients of the complex and free b2 are close enough to prevent their separation) was seen. By measuring the decrease of the concentration of slowly sedimenting species as a function of the concentration of added b2, one could determine precisely the concentration of b2 corresponding to the saturation of a. The result unambiguously confirmed a stoichiometry of two a chains per b2 subunit. Monsieur Monod was right! b2 was indeed a symmetrical dimer, and the complex was symmetrical too. In the mean time, Tom Creighton, in Charles Yanofsky’s laboratory, had performed somehow similar experiments using sucrose gradient centrifugation to monitor the fate of the a chains. He could show that, in the presence of a large excess of b2 over a, the a1b2 intermediate species could be observed, indicating that the binding of a to b2 was not strongly cooperative. We published these results in a common paper, my first publication. And in that paper, I was in good company: Tom Creighton, Charles Yanofsky, and Buzz Baldwin [10]. The next step was to find out a convenient, sensitive and rapid, optical signal for monitoring the association of a and b2 at low protein concentrations. No change in absorbance or in fluorescence could be observed upon mixing the two subunits. Because I was not certain that the complex is stable at low protein concentration and because Tom Creighton had shown that preincubation in the presence of the coenzyme pyridoxal-5uphosphate (PLP) and of the substrate L-serine the complex is very


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stable, I added PLP and L-serine to a mixture of a and b2 subunits in order to strengthen their association. And there I got a superb, intense absorption signal with a band centered at 468 nm. This signal however turned out not to be characteristic of the association itself. Rather, I could show that it corresponds to an intermediate in the reaction (i.e. the condensation of indole and L-serine to form L-tryptophan) catalyzed by the b2 subunit. This signal appeared by far more intense for the complex than for isolated b2, presumably because the intermediate is stabilized in the complex. I also observed that the signal is much strengthened in the presence of b-mercaptoethanol and that, in the absence of indole, b2 was able to catalyze a reaction involving b-mercaptoethanol and L-serine. At that stage, Buzz told me that Ann Norris (a brilliant post-doc with Arthur Kornberg whom Buzz had just married) had suggested that the intermediate we were observing might be the enzyme-bound Shiff’s base between a-aminoacrylic acid and PLP. From this, one could infer that the product of the reaction between mercaptoethanol and serine would be S-hydroxyethyl-L-cysteine. This was verified thanks to George Stark who helped me in the chemical synthesis of this compound and showed that the synthetic compound and the product of the ‘‘parasite’’ enzymatic reaction co-migrate in an amino acid analyzer. Other nucleophilic molecules were also shown to enter such b2-catalyzed condensation reactions with L-serine. Thus, while my search for an association-specific signal was a failure, I had succeeded in identifying and characterizing an intermediate in the catalytic cycle of tryptophan synthase. We coined the name ‘‘amber complex’’ onto it, as a reminder of its amber color. Buzz took me to Berkeley to visit Esmond Snell and told him about this complex. Snell was the pope of pyridoxal catalysis. He was indeed interested in our findings and told us of similar observations and conclusions that he and his coworker William Newton had reached on another PLP enzyme, tryptophanase. Esmond Snell encouraged us to publish our results, which he considered as a breakthrough in understanding the catalytic mechanism of b2. I started writing the manuscript on the amber complex. But, since I had not given up the hope of finding an association-specific optical signal, and because there were only a couple of months left before my return to France I simultaneously started some fluorescent studies on Lubert Stryer’s fluorometer. Again, no

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specific signal could be observed upon mixing the two subunits. Lubert, who was supervising me while I was using his machine, made a wise suggestion. Since the a subunit does not contain any tryptophan residue while the b2 subunit does, one could imagine that an energy transfer between tryptophan residues of b2 and a fluorescent probe attached to a might show up upon subunit association. Following his suggestion, I attached dansyl residues on a chains, mixed them with b2 and recorded the resulting fluorescence emission spectrum when exciting the tryptophans at 280 nm. No energy transfer could be detected. Again, I added PLP and L-serine to ensure that the subunits would associate. And here again, a superb fluorescence signal would appear. But I could show that the signal did not correspond to that of the expected energy transfer and that the same signal appeared without the labeled a chains. Lubert and I worked uninterruptedly on this signal for about 2 weeks. We practically never switched off the fluorometer. We had to do as much as we could to understand the nature of this signal, since my visit at Stanford was coming to an end. Following exactly the same strategy as I had worked out for the amber complex, we could within these 2 weeks demonstrate that (1) the signal, which we named the ‘‘aqua complex,’’ was seen with b2 only in the absence of active a, (2) it could be observed with labeled a because the labeling of a inactivated it and prevented it from associating with b2, (3) the signal corresponds to an intermediate in the catalytic cycle of b2, and (4) it accumulates on isolated b2 but disappears upon addition of a. After I left Stanford, Lubert and one of his students, Sheldon York, completed this study, which resulted in my third paper. My two papers on the amber and aqua complexes [11–12] pioneered the way for many subsequent studies, mostly by the group of Edith Miles at the NIH (National Institutes of Health), on the catalytic mechanism of tryptophan synthase. Just before I left back to France, Buzz asked me to present my results at a Wednesday noon seminar. Only the senior members of the Department were invited to these seminars. I had not expected to talk in front of this distinguished audience, and had good reasons to apprehend a confrontation with the senior staff of the Department. The whole Department was involved in topics related to DNA replication. Tryptophan synthase was not their cup of tea. Furthermore, my working hours were rather atypical


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and I knew that some people did not like it. Yet, in spite of the initial tension, my talk went easily. The interest of the audience rose rapidly. Pertinent and constructive questions were asked, to which I could provide competent answers. And the seminar ended with the following comment from Arthur Kornberg: ‘‘Well, Michel, after all, you did achieve somethingy’’ Arthur Kornberg’s comment meant a lot for me. During my 2 years at Stanford, I had been torn between two conflicting poles. One was my deep interest in my work and my anxiety to gather enough results to write a thesis when returning to France. The second was my wife. We had been married for only a year before coming to the US. She had interrupted her studies in France, left her family and friends behind. I could not let her wait at home alone. I therefore had decided to limit my working hours and devote a significant fraction of my time to her. I would thus work every day from 8.30 am till 7 pm, with a 1 hour interruption for a quick lunch that I usually would take with her. On Saturdays, I worked only in the morning, and never on Sundays. Only occasionally would I briefly appear in the lab during the night or the week-end to change tubes in a fraction collector. Moreover, I regularly took a few days of vacations and even spent a month in France during the summer of 1965. Back in the 1960s, this was not the way one would behave in the Biochemistry Department at Stanford. The laboratories were buzzing with activity until well after midnight, and the main difference between week-ends and working days was that the students and post-docs would come to work wearing shorts rather than pants. I remember PhD students and post-docs walking along the walls, trying to hide their skis, asking the help of friends not to be seen by their boss when escaping for a week-end in the mountains. This contrast between the common behavior and mine had elicited some negative feelings against my presence in the department. That I had been able to produce three respectable papers and to be awarded Arthur Kornberg’s compliment comforted me and showed me that I had been right in the way I had organized our life in California. Indeed, in addition to being productive in terms of research and training, my stay at Stanford turned out to be an extremely rewarding experience for my wife and I. We discovered the pleasures of outdoor life in California: sailing on lake Lagunita, horse riding, skiing in Yosemite, hiking along the Pacific coast,

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or in the nearby woods. We spent hours in Carmel, Big Sur, Lake Arrowhead, and Nappa Valley. We fell in love with San Francisco. During our second year, we went at least twice a week to the city. At the San Francisco Opera, among many excellent other performances, we saw the Bolchoi Ballet and attended a memorable concert of Stravinsky himself performing his Symphony of Psalms. We heard outstanding jazz concerts, in San Francisco as well as on the Stanford campus: Eroll Garner, the Modern Jazz Quartet, Louis Armstrong, Duke Ellington, and Ray Charles. We heard a fantastic koto concert by Kimio Eto and an unforgettable sitar performance by Ravi Shankar. We discovered the Far East through the Chinese and Japanese communities in San Francisco: shops, music, food. We visited numerous times the De Young Collection of Asian Art in the Golden Gate Park. This collection was of particular interest to us since, for her pleasure, my wife had taken a course on Far Eastern Art at Stanford with an outstanding professor, Mr. Laplante. He gave her a life lasting passion for ancient Chinese bronzes and for paintings of the Sung dynasty. Well, San Francisco became, and still is, our second home. But we also traveled to Mexico, where we visited several of the most renowned Maya sites. During our last month in the US, we traveled to Oregon and Washington State. In Seattle, I gave a seminar on my work on tryptophan synthase in front of a highly distinguished audience, among which were Eddie Fischer (whom I had met at the Institut Pasteur before I left France), Hans Neurath, the Coris, etc. Well, our 2 years at Stanford transformed us. They sealed our couple. They opened our eyes and minds to other cultures. We had come into the US as real Frenchies, we left as citizens of the world. During my 2 years in California, I had had sparse contacts with Monsieur Monod. We had corresponded occasionally, particularly after he got the Nobel Prize in 1965. I had heard the news on the radio in the morning, but still was extremely surprised when I arrived in the laboratory to be greeted and congratulated as if I had been responsible for this success. On that morning, I realized (at last) how immense Monsieur Monod’s prestige was in the scientific community. I felt so happy to belong to his group. And sad, so terribly sad, not to be in Paris and share my happiness with him. I wrote him a letter, and he soon answered, asking about my work. My report was not very optimistic, since at that time I had not yet observed the amber complex. But soon after, in


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February of 1966, he came to California to attend the yearly meeting of the scientific advisors of the Salk Institute. He called me from La Jolla, told me that he would come to see me on Saturday, and asked me to pick him up at the airport at noontime. My wife anxiously prepared an elaborate French meal for lunch. She was terrified to host my boss, a Nobel laureate, in our modest household: we did not have with us our Limoges porcelain plates, silver ware, crystal glasses that we had left in Paris. Yet, she felt very disappointed when I called her from the airport telling her that Monsieur Monod had been invited for lunch by Gunter Stent in Berkeley and was taking me with him. This lunch at the Stents was for me the occasion to discover Monsieur Monod’s talent as a musician. I knew that, in his 20s, he had hesitated between science and music. I knew that he had been a dynamic and creative orchestra conductor, and that he played the cello. But I had never heard him playing. At the Stent’s, while Gunter was preparing coffee and Mrs Stent cleaning the table, Monsieur Monod and I were silently sitting in deep comfortable armchairs, looking through the window at a superb sunset on the San Francisco Bay. Just when the red disk of the sun, shining on the electric blue background of the sky and the sea was reaching the top of the Golden Gate Bridge, I felt a movement on my side, turned my head and saw Monsieur Monod diving under the grand-piano and dragging out a cello. He tuned it in a matter of seconds and started playing Bach’s second Suite for cello. The quality of his technique, his concentration, his inspiration made this moment of communion between his sensitivity and the beauty of the landscape unforgettable. Soon after this unique moment of emotion, I drove him back to the airport and we had time to discuss my work and plans for the months to come (Figure 3). As soon as I was back in Paris, Monsieur Monod enquired about my results and about my training in terms of methodologies. He was interested by the experimental techniques I had learnt more than by my research. He liked the fact that I had acquired some expertise in ultracentrifugation, in differential spectroscopy, in fluorescence spectroscopy including polarization and energy transfer, and even in ORD (optical rotatory dispersion). He asked me to give a seminar and, just after the seminar, he concluded that I would have to perform just a few additional experiments to determine some kinetic constants

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Fig. 3. Monsieur Monod tuning the cello during a visit to Eddie (Edmond) Fischer – Seattle 1969.

for b2. I would then have enough material to start writing my PhD dissertation. Writing a first draft of my dissertation took me about a month. I showed the manuscript to Monsieur Monod. Two days later, he turned it back to me with just a few suggestions and corrections, which were easy to take into account. The corrected manuscript was submitted to the three members of the jury: Monsieur Monod, Raymond Dedonder (a microbiologist who was to become the Director of the Institut Pasteur in the 80s), and Bernard Labouesse, a young and brilliant enzymologist.


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Back and Deep into Protein Folding: The ‘‘Globule’’ Model My work at Stanford had taken me away from my original questions about the folding of tryptophan synthase. It dealt with its catalytic mechanisms. And I probably would have continued on this subject if the defense of my thesis would not have taken me back to protein folding. Indeed, the custom in French universities was that, while the PhD Dissertation was being evaluated by the jury, the chairman of the jury proposed to the candidate a ‘‘minithesis.’’ It consisted in a question that the candidate had to answer by reading the literature. The theme of this bibliography search was proposed to the candidate some 2–4 weeks before the defense. The chairman of my jury, Raymond Dedonder, suggested that Monsieur Monod would choose the subject. The question, which Monsieur Monod asked me to answer, was ‘‘Can one predict the 3D structure of a protein from its amino acid sequence?’’ Looking into this question decided on the rest of my scientific trajectory. I red all the papers I could find on protein folding. There were not many, so the search did not take long. I wrote a 10 pages report which ended with the following conclusion: yes, with many more 3D structures solved by X-ray crystallography and if significant progress is made in the speed and power of computers, it should become possible (within the next 10–20 years) to predict the 3D structure of a native protein from its amino acid sequence. The ultimate conclusion was correct, but the time span much too optimistic y On January 7, 1967 I defended my thesis. Mrs Giraudeau was in the audience during the defense. A few days earlier, I had knocked at her door and told her the major impact of her teaching on my decision to become a biochemist. While she had always behaved, in front of her students, as a rather harsh person, she burst into tears and embraced me. And after the defense, she congratulated me and told me that, though she could hardly understand what I had explained, she felt proud of having somehow contributed to bringing me to this stage. Now that I had got rid of the burden of obtaining my PhD within a 5 years limit, I felt free to think of my future work. Of course, I discussed it with Monsieur Monod. He was eager to test ideas he had about the formation of oligomeric proteins. Based on evolutionary and energetic considerations, he had predicted that homologous interactions between subunits should prevail over

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heterologous interactions. He therefore thought that most oligomers should contain a number of protomers equal to a power of two: dimers, tetramers, octamers, and hexadecamers. To test this idea, he suggested that I should determine the number of protomers in several oligomeric proteins. The approach seemed simple to him: use the analytical ultracentrifuge to determine the molecular weight of the native protein, that of the dissociated subunit (in urea or guanidine, for instance) and divide the former by the latter. I argued that this was not easy to do because such molecular weight measurements should be done with a precision greater than a few percent for oligomers of an order equal to or higher than four. Indeed, to distinguish between a tetramer and a pentamer requires a precision of better than 10% on the absolute values of the monomer and oligomer molecular weights. Such a precision was difficult to achieve with native proteins, and impossible to reach for isolated monomers in the presence of 8 M urea or 6 M guanidine because of the uncertainties on the partial specific volume of the protein in the presence of concentrated denaturing agents. Monsieur Monod had no idea about what the partial specific volume was. When I explained it to him and talked about the preferential solvation or hydration that a protein may undergo in binary solvents, he found an unexpected reason to overcome my hesitations. Once unfolded in urea or guanidine, he argued, a protein loses its specific properties. Its behavior should be dictated essentially by its amino acid content. Because globular proteins show rather similar overall relative amino acid compositions, their degrees of preferential solvation or hydration and their partial specific volumes v* in the presence of urea or guanidine should be more or less the same. This intuitive reasoning seemed plausible. Together with Agne`s Ullmann, we undertook testing it. Six proteins made of polypeptide chains of known molecular weights ranging from about 15.000 to about 135.000 were denatured in 6 M guanidine and submitted to analytical centrifugation at three to four different protein concentrations. Agne`s Ullmann and I worked in alternating teams. The two analytical ultracentrifuges were spinning day and night. For each sample the experiment provided the value of the apparent molecular weight times the flotation factor (1 – v*r). This value was plotted as a function of the protein concentration and extrapolated to zero protein concentration so as to minimize the non-ideality of the solution. It was hard work, but it was


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worth it. The end result was that for these six proteins the flotation factors, and hence the apparent partial specific volumes, in the presence of 6 M guanidine were practically the same. When applying a correction aimed at taking into account the partial specific volume of the protein in water from its amino acid composition, we found an agreement to better than 2% between the known molecular weights and the molecular weights determined in guanidine. These findings open the way to an accurate determination of the molecular weights of isolated subunits by centrifugation in guanidine. Because he had given the original idea that was the basis of this research, I managed to convince Monsieur Monod to sign the paper that we wrote. He reluctantly accepted to be the senior author of this paper, which was submitted for publication in July of 1967 [13]. This was the single paper I ever published together with Monsieur Monod. Its publication prompted an anonymous violent editorial in Nature that criticized Monsieur Monod for having ‘‘rediscovered’’ centrifugation in guanidine. Indeed, after we had submitted our manuscript, a paper by Hade and Tanford [14] had appeared that dealt with protein solvation and hydration in guanidine. Monsieur Monod’s answer to the editorial was harsh. He did not tolerate its brutal and unjustified irony and a criticism based on the fact that we had ignored a paper y that was not published when we submitted ours. Though I think he was basically right, I regret that he did not make it sufficiently clear that while Hade and Tanford’s results were certainly more rigorous than ours, our approach made it much easier to interpret experimental results from centrifugation in guanidine, then practically the unique method for measuring accurately the molecular weight of protein subunits. It may be interesting for the reader to compare my account of this controversy with that by Tanford on pages 25–27 in a previous volume of this series [15]. For a short period, several groups used our experimental approach. Quite successfully as long as they did not omit to extrapolate their molecular weight determinations to zero protein concentration. But soon, electrophoresis on polyacrylamide gels in the presence of SDS (sodium dodecyl sulfate) was discovered and centrifugation in guanidine was abandoned. In the mean time however, I had used centrifugation in 6 M guanidine to answer another question asked by Monsieur Monod. During my stay at Stanford, together with Agne`s Ullmann, David Perrin, and

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Franc- ois Jacob, Monsieur Monod had identified a peptide fragment of galactosidase, named o, that was able to complement, both in vivo and in vitro, inactive galactosidase mutants (named o-acceptors) carrying deletions of various lengths in the operator distal region of the gene [16]. The stoichiometry of the o and oacceptor polypeptide chains in the complemented enzyme had to be determined. To do that, the complemented enzyme was purified to homogeneity from a strain expressing the o fragment on an episome and an o-acceptor on its chromosome. The enzyme was submitted to sedimentation-diffusion equilibrium in 6 M guanidine and the protein distribution was analyzed using the interference optics. Stuart Edelstein, a former student of Howard Schachman who was a world famous expert in analytical centrifugation, had helped me in the very delicate task of adjusting and calibrating the interference optics and showed me how to interpret the diagrams. Analysis of the protein distribution at equilibrium enabled us to determine with good precision the molecular weights of the o and o-acceptor peptides and to determine a one o to one acceptor stoichiometry [17]. How could these polypeptide chains assemble to generate an enzyme native-like enough to be active? I felt that answering this question might bring some important insight on the protein folding problem. Indeed, one could imagine that what we were observing for an artificially cleaved polypeptide chain might be of general significance: assuming that each fragment would fold into a native-like conformation, and then assemble as subunits do in an oligomer, would change the problem of folding a very large polypeptide chain to the simpler problem of folding two smaller regions of that chain. And if this would be correct, one then could imagine that the o-acceptor itself might be built of several independently folding regions. Thus, if one would dare extend this model to other large proteins, the complexity of folding large polypeptide chains would be reduced to the folding of small proteins. The model seemed elegant, and o-complemented galactosidase seemed an appropriate system to test it. By ultracentrifugation of the purified complemented galactosidase in non-denaturing buffer, I could determine the molecular weight of the complemented enzyme and show that it contains four o and four o-acceptor chains. Furthermore, I showed that a mild proteolytic treatment with papain reduced the molecular weight of the complemented enzyme to that of the wild-type


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enzyme, which suggested that the region of the acceptor overlapping with o was highly susceptible to proteolysis, and therefore probably not folded. Conversely, based on the fact that the isolated o-acceptor had been shown by David Perrin to strongly react with an immune-serum raised against native galactosidase, I concluded that even in the absence of o it can fold into a native like conformation. From the axial ratio of the isolated o fragment (as determined from its molecular weight and sedimentation coefficient) I concluded that it is folded in a condensed globular structure and inferred that it, too, is likely to fold in a native-like conformation even in the absence of acceptor. From this, I proposed that association between the o and o-acceptor occurs through quaternary interactions between stereospecific association areas formed on their surfaces. Based on these considerations, I suggested that the wild-type monomer might be made of two compact ‘‘globules,’’ one corresponding to the o region and the other to the structured (papain insensitive) part of the o-acceptor. Each globule would fold around an independent nucleation center, thus creating complementary stereospecific association areas. The two globules would then associate via intrachain inter-globule contacts to generate the final native tertiary conformation. This ‘‘globule model’’ was proposed in a paper published in the then very prestigious Journal of Molecular Biology [18]. Monsieur Monod was thrilled by my observations and helped me a lot in the formulation of this model. Following his suggestion (he never told me that, but it could not have been otherwise) I was invited to present this model at the ‘‘Lactose Operon’’ meeting organized by Jonathan Beckwith and David Zipser at Cold Spring Harbor in September 1969. One day before I had to leave for this meeting, my father was taken to the hospital in a critical condition. I had to postpone my departure and could not arrive in time for my lecture. Agne`s Ullmann was kind enough to replace me and deliver an unprepared short talk about the globule model which, she told me, was well received. I had to write a paper for the proceedings of this meeting. It included additional studies on a complemented enzyme made of o and a longer o-acceptor, which confirmed the results previously published with the shorter acceptor. In the discussion of the manuscript I submitted, I had introduced a speculative section in which I tried to generalize this model by suggesting that polypeptide chains of a size larger than 15,000 kD

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should be made up of distinct globules. I also proposed that globules might be structural elements used as building blocks by evolution to construct new functions by reshuffling sub-sites. The latter assumption was based on my observations on tryptophan synthase whereby the reaction could be divided in two main steps: the activation of L-serine by PLP followed by the nucleophilic attack of indole on the b-carbon of serine. I suggested as an example that the b-chain of tryptophan synthase might be made of two globules, one carrying the binding sites for indole and L-serine, the second carrying the binding site for indole. I thus proposed that tryptophan synthase and other amino acid synthases might share a common serine-activating globule. Replacing the indolespecific globule of the tryptophan synthase b-chain with a globule carrying a site for hydrogen sulfide would create a new enzyme able to catalyze the ‘‘parasite’’ cysteine synthase reaction of tryptophan synthase with a good affinity for hydrogen sulfide. The editors returned my manuscript with the request of deleting the speculative section and restricting the Discussion to the mere facts related to o-complemented galactosidase. I was young, somehow shy and eager to publish one paper. I gave up my speculations and promptly returned the revised manuscript, which was immediately accepted. In retrospect, I regret my weakness. Indeed, assuming that large polypeptide chains are made of several globules folded around distinct nucleation centers was the essence of the ‘‘domain’’ model proposed by Donald Wetlaufer [19] 4 years after my paper on the globules in galactosidase. And my (unpublished) speculations about globules being possible functional building blocks during evolution was superbly supported 5 years later by the observation of a common NAD-binding domain in several dehydrogenases [20]. That domains are both autonomously folding regions and structural elements carrying functional subsites are now two widely accepted concepts. I think it of interest to wonder, in retrospect, why these two aspects of the ‘‘globule model’’ had so little impact on the scientific community in spite of a paper published in the Journal of Molecular Biology [18] and of several seminars I gave on the subject. One reason, I think, is that reviewers and editors too often (in my opinion) refuse to publish speculations, even when they are specifically referred to as such, derived from experimental results. This is what happened to my Cold Spring Harbor paper. The second reason is that too many


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scientist believe in what they ‘‘see’’ more than in what they ‘‘think.’’ Indeed, Wetlaufer’s proposal that domains are folding units [19] was as speculative as mine. Wetlaufer could attract the attention of the scientific community because he was able to show images of distinct globular domains in native proteins. Yet he failed to demonstrate that they are able to fold independently of one another. On the contrary, we clearly demonstrated that globules are able to fold independently into compact globular structures, but our results failed to convince people because we were unable to show images of the folded isolated domains. Neither of our approaches was fully convincing, but there were images of domains that stayed in the peoples’ minds. I believe that this is why domains prevailed over globules. It took many years to solve the 3D structure of galactosidase. But when it was finally published [21] I was happy to see that the autonomously folding o-globule we had identified appeared as a distinct structural domain as defined by Wetlaufer. Our two approaches had finally fully converged. In spite of the little initial impact of the globule model, two persons were highly supportive. Buzz Baldwin and Monsieur Monod. In September 1969, I was invited to attend a Battelle Meeting in Seattle. A prestigious assembly of physical-chemists was gathered there to discuss the properties of biological macromolecules. I gave a lecture on the globule model. After my talk, Buzz Baldwin congratulated me with unusually warm words. He added that he was happy to see me get involved in protein folding because, he said, this was the subject of the future. I asked him why, then, did he not switch to that subject himself. He replied that he was too old for that and should leave it to young people like me. Yet, to my surprise, less than 2 years later I saw Buzz’s first paper [22] of a spectacular series of outstanding publications that made him one of the world leaders in the field of protein folding. Were it only for contributing to turn Buzz into a protein folder, the globule model had after all had some impact on that research area. The second person to strongly support me and show a strong interest in the globule model was Monsieur Monod. He had good reasons for that. In 1967, he had been elected Professor at the Colle`ge de France, the highest possible academic position in France. While preparing a series of lectures on proteins for the 1968–1969 academic year, he spent much effort trying to answer simple, general questions about proteins.

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Like what makes a protein stable? He liked Kautzman’s oil drop model very much. Or why are proteins so large? Together with the future Nobel laureate in physics Pierre-Gilles Degenne and based on the assumption that about five residues contribute to building an active site, that the peptide bond has a limited flexibility, that enough hydrophobic residues must be buried in the core of the oil drop and enough polar residues exposed on its surface to ensure solubility, he estimated that about 100 residues would be needed to build up a stable functional globular protein – a fairly good prediction! He was struck by a paper by Fisher [23] which reported that the ratio of polar over non polar residues in proteins, which should have decreased to zero with increasing molecular weights if protein obeyed the oil drop model, in fact reached a limiting value corresponding to an oil drop-like globular protein of molecular weight about 15–20 kD. From this he inferred that polypeptide chains larger than 20–30 kD should be made of several oil drops, each folding around a distinct hydrophobic nucleation center, which then assemble by means of stereospecific interactions into the native tertiary structure (see Scheme 1). This view was in perfect agreement with my globule model and was supported by my observations on o-complemented galactosidase. Monsieur Monod therefore encouraged me to try and extend my observations along two lines. One was based on the assumption that the hinge between domains was loosely structured and hence particularly sensitive to proteolysis. One might therefore try to nick the polypeptide chain in the hinge and separate the domains after denaturation in urea or guanidine. The second was aimed at testing the validity of the globule model in proteins other than galactosidase. I first tried to obtain the o and acceptor fragments by mild proteolysis of wild-type galactosidase. With limited success since only small amounts of complementing material could be obtained. I therefore decided to change enzyme. The allosteric enzyme aspartate transcarbamylase had just been shown by Howard Schachman and his collaborators to be made of two types of polypeptide chains corresponding to distinct catalytic and regulatory subunits [24]. I reasoned that allosteric proteins made of only one type of polypeptide chains might contain a catalytic and a regulatory globule that evolution could have fused into a single chain. I asked a young graduate student, Olivier Raibaud, to test this idea on rabbit muscle phosphorylase, a protein that


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Scheme 1. Notes handwritten by Monsieur Monod for his course on Proteins at the Colle`ge de France: These notes can be translated as follows: For the scheme on the left: ‘‘which, for a unique polypeptide, implies several hydrophobic nucleation centers.’’ For the scheme on the right: ‘‘or association between oil drops.’’

was extensively studied in Monsieur Monod’s laboratory by Henri Buc. Within a year, Olivier managed to set up a controlled proteolytic treatment that cleaved the enzyme into two complementary fragments, to separate and purify them after denaturation in guanidine and to demonstrate that the smaller fragment corresponds to the N-terminal portion of the intact protein. We published this result [25], which turned out to be of considerable help for the Seattle group who was trying to determine the sequence of the whole phosphorylase chain and had trouble aligning its tryptic fragments. Olivier then showed that the N-terminal fragment could be dialyzed against a urea-free buffer without precipitation and that it looked monodisperse by analytical ultracentrifugation. This was a good preliminary sign that the fragment was able to refold into a compact globule. I asked Olivier to study its secondary structure by circular dichroism and to investigate by equilibrium dialysis its ability

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to bind ligands of phosphorylase. But Olivier, who now was working for his PhD, thought it too daring to invest more time on the globule model. He did not trust it too much. Radioactive regulatory ligands were not easily available for performing binding studies, and circular dichroism was not available at the Institut Pasteur. I reluctantly surrendered to his objections and we abandoned phosphorylase. We failed to bring our study to completion and had to wait for the publication of the 3D structure of phosphorylase to have the confirmation that the globule we had prepared is indeed a structural domain of the native enzyme and corresponds to a regulatory domain as it carries the binding sites for regulatory ligands [26]. Though Olivier had refused to go on working on the globule model, he was indirectly of great help in the continuation of our studies on this subject. During his first year in my lab, he was working side by side with a young Swedish technician, Anna Ho¨gberg, who had come for about a year to work with Franco Celada during his sabbatical with Monsieur Monod. The two youngsters fell in love and, when Anna’s grant came to an end, both were in despair. I had noticed that Anna was outstandingly talented and motivated. I had just been appointed as professor at the Paris University, which interfered with my experimental work and felt that I needed a technician if I wanted to pursue my own projects. I asked Monsieur Monod, who had become the Director of the Institut Pasteur, if I could get a position to keep Anna as my technician. He accepted. Anna and Olivier got married soon after. During a couple of years Anna worked with me, then with Olivier, on Tryptophanase, a project to which I shall come back later. I then managed to convince her to work on globules. Aware of the fact that one needs a convenient, sensitive way to monitor the functional properties of isolated domains, I asked Anna to use the b2 subunit of E. coli tryptophan synthase as a new model to isolate and characterize globules. Indeed, in view of the spectroscopic signals that appear upon PLP binding, of the aqua band generated upon L-serine binding and of the ease with which one could obtain radioactive indole, I reasoned that it would be easy to monitor the binding of ligands to putative functional globules. Using the strategy developed by Olivier for phosphorylase, Anna quickly found an easy and reproducible way to nick b2 at the hinge between two complementary fragments, named F1 and F2. She could separate the fragments in guanidine


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and refold the fragments into conformations that showed hydrodynamic properties compatible with compact globules and had significant contents in stable secondary structures as evidenced by far UV circular dichroism. Furthermore, F1 was shown to be a dimer. The fluorescence emission spectrum of its tryptophan residue, characteristic of a buried side chain, was very similar to that of the native enzyme. And two out of the four cysteines of F1 had low reactivities indicating that, as in native b2, they were buried in an hydrophobic core. Taken together, these properties clearly pointed to native-like structures, in particular for F1. And last but not least, mixing the F1 and F2 fragments resulted in a protein with exactly the same hydrodynamic, optical and functional properties as the nicked enzyme before denaturation and separation of the fragments [27]. This was so perfectly in agreement with the globule model that, as soon as we could, for the first time, reconstitute native nicked b2 from F1 and F2 fragments pre-folded separately, I ran to Monsieur Monod’s secretary and asked to see him as soon as possible. She knocked at the door of the Director’s office, whispered my name, and I was invited to come in. Monsieur Monod asked me what was happening that needed such immediate care. I told him about our results. He looked happy. In spite of the fact that he was overloaded with administrative tasks, he took the time to question me at length about the properties of the isolated fragments, about the nicked enzyme. He finally told me ‘‘C’est superbe mon petit Michel.’’ We went on chatting for a while about my future projects. I felt so comforted by his warm, indefectible support, particularly because at that time so few people were interested in protein folding. He briefly told me about his own projects. When I left his office, I felt so happy of his support and friendship. I did not know that this was the last time I would see him. Less than a month later, he lost the long fight he had fought against medullar aplasia and passed away in his villa at Cannes. I shall come back later on the practical consequences that Monsieur Monod’s decease had on my career. But I can not escape telling how lonely I felt, from then on, without the inspiring and comforting discussions I had regularly had with Monsieur Monod. Since that day of May 1976, I had to find in myself, without my mentor’s incentive, the drive to go on working on protein folding, a domain of science that was entirely neglected by French

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biochemists and granting agencies who thought only in terms of ‘‘Molecular Biology.’’

Without Monsieur Monod: Globules, Molten Globules, and the Tryptophan Synthase Folding Pathway After the shock of Monsieur Monod’s sudden death, Anna and I completed the characterization of the F1 and F2 isolated fragments and of the complemented enzyme [27]. With Monique Decastel, a graduate student who worked in my laboratory for 2 years, we fully characterized the ligand binding specificity of the nicked b2-protein and identified the F1 fragment as corresponding to the N-terminal side and F2 to the C-terminal end, respectively, of the b chain. The latter conclusion was reached through a close collaboration we had with Irving Crawford just before he left the Scripps Clinics in La Jolla and moved to the University of Iowa. This collaboration marked the beginning of decades of a friendship that culminated during a sabbatical year that Irving spent in my laboratory, years later. Anna then undertook a careful investigation of the ligand binding properties of the isolated fragments. She failed to detect any spectral change related to PLP binding to either F1 or F2. Equilibrium dialysis with tritium-labeled PLP or 14C-labeled indole failed to show any binding to either fragment alone. Finally, neither L-serine nor indole or PLP induced any protection of the exposed cysteins in either fragment. From this, we had to conclude that neither fragment alone was able to bind these ligands. Our globules thus did not appear to be the functional building blocks of tryptophan synthase we had expected. I therefore decided to concentrate our efforts in investigating their possible role as folding intermediates. At this stage, Anna left the laboratory. She had achieved a remarkable and complete set of experiments, with outstanding precision, rigor, and understanding. She had published six papers, five of which as the first author. Though she was ‘‘only’’ a technician, she wrote her dissertation and obtained her PhD from the Paris University. She is now an independent research associate at the Institut Pasteur. Her studies on the tryptophan synthase fragments was taken over by Carlos Zetina, a charming, highly competent graduate student from Mexico who had come to


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France to study for his PhD and had ended up in my laboratory. Carlos studied the guanidine-induced reversible unfolding of intact b2, the nicked b2-protein and the F1 and F2 fragments. He showed that the transitions of the intact and nicked proteins are identical and appear bimodal. Each fragment showed a single transition. Each of these transitions coincided with one of the modes observed for the complete protein. From this we concluded that the transition of each domain is the same in the complete protein as it is for the fragment alone, thus indicating that the domains are indeed autonomously folding regions of the complete b chain [28]. Carlos then analyzed the kinetics of reactivation and self-assembly of intact b2 and of its fragments and showed that the folded fragments appeared as intermediates on the folding pathway of the intact enzyme [29]. This work was published in 1982. Among other findings this paper showed that the renaturation of the intact protein can be described as a monomolecular reaction corresponding to the folding of F1, followed by a very rapid assembly of two F1 and two F2 fragments, followed by an isomerization of the assembled complex. It may be of interest for the young readers accustomed to the use of modern computers to note that we had no computer to find the best fit between experimental data and a theoretical model. All I had was an Apple IIe computer, the power and speed of which were certainly much lower than those of a modern pocket calculator. I wrote a simulation program based on the differential equations for two coupled monomolecular reactions, introduced a pair of rate constants for the two components, run the simulation, and changed the pair of parameters until the fit looked correct. The ‘‘optimization’’ was made by eye, comparing the simulated plot with the experimental data. Finding the optimal simulation took me about 2 days y Yet, after these painstaking efforts, it seemed well established that folded globules were intermediates in the complete folding reaction, with F2 folding extremely fast while the folding of F1 was the rate limiting step in the formation of the association competent monomer [29]. After having completed these studies, Carlos wrote his dissertation and left Paris for a post-doc in the US. He had opened the way to a long series of investigations aimed at detecting and characterizing intermediates on the folding pathway of tryptophan synthase. Indeed, Carlos Zetina’s kinetic approach was taken over by a very young technician, Sylvie Blond, who joined the laboratory in

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May 1981 at the age of 18, shortly after having finished high school. This was her first position. She had very little practice, but an incredible energy and a strong motivation. While working full time in the laboratory, she managed to take Biochemistry courses at the University, and obtain her BSc, MSc, and PhD. After her PhD, she left for a post-doc in the US, where she stayed and became a University Professor in Chicago. During her first year at the Institut Pasteur, I trained her in basic biochemical techniques like protein purification, enzymatic assay, and spectroscopy. Inspired by the energy transfer and fluorescence polarization experiments I had learnt with Lubert Stryer, I asked her to introduce various fluorescent labels on the b2 protein and use them to study the kinetics of monomer folding and assembly during the renaturation of intact b2. She used hand mixing, and then stopped-flow techniques, to initiate the renaturation of the guanidine unfolded, labeled protein (or its fragments). She monitored all sorts of signals: fluorescence polarization of fluorescent groups linked to the N-terminus of the b chain, to F1, or to F2, energy transfer between these markers, fluorescence quenching of the tryptophan residue by PLP, changes in the intrinsic fluorescence of the tryptophan residue, fluorescence of the aqua-complex, etc. She thus could follow the kinetics of formation of several folding intermediates and measure the rate of dimerization of the folded monomers [30]. Using monoclonal antibodies specific of b2 and monitoring in the stopped-flow their association with b2 during its renaturation, she could demonstrate that these antibodies recognized a very early intermediate [31]. Anne Murry-Brelier could determine the rate constant of the folding step leading to this immunoreactive intermediate and show that it has an affinity for the antibody close to that of the native enzyme. The epitope on the intermediate thus appeared to have a native-like conformation. Oleg Ptitsyn, the inventor of the ‘‘molten globule,’’ was spending a few months in my laboratory in 1988. He got excited about these very early immunoreactive intermediates and was interested in knowing whether or not they corresponded to a molten globule or to a later folding stage. Indeed, one of the essential features of a molten globule as initially defined by Oleg and his coworkers was that it should have a native secondary structure and a close-to-native super secondary arrangement. Because of the very transient and unstable nature of the molten globules thus far characterized, they had


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never been shown to exhibit such a feature. Hence Oleg’s interest in the ‘‘native-like’’ immunoreactivity of our intermediates. He took some b2 protein back to Pushchino (USSR) and arranged that a sample be analyzed on the circular-dichroism stopped-flow machine that he had set up by Kunihiro Kuwajima in Sapporo (Japan), then the single CD stopped-flow in the world. The kinetics of regain of the far UV CD signal during the renaturation of urea unfolded b2 clearly showed that the immunoreactivity appeared at the molten globule stage, that is at a stage where a large fraction of the far UV CD signal is already formed and ANS binds in large amounts, and well before the appearance of a near UV CD signal and the release of ANS characteristic of the compact native state. This led us to conclude that the early immunoreactive intermediate we had observed was indeed a molten globule [32]. Oleg Ptitsyn was enthusiastic about this finding. He considered our experiments as the ultimate proof that the molten globule is a universal intermediate in protein folding. ¨gbergThus, after years of efforts by Carlos Zetina, Anna Ho Raibaud, Sylvie Blond, Anne Murry-Brelier, and Guenady Semisotnov devoted to investigating the kinetics of folding of b2 we ended up with a folding pathway in which seven distinct intermediates, including a molten globule, had been identified and characterized. We had gone a long way from the initial credo that the folding of a globular protein is a one step event! In the process of this study, we faced a situation which is worth describing, since it shows how difficult it may sometimes be to go against the ‘‘common knowledge.’’ Sylvie Blond had first identified these intermediates by hand mixing experiments. Because their appearance was fast, one had to use a stoppedflow in order to determine their rate of appearance. We therefore acquired a very simple, cheap, hand activated stopped-flow. It could be fit to our fluorometer. It had two syringes of equal volume and therefore could not be used for renaturing guanidine denatured proteins, where a dilution of at least 20-fold was required. Hence our switch to acid denatured b2: renaturation could be triggered by a simple pH jump. Anne Murry-Brelier, a PhD student who had been trained as an engineer in the Ecole Centrale, showed that acid denatured b2, though showing a distinct far UV spectrum, is indeed unfolded and that it can be renatured by increasing the pH. In order to demonstrate that the pH induced folding process was the same as during refolding

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from guanidine, she repeated the experiments made by Sylvie Blond. And to our surprise, we observed that one phase that preceded another one during refolding from guanidine occurred after the other one when refolding from acid. Thus the order of these two steps was inverted, indicating that there was not an obligatory order in the observed steps, which thus appeared as independent events. We submitted a manuscript to Biochemistry. It was rejected by the editors and we received a harsh comment from one of the reviewers who stated that ‘‘If Goldberg wants to publish more papers, he has to do more work, not to repeat the same ones again and again.’’ The reviewers had obviously not understood the point we were trying to make. So we wrote a more explicit version of the manuscript, included our main conclusion in the title, and submitted the new manuscript to Proteins: Structure, Function and Genetics. The editor, Tom Creighton, sent us back the paper with some negative comments and suggested that we submit a revised version with some modifications in the discussion. We followed his advice and modified the discussion accordingly, but without changing our conclusions. Again, modifications in the discussion were requested centered on the fact that our conclusions went against the common view and that we should be less general. We made some changes, but kept our conclusion relative to b2. The third version was also rejected, with suggestions that would bring the manuscript back to the first version. I became furious, wrote a harsh letter to the editor in which I stated that in not a single place had the reviewers criticized the experiments or detected a flaw in our interpretations of our results. That the single blame I could read was that our conclusions were unorthodox. That a scientific journal should not bar a paper on this basis. That I was ready to accept any sound objection on either our experimental approach or our interpretation, and that I would accept that our unorthodox conclusion should be rejected if another interpretation, compatible with the current view, could be proposed by the reviewers. In the absence of such objections or of an alternative interpretation, I claimed that our paper be published. In return, I received a brave and elegant letter from Tom telling me that I was right and that the paper would be published as it was [33]. This was not enough, yet, to convince the scientific community that multiple folding pathways can exist. A couple of months after our paper was published, an editorial by Buzz Baldwin in Nature stated that


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‘‘the ghost of multiple folding pathways had been set to rest.’’ It took the characterization by NMR (nuclear magnetic resonance) of an intermediate on an alternate lysozyme folding pathway [34] to make protein folding experts finally accept the idea of multiple folding pathways.

From Molten Globules to Pre-Molten Globules Those were the years when the molten globule became in the fashion. Though our studies on tryptophan synthase had contributed to it, I did not share this infatuation for this ‘‘universal’’ intermediate. I saw with dismay a flourishing literature describing new molten globules every now and then. But the evidence for these species being real molten globules was very scarce. Usually, the existence of some negative ellipticity around 220 nm and the ability to transiently bind ANS was enough for the authors to conclude that they had detected a molten globule. In the best cases, a decrease in the Stokes radius was added as a final proof. This did not satisfy me at all, since none of this data brought the evidence that the secondary structure was the native one, and the super-secondary structure was native-like. I considered (and still consider) that these features – which were explicitly included in the initial definition of the molten globule – are an absolutely essential element of the molten globule model. Some secondary structure as evidenced by ellipticity at 220 nm, a somehow accessible hydrophobic core as evidenced by ANS binding, some condensation as evidenced by a decrease in the Stokes radius are enough to characterize a hydrophobic collapse, but surely not to demonstrate the nativelike characteristics of the resulting conformation. I therefore thought it of importance to investigate in details some molten globules. Here again, on two occasions, Oleg Ptitsyn’s presence in the laboratory was of considerable help. While Oleg was in my laboratory, Roger Pain, from Newcastle came to visit him. The three of us had a long, vivid discussion comparing the merits of the ‘‘frame work’’ model and of the ‘‘molten globule’’ model. During this discussion, I realized that, according to the former model, the native secondary structures should form first and then only assemble into the native tertiary structure. According to the latter, the secondary structure elements appear only because

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they are stabilized by the native super secondary structure. From this, one could make a strong prediction: in the frame work model, the native far UV CD ellipticity should appear before the ANS binding characteristic of the condensed state, while in the molten globule, both far UV ellipticity and ANS binding should appear simultaneously. Testing these predictions experimentally required however a CD stopped-flow with a very short dead-time, that did not exist in 1988. I contacted two French companies, the stopped-flow manufacturer BioLogic and the dichrograph manufacturer Jobin-Yvon, and prompted them to join effort to adapt a far UV CD detector to a CD spectrometer. They did it and provided us with their first prototype in 1990. Thanks to this machine, which was extensively exploited by my co-worker Alain Chaffotte, we did several pioneering experiments on the early appearance of secondary structure during protein folding. Another important contribution that was triggered by Oleg Ptitsyn originated from a letter I received from Edith Miles, at the NIH while Oleg was with us. Edith had worked extensively on the catalytic mechanism of tryptophan synthase, building up on my initial observations with the amber and aqua complexes. We had met several times, and become friendly. After many unsuccessful attempts by several groups, including mine, to crystallize either the E. coli tryptophan synthase complex or its isolated subunits, Edith had thought of giving a try with the complex from S. typhimurium. Her letter reported her spectacular success in obtaining crystals and solving the structure [35]. Receiving a color picture of this protein, with which I had been acquainted for more than 24 years, was a terrific excitement. It was as if I had met a girl on the Internet (then, by mail y) corresponded with her for years, fallen a love with her and suddenly see her showing up in my office, unveiled, face to face! I remember receiving this letter just before giving a lecture to my students at the University. I was so excited that I could not resist showing them the 3D structure of tryptophan synthase and sharing my emotion with them. Years later, I heard from students who had attended this event that they had sensed my excitement and really felt my passion for science. Back in the lab, I showed the photographs to Oleg Ptitsyn. He immediately noticed something striking. In her representation of the protein, Edith Miles had colored the F1 and F2 domains in different colors. They were easily identified. Oleg noticed that the F2 domain contained a b-sheet, one strand


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of which belonged to the F1 domain and was ‘‘sandwiched’’ between two strands from F2. Isolated F2 thus could not, in the absence of F1, build up the native b-sheet. From this he concluded that the folding of isolated F2 should be blocked at the stage of the molten globule. This was no surprise to me, since Oleg would see molten globules everywhere. But his observation led me to consider F2 as a good system to test the molten globule hypothesis. Indeed, because F2 lacked one of the central strands of its b-sheet, it could not achieve a native-like super secondary structure and therefore could not be an authentic molten globule. We first confirmed, by a variety of experimental approaches, that F2 was not ‘‘native-like.’’ Indeed, using monoclonal antibodies raised against native b2, we had shown earlier that both F1 and F2 undergo conformational changes upon their assembly into nicked b2. Furthermore, by using analytical ultracentrifugation, NMR, proton exchange rates, differential scanning microcalorimetry, far and near UV circular dichroism we demonstrated that F2 folded into a stable, organized globular conformation which, like a molten globule, was condensed and showed a significant amount of secondary structure, no tight packing, a non cooperative thermal unfolding transition and an NMR spectrum and proton exchange rates characteristic of several conformations in rapid equilibrium. However, isolated F2 appeared more hydrated and expanded than an authentic molten globule and its predicted contents in a-helices and b-structure differed widely from that of the F2 region in native tryptophan synthase [36]. Kinetic studies in our new CD stopped-flow apparatus on the rate of appearance of the far UV CD signal during the renaturation of guanidine unfolded F2 showed that the secondary structure was formed within less than 4 milliseconds, the dead time of our machine. Similarly, ANS binding occurred during the 4 millisecond dead-time of the fluorescence stopped-flow [37]. We coined the name of ‘‘pre-molten globule’’ to this non-native like, collapsed intermediate, which we further characterized by NMR and infrared spectroscopy in two friendly collaborations, one with Inaki Guijarro and Muriel Delepierre in the NMR unit at the Institut Pasteur, the other with Michael Jackson and Henry Mantsch at the Institute for Biodiagnostics of the National Research Council in Winnipeg (Canada). Finally, together with Alain Chaffotte and colleagues from the Max Delbru ¨ ck Center for Molecular Medicine in Berlin (Germany) we showed that in this

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pre-molten globule, the secondary structure and the collapse are completely uncoupled, demonstrating that, unlike the molten globule, its secondary structure is not stabilized by specific hydrophobic contacts [38]. This pre-molten globule, formed in less than 4 milliseconds, was the most upstream folding intermediate accessible to the experimental techniques then available. This was the end of our studies on the folding pathway(s) of tryptophan synthase. From our long walk along the folding pathway of the tryptophan synthase b2 subunit, we brought a good crop of original results that were of interest for protein folding in general, some of which went clearly against the dogma generally accepted in the scientific community. Though Oleg Ptitsyn later used this name and popularized the corresponding concept, we were the first to identify, coin the name onto, and characterize the pre-molten globule. We showed that non-native secondary structures are formed at very early stages of the folding process. We challenged the widely accepted view that some far UV CD and ANS transient binding are enough to identify a molten globule. We introduced the concept of independently folding regions (globules or domains) in large polypeptide chains. We showed that the folding, rather than being a two state process, involves several identifiable intermediates and demonstrated that a given protein can follow alternate folding pathways. But that was not all. In parallel with our physical-chemical studies based essentially on absorption, fluorescence, circular dichroism, infrared and NMR spectroscopy, stopped-flow, calorimetry, and analytical centrifugation, we developed a whole strategy aimed at studying some specific aspects of the b2 protein folding by means of monoclonal antibodies.

Probing the Conformation of Proteins and Folding Intermediates with Monoclonal Antibodies From now on, the reader will certainly find it more difficult to follow the chronology of the events I am going to narrate. Indeed, in order to outline the scientific context as well as the logics underlying the popperian succession of hypothesis, experiments and conclusions that oriented my research, I have deliberately


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chosen to report my activities by theme rather than in a purely chronological order. So, let’s go back in time y As mentioned above, Carlos Zetina had observed that the assembly of the independently folded F1 and F2 fragments was FOLLOWED in time by an isomerization step [29]. Except for the ability of the reconstituted nicked protein to elicit the fluorescence of the aqua complex in the presence of L-serine and PLP, we could find no signal that would give us any information on the nature of this isomerization. Was it a change in the structure of one, or of the two fragments, or did the folded fragments behave as solid bodies, in which case the isomerization would correspond to a rearrangement of solid bodies within the oligomer. While on duty as the Scientific Director of the Institut Pasteur (see below) I had been in charge of setting up a new Department of Immunology (which meant designing a building and hiring new research teams), I became aware of the recent discovery of monoclonal antibodies by Kohler and Milstein [39]. I thought of using monoclonal antibodies (mAbs) specific of native b2 to probe the conformations of F1 and F2 either in isolation or assembled within the nicked protein. To do that, one ‘‘just’’ had to determine the affinities of a panel of mAbs for the isolated fragments and for the native whole protein. It seemed extremely simple and straightforward. But it was the beginning of a real saga, which lasted for two decades and taught us at least as much about mAbs as it did about the folding of b2. The persons who really led this project were Lisa DjavadiOhaniance and her student Bertrand Friguet, now a Professor at the University of Paris. Lisa had fled Iran to escape the turmoil of the Islamic Revolution and joined the Institut Pasteur during the autumn of 1979. She originally came to my laboratory for a few months, but the fit between us was so good that she decided to stay, and I asked her to take the leadership of the mAb project. I shall not dwell too long on this project, and just describe our main findings. Lisa isolated some 40 independent clones that secreted mAbs recognizing native b2 coated on ELISA (Enzyme Linked Immuno Sorbent Assay) plates. Together with Bertrand Friguet, she started characterizing these mAbs. Their first unexpected observation was that, though the protein used to immunize the mice and to coat the ELISA plates for the screenings was nicely purified native b2, only about half of the mAbs recognized the native protein in solution while the other half recognized only

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unfolded b2. This was due to the fact that coating the globular protein onto the ELISA plate, at the interface between the plastic of the plate and the solvent, results in its partial denaturation, thus leading to ‘‘false positives’’ during the screening of the mAbs. This observation gave at last a reasonable interpretation for several odd reports according to which mAbs specific of epitopes that are buried in the core of globular proteins still could react with the ‘‘native’’ protein. It also led us to develop a rapid, convenient, ELISA-based test to discriminate mAbs that bind to native proteins from those who bind to unfolded proteins. A second unexpected finding was that a perfectly monoclonal hybridoma could secrete heterogeneous antibodies. Indeed, we showed that one of our hybridoma resulted from the fusion between a non secreting lymphoma and two distinct secreting lymphocytes, thus producing antibodies made by the random combination of distinct heavy chains and light chains. In order to analyze with thermodynamic rigor the antigen– antibody association, we developed a series of ‘‘competition’’ ELISA-based tests. Indeed, the ELISA tests that were routinely used to measure the affinity between a mAb and its protein antigen were made with antigen immobilized on the ELISA plate. They had the merit of being very convenient, but we found them to be irreproducible and to give grossly false results, essentially because the same laws as in solution do not rule the binding equilibrium at the solid/liquid interface. We therefore developed a new ELISA-based competition test that gave the real affinity constant in solution and could be used with minute amounts of unpurified antigen or mAb. This test has since become extremely popular and the paper describing it is by far the most frequently cited of all my publications. We also developed ELISA-based competition tests to monitor the kinetics of association and of dissociation of protein–antibody complexes in solution. Once these tests were developed, several mAbs specific of native b2 that can bind simultaneously to the protein, and thus recognize distinct epitopes scattered on the protein surface, were used to study various aspects of the folding of b2 and its fragments. Their affinities for the isolated F1 and F2 fragments were significantly lower than for the nicked protein. This showed that both the F1 and F2 fragment undergo a global conformational rearrangement upon associating to one another. Furthermore, the ratios of the affinities of the mAbs for the nicked protein and for the isolated


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fragment it recognizes were not the same for the various antibodies, indicating that the change in conformation undergone by the fragments is not a shift in equilibrium between an unreactive and an immunoreactive form. Using small angle X-ray scattering with the help of Ingrid Pilz in the University of Gratz (Austria), we could localize the epitopes recognized by an anti-F1 and an anti-F2 domain, thus enabling us to determine the relative positions of the domains in the native protein (this was, of course, before the X-ray structure was solved). We also used some of these mAbs to identify and characterize several early immunoreactive intermediates during the folding of b2 (see above). This enabled us to demonstrate that different epitopes appear at different rates during the folding process. We thought that, because of their high specificity and high affinity for the native conformation, mAbs could be a powerful tool to investigate the folding of nascent polypeptide chains during their biosynthesis on the ribosome. Let me briefly tell the story of one of the most challenging series of experiments, and certainly the most disappointing one, we ever undertook in my laboratory. Sylvie Blond and Anne Murry-Brelier had characterized a monoclonal antibody, mAb19, that had the following characteristics: when the renaturation of unfolded b chains was triggered by dilution into a folding buffer containing mAb19, the antibody did not bind to the antigen until a folding intermediate was formed. The affinity of mAb19 for this folding intermediate was close to that of mAb19 for the native protein. From this we concluded that the antibody detected a folding step leading to the appearance of a native-like epitope on the protein surface. Furthermore, the cognate epitope had been shown to be carried by F2, that is to be localized on the N-terminal third of the b-chain. We therefore decided to use this antibody in a cell-free protein biosynthesis system to find out whether or not the N-terminal domain of the b polypeptide chain could fold on the ribosome during chain elongation. To do that, we invited Alexei Fedorov from the laboratory of Alexander Spirin in Pushchino (Russia) – a pioneer and world leader in cell-free protein biosynthesis – to spend a few months at the Institut Pasteur and help us launch the project. He prepared the extracts for synthesis and engineered the proper DNA templates. Together with Bertrand Friguet, Alexei, Bertrand first showed that peptides of a size larger than about 100 residues, still bound to the ribosomes

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are immunoreactive to mAb19, indicating that the epitope for mAb19 is present on the ribosome-bound nascent polypeptide chain. Using a bi-dimensional radioactivity scanner with high sensitivity and low background to detect low energy b-emitting isotopes developed for us by Georges Charpak – a Nobel laureate in Physics – we showed that these nascent chains had the same affinity for mAb19 as the complete native protein in solution, indicating that the native-like epitope is spontaneously present on the nascent chain, and does not result from a mAbinduced conformation [40]. But the experimental approach used did not enable us to decide whether or not the folding was co translational. Indeed, the procedure used to detect the immunoreactive species involved several incubation and separation steps that lasted much longer than the synthesis of the polypeptide chain. Bertrand Friguet and a brilliant post-doctoral fellow, Kostas Tokatlidis, therefore designed a fast immunochemical pulse-labeling method whose time resolution was much shorter than the chain elongation time. By pulse-labeling the nascent chains during their elongation on the ribosomes, isolating the ribosomes by ultra-fast centrifugation in an Airfuge to get rid of chains released from the ribosomes, trapping the mAb-bound nascent chains with anti-IgG beads and analyzing the length of the immunoreactive ribosome-bound chains by SDS polyacryl amide gel, we could establish the kinetics of appearance of immunoreactive chains of a given length and show that, within the time resolution of the pulse, it coincided with the kinetics of appearance of chains of that length. These results clearly demonstrated that the appearance of the immunoreactivity was co translational. For the first time, one was able to clearly establish that the folding of a polypeptide chain longer than some 50 residues occurs on the ribosome and is indeed co translational. We immediately wrote a manuscript and submitted it to the Journal of Molecular Biology. The novelty of our experimental approach and the importance of our conclusion made it that the manuscript was quickly accepted. On the very same day when I received the printed proofs, a devastating result was finally obtained. For months, we had tried hard to define the residues involved in the epitope recognized by mAb19. On that day, it became clear that the epitope was entirely comprised in a short sequence of eight residues carried by the N-terminal end of the b chain which, in the isolated b2 subunit, is highly susceptible to


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proteases and therefore presumably unstructured and exposed to the solvent. Indeed, the corresponding synthetic octapeptide and the native b2 protein had the same affinity for mAb19. Thus, as opposed to our previous belief, mAb19 was not a conformation dependent mAb. We later demonstrated that the ‘‘folding’’ step leading to the first immunoreactive folding intermediate that we had observed could be interpreted as follows: during an extremely rapid initial collapse, the N-terminal end of b gets transiently buried in the hydrophobic core of the collapsed chain and is therefore inaccessible to the antibody. In a second step, the chain condenses further, thus expelling its N-terminal end out of the core and exposing the epitope to the solvent and to the mAb. While this finding did not invalidate our earlier conclusions on the folding of the b2 protein in vitro – mAb19 had indeed been able to trace an early folding event – it devastated our work on the immunoreactivity of nascent chains since mAb19 was unable to distinguish the conformations of the fully unfolded and the native chain. All this superb (technically) work had been done in vain! My sole comfort was that we could withdraw our manuscript before it was published. This was a terrible blow for Kostas Tokatlidis at this very early stage of his career. He had left the laboratory after one year of very hard and passionate work, and had no publication. Yet, he accepted without discussion my decision to withdraw the manuscript. This was a brave, honest, and noble attitude, for which I still admire him and am grateful to him. Fortunately, he was much more lucky with his other postdocs and is now a Professor at the University of Crete in Heraklion. Our disastrous attempt to use mAbs as probes for the folding of nascent chains marked the end of our studies on the folding of tryptophan synthase. I had learnt the lesson: first get an undisputable proof that a mAb is conformation dependent before using it as a conformation probe! In addition to using mAbs for detecting and characterizing protein folding intermediates, we showed that mAbs could also be used to investigate the conformational dynamics of a native protein. Thus, by monitoring the effects of ligands of b2 on the affinities of mAbs for the native protein, we showed that several of the ligands induce a change in the enzyme conformation. By looking at the spectral properties of b2 in the presence of various ligands and of the mAbs, we observed that different mAbs could freeze the conformation of the protein at different stages of the

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catalytic cycle. We showed that mAbs recognizing the unfolded protein but not the native one can be used to quantitatively analyze the kinetics and equilibrium of ‘‘breathing’’ (transient unfolding) of the protein and study the effects of specific ligands on the breathing. Finally, we clarified some general questions related to the association of an antibody to a protein antigen. One was to demonstrate that, in some instances, the binding of monoclonal antibody to a protein antigen could induce a conformational change of the antigen. While this had been sometimes envisaged, we brought what I think was the first direct kinetic evidence showing that a change in conformation of the antigen was triggered by, and occurred after, the binding of the mAb to the antigen. Using polypeptide chains of various lengths that carry the epitope for a given mAb and show different affinities for that mAb (the longer the chain, the closer the affinity to that of the native protein), we could demonstrate that the association rate constants of the different forms of the antigen did not change significantly, while the dissociation rate constants increased with decreasing size and affinity. This demonstrated that the affinity of the mAb for the antigen was essentially controlled by the dissociation rate constant of the antigen–antibody complex. Moreover, we showed that, while large, conformationally stable antigens bind according to a simple molecular mechanism (the affinity is equal to the ratio of the on and off rate constants), this is no longer true for small peptides, for which a conformational adaptation seemed involved. These conclusions were reached for all the mAbs we investigated. We wrote a manuscript on these results and submitted it to the EMBO Journal who tried to widen the scope of its publications and had just asked EMBO members to submit papers on topics related to protein structure, immunology, enzymology, etc. During 3 months, we got no answer. I sent a message to the editor who answered that one referee had not yet sent his report. More than 1 month later, there still was no answer. I sent a new, pressing message to the editor. After one more month or so, the paper was rejected with a one line comment from the reviewer: this paper has little interest and no originality since similar results have already been published. I knew, and had quoted in our manuscript, a related work showing that the affinity of small haptens for antibodies was controlled by the dissociation rate constant. But nothing similar had been published, as far as I knew, for a large


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protein. I wrote a letter to John Tooze, the editor who had been in charge of our paper. I complained about the long time it had taken to obtain such a short and non-constructive comment from the reviewer. I did not challenge his decision but asked John Tooze to get from the reviewer the references of the papers describing ‘‘similar results’’ that had been published previously according to the reviewer’s comment. I never got the answer to my query. This discouraged me from publishing my research in the EMBO Journal, and I never again submitted a manuscript to this journal. Our work on the dissociation-controlled affinity ended up in Research in Immunology, one of the publications of the Institut Pasteur [41]. In view of the number of reprint requests I got from well-known immunochemists, I understood that our work had more interest and relevance than the EMBO Journal’s reviewer had thought. Thus, our incursion into the world of monoclonal antibodies had been much longer, but by far much more productive than originally thought. Not only had we been able to reach our initial goal (the characterization of the isomerization undergone by F1 and F2 upon their association) and to identify and characterize early immunoreactive intermediates on the folding pathway of tryptophan synthase. We also developed original, widely used methods to determine the affinity and the association and dissociation rate constants of the immune complex. We improved the screening procedures for obtaining mAbs that recognize the native antigen. We improved our understanding of the molecular mechanisms involved in the binding of a mAb to a protein antigen. We showed how mAbs could be used to investigate the conformational dynamics of a protein in its native state as well as during its catalytic cycle. And though our attempts to investigate co translational folding were unsuccessful, they led us to design immunochemical labeling techniques that we later used, together with more classical methods, to characterize the folding pathway of proteins other than tryptophan synthase (Figure 4).

Analogies between the Folding Mechanisms of Tryptophan Synthase and other Proteins I related earlier how the discussions I had with Oleg Ptitsyn and Roger Pain in 1988 had led me to collaborate with two French

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Fig. 4. Michael Sela (co-worker of C. Anfinsen in the fundamental RNase renaturation experiments, former President of the Weizmann Institute, former chairman of the Pasteur-Weizmann Scientific Council – see Ref. [63]), while in sabbatical at the Institut Pasteur, delivers a speech during a party organized as a surprise for the author’s 60th birthday. From left to right: Michel Goldberg, Daniel Ladant, Mariangela Conconi, Yves Gogue´, Yvonne Guilloux, Michael Sela, and Lisa Dajavadi.

companies on the design and setting up of a circular dichroism stopped-flow with a dead-time of 4 milliseconds. This machine enabled us to look at the formation of secondary structure in very early folding intermediates. When the CD stopped-flow was assembled in our laboratory, in 1991, my co-worker Alain Chaffotte and I started testing its capabilities. To do that, we decided to use as a test protein hen egg white lysozyme (HEWL). This choice was based on several reasons. I had become familiar with HEWL renaturation during a half-sabbatical year spent in Regensburg (Germany) a year earlier. It was a cheap protein, commercially available in grams, and its folding properties had already been extensively studied. Fluorescence stopped-flow investigations had shown rapid phases, the rate constants of which fell perfectly within the range expected for the molten globule appearance and disappearance. Finally, HEWL showed very strong sequence and 3D-structure homologies with human a-lactalbumin, a protein for which good experimental evidence


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showed the presence of an authentic molten globule as an intermediate in the folding/unfolding transition at equilibrium. The refolding kinetics of HEWL with its disulfide bonds intact were thus investigated by a 50–60-fold dilution of guanidine unfolded, non-reduced HEWL into guanidine-free buffer, monitoring the regain of ellipticity at 222 nm (to detect secondary structure formation) and at 280 nm (to detect the formation of the tight packing of side chains in the core of the native molecule). In addition to two kinetic phases observed both in the near and far UV, that had already been reported using other methods, the far UV CD signal showed a large ‘‘burst’’ of secondary structure, occurring during the dead time of the stopped-flow. In order to better characterize the amplitude of this burst, we introduced a continuous-flow phase of injection in the mixing protocol and analyzed the CD signal during the flow. This enabled us to average over 500 milliseconds the far UV CD signal, thus improving considerably the signal-to-noise ratio. Repeating this experiment at different wavelengths enabled us to construct the CD spectrum of the 4 milliseconds intermediate between 214 and 240 nm. Analyzing the evolution of the spectrum during the rest of the folding reaction indicated that all further changes in the far UV CD spectrum could be interpreted as contributions from disulfides becoming distorted, which suggested that the secondary structure did not evolve significantly after the first 4 milliseconds of folding. This conclusion was further supported by the excellent fit between the far UV CD spectrum of the burst intermediate and that calculated from the known secondary structure of native lysozyme. We therefore concluded that, during the refolding of non-reduced lysozyme, the native secondary structure is formed during the burst phase, that is in less than 4 milliseconds [42]. The publication of this piece of work had an immediate impact. It brought a lot of support to the molten globule hypothesis since, for the first time, we could prove that a kinetic intermediate had a native-like secondary structure. But it also marked us out, for a few years, as leaders in the use of CD stopped-flow, which triggered a large number of collaborations that I shall briefly enumerate later. In spite of the impact of our finding on the secondary structure of the HEWL burst intermediate, I was a bit skeptical about it real significance in terms of protein folding. Indeed, I always considered that studies on the folding of proteins that contain

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native disulfide bonds in the so-called ‘‘unfolded’’ state were biased. Because, from a purely thermodynamic point of view, the native disulfides introduce a considerable reduction in the entropy of the polypeptide chain. And because the folding of the protein in vivo starts from the reduced state. As Chris Anfinsen did in his pioneering experiments on the refolding of ribonuclease, I considered that the relevant studies should be those on the renaturation of reduced polypeptide chains. I therefore thought it important to test whether or not reduced HEWL would be able to rapidly recover its native secondary structure in the same way as the oxidized protein. Simple stopped-flow experiments could however not be envisaged, since reduced HEWL heavily aggregates when diluted into a guanidine-free buffer at the high protein concentrations used in a stopped-flow. To circumvent this problem, I used the continuous-flow protocol we had designed for oxidized HEWL, but the injection of protein was immediately followed by an extensive injection of buffer. This washed out the protein that never stopped in the machine. The aggregates would thus form in the waste and neither interfere with the observation of the sample nor clog the stopped-flow. This enabled us to record the far UV CD of reduced HEWL 4 milliseconds after its dilution in denaturant-free buffer. The result was unambiguous. No secondary structure formation could be detected at this early stage of the folding. The native disulfides thus appeared to have a crucial role in either speeding up the formation of the secondary structure, or in stabilizing it. This initiated a series of experiments aimed at understanding in details the coupling between disulfide and secondary formation in HEWL. For years we tried to collaborate with colleagues who had prepared HEWL variants lacking one of the four native disulfides, but failed to convince them of providing either the strains, or extracts, or purified protein to find out which SS bonds were essential for the burst of secondary structure. I convinced Vale´rie Guez, a post-doc in my laboratory, to construct the three SS variants of lysozymes, each having the two cysteins of one of the disulfides replaced with alanines. She then expressed the variant proteins, purified them, characterized them in terms of spectral properties and activity, and studied their folding kinetics in the stopped-flow using intrinsic fluorescence, ANS binding and far UV CD. No individual disulfide bond turned out to be


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indispensable for the rapid formation of the native-like secondary structure. But their individual roles in controlling the stability of a trapped intermediate could be assigned. Besides our continuous-flow studies of the very early stages of reduced HEWL folding, we also studied the course of its oxidative renaturation, monitoring the kinetics of regain of the intrinsic fluorescence, the far UV CD, the number of disulfide bonds (in an exciting collaboration with Margherita Ruoppolo in Naples) and the immunoreactivity for two conformation dependent monoclonal antibodies recognizing discontinuous epitopes located one on the a-domain and the other on the b-domain of HEWL. To analyze the immunoreactivity of folding intermediates Nicole Jarrett, a young undergraduate student from the University of Arizona, used the pulsed immunochemical labeling method we had developed previously for our studies on the co translational folding. From the results we obtained, we could conclude that the a-domain folds first, together with the formation of the two intraa-domain disulfides, followed by the b-domain which folds together with the intra-b-domain disulfide. Finally, the structure reaches its fully closed structure and its full activity with the formation of the inter-domain disulfide [43]. With this set of observations, we demonstrated that the order in which the domains fold during the oxidative folding was the same as for the folding of the oxidized protein, suggesting that the relative rates of folding of the domain are indeed directed by the polypeptide chain itself, not by the disulfides. We also brought solid evidence in support of the role of ‘‘disulfide secure species’’ in the folding of disulfide containing proteins as proposed by Welker et al. [44]. The expertise we had gained with Alain Chaffotte on the use of our stopped-flow machines, particularly with respect to far UV ellipticity fast kinetics, as well as with Lisa Djavadi and Bertrand Friguet in the use of monoclonal antibodies as conformational probes, attracted to our laboratory many scientists who offered us to collaborate on investigating their own ‘‘model protein.’’. With Heinrich Roder (University of Pennsylvania, Philadelphia, USA) we studied the early steps of secondary structure formation during the folding of horse ferricytochrome c, showed that a ‘‘burst’’ intermediate is formed within the 4 millisecond dead time of the stopped-flow. This intermediate has a very substantial amount of secondary structure, as evidenced by a far UV ellipticity nearly half that of the native protein. Yet, like the

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isolated F2 domain, or like the burst intermediate of lysozyme, it shows no protection of amide protons against exchange with the solvent. Such burst intermediates, observed in three different proteins, thus appear as partly condensed structures (essentially a-helices for HEWL and cytochrome c) with a fluctuating core, a significant amount of secondary structure, but no stable hydrogen bonds [45]. The contradiction between the existence of secondary structure and the lack of proton protection was later solved by our work on the structure of the isolated F2 fragment demonstrating that amide protons are in fact slightly protected against exchange, with protection factors too low to be detected by the pulsed proton exchange method used in rapid kinetics studies, but high enough to provide some stability to secondary structure elements [46]. With Roxana Georgescu and Maria-Luisa Tasayco (City College of the City University of New York, USA) we investigated the kinetics of assembly of two disordered complementing fragments of E. coli thioredoxin with the aim of understanding the mechanism of recognition between disordered states. We also showed that the intact thioredoxin polypeptide chain folds along heterogeneous pathways, and we identified and characterized an early intermediate that accumulates on one of the pathways. Myriam Ziegler and Thomas Baldwin (Texas A&M University, College Station, USA) spent a sabbatical year with us in 1991–1992, investigating the folding pathway of the heterodimeric bacterial luciferase, identifying folding intermediates both before and after the assembly step. They also demonstrated that the formation of the ab heterodimer is a kinetic trap on the folding pathway of the individual subunits [47]. Jonatan King (MIT, Cambridge, USA) also spent a sabbatical with us, mainly interested in monoclonal antibodies. Lisa Djavadi prepared and cloned a series of hybridoma secreting mAbs against the homotrimeric tail spike endorhamnosidase from bacteriophage P22. Together with Jon and with Cameron Haase-Pettingel in Jon’s laboratory, they selected several of these mAbs for their ability to differentially recognize the wild type protein and different variant proteins blocked at different stages of the protein folding or assembly. Bertand Friguet then spent a summer in Jon’s laboratory and used these mAbs to demonstrate the existence of ribosome-bound tail spike protein folding intermediates [48].


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We came across several other proteins or peptides on which we worked either in collaboration with other groups, or within my laboratory. It would be boring and of little use to describe all of them. There is however one relatively recent set of experiments on protein folding and assembly that I wish to briefly report. It deals with the homotetrameric dihydrofolate reductase (DHFR) encoded by the R67 plasmid that confers trimethoprim resistance to E. coli. This study was initiated in my group by a young and brilliant scientist, Arnaud Blondel, who had been trained for his PhD in molecular biology by Hugues Bedouelle, an independent worker in my unit at the Institut Pasteur. Arnaud had shown a strong interest for protein modeling. I had helped him arrange a post-doctoral visit with Martin Karplus at Harvard, then in Strasbourg, and he got an excellent training in the use of molecular dynamics for predicting structural and thermodynamic properties of proteins. I did my best to attract Arnaud back to the Institut Pasteur. After several attempts, we finally managed to obtain a position for him with the idea of starting a group on protein modeling in our institute. Arnaud came to my lab, rapidly set up the computer facility he needed, and started investigating the effect of single point mutations on the pH dependent dimer– tetramer equilibrium of R67 DHFR. The idea was to predict the changes in the free energy of association caused by the mutation, and compare it to the experimental value determined from equilibrium constants measurements. This would allow testing the quality of the prediction algorithm and hopefully improving the quality of the various components of the force field introduced in the algorithm. Arnaud and his student Julie Dam constructed a whole set of point mutants with amino acid replacements located in the dimer–dimer interface. They showed that none of these mutants was able to form tetramers anymore. Indeed, because of the symmetry of the protein, each mutation disrupts four contacts in the dimer–dimer interface, which results in a dramatic decrease of the association energy. They however observed that pairs of different dimeric mutants could complement and form tetramers. They developed a very clever procedure to determine the association–dissociation equilibrium constant of tightly associated complexes by iso-fluorescence titration, and determined the dissociation constants of several of these heterotetramers [49]. In parallel, Arnaud Blondel developed a powerful algorithm to predict the effect of the mutations on the association

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energy and, by comparing the predicted value with the experimentally determined ones, he showed that the algorithm must allow for conformational changes of the polypeptide chain near the mutation site. Including these local relaxations in the prediction procedure resulted in an excellent agreement between prediction and experimental measurements [50]. In parallel, Annick Me´jean and her student in the laboratory studied the kinetics of association of the dimers and of dissociation of the tetramer induced by pH jumps in the stopped-flow and showed how the protonation of histidines in the interface in the tetramer is responsible for triggering its dissociation, while association involves only unprotonated dimers. They also characterized the folding pathway of the protein, identifying the dimerization and tetramerization steps and focusing on how the oligomerization steps are coordinated with the formation of secondary and tertiary structure all along the folding pathway. This study was of particular interest because the polypeptide chain of R67 DHFR is among the shortest ones (it contains only 78 residues) that have been reported to give rise to a globular conformation. It is noteworthy that, in spite of its very small size and the absence of any stabilizing disulfide bond, DHFR shows a burst of secondary structure with about 50% of the native ellipticity at 222 nm already present within the first 4 milliseconds of folding. Taken together, the kinetic studies to which we contributed involved a range of proteins with widely different structural properties. Their polypeptide chain lengths varied from 78 (DHFR) to over 1,000 (b-galactosidase) residues. Some were monomeric (HEWL, cytochrome c, thioredoxin), others were homo-oligomers (DHFR, b2, b-galactosidase), or hetero-oligomers (luciferase). Some had a very high a-helical content (cytochrome c) others were very rich in b-structure (thioredoxin). Some had internal disulfide bonds (HEWL), the others not. Some were from bacteria, others from mammalian origin. Yet we observed that they share several common features in their folding mechanisms, in particular at early stages of the folding process. In this respect, I consider that by reporting the presence of ‘‘pre-molten globules’’ at early stages of the folding of a variety of proteins, by contributing to break the dogma of the unicity of the folding pathway and by showing the plasticity of folding intermediates even at advanced stages of the folding process, we have participated significantly in narrowing the gap between the


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experimentalists’ and the theoricians’ understanding of protein folding, thus contributing to the ‘‘New View’’ of protein folding that emerged in the early 1990s [51].

From Aggregates to Prions: Back to Early Concerns All along our studies on protein folding in vitro, we have been facing the problem of aggregation. This had started with my very early studies on the renaturation of urea unfolded b-galactosidase and the discovery of the ‘‘4 M urea trough’’ which I had described in my MSc thesis. I also faced it with the renaturation of the b2 subunit of tryptophan synthase, which could be efficiently achieved only at low temperatures and rather low protein concentrations. While I was at Stanford, Agne`s Ullmann took over my finding on the 4 M urea trough, studied in more details the renaturation yield as a function of the intermediate urea concentration in the two-step renaturation procedure and found that the concentration range leading to loss of activity was very narrow. She also discovered that, though both manganese and magnesium ions are required for galactosidase to be active and bind strongly to the native protein, their presence in the renaturation buffer was highly deleterious for the renaturation. She reported this finding in an elegant paper she wrote with Monsieur Monod [52]. Several statements made in this paper are worth mentioning. ‘‘y two entirely different pathways may be followed by a solution of denatured protein during removal of the denaturing agent.’’ Or ‘‘y interactions between chains may occur leading to an inactive precipitate.’’ Or, discussing the two possible outcomes of the removal of the denaturant, that is the native and aggregated states, ‘‘One of these two states should, presumably, be considered as metastable with respect to the other. But it seems difficult to decide which of the two constitute a kinetic trap.’’ In 1969, these were visionary statements. They had however no impact on the community of protein folders. While the facts were not discussed, the conclusions were considered irrelevant by most scientists who considered aggregates of no interest and the effect of divalent ions a trivial artifact due to the formation of non-specific chelates involving the divalent ions and negatively charged side chains of the unfolded protein. I did not share this opinion, having been involved in the initial

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experimentation and in many discussions with Agne`s and Monsieur Monod that led to their conclusions. But when I again came across aggregation during the renaturation of two other proteins, E. coli tryptophanase and chymotrypsinogen, I realized that this general behavior may teach us something about the interactions involved in the folding process, and I decided to further investigate the formation of aggregates during protein folding. Tryptophanase was an important subject in our laboratory for many years, and my two first PhD students, Jacqueline London (then a very young assistant, now a full Professor at the Paris University) and Olivier Raibaud (who unfortunately was taken away by leukemia at a young age) worked on it extensively. Again, Monsieur Monod was responsible for introducing this enzyme in the laboratory. He once showed me a paper reporting that tryptophanase was an octamer made of identical polypeptide chains, but behaved functionally as a tetramer. To his knowledge, such ‘‘half of the sites reactivity’’ had not been reported previously and, immersed as he was in the symmetry in oligomers, he asked me to try and find out what was the structural basis of the differentiation between ‘‘active’’ and ‘‘inactive’’ subunits. After extensive purification of the enzyme, Jacqueline and I used analytical centrifugation as well as the first SDS polyacrylamide gels ever performed at the Institut Pasteur to determine the molecular weight of the tryptophanase chain. It turned out to be twice that estimated previously by others. Tryptophanase thus returned to the classical status of a structurally and functionally tetrameric protein. Olivier Raibaud, reluctant to pursue his studies on globules in rabbit muscle phosphorylase, turned to tryptophanase and made a number of most interesting observations. He found that, while the binding of PLP to the apo-enzyme showed no cooperativity whatsoever at equilibrium, the binding kinetics were biphasic, with the two first coenzyme molecules binding about 10-fold more rapidly than the two last molecules. He demonstrated that the association of two dimers into a tetrameric enzyme confers to the protein three important properties: a large increase in heat stability, a good protease resistance, and activity. These properties justified that the tetramer had been selected during evolution. He showed that the binding of two or three coenzyme molecules per tetramer were enough to confer to the unsaturated protomers the heat stability and active conformation


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of the holo-protomers. He finally demonstrated that the tetramerization step must occur first in order for the polypeptide chain to be able to reach its native conformation, thus introducing the concept of ‘‘conformational feedback’’ at a time when, under the influence of the crystallographers, folded proteins like the tryptophanase dimer were considered as solid blocks. But the most striking observation we made resulted from studies by Jacqueline London on tryptophanase renaturation. She first noted that the yield in active protein was strongly dependent on the protein concentration during the renaturation, both in direct dilution and in dialysis experiments. Decent yields of around 30–50% could be reached only at protein concentrations below about 50 mg/ml. She then repeated on tryptophanase the ‘‘two-step’’ renaturation studies that had uncovered the 4 M urea trough with galactosidase. She found a similar behavior, though at a lower urea concentration, with tryptophanase. She also observed that the higher the protein concentration during incubation at intermediate urea concentrations, the wider and deeper the urea trough. The concentration dependence of the yield suggested that intermolecular interactions were involved in the formation of the inactive species, which we demonstrated by showing that, while all the activity recovered was contained in native tetramers, all the inactive protein ended up in aggregates. What was the nature of these intermolecular interactions? We showed that they were not covalent (S–S) bonds, that they were fully reversible in the presence of denaturant, and that the redissolved protein could be renatured again with good yield at low protein concentration. Unlike the experiments reported by Ullmann and Monod [52], these refolding studies were performed in the absence of metal ions, which excluded the presence of metal chelates. I thus thought that stereospecific interactions between partly folded intermediates might be involved in the formation of aggregates. To test the specificity of the illegitimate interchain interactions responsible for the aggregates, I asked Jacqueline to test the influence of the presence of folded or unfolded ‘‘foreign’’ proteins (i.e. proteins other than tryptophanase) in the folding buffer. The result was clear-cut: neither serum albumin at 1/2 mg/ml, nor 2 mg/ml of a crude extract of E. coli uninduced for tryptophanase had any effect on the renaturation, while tryptophanase at a concentration as low as 250 mg/ml already failed to refold efficiently. The illegitimate interchain interactions thus appeared to be specific

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[53]. To account for this phenomenon, we proposed a model according to which specific interactions between folded globules can form either between two complementary globules belonging to the same chain, thus leading to native protein, or between globules belonging to different chains, thus leading to aggregates via a globule exchange mechanism. We later used this model to account for the formation of aggregates upon incubation of the b2 subunit of tryptophan synthase at guanidine concentrations close to the transition zone [54]. Though we had not coined a name to this model, we had introduced – as a natural extension of our globule model – the concept known nowadays as ‘‘domain swapping.’’ At about the same time, another PhD student, Gilbert Orsini, had joined my group to study the effects of disulfide bonds on the renaturation of proteins, a subject that haunted me for years. Because Gilbert was familiar with chymotrypsin and chymotrypsinogen (CTG), on which he had been working for his MSc, he tried to find optimal conditions for the oxidative renaturation of unfolded and reduced chymotrypsinogen. At first, and in spite of numerous tedious attempts, he could recover no native CTG as judged by its ability to be activated into chymotrypsin. Furthermore, all the protein was recovered as aggregates. Gilbert thought of trying to avoid aggregation by rendering the polypeptide chain more soluble. A biochemical trick that had been reported to efficiently increase the solubility was to acylate the protein’s lysyl amino groups with N-carboxyanhydrides of D- and L-alanine, resulting in a poly-D-L-alanylated protein [55]. Gilbert could indeed renature polyalanyl-CTG to a low, but measurable extent. When trying to optimize the renaturation of reduced unfolded polyalanyl-CTG, he found that moderate, sub denaturing concentrations of a denaturant like urea or guanidine greatly improved the efficiency of the oxidative renaturation. Gilbert then looked at the oxidative renaturation of reduced CTG in the presence of various concentrations of guanidine to see whether the solubilizing effect of the denaturant might suffice to prevent aggregation. He found that, in the presence of 1.2 M guanidine, over 50% of the protein could be recovered as activable zymogen. The observed renaturing efficiency, that is the ratio of activatable CTG over aggregated protein, strongly decreased with increasing CTG concentrations in the renaturation mixture [56]. These two observations (effect of sub denaturing concentrations of


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denaturant and inhibition of renaturation by high protein concentrations) had very important consequences. One was to make me aware that the decrease in renaturation yields with increasing protein concentrations during the folding, that Agne`s Ullmann and Monsieur Monod had reported for galactosidase and that I had encountered with tryptophanase, was a general phenomenon, even if the concentration range where this effect becomes crucial depends on the protein. It is observed at very low protein concentrations for reduced CTG (sub-mg/ml), at medium tryptophanase concentrations (5–100 mg/ml) and high galactosidase concentrations (0.1–1 mg/ml). Based on these observations, we proposed that the outcome of the renaturation process is controlled by a competition between two pathways with different kinetic properties: one leading to the native protein, involves only first order intramolecular isomerization reactions, and thus is kinetically independent of the protein concentration. The other pathway, leading to aggregates, involves intermolecular, higher order association reactions and is therefore favored at high protein concentrations. This ‘‘kinetic competition’’ model [56] contained all the ingredients of the ‘‘kinetic partitioning’’ model, later rediscovered by others and now widely used to interpret the mechanisms by which chaperones help protein fold by preventing their aggregation. The second consequence of our observations on the competition between folding and aggregation was in the area of biotechnology, when the first successes of genetic engineering ended up in the disappointing finding that most proteins expressed at high levels in E. coli were recovered as ‘‘inclusion bodies’’ – that is highly insoluble, inactive aggregates. Alain Rambach, a young and brilliant geneticist trained at the Institut Pasteur with Franc- ois Jacob, was to my knowledge the first French scientist to become aware of the revolution that genetic engineering was about to bring about. After trying in vain to convince the Direction of the Institut Pasteur (including myself) to get involved in industrial projects based on this novel technology, he launched a joint venture with the powerful French industrial group RhoˆnePoulenc. Together, they created a biotechnology company, GENETICA. Their two first projects involved the cloning and expression of two human proteins, human serum albumin (HSA) to be used in the production of artificial human serum, and tissuespecific plasminogen activator (tPA) to be used in the treatment

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of heart infarct. Quite rapidly, they cloned the two genes and got very high levels of expression of these proteins. However, in both cases, the protein was found in inclusion bodies. They worked for quite a time trying to avoid aggregation. To no effect however, until Alain Rambach, who had worked for years in the same department as me and was aware of my research, came to me asking whether I thought such inclusion bodies could be converted into native proteins. Based on our observations that aggregated proteins formed in vitro could be rescued after solubilization in urea or guanidine, I was confident that similar procedures should be able to rescue proteins from inclusion bodies. Moreover, the kinetic competition model and the favorable effect on renaturation of sub denaturing urea or guanidine concentrations were providing a solid basis to imagine which parameters should be tested in order to optimize the renaturation yield. Indeed, one could predict that folding would be favored by the following factors: low protein concentrations to slow down intermolecular interactions, low temperatures to minimize the hydrophobic interactions mostly responsible for aggregation, sub denaturing concentrations of denaturants to minimize intermolecular interactions without disrupting native structures, nondenaturing pH as far away as possible from the isoelectric point so as to increase electrostatic repulsion between chains, etc. With these guide lines, it took GENETICA only a few weeks to convert inclusion bodies of totally inactive tPA into active tPA with a 30% yield, a spectacular result which could probably have been improved by further optimization. Similarly, inclusion bodies of HSA were converted into mostly soluble protein. However, due to internal conflicts that resulted in a ‘‘divorce’’ between Alain Rambach and GENETICA, our collaboration came to an end and these projects were abandoned. It is however a tremendous satisfaction for me to know that several industrial processes still used for the production of recombinant proteins (like human insulin, human growth hormone, or bovine growth hormone for instance) are based on our early reports on the renaturation of proteins at sub denaturing concentrations of urea or guanidine, and on several of our later findings derived from the kinetic competition model. If I had predicted that genetic engineering would stumble on inclusion bodies, and had I patented the principles of the renaturation procedure we devised with Gilbert Orsini for chymotrypsinogen, I would be a rich man today y But


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such moneymaking considerations were so far from our minds in 1978! The third consequence of our investigations on folding versus aggregation in vitro is of a completely different nature. It deals with my personal relation to Germany and to my past. As the reader may have understood from the first section of my narration, the sufferings and devastations inflicted by the Nazi regime onto my family, the effects it had had on me as a child and the perception of Germany I had constructed in my mind through the stories I had heard about the Gestapo and about the concentration camps, had made it unconceivable for me during all my youth to shake hands or even speak to a German. For years after the war, we would not buy a German product at home. Though I learnt German at high school (our teacher was French) I never spoke to the young assistant from Saarbru ¨ ck with whom we were supposed to practice German. And obviously, I refused to visit Germany. In the summer of 1958, together with a cousin of my age, we drove across Germany from Belgium straight to Denmark. From the second we crossed the boarder into Germany until we left, we felt oppressed. We hardly could speak to each other. We stopped only to buy gasoline and felt really relieved when we entered Denmark. During my years at Stanford, I met a few young German scientists. They were more or less of my generation. They had been babies or very young children during the Nazi period and therefore could not have done any harm or be considered as responsible for the deeds of the Nazis. I made real efforts to try and overcome my initial aversion. And I came to really appreciate some of them, with whom I dare say we became friends like Walter Doerfler and his wife. But these were individual cases. My instinctive reactions against Germans still persisted. How does this relate to protein folding and aggregation? In July 1975, I was invited to give a talk at the 10th Meeting of the Federation of European Biochemical Societies that was held in Paris. At lunch, just after my talk which had dealt with the functional role of subunit interactions in oligomeric proteins, a gentleman sat near me and introduced himself: ‘‘Jaenicke, from Regensburg University.’’ He was German. I forced myself to be polite. He started talking about my lecture, and I asked about his work. We soon found much common interest. His experimental approaches, his ideas, his difficulties, the problems he was addressing, were very similar to mine. The next day, he sat again

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at my table. The discussion became more personal. I very soon told him about my Jewish origin, the story of my family and my childhood in occupied France. He told me about his family and his childhood in Nazi Germany. How, because his father had been an early opponent to the Nazi regime and because one of his remote ancestors was Jewish, he had been living hidden for years in a cellar. How his humanity teacher from high school, a German gentile, had come secretly for years to help him continue studying. How the little girl who later became his wife, Gartie, had had a similar experience and how they played music together, hidden in their cellar. This had a terrific impact on me. Here I saw a German who had lived his youth, as I had, as a hidden child. Whom Germans had helped at the risk of their lives. Who spoke of Nazism with the same hatred as I did. And who, last but not least, seemed so bright, so open minded, so gentle, and so generous. Rainer Jaenicke invited me to visit him in Regensburg, were he had just moved and created a laboratory (which would become one of the most active protein folding research centers in Europe). The strength of the human relation that was developing between us was such that I immediately accepted and, soon after, made my fist visit to Germany. A very short one that included only one day in Regensburg with Rainer Jaenicke and one day in Wu ¨ rtzburg with Ernst Helmreich, whom I had met at the Institut Pasteur and had also invited me to visit him. Though I was struck by the beauty and historical interest of the two places I visited, and by the warmth of my two hosts’ hospitality, I did not feel at ease in Germany. There were too many people I crossed everywhere in the streets, in the trains, in the restaurants, that were old enough to have been active members of the Nazi party, of the Wehrmacht, or of the Gestapo. There were policemen in uniforms waking up dormant frights. There were people loudly speaking a language that, scared to death, I had heard shouted in the streets of France. Yet, I had stridden my first steps in Germany. And that opened the way to many visits, to many fruitful collaborations, to deep-rooted friendships, and to many happy moments in Germany. The most memorable ones are all directly related to Rainer Jaenicke. Sometime in 1988, Rainer told me that he was thinking of me as a possible candidate for an Alexander von Humboldt Award. But before proposing my name, he wanted to make sure that, in case I would be elected, I would accept the award, which implied that I would have to spend some time in a


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laboratory in Germany. After long hesitations and discussions with my wife, and probably influenced by the thought that this award was too prestigious for me to get it, I finally accepted. To my surprise, I was offered the award and funded for up to 1 year in Germany. For family reasons (my wife could not leave Paris and I did not feel like leaving her behind for such a long time) as well as professional obligations (I had my teaching at the University, and students in my laboratory preparing their PhDs) I chose to spend only 6 months in Germany. And I decided that, except for a few visits and seminars to other Universities, I would spend all my time in Rainer’s laboratory. During my first month in Regensburg, in December 1989, I gave a series of seminars entitled ‘‘The Goldberg Variations’’ on the various activities of my laboratory (protein folding, monoclonal antibodies, cellsorting, chromosome sorting, etc.) and discussed with Rainer the research project I though of conducting during my stay in his laboratory. Shortly after our paper describing the kinetic competition between folding and aggregation, Rainer Jaenicke and his collaborators had reached similar conclusions [57]. Because of our common interest for this model, I chose to work on it again, asking a question that was recurrently coming to my mind but that I had never dealt with for lack of time. The idea was to find out at what stage of the folding process the protein becomes irreversibly committed to form inactive aggregates. Because HEWL was routinely used by Rainer and his coworkers in their practical courses for Biochemistry students, because the protocol for oxidative renaturation of HEWL had been very well worked out, and because the oxidative renaturation of reduced HEWL gave rise to aggregates, I chose to work on HEWL. I spent 5 most exciting months performing experiments like a young post-doc again. I had no technician. I prepared my buffers and solutions all by myself. I had no administrative task, no telephone call, no student interrupting me. I could work as long as I wanted in the evenings and the weekend. And the project progressed beautifully. Within a month I had set up a highly reproducible protocol for renaturing reduced guanidine-unfolded HEWL by dilution into guanidine-free buffer, established the kinetics of reactivation and studied the concentration dependence of the renaturation yield of HEWL. To find out when HEWL became irreversibly committed to aggregate, the following procedure was used: a concentrated solution of reduced unfolded HEWL was

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diluted 10-fold in denaturant-free buffer, thus initiating the folding at ‘‘high’’ protein concentration. At various times after this initial dilution, aliquots were taken and diluted 10-fold again, resulting in ‘‘low’’ protein concentration. Renaturation was then allowed to go to completion and the samples were assayed for activity. My results clearly showed that HEWL becomes irreversibly committed to aggregate within the first 5 seconds of refolding – the dead-time of my hand-mixing experiments. I was quite happy. In less than 2 months, I had answered my question: the commitment to form aggregates occurs very early in the process when compared to the half life of about 4–5 minutes of the reactivation reaction under these experimental conditions. Since I had 3 more months to spend in Regensburg, I started thinking finding out when does the protein become irreversibly committed to fold into the native conformation. In theory, a similar strategy seemed appropriate: first dilute reduced unfolded HEWL 100-fold to initiate the folding at ‘‘low’’ protein concentration, incubate it for various time intervals, then concentrate it rapidly 10-fold and let the folding go to completion at ‘‘high’’ protein concentration. There was however a technical difficulty. How could one concentrate the protein 10-fold in a matter of seconds? I stumbled on this problem for days. I thought of it day and night. I tried to imagine all kinds of techniques, none of which could work without perturbing the folding process. Because I could imagine no physical way of achieving such a rapid concentration step, I thought of a ‘‘biochemical’’ way. The idea was to add to the dilute HEWL an excess of a modified form of lysozyme that would behave as unmodified lysozyme with respect to the folding process but would not interfere with the assay of the unmodified lysozyme. For days and nights again, I tried building on this idea. Nothing satisfactory emerged until, in a flash, all the pieces of the puzzle came together into a dazzling solution: dilute 100fold reduced turkey lysozyme to initiate its folding at low concentration, at various times add reduced hen lysozyme, let the mixture refold to completion, add a monoclonal antibody that inactivates HEWL but does not react with turkey lysozyme, and determine the activity of turkey lysozyme. I knew that Roberto Poljak, at the Institut Pasteur, had isolated such an antibody and that he would provide me with as much of it as I might need. I rarely experienced an excitement comparable to that I felt when this idea came to my mind. I was sure that it would work.


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On the spot, I remembered what Monsieur Monod would sometimes tell: ‘‘It is too elegant not to be true.’’ And indeed it worked out very nicely. Turkey and hen lysozymes turned out to have the same oxidative folding kinetics and concentration dependence and to form heterologous aggregates. MAb D1.3 completely inhibited HEWL without affecting turkey lysozyme, and the irreversible commitment of turkey lysozyme to fold into the native conformation could thus be shown to occur at a late stage of the folding process. During my last month in Regensburg, I wrote a paper reporting this study [58]. When I showed the manuscript to Rainer, asking for his criticisms and suggestions, his comment was: ‘‘Only a Frenchman could have written such a manuscript. It is so Cartesian y’’ This piece of work holds a very special place in my heart. The simplicity and originality of the experimental approach give it a touch of elegance. I did this work all on my own, which impressed my colleagues and students at the Institut Pasteur. When I came back to Paris and gave a lecture on my work, I could see, with some pride, that all were surprised to see that ‘‘the old one’’ was still capable of working at the bench. But the main reasons why I like thinking of this project is the intense pleasure I had working on it and being in Rainer Jaenicke’s laboratory. During my 6 months in Regensburg, I lived moments of intense scientific excitement. But I also lived unforgettable human experiences. First, coming to know Rainer Jaenicke more closely and discovering the many facets of his personality and activities. Not only as a prominent scientist. But also as an enthusiastic, modest, devoted founder of a lineage of outstanding protein folders, of which I met several during my visit there: Rainer Rudolph, Robert Seckler, Johannes Buchner, Thomas Kiefhaber, and Andreas Plu ¨ ckthun. But also as a Professor entirely devoted to his student. As a man full of humanity tirelessly working at breaking boarders between countries, regimes, races, or creeds. As a man of culture. As a musician. Second, I also discovered that life in Regensburg could be very pleasant. When, for some periods, my wife, my children, or friends came to stay with me, we enjoyed the country side, the ‘‘bier garten,’’ the superb museums in Munich and Nuremberg, the omnipresence of classical music in the streets and concert halls. We visited lots of places and were impressed by the historical load of every city we came across. And to my surprise, I as well as my

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wife and children, in spite of our prejudiced feelings against Germany, really enjoyed being there. Third, my relation to Germany and the German people really changed. This was made possible only because I could on many occasions witness the efforts that Germany as a country, and many Germans as individuals, had made and were still making to face their past and recognize the responsibility of their country in the dramatic events of World War II. Efforts that France has started making only recently, and other countries – like Austria or Poland – have not yet made. Among the numerous occasions where I could see exhibits, read books, or hear discussions related to the Holocaust, I remember with particular emotion a talk delivered in December 1989 by Benno Mu ¨ ller-Hill as part of the curriculum in Biochemistry and Medicine for students at the University of Regensburg. Benno is a well-known molecular biologist who became world famous when he isolated the repressor of the lac operon in 1966. He had visited several times the Institut Pasteur and we knew each other. Besides his scientific activities, Benno has spent a lot of time and energy trying to detect and eradicate remnants of the Nazi regime still present in the German academic system. The topics of the lecture he gave in Regensburg was to analyze the role of German geneticists in support of the Nazi ideology. I was sitting in the last row of the auditorium, listening at him, but also watching the two hundred or so students in their early 20s, respectfully and sternly concentrated on Benno’s lecture. Except for Benno’s voice, there was a total silence. I kept thinking how times had changed. Here I was, one among the few lucky ones who escaped annihilation by the Nazis. Here I was, honored by an award from the German Alexander von Humboldt Foundation, hearing this noble German scientist battling against the ideas of those, dead or alive, who were responsible for the humanitarian disaster that had swept away nearly all my people. When Benno finished his talk, questions were asked by the audience. All focused on one idea: how to prevent such things from happening again. When there were no more questions, I came to Benno who was still surrounded by a dozen of students. I thanked him for what he was doing and wanted to tell him that, through his action, History was taking revenge over Hitler. But I could not complete my sentence. Both Benno and I burst into tears and embraced each other. I’ll never forget that moment. Nor shall I forget the look of


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the students, petrified, motionless at the sight of these two respected Professors, the tall sturdy German and the little frail Jew, swamped by a wave of emotion. Thus, my stay in Regensburg was a real success. I’ll never find enough words to thank the Alexander von Humboldt Stiftung and Rainer Jaenicke for the opportunity they gave me to enjoy science and change my view of contemporary Germany. Not only did this visit result in one of my most frequently cited papers. It also opened my heart to a people I had thus far rejected because it had, in the past, rejected my people. Since my days in Regensburg, I have visited Germany several times. For international meetings, for official reasons, or just for pleasure. I have had several fruitful collaborations with German groups. I have invited several German scientists and students in my laboratory. I have even made German friends whom I met here, in France. In this respect I think that the von Humboldt Foundation has made a good investment, in terms of both science and international relations (Figure 5). My friendly collaboration with Rainer Jaenicke culminated when both of us climbed on the stage of the Festsaal of the Friedrich Wilhelm University in Bonn, in 1994, to receive a Max Planck Research Award from the hands of the Chairman of the

Fig. 5. The author and Rainer Jaenicke receive the Max Planck Award in Bonn in 1994.

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Max Planck Society and of the German Minister of Research and Technology. While receiving this honor, I could not refrain from thinking of how and why my grandparents went to ashes, and of how the tides of History go back and forth. In retrospect, I can not help finding it somehow strange that Germany honored me with two prestigious awards for my achievements in the study of protein folding and aggregation, while I did not receive any award, even a minor one, in my own country y My involvement in research on protein folding and aggregation and my indefectible fidelity to the memory of Monsieur Monod strongly influenced my scientific life even during my last years of activity at the Institut Pasteur. Both were at the origin of my short incursion into prion research between 2001 and 2004. It all started with an editorial written in 1996 as an introduction to a special issue of Science devoted to misfolding diseases [59]. The author insistently referred to our old studies on aggregation [53,56] as being at the basis of our understanding of amyloid and prion-related diseases. I felt flattered by such statements and extremely happy to find out that our work might perhaps be at the origin of future medical applications. But I did not see how I might be of any help in such medical studies. Shortly after I became aware of this editorial, I was approached by Maxime Schwartz – then Director of the Institut Pasteur – and Agne`s Ullmann who were trying to organize a Memorial Lecture on the occasion of the 20th anniversary of Monsieur Monod’s death. They offered me to give this lecture. All the previous Monod Memorial Lectures – one every year between 1977 and 1987 – had been delivered by highly prestigious scientists, mostly Nobel laureates, who either had collaborated with Monsieur Monod or worked on subjects directly related to his two well-known contributions: allostery and gene regulation. I was surprised by Maxime and Agne`s’s idea. I was not a scientist with a reputation comparable to that of the previous lecturers. Moreover, protein folding was not a subject for which the French scientific community had shown much interest. I therefore thought that a conference on Protein Folding by Michel Goldberg would not attract an audience respectable enough – in terms of quantity and quality – to honor the memory of Monsieur Monod. I first refused Maxime and Agne`s’s offer. They insisted, putting forward a variety of reasons (political, personal, financial, scientific) for which they thought I should accept. I was not convinced, but


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promised to think it over and give them my final answer on the next day. On the one hand, I felt honored by the offer and found in it an outstanding opportunity to express my gratitude and admiration for Jacques Monod. On the other hand, an empty auditorium for a Memorial Lecture would be an insult to the memory of the man we wanted to honor. I was about to give up the idea of accepting this lecture when, during the night, I got the idea that changed my mind. Taubes’s editorial came back to my mind. Europe was shaken by the Mad Cow crisis, and people’s sudden interest for prions was likely to attract a crowd eager to learn more about this unconventional pathogen. I thought of introducing the word ‘‘Prion’’ in the title of the memorial lecture, focus part of the talk on our studies on protein aggregation, and show how these studies were related to the prion-induced appearance of protein aggregates. Maxime Schwartz and Agne`s Ullmann were happy with this idea. Was it due to the pompous title of my lecture Selective interactions and protein polymorphism: From allosteric proteins to the prion [60] or to the prestige of Monsieur Monod and the faithfulness of those who had known him? Probably a bit of both y . Whatever the reason, the end result was that on May 31, 1996 the Grand Lecture Hall of the Institut Pasteur was absolutely crammed with people, many sitting on the stairs, many standing behind the last row of chairs, others unable to even enter the hall. And my lecture was the best I ever delivered. I owed it to Monsieur Monod y I had worked a full month preparing this lecture. Much of the time had been devoted learning and thinking about the prion. A few months later, the Direction of the Institut Pasteur organized a brain storming meeting between a few scientists of the campus with the aim of deciding whether or not the institute might undertake some research on the prion diseases. I submitted a modest project, based on our expertise in the production and use of conformation specific monoclonal antibodies, aimed at investigating the structural differences between the normal and pathogenic states of the PrP protein. I received a brutal rebuff from Jean-Pierre Changeux, the highly considered French neurobiologist and co-inventor of allostery, who contemptuously mocked me for being naı¨ve enough to believe Stanley Prusiner’s ridiculous hypothesis of a protein-induced irreversible change in conformation. Needless to say how happy I was, a few years later, when I heard that Prusiner was awarded the Nobel Prize for his

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‘‘ridiculous’’ hypothesis y . For a variety of reasons that appeared to me well grounded, the Direction of the institute decided not to launch any prion-related project, and my enthusiasm for the subject faded away. However in 2001, soon after he became the Director of the Institut Pasteur, I discussed with Philippe Kourilsky the future of my group in anticipation of my retirement scheduled for 2005. Philippe Kourilsky prompted me to spend my last years setting up a research group focused on the prion. He promised to give all the support I would need – in terms of personnel, equipment, and budget – to create such a group and encouraged me to submit a project as soon as I could. Within a couple of months, I came with a project which was approved by the Scientific Council of the institute and received strong financial support from the French Ministry of Research and from a pharmaceutical company. In December of 2001, I got the OK from the Direction of the institute. In less than a year, I recruited a bunch of six enthusiastic coworkers, implemented all the techniques required for the biochemical and physiological approaches we envisaged, collected all the biological material we needed (transgenic mice, cell lines, prion infected brains, and PrP-specific monoclonal antibodies), established local colonies of transgenic mice, expressed and purified recombinant PrP and set up a P3 facility. Getting access to a P3, which became available only in November 2002, was the rate limiting step in starting the project. Within a year and a half, we obtained 26 different clones of mAbs with specificities against seven different peptides of the PrP, and developed a rapid spot test to screen the specificity of mAbs with respect to the normal or pathogenic forms of PrP. We isolated a PrP-hyper producing cell-line capable of propagating the infectious form of the PrP in amounts such that obtaining 1 mg of infectious prion from cell cultures in the laboratory became possible. From such prion-infected cultures grown in the presence of a glycosylation inhibitor, we obtained non-glycosylated, protease resistant PrP and showed that it was able to infect mice, thus demonstrating that glycosylation of PrP is not essential for its infectiosity. And we set the basis for a hypersensitive prion detection test in potentially infected tissues. The project was having an unexpectedly rapid and successful start. It was however abruptly stopped because of maintenance problems due to a misconception of the P3, of a drastic decrease in the Direction’s interest for prions, and of internal problems that


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opposed the Direction and the scientists on the campus and led to the resignation of the Board of Administrators and of the Director of the Institute. This sad period of internal turmoil within the Institut Pasteur and of dismantling of the prion group came to an end in September 2005 with the election of a new Director, Alice Dautry, and with my retirement. Indeed, Alice Dautry took her position on October 1, 2005, the exact date when my retirement became effective since I was allowed to work until the last day of the month of my 67th birthday – a privilege when compared to the normal age (65) of mandatory retirement at the Institut Pasteur. I was sad to see the prion activity of our group halted. Our brief attempts to contribute to solving this mysterious pathogen had raised so much enthusiasm among my collaborators and myself! Yet I felt proud of having been able, at such a late stage of my career, to start a new project in a productive way. I felt grateful, too, to the Institut Pasteur and to my collaborators who embarked with me for this adventure for having allowed me to spend my last years of scientific activities on such a thrilling project.

My Proteinless Studies Protein folding, misfolding, and aggregation have undoubtedly occupied most of my time and of my thoughts during my years in research. However, some ‘‘side studies’’ also gave me a lot of fun. Some were quite successful. Some were useful to others. Some gave rather spectacular results (Figure 6). An exciting episode was to set up, in 1980, a flow-cytometry facility as a joint venture between the Institut Pasteur and INSERM (the French National Institute of Health and Medical Research). At a time when I thought I was about to end my studies on protein folding and was starting to think of an alternative subject, I was asked by Franc- ois Gros, then Director of the Institut Pasteur, to organize and supervise a facility aimed at making a novel technique ‘‘flow-cytometry’’ available to the scientific community. He did not know what flow-cytometry was. Neither did I. I asked colleagues to help me find information about it. Very few people in France had heard about it. No one I could identify had practiced it. Information came from abroad. When I realized the complexity of the equipment that involved

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The author working on the cell-sorter with Philippe Me´te´zeau.

fluidics, optics, lasers, light scattering, fluorescence, electrostatics, and computer, and tried to identify a scientist on the campus who could master these various aspects of the machine, I could think only of Henry Buc and myself. Henri was not interested. I decided to give it a try, thinking that it would be a good way to establish collaborations from which my future scientific orientation would perhaps emerge. I therefore accepted to set up the ‘‘INSERM-Pasteur Cell-sorting Facility’’ and to be its Director, a position I kept from 1979 till 1993. I set however a condition: I agreed that the main aim of the facility was to run experiments for others, but requested that 30% of the time should be devoted to ‘‘personal’’ projects. Indeed, I considered that a facility open to the public could run efficiently and stay in the forefront of the technological progress only if the persons in charge are personally involved in research projects that call for the most modern developments of the method. This request was accepted. I carefully selected the first machine we purchased, a cell-sorter from Ortho (USA) and the person who would help me in running the facility, Philippe Me´te´zeau, a young engineer at the Institut Pasteur who was about to lose his job because his boss


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was retiring. Reluctantly, he accepted this new job, but very soon became passionate with it. Philippe became one of the best European experts in flow-cytometry, wrote several reviews and books about this method, and took over the Direction of the facility in 1993 until he retired. Together, we discovered the difficulties and advantages of flow-cytometry, convinced our colleagues of its fantastic capabilities (in terms of precision, reproducibility, and speed in the analysis of cell populations), performed the first successful cell-sorting experiments in France, participated to hundreds of projects with many teams from Pasteur and INSERM throughout the country. We introduced new developments, like a procedure to improve the discrimination between bull spermatozoa carrying the X or the Y chromosome. Or like a gadget to greatly improve the signal-to-noise ratio in spot tests performed on sorted chromosomes. We designed a procedure to monitor, on a cell-by-cell basis, the kinetics and extent of receptor-mediated endocytosis, which dragged me into an unexpected collaboration with the well-known French manufacturer ‘‘Parfums Christian Dior.’’ A schoolmate from the Ecole Polytechnique, Jean-Pierre Me´gnin, had become the Deputy Director in Charge of Development for an industrial group dealing with luxury products. In 1980, he consulted me about a possible implication of his group in Biotechnology, a word he had heard a lot but wanted to know more about. During our discussions, he once mentioned the interest of his group for ‘‘a scientific approach to skin aging. Not a miraculous product. Just a really scientific approach.’’ About 6 months later, I attended a meeting in Montpellier. The meeting was on ‘‘Molecular Markers of the Cell-Membrane Dynamics.’’ During this meeting, I heard three talks. One reported that hepatocytes in culture accumulated cholesterol in their plasma membrane, which became less fluid. A second was that the plasma membrane of cells becomes more fluid during cell division. The third one was that one could modulate at will the membrane fluidity of cells by manipulating their lipid contents. Put together, these reports triggered the idea that changes in membrane fluidity might be involved in skin aging. Back in Paris I called Jean-Pierre Me´gnin. He showed much interest in the idea and organized a lunch with the Research Director of Parfums Christian Dior. When I told them that changing the membrane fluidity could easily be achieved by means of liposomes, they became really excited: they had just

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patented a new procedure for the industrial production and the stabilization of liposomes. On the basis of my idea – given without any financial counterpart – and of their procedure, Parfums Christian Dior developed one of the most successful products in the history of cosmetics ‘‘Capture’’, the first liposome-based antiaging product. Capture is still present, some 25 years later, on the shelves of beauty shops throughout the world. I do not know how good it is at slowing down skin aging. My mother used Capture until her last day, considering it as a fantastic product, but I am sure she was totally biased by the fact that I had been at its origin. I do not consider my contribution to Capture as a great scientific achievement. Yet, it conferred on me the status of a ‘‘productive’’ scientist because my collaboration with Christian Dior had resulted in a spectacular commercial success. From then on, all my grant applications to the Ministry of Science and Technology were approved. My name appeared in newspapers in the Far East, Europe, and North America, something that had never happened for any of my really scientific contributions. The ways to fame are unpredictable, and not always as gleaming as one would hope y Another piece of work based on flow-cytometry, which had nothing to do with protein folding but which I particularly enjoyed, was the successful sorting of mouse chromosomes and the construction of the first mouse chromosome-specific gene library. One of the reasons why the Institut Pasteur had decided to set up the flow-cytometry facility was Franc- ois Jacob’s interest in obtaining chromosome-specific gene libraries for his investigations on the development of mouse embryos. He had heard about promising attempts to sort human chromosomes by means of a cell-sorter and had put pressure on the Direction of the Institut Pasteur to get such a machine. Each time we met after I started running the flow-cytometry facility, Franc- ois would ask me about mouse chromosome sorting. I kept answering him that, unlike human chromosomes, mouse chromosomes are grouped in a small number of ‘‘clusters’’ – in terms of size and base composition – that are too similar for them to be individually identified by flowcytometry. This lasted until a lunch I had with Jean-Louis Guenet, an outstanding mouse geneticist who was also in charge of the animal facilities at the Institut Pasteur. We were good friends. Like me, he had been attracted to the Institut Pasteur by Monsieur Monod, whose memory we often called up together. We were talking casually about Jean-Louis’s work when he


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mentioned studies on ‘‘Robertsonian translocations’’ in mice. My training in math and physics had obviously not taught me what such translocations were. I shamelessly asked Jean-Louis what he was talking about and he explained, in simple words that even a layman like me could understand, that these were stable ‘‘accidents’’ occurring during somatic cell division which results, as a first approximation, in a fusion of two chromosomes at the level of their centromers. Because, in mice, all the chromosomes except for the X have their centromer at one extremity (acrocentric chromosomes), there is no significant loss in genetic information and the mice with this defect are viable. I immediately realized the advantage one could get from such mice. Indeed, a ‘‘Robertsonian’’ chromosome is much larger than the original chromosomes, and should be easily distinguished – and therefore sorted – in a flow-cytometer. We built up on this idea. Bruno Baron, an undergraduate student who joined the laboratory initially for a 1-year training, first analyzed and sorted chromosomes from a cell line with an abnormally large chromosome 1. These large chromosomes could indeed be sorted, but they were contaminated by aggregates (aggregates again!!!) of normal chromosomes. We therefore slightly changed our strategy. Rather than sorting large translocated chromosomes likely to be contaminated by aggregates, we thought of using mice strains in which all the chromosomes with a size similar to the one we wanted to isolate would be involved in translocations, thus leaving the desired chromosome alone in its size range. With Jean-Louis Guenet’s help, we got a mouse strain with all its chromosomes, except chromosome 19 and the sexual chromosomes, involved in Robertsonian translocations. This strain contains three classes of chromosomes: one with very small chromosomes of the same size (Y and 19), one with medium size chromosomes (X), and one with all the large translocated chromosomes. Bruno Baron cut a small piece of ear from such a mouse, obtained a primary culture of its fibroblasts, eternalized them with a non-transforming defective SV40 virus with the help of Franc- oise Kelly, selected a stable permanent cell line of fibroblasts, checked that they had kept the translocation, and learnt from Alain Bernheim and Roland Berger (Hoˆpital SaintLouis, Paris) how to accumulate cells in mitosis and to extract chromosomes. The chromosomes were then labeled with ethidium bromide and run in the flow-cytometer. The histograms thus

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obtained with cells from a female mouse showed the three peaks expected (19, X, and translocated) thus leading to the first successful sorting of pure mouse chromosomes [61]. The same approach was then applied, using a piece of ear from a male mouse hosting a single Robertsonian translocation involving chromosomes 9 and 19 (Rb 9.19). In this strain cells from males contain only one very small chromosome, the Y chromosome. This enabled Bruno to sort a million of pure murine chromosomes Y from which a Y-specific gene library was constructed [62]. The three mouse chromosome-specific gene libraries (X, Y, and 19) that we thus prepared were the first to be obtained and were amply used by Philip Avner and Colin Bishop at the Institut Pasteur to study several aspects related to gene silencing, sex determination, and sex-related pathologies, as well as by Bruno Baron to establish the first physical map of chromosome 19. This approach would certainly have been applied the study of other mouse chromosomes if there had not been the technical revolution brought about by ‘‘walking on the chromosomes,’’ PCR (polymerase chain reaction) and DNA sequencing. In spite of its limited impact, this project brought me the immense satisfaction of having solved a tough technical problem, of having met Franc- ois Jacob’s expectations, of having initiated such an original project involving so many different aspects of modern biology, and of having participated in such a friendly, efficient and productive collaboration. During a meeting that was organized at the NIH in Bethesda to seal the reconciliation between the NIH and the Institut Pasteur after their fight over the HIV (human immunodeficiency virus), I gave a lecture on our chromosome sorting studies and on the way in which Jean-Louis Guenet used the chromosome-specific probes and his novel approach of restriction fragment polymorphism segregation to map genes on mouse chromosomes. After the meeting, I dropped into the laboratory of Edith Miles. She was still actively working on tryptophan synthase. When I told her that I was here because of the NIH-Pasteur meeting, she said that she had seen its program and was astonished that there was another Michel Goldberg at the Institut Pasteur. She could not imagine that I was the person lecturing on such an odd subject y To complete this bird’s eye sight of my ‘‘proteinless’’ scientific contribution, I would like to briefly mention an exciting collaboration I had with a bunch of outstanding physicists, trying


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to solve one of the big, still unanswered questions in the field of nuclear physics and cosmology: what is the nature of the missing mass of the universe? Briefly, according to the current model for the formation of the universe, its mass should be about 10-fold larger than its observed mass. My very dear brother-in-law, Michel Spiro, a brilliant nuclear physicist – now Head of the IN2P3, that is the Department of Nuclear Physics and Particle Physics of the CNRS – wanted to test one attractive hypothesis that had been put forward to account for this missing mass. The hypothesis was that there may exist ‘‘super-heavy’’ particles, included in super-heavy nuclei that belong to super-heavy atoms. To test this hypothesis, Michel Spiro had designed an experiment and set up a collaboration with the Laboratory of Hertzian Spectroscopy of the Ecole Normale Supe´rieure. They developed a fluorescence spectrometer that should have been able to detect the presence of super-heavy hydrogen atoms in water. The sensitivity of the machine was however too low to detect superheavy water molecules in the concentration range sufficient to account for the missing mass of the universe. Hence the need to ‘‘concentrate’’ such putative super-heavy water. Michel Spiro called me on the phone one evening and consulted me on the possibility of using ultracentrifugation for that purpose. We spent a couple of hours on the phone estimating the sedimentation coefficients, frictional coefficients, diffusion coefficients, volumes needed, times of centrifugation y and ended up with a positive conclusion. As a result, I set up an iterative centrifugation procedure of ultracentrifugation in swinging buckets: a water sample was deposited in a tube over a thin layer of concentrated sucrose. The sample was centrifuged overnight and the water overlay carefully removed. A fresh water sample was deposited over the sugar solution, which putatively contained the superheavy water from the previous centrifugation, and a new overnight centrifugation performed. This procedure was repeated many times. Sometimes, the sucrose cushion was diluted with fresh water and centrifuged over a fresh cushion. Cushions from several tubes were mixed, diluted with water, and centrifuged over a single cushion to bring all the putative super-heavy water molecules into the same small volume. In this way, we ‘‘concentrated’’ the putative super-heavy molecules from a total of 12.5 liters of water from six different origins – from the Indian Ocean near the Kerguelen Islands, or near the Comoro Islands,

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deep sea water from the Mediterranean, water from the Dead Sea and snow from the French Alps, and distilled water – into a final volume of 180 ml, that is by a factor 7 104. This considerable concentration factor brought the theoretical concentration of super-heavy water molecule in a range easily detectable by the fluorometer. Yet, no such molecule could be detected, thus ruling out that super-heavy particles might account for the missing mass of the universe. This nice experimental approach, though failing to bring an explanation, had the merit of discarding an attractive hypothesis that had been hanging around for decades. Development of an anti-aging cosmetic drug, mouse chromosome sorting, centrifugation of imaginary nuclear particles accounting for the missing mass of the universe, studies on a skin autoimmune disease (pemphigus) by flow-cytometry were my main scientific excursions out of the realm of protein folding and aggregation. The diversity of the topics I was led to investigate and of my experimental approaches show how useful it can be to keep a curious mind, spend some time and efforts on daring projects and talk to colleagues interested in different subjects. Having had a basic training in ‘‘hard sciences’’ like physics and mathematics certainly helped me dialogue with physicists like Michel Spiro (super-heavy particle project) or Georges Charpak (development of the b-imager radioactivity scanner). Together with my love for tinkering about, it also helped me a lot in quickly mastering the various technical aspects of our cell-sorter and circular dichroism stopped-flow experiments. In this respect, Monsieur Monod’s choice of attracting a student in physics to his laboratory turned out to be of some use.

Administration and Transmission of Science I cannot bring to an end this narration of my scientific life without briefly reporting some of the most significant sciencerelated activities, several of which were also strongly influenced by Monsieur Monod. Taking American universities as a model, he had conceived and planned the ‘‘Institut de Biologie Molećulaire’’ – now renamed ‘‘Institut Jacques Monod’’ – a research institute based on the (then) modern campus of the Paris Faculty of Sciences, with research units led by Professors involved in teaching, and widely


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open to students. He had to take the Direction of this institute. However, during its construction, he was appointed Professor at the Colle`ge de France and the leadership of the institute was handed over to Raymond Dedonder. The organizing committee wanted to hire a Professor in Biophysics. In early 1968, I was asked by Raymond Dedonder to apply for that position. I was not yet 30 years old, had obtained my PhD just a year earlier, and had no more than three published papers. An extremely modest curriculum for a professor. I was about to refuse, but Monsieur Monod insisted that I should accept this offer. I sent out my application file a month before the student upheaval that shook France in May 1968. The appointment committee of the university did not accept my application because there was only one candidate. They wanted an open competition. A second applicant was therefore stimulated. The two applications were reviewed and, in September 1968, I became the youngest professor at the Paris University. I gave my first lecture in October. And I kept this position until September 1998. Though I suffered from the inertia of the French academic system, from the lack of selection of the students, from the incoherence of the sequence of reformation of the courses imposed by nearly each new minister of education, from the useless and endless faculty meetings, I loved teaching to students. Teaching Biophysics and Advanced Enzymology – two disciplines relying heavily on Mathematics and Physics – to students who very often chose to study Biochemistry to escape Mathematics and Physics was a challenge. But I dare say that my passion for research and my pedagogic approach much inspired by my experience as Laurent Schwartz’s student at the Ecole Polytechnique favorably impressed most of my students. Until today, I receive testimonies of former students who acknowledge the impact of my teaching on their vision of science and of research. After 30 years of teaching to over 7,000 students, I decided to retire from my position at the university. I had a conflict with the University and the Ministry of Education about the organization of the Master and PhD programs on ‘‘Protein Structure and Function’’ that I had created and of which I had been in charge for over 20 years. Based on technocratic reasons, the ministry wanted me to take the responsibility of PhD students that came from training programs in Microbiology, in Molecular Biology and in Developmental Biology. I had no control on the registration of these

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students, on the choice of their laboratories and research projects, on granting their fellowships, and on their fate after their thesis. Moreover, I felt unable to supervise their research projects in research areas unrelated to my field of expertise. Rather than leaning to the pressures put onto me by the Ministry and by the Direction of the University, I decided to retire on the date of my 60th birthday. During my last lecture to my students, I told them about my decision, explained its reasons, thanked them for the magnificent moments I had experienced with them and their predecessors and ended with a farewell full of emotion. I had sobs in my voice, and tears blurred my eyes. This decision meant much to me. It meant a severe financial loss during the 8 years I would have been able to go on teaching, as well as for my retirement pension. It also meant loosing contact with the students who, for so many years, had been a source of rejuvenating vigor for me. But it also meant the possibility of devoting my last years of scientific activity entirely to research at the Institut Pasteur, without the time and energy-consuming burdens related to the university. I missed, and still miss, the contact with the students. But I do not regret my decision. I enjoyed so much being full time in the laboratory, and having time to perform experiments with my own hands. Though my employer was the Paris University and my teaching at the undergraduate level was on the campus of the University, my laboratory and all my research activities were located at the Institut Pasteur. And when I decided to include a practical course in our Master’s program in Protein Science, I organized it in the Teaching Department at Pasteur. I therefore never left the Institut Pasteur and was in charge of a variety of jobs there. Shortly after he became Director of the Institut Pasteur, Monsieur Monod created an industrial branch, Institut Pasteur Production, for the production and sales of sera, vaccines, and diagnostic products derived from the pasteurian research. He entrusted me with a nomination as a member of the Board of Administrators of this company, a position I kept for several years, until it was taken over by the French pharmaceutical group Sanofi. When Monsieur Monod passed away in May 1976, I was asked by the Scientific Council of the Institut Pasteur to be one of three members of a search committee in charge of identifying possible future candidates for the Direction of the institute. I contributed


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to convince Franc- ois Gros of accepting this position. When the Board of Administrators of the institute elected him, he asked me to join his team as the Deputy Scientific Director. I argued that my knowledge of biology was too limited to endorse such a responsibility that I was too young and inexperienced for it. He replied that I had twisted his arm to make him accept the direction of the institute and had promised him that all of us would help him. I therefore should feel compelled to help him. Furthermore, he added that shortly before passing away Monsieur Monod had told him that he (Franc- ois Gros) should accept becoming the director and that he should ask me to help him in the scientific direction of the institute. There was no way out. In view of these two reasons, I had to accept. During a year I acted as Deputy Scientific Director, in charge of the departments involved in molecular research, while Yves Chabbert, the Scientific Director, was dealing with the departments involved in microbiology and microbial diseases. After a year, Yves Chabbert retired and I became Scientific Director, which in our institute is the Deputy Director. I hated this job. I did not feel mature enough to evaluate, judge, influence eminent scientists who knew so much more than I did. I worked hard, learnt a lot, and enormously widened my vision of biology. But all this dragged me away from research, from my laboratory, from my research group. I soon realized that I had to choose between science and the administration of science. I had promised Franc- ois Gros to help him during 3 years and to continue for the 3 other years of his term as Director only if I managed to keep my laboratory active. I completed my 3 years, resigned from my administrative position and went back to my laboratory and my teaching. Becoming the Scientific Director of this prestigious institute had been a tremendous honor. Serving as the Scientific Director taught me a lot about science, about the administration of science, and about scientists as human beings. In that position, I saw only people who had problems, complaints, unsatisfied ambitions, relation problems. I never saw the good scientists, happy with their status, with their laboratory, with their budget. I learnt from this experience that when you are happy with your fate, you should tell it to the people to whom you owe it. I also discovered how people’s attitudes could change depending on whether or not you have the power. From the very day I left my position as a Scientific Director, I became transparent again for many of my

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colleagues. They no longer came to shake hand or salute me when we met. Only a very small minority of those who had become so ‘‘friendly’’ when I was in business seemed to even notice my presence. Fortunately, a few solid, long lasting friendships highlighted this period, during which I discovered some shy, modest, yet fantastic personalities. At the Institut Pasteur, I spent all my time within 50 yards of my bench. Even when I became the Scientific Director, I kept my small office in the Unit of Cellular Biochemistry, created and previously chaired by Monsieur Monod. When Monsieur Monod became the Director of the institute, Georges Cohen took over the Direction of the unit. Agne`s Ullmann and I stayed in it unit, while most of its former members moved to the newly built Department of Molecular Biology. Georges Cohen’s unit later was split in two, one under his direction, and the other under Agne`s Ullmann’s direction. I joined that of Agne`s Ullmann, still in the same location. My three unit leaders, Monsieur Monod, Georges Cohen, and Agne`s Ullmann were fantastic persons. All of them gave me complete freedom. All of them trusted me in all respects. All of them took on their shoulders all the administrative burdens and made life so easy for me. Thanks to their generous protective attitude, I could concentrate entirely on my research and my teaching. But when Georges Cohen retired in 1989, I could no longer escape my responsibilities and had to become, in turn, the head of the Unit of Cellular Biochemistry. When Georges Cohen left, the office had to be renovated. I asked the architect to paint it the color that Monsieur Monod had chosen, to replace the wall-towall carpeting with one similar to the original, and to keep everything else exactly as it had been before. Except for the sofa and armchairs that could not be replaced with similar ones, the office found its original look again. When the renovation was completed, I stepped into it, locked the door, sat at Monsieur Monod’s desk during a long, very long time and meditated on the unpredictable, unplanned succession of events that had led me to sit there, head of Monsieur Monod’s unit, in his office. And I felt overwhelmed by the responsibility I was facing. In addition to being head of the Cellular Biochemistry Unit and Scientific Director, I had many responsibilities, which gave me a chance to somehow influence the Institut Pasteur’s policy. Thus, I served as Chairman of the Scientific Council, member and secretary of the Board of Administrators, and I sat on many


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committees. If one would want to define the trace I left on the life of the Pasteur, I think it would be the development of Physical Biochemistry in the institute. As the Scientific Director, I managed to attract Roberto Poljak and several of his collaborators to Pasteur, and fought to give them the space and equipment that enabled them to start and develop a superb X-ray crystallography group. I decided and organized the implementation of the first Central Computer Facility at Pasteur. I twisted the Direction’s arm to get the money for hooking our central computer onto ‘‘Bitnet,’’ the first electronic network – a precursor of the Webdesigned by IBM. With the help of my brother, a computer expert who worked at the Technion in Israel, my technician’s son wrote an emulation program to interface our non-IBM computer onto Bitnet. Pasteur was thus one of the very first academic centers in France to be on the net, and I got the first e-mail address at the Institut Pasteur. As chairman of the Scientific Council, I managed to attract Muriel Delepierre to the Institut Pasteur, where she implemented a strong NMR unit. As head of the INSERMPasteur Cytometry Facility, I introduced quantitative fluorescence analysis by means of flow-cytometry and fluorescence scanning microscopy. I brought Arnaud Blondel into the institute and helped him develop the first research group at the Institut Pasteur on molecular modeling. And several of the methods and equipments I introduced and run throughout the years in the laboratory have been transferred either to a technical platform (analytical ultracentrifugation, circular dichroism, and infrared spectroscopy) or taken over by Alain Chaffotte (fluorescence and circular-dichroism stopped-flows) and are still in use. Last but not least, I got involved in a fund raising institution, the Pasteur-Weizmann Council for Cancer Research, which aims at promoting and supporting effective scientific collaborations between the Institut Pasteur in Paris and the Weizmann Institute in Rehovot (Israel). Recalling that my decision to become a scientist was born during my visit to the Weizmann Institute in 1955, and that I am now its chairman, one might imagine that I have had an active role in the creation of this institution. Yet I have not. Pasteur-Weizmann was initiated as a result of a decision by the UNESCO to expel Israel from its General Assembly and to boycott Israel. Andre Lwoff, Professor at the Institut Pasteur and Nobel laureate, was shocked by this decision. He claimed that, because Israel was doing so much for

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education, art, culture, and science, and producing so many outstanding musicians, writers, painters, and scientists, Israel’s exclusion and boycott went against the very aims of the UNESCO. He angrily reacted against this decision by first creating a ‘‘Committee for the Universality of UNESCO’’ joined by many Nobel laureates and intellectuals. He then met Robert Parienti a young envoy of the Weizmann Institute. Robert had just settled in France to organize the fund raising for this institute in Europe. Building on a suggestion made by Mrs Simone Veil, then Ministry of Health of the French Government, Andre´ Lwoff and Robert Parienti came up with the idea of a fund raising institution that would not only collect funds, but also work on giving to the public opinion an image of the relations between France and Israel other than that of the freezing-cold diplomatic relations as reflected in the media. The Pasteur-Weizmann Council for Cancer Research was thus created in 1975, with the full support of the Director of the Institut Pasteur, Monsieur Monod, a faithful supporter of the Weizmann Institute and friend of many of its prominent scientists. Robert Parienti, with a fantastic creativity, organized galas, concerts, shows that not only brought in money to support research in the two institutes, but also greatly contributed to the reputation of the Weizmann Institute in France and Europe, and to the image of Israel. I became involved in the PasteurWeizmann Council only in 1988. Because the sums collected by Pasteur-Weizmann had become significant Raymond Dedonder, then Director of the Institut Pasteur, thought that it would be wise to codify the way in which the money collected would be distributed and shared between the two institutes. He called me in his office and told me that, because he was aware that I had many friends in the Weizmann Institute and knew how faithful I was to the Institut Pasteur, he wanted me to establish a charter to rule the scientific and financial interactions between the Institut Pasteur, the Weizmann Institute and the Pasteur-Weizmann Council. I accepted this mission but requested that a scientist from the Weizmann Institute be commissioned by his institute to work with me on this project. Benny Geiger and I thus established the Pasteur-Weizmann Charter. We defined the types of programs that would be supported: fellowships, symposia, scientific visits, and joint research projects. Funds provided by the PasteurWeizmann Council would be shared on a 50-50 basis between the two institutes. Evaluation committees were organized.


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The respective roles of the Board of Administration, the Scientific Council, and the Evaluation Committees of Pasteur-Weizmann were defined. Reports on the financial and scientific activities were written and discussed every year. This rigorous organization provided the activities of Pasteur-Weizmann with complete transparency. It gave to the two institutes as well as to our generous donors full confidence and enabled the PasteurWeizmann to develop and flourish. For several years, Benny Geiger and I helped implement the new organization. But when Michael Sela completed his two terms as Chairman of the Scientific Council of Pasteur-Weizmann in 1996, I was elected as his successor. And when in 2003 Franc- ois Gros completed his last term as Chairman of the Council’s Board of Administration, I took over this responsibility. Being involved in the PasteurWeizmann Council brought me a lot of satisfaction. Working with, and becoming friend with Robert Parienti has been a magnificent reward for the time I devoted to this institution. But also, meeting the many partners who help and support our action: our generous donors who, sometimes since the birth of Pasteur-Weizmann, bring us their financial contributions; artists who accept, often for no money, to participate in our galas; politicians whose presence highlights our meetings. Thanks to them, the reputation of Pasteur-Weizmann has crossed the boarders. Our Council is quoted as an outstanding example of a successful, long-lasting collaboration between scientific institutions. Spectacular advances have been made in several areas, such as cancer research and vaccinology. Friendly collaborations, that are lasting for over a decade, have been initiated between scientists in France and Israel, in spite of political divergences that have often separated their countries. Now that I am no longer active as a researcher, being able to help others work at such noble tasks is a mere blessing for me (Figure 7). And the circle thus closes. My interest in research started with a visit to the Weizmann Institute and I became a scientist at the Institut Pasteur. Much later, I became a member of the Administration Council of the Institut Pasteur and recently was elected member of the Board of Governors of the Weizmann Institute. All that I have done, and shall do, to help Pasteur and Weizmann take advantage of their remarkable complementarities will never suffice to repay these two top level research institutes for the fantastic life they offered me.

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Fig. 7. The author delivers the Gerhard Schmidt Memorial Lecture at the Weizmann Institute in June 2004. A visit to Gerhard Schmidt in 1955 was at the origin of the author’s decision to become a scientist.

Concluding Remarks When I was asked to write these ‘‘personal recollections’’ I wondered how I could fill the minimum of 50 pages requested by the editors. With my writing drawing to a close, pressed by the need to stay within the upper limit of 150 pages, I have had to omit so many things, so many events, so many people that yet have been so important in my professional life. I tried to mention every aspect of it: basic research, applied research, industrial projects, technical developments, teaching at the university, fund raising, and administration. I hope that, in spite of the heterogeneity of the activities and events related in these pages, and the lack of chronological order in the description of our various research projects, the reader will have perceived some features that were common to the different phases of my scientific life. To begin with, how passionate and happy I have been during these 45 years devoted to science. I was never interested in money and was fortunate enough that my wife and children considered my love for research more important to


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them than the luxury that more money would have given them. Only once was I briefly tempted. By the end of 1966, just before I defended my PhD thesis, I was approached by the Director of Research for Europe of British Petroleum (BP). BP had started a vast economical and humanitarian project aimed at producing steaks out of oil. Oil was cheap, then. The project was to grow bacteria on oil, feed cows with proteins extracted from these bacteria, and get meat from these cows. BP-Europe had already set up fermentation and protein extraction procedures and was about to build a pilot plant near Paris. They were trying to hire an engineer able to cope with bacterial growth and protein purification. My training as an engineer at the Ecole Polytechnique and my work on a bacterial protein for my PhD corresponded exactly to the profile they were looking for. I was extremely interested in BP’s project, not realizing that, with the rise in the cost of oil that was about to occur, the factory would have to work backwards, transforming steaks into oil y My salary as a young research assistant was 1,800 French Francs, equivalent to about 350 US $. To convince me of accepting the job, I was offered up to 20,000 French Francs, 11-fold my current salary. In spite of my interest for the project and for this fantastic salary, I refused the job. But on my way home, I anxiously questioned myself as to my right to deprive my wife – and the children we planned to have in a near future – of the materialistic comfort and security that BP’s offer would bring them. But Cećile’s choice was clear-cut and immediate. I should choose the work I like, not that which brings in more. I am grateful to her and convinced that our choice was the best. I was not interested by fame more than by money. I never planned a career. I never looked for a promotion, for responsibilities, for honors. All that guided me was my initial desire to study the mechanisms of life at a molecular level. But from the moment I entered the Institut Pasteur, I was dragged along by my curiosity and my will to answer questions, to solve problems, to overcome difficulties, one after the other. The lack of planning is certainly responsible for the apparent heterogeneity of the subjects I have been touching. I am not sure however that a carefully planned scientific strategy would necessarily have been more productive and/or interesting. And for sure, it would not have been as exciting. Each time I touched a new technique (analytical centrifugation, zonal centrifugation, rapid kinetics,

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flow cytometry, monoclonal antibodies, infrared spectroscopy) I felt like a kid discovering a new toy. Each time I entered a new scientific field (protein folding, aggregation, conformational changes, immunoreactivity, molten and pre-molten globules, prion action) I felt the same enthusiasm, the same avidity for understanding the underlying basic molecular mechanisms. And each time I was fortunate enough to walk one step forward on the way of understanding, I felt first an uncontrollable delight and then a quiet, profound satisfaction which lasted y until the next question came to my mind. It never lasted for more than 1 or 2 days. But the quest for these moments of deep happiness, the satisfaction of discovery, was certainly one of the essential driving forces that constantly pushed me ahead and never left me until I retired. Only now, looking back at the ensemble of my contributions to protein science, do I consider them as a whole and do I get the feeling that, as Arthur Kornberg stated at the end of my stay at Stanford, ‘‘After all you did achieve something, Michel.’’ I hope that the reader will also have sensed how important the influence of Monsieur Monod has been on me. The first questions I worked on were asked by Monsieur Monod. Those which came later were often inspired by his ideas. I took over his chair at the Paris University and tried to give my students as much attention, as much care, as much love as Monsieur Monod had given us. I tried to be as selfless, as generous, as helpful to my co-workers as he had been to me. I tried to contribute to the development of the Institut Pasteur and of the University with as much devotion and generosity as Monsieur Monod had offered them. Writing this account of my scientific life has given me the rare opportunity of acknowledging my immense debt to Monsieur Monod. It also gave me the opportunity to show how this immense scientist, who gained world fame for his determining contributions to understanding the regulation of gene expression – through the operon model – and the regulation of enzymatic activity – through the allostery model – also had a profound interest for the problem of protein folding. Without his personal incentives I probably would not have entered that game, and without his stimulating innovative ideas, I would not have brought most of my original contributions to this problem that nowadays stands at the forefront of modern biological research. Being trained by Monsieur Monod, inspired by him, working at his side for years, receiving his encouraging words – C’est bien


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mon petit Michel – and being able to rely on his example when difficult choices had to be made enlightened all my active life. Yes indeed, great men of that kind are rare and do a lot of good. It was my luck, my immense luck, that fate, or destiny, led me to his laboratory and allowed me to grow in his shade. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

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[15]

Douzou, P. (1980) Cryoenzymology in aqueous media. Adv. Enzymol. Relat. Areas. Mol. Biol. 51, 1–74. Cohen, R. and Mire, M. (1971) Analytical-band centrifugation of an active enzyme-substrate complex. 1. Principle and practice of the centrifugation. Eur. J. Biochem. 23, 267–275. Messelsohn, M. and Stahl, F.W. (1958) The replication of DNA in E. coli. Proc. Natl. Acad. Sci. USA 44, 671–682. Jacob, F. and Wollman, E. (1960) Sex induction and functional analysis in bacteria. C R Seances Soc. Biol. Fil. 154, 1960–1963. Monod, J., Changeux, J.P. and Jacob, F. (1963) Allosteric proteins and cellular control systems. J. Mol. Biol. 6, 306–329. Crick, F.H. and Orgel, L.E. (1964) The theory of inter-allelic complementation. J. Mol. Biol. 37, 161–165. Perrin, D. and Monod, J. (1963) On the reversibility by treatment with urea of the thermal inactivation of E. coli beta-D-galactosidase. Biochem. Biophys. Res. Commun. 12, 425–428. Carlton, B.C. and Yanofsky, C. (1963) Studies on the position of six amino acid substitutions in the tryptophan synthetase A protein. J. Biol. Chem. 238, 2390–2392. Goldberg, M.E. and Chaffotte, A.F. (2006) Undistorted structural analysis of soluble proteins by attenuated total reflectance infrared spectroscopy. Protein Sci. 14, 2781–2792. Goldberg, M.E., Creighton, T.E., Baldwin, R.L. and Yanofsky, C. (1966) Subunit structure of the tryptophan synthetase of Escherichia coli. J. Mol. Biol. 21, 71–82. Goldberg, M.E. and Baldwin, R.L. (1967) Interactions between the subunits of the tryptophan synthetase of Escherichia coli. Optical properties of an intermediate bound to the a2b2 complex. Biochemistry 6, 2113–2119. Goldberg, M.E., York, S. and Stryer, L. (1967) Fluorescence studies of substrate and subunit interactions in the b2 protein of Escherichia coli tryptophan synthetase. Biochemistry 7, 3662–3667. Ullmann, A., Goldberg, M.E., Perrin, D. and Monod, J. (1968) On the determination of molecular weights of proteins and protein subunits in the presence of 6 M guanidine hydrochloride. Biochemistry 7, 261–265. Hade, E.P. and Tanford, C. (1967) Isopiestic compositions as a measure of preferential interactions of macromolecules in two-component solvents. Application to proteins in concentrated aqueous cesium chloride and guanidine hydrochloride. J. Am. Chem. Soc. 89, 5034–5040. Tanford, C. (2003) Fifty years in the world of proteins. In Comprehensive Biochemistry, Vol. 42. Personal Recollections (Semenza, G. and Turner, A.J., eds.), VII, pp. 1–52. Amsterdam, Elsevier.

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[16] Ullmann, A., Perrin, D., Jacob, F. and Monod, J. (1965) Identification, by in vitro complementation and purification, of a peptide fraction of Escherichia coli beta-galactosidase. J. Mol. Biol. 12, 918–923. [17] Goldberg, M.E. and Edelstein, S.J. (1969) Sedimentation equilibrium of Paucidisperse systems. Subunit structure of complemented b-galactosidase. J. Mol. Biol. 46, 431–440. [18] Goldberg, M.E. (1969) Tertiary structure of Escherichia coli b-Dgalactosidase. J. Mol. Biol. 46, 441–446. [19] Wetlaufer, D.B. (1973) Nucleation, rapid folding, and globular intrachain regions in proteins. Proc. Natl. Acad. Sci. USA 70, 697–701. [20] Rossmann, M.G., Moras, D. and Olsen, K.W. (1974) Chemical and biological evolution of a nucleotide-binding protein. Nature 250, 194–199. [21] Jacobson, R.H., Zhang, X.-J., DuBose, R.F. and Matthews, B.W. (1994) Three-dimensional structure of beta-galactosidase from E. coli. Nature 369, 761–766. [22] Tsong, T.Y., Baldwin, R.L. and Elson, E.L. (1971) The sequential unfolding of ribonuclease A: Detection of a fast initial phase in the kinetics of unfolding. Proc. Natl. Acad. Sci. USA 68, 2712–2715. [23] Fisher, H.F. (1964) A limiting law relating the size and shape of protein molecules to their composition. PNAS 51, 1285–1291. [24] Changeux, J.P., Gerhart, J.C. and Schachman, H.K. (1968) Allosteric interactions in aspartate transcarbamylase. I. Binding of specific ligands to the native enzyme and its isolated subunits. Biochemistry 7, 531–538. [25] Raibaud, O. and Goldberg, M.E. (1973) Characterization of two complementary polypeptide chains obtained by proteolytis of rabbit muscle phosphorylase. Biochemistry 12, 5154–5160. [26] Fletterick, R.J., Sygusch, J., Semple, H. and Madsen, N.B. (1976) ˚ resolution and its ligand Structure of glycogen phosphorylase a at 3.0 A ˚ . J. Biol. Chem. 251, 6142–6146. binding sites at 6 A [27] Ho¨gberg-Raibaud, A. and Goldberg, M.E. (1977) Isolation and characterization of independently folding regions of the b chain of Escherichia coli tryptophan synthase. Biochemistry 16, 4014–4020. [28] Zetina, C.R. and Goldberg, M.E. (1980) Reversible unfolding of the b2 subunit of Escherichia coli tryptophan synthetase and its proteolytic fragments. J. Mol. Biol. 137, 401–414. [29] Zetina, C.R. and Goldberg, M.E. (1982) Kinetics of renaturation and selfassembly of intermediates on the pathway of folding of the b2-subunit of Escherichia coli tryptophan synthase. J. Mol. Biol. 157, 133–148. [30] Blond, S. and Goldberg, M.E. (1986) Kinetic characterization of early intermediates in the folding of Escherichia coli tryptophan synthase b2 subunit. Proteins: Structure, Function and Genetics 1, 247–255. [31] Blond, S. and Goldberg, M.E. (1987) Partly native epitopes are already present on early intermediates in the folding of tryptophan synthase. Proc. Natl. Acad. Sci. USA 84, 1147–1151. [32] Goldberg, M.E., Semisotnov, G.V., Friguet, B., Kuwajima, K., Ptitsyn, O.B. and Sugai, S. (1990) An early immunoreactive folding intermediate of the tryptophan synthase b2 subunit is a ‘‘Molten Globule’’. FEBS Lett. 263, 51–56. [33] Murry-Brelier, A. and Goldberg, M.E. (1989) Alternate succession of steps can lead to the folding of a multidomain oligomeric protein. Proteins: Structure, Function, and Genetics 6, 395–404.


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[34] Radford, S.E., Dobson, C.M. and Evans, P.A. (1992) The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature 358, 302–307. [35] Hyde, C.C., Ahmed, S.A., Padlan, E.A., Miles, E.W. and Davies, D.R. (1988) Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium. J. Biol. Chem. 263, 17857–17871. [36] Chaffotte, A., Guillou, Y., Delepierre, M., Hinz, H.J. and Goldberg, M.E. (1991) The isolated C-terminal (F2) fragment of the E. coli tryptophan synthase b2 subunit folds into a stable, organized non-native conformation. Biochemistry 30, 8067–8074. [37] Chaffotte, A., Cadieux, C., Guillou, Y. and Goldberg, M.E. (1992) A possible precursor to the ‘‘molten globule’’: The C-terminal proteolytic domain of tryptophan synthase b chains folds in less than 4 milliseconds into a hydrophobic condensed state with non native-like secondary structure. Biochemistry 31, 4303–4308. [38] Gast, K., Chaffotte, A.-F., Zirwer, D., Guillou, Y., Mueller-Frohne, M., Cadieux, C., Hodges, M., Damaschun, G. and Goldberg, M.E. (1997) Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase b chain. Protein Sci. 6, 2578–2588. [39] Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497. [40] Fedorov, A.N., Friguet, B., Djavadi-Ohaniance, L., Alakhov, Y.B. and Goldberg, M.E. (1992) Folding on the ribosome of Escherichia coli tryptophan synthase b subunit nascent chains probed with a conformation-dependent monoclonal antibody. J. Mol. Biol. 228, 351–358. [41] Friguet, B., Djavadi-Ohaniance, L. and Goldberg, M.E. (1989) Polypeptide-antibody binding mechanism: Conformational adaptation investigated by equilibrium and kinetic analysis. Res. Immunol. 140, 355–376. [42] Chaffotte, A.F., Guillou, Y. and Goldberg, M.E. (1992) Kinetic resolution of peptide bond and side chain far-UV circular dichroism during the folding of hen egg white lysozyme. Biochemistry 31, 9694–9702. [43] Jarrett, N.M., Djavadi-Ohaniance, L., Willson, R.C., Tachibana, H. and Goldberg, M.E. (2002) Immunochemical pulsed-labeling characterization of intermediates during hen lysozyme oxidative folding. Protein Sci. 11, 2584–2595. [44] Welker, E., Narayan, M., Wedemeyer, W.J. and Scheraga, H.A. (2001) Structural determinants of oxidative folding in proteins. Proc. Natl. Acad. Sci. USA 98, 2312–2316. [45] Elo¨ve, G.A., Chaffotte, A.F., Roder, H. and Goldberg, M.E. (1992) Early steps in cytochrome c folding probed by time-resolved circular dichroism and fluorescence spectroscopy. Biochemistry 31, 6876–6883. [46] Guijarro, J.I., Jackson, M., Chaffotte, A.F., Delepierre, M., Mantsch, H.H. and Goldberg, M.E. (1995) Protein folding intermediates with rapidly exchangeable amide protons contain authentic hydrogen bonded secondary structures. Biochemistry 34, 2998–3008. [47] Baldwin, T.O., Ziegler, M.M., Chaffotte, A.F. and Goldberg, M.E. (1993) Contribution of folding steps involving the individual subunits of bacterial Luciferase to the assembly of the active heterodimeric enzyme. J. Biol. Chem. 268, 10766–10772.

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[48] Friguet, B., Djavadi-Ohaniance, L., King, J. and Goldberg, M.E. (1994) In vitro and ribosome-bound folding intermediates of P22 tailspike protein detected with monoclonal antibodies. J. Biol. Chem. 269, 15945–15949. [49] Dam, J., Rose, T., Goldberg, M.E. and Blondel, A. (2000) Complementation between dimeric mutants as a probe of dimer-dimer interactions in tetrameric dihydrofolate reductase encoded by R67 plasmid of E. coli. J. Mol. Biol. 302, 235–250. [50] Blondel, A. and Dam, J. (2003) Impact of mutations on the association of R67 DHFR subunits: Can free energy calculation approach experimental accuracy and speed ? Protein Sci. 12(suppl. 1), 60. [51] Baldwin, R.L. (1995) The nature of protein folding pathways: the classical versus the new view. J. Biomol. NMR. 5, 103–109. [52] Ullmann, A. and Monod, J. (1969) On the effect of divalent cations and protein concentration upon renaturation of b-galactosidase from E. coli. Biochem. Biophys. Res. Com. 35, 35–42. [53] London, J., Skrzynia, C. and Goldberg, M.E. (1974) Renaturation Escherichia coli tryptophanase after exposure to 8M urea: Evidence for the existence of nucleation centers. Eur. J. Biochem. 47, 409–415. [54] Goldberg, M.E. and Zetina, C.R. (1980) Importance of interdomain interactions in the structure function and stability of the F1 and F2 domains isolated from the b2 subunit of E.coli tryptophan synthetase. In Protein Folding (Jaenicke, R., ed.), pp. 469–484. Amsterdam, New York, Elsevier/North-Holland Biomedical Press. [55] Sela, M. and Arnon, R. (1972) Reaction with N-carboxy-a-amino acid anhydrides. Methods Enzymol. 25 B, 553–558. [56] Orsini, G. and Goldberg, M.E. (1978) The renaturation of reduced chymotrypsinogen A in Guanidine Hcl – Refolding versus aggregation. J. Biol. Chem. 253, 3453–3458. [57] Rudolph, R., Zettlmeissl, G. and Jaenicke, R. (1979) Reconstitution of lactic dehydrogenase. Noncovalent aggregation vs. reactivation. 2. Reactivation of irreversibly denatured aggregates. Biochemistry 18, 5572–5575. [58] Goldberg, M.E., Rudolph, R. and Jaenicke, R. (1991) A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysosyme. Biochemistry 30, 2790–2797. [59] Taubes, G. (1996) Misfolding the way toward disease. Science 271, 1493–1495. [60] Goldberg, M. (1996) Selective interactions and protein polymorphism: From allosteric proteins to the prion. Bull. Inst. Pasteur. 94, 157–171. [61] Baron, B., Me´te´zeau, P., Kelly, F., Bernheim, A., Berger, R., Gueńet, J.L. and Goldberg, M.E. (1984) Flow cytometry isolation and improved visualization of sorted mouse chromosomes: Purification of chromosomes X and iso-1 from cell lines with Robertsonian translocations. Exp. Cell Res. 152, 220–230. [62] Baron, B., Me´te´zeau, P., Hatat, D., Roberts, C., Goldberg, M.E. and Bishop, C. (1986) Cloning of DNA libraries from mouse Y chromosomes purified by flow cytometry. Somatic Cell and Molecular Genetics 12, 289–295. [63] Sela, M. (2004) My world through science. In Comprehensive Biochemistry (Semenza, G. and Turner, A. J., eds.), Vol. 43, VIII of Personal Recollections, pp. 1–100.

V.P. Skulachev and G. Semenza (Eds.) Stories of Success – Personal Recollections. XI (Comprehensive Biochemistry Vol. 46) r 2008 Elsevier B.V. All rights reserved. DOI: 10.1016/S0069-8032(08)00003-X

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Chapter 3

Explosive Extracellular Matrix Research from 1960 to 2000. A Personal Recollection of the Work Pioneered by Rupert Timpl ¨ RGEN ENGEL JU Biozentrum University of Basel, Switzerland

Abstract During the period of my active research, I witnessed the introduction of new techniques and interdisciplinary approaches, which resulted in completely novel views of the structure and function of the extracellular matrix (ECM), which at this time was still called connective tissue. I memorize my shock and surprise when about 15 years ago my friend and colleague Darwin Prockop smuggled a few much older slides into an at this time modern research seminar, which I presented at his department. These slides originated from the time, when Darwin Prockop and I first met at one of the first ECM meeting in St. Margherita in 1966. The difference between the new research and the historical slides dramatically demonstrated the high pace of research. With these thoughts in mind I decided to write a chapter on the history of ECM research, which I experienced as a member of a group of scientists located in many different countries and disciplines, who all intensely collaborated with a pioneer in the field, Rupert Timpl. My personal review deals with the turn from a purely mechanical role of the connective tissue to the understanding of the importance of the ECM in cell differentiation and development. The discovery and characterization of a large repertoire of complex multifunctional proteins involved as major

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players in these events is described. Rupert passed away in October 2003 and this chapter is also a memorial to him. Keywords: Connective tissue; Laminin; Basement membrane; Collagen IV; Fibulin

Biographical Notes Rupert Timpl grew up at the hard time when Europe was deeply shaken by the World War II. He was born on March 4, 1936 in Friedrichsdorf near Iglau (Czech name Jihlava) in the Sudetenland. From 1918 to 1938 the region belonged to Czechoslovakia and in October 1938 it was annexed by Nazi-Germany. It was returned to Czechoslovakia after Germany lost the war. Up until 1946 the majority of the population of Sudetenland were Germans and the region had a mixed Austrian and German history. The other large group of people living there were of Czech origin, but Polish and Jiddish were also frequently spoken. Rupert’s father, Engelbert, was a merchant and his mother Anna was a tailor. In 1937 his father bought a shop in Reichenberg (Liberec). Soon after he was drawn into the war as a German soldier. In September 1939 the World War started with the invasion of Poland, first by German and then later by Soviet troops. The war, to date the largest in history, lasted almost 6 years and about 50 million people were killed. Engelbert Timpl was one of the victims in June 1943. In 1946, nearly the entire German population was expelled from the Sudetenland. The widow Anna Timpl was transfered with her children, her two younger sisters and her father, to Dehlitz an der Saale in East Germany. The mother of Anna, Rupert’s grandmother, her sister Mitzi Resek and her sister’s husband, Hans Resek, were expelled towards Austria. They later moved to Vienna, a fact which would be decisive for Rupert’s later life. In Dehlitz, Rupert’s mother somehow earned the money for a very modest life by tailoring dresses for the wives of nearby farmers. At this time food was rationed and sparse. It was very common that people worked for farmers and were paid by egg and other foodstuffs. The sister of Rupert’s mother worked in the ammonium production of the nearby Leuna Company in heavy 12-hour shifts. Rupert went to the very primitive village school of Dehlitz in which pupils of all ages were educated in a single

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classroom. The sole teacher of the school was named Herr Donner. Rupert’s sister Liesel Hoffmann remembers that he mentioned many years later that Rupert was his best student. After this elementary school, Rupert joined the high school of Droissig. Also there his unusual intelligence was recognized. During the last year students were allowed to go home after solving a mathematical problem and usually Rupert was the winner. I should mention that Rupert did not tell me much about his life up to the age of 15. Personally I know the town of Iglau only from the beautiful children’s book by Janosch, ‘‘Komm nach Iglau, Krokodil’’ (Come to Iglau, crocodile). This book certainly did not help me in writing these notes but Rupert’s second sister Liesel Hoffmann, who lives in Merseburg now, has kindly given me a lot of information and also contributed Figures 1 and 2. I assume from the limited information that I have on the family history, that Rupert’s youth was not easy. I remember my own almost permanent feeling of hunger during the last years of the war and the first post-war years. As a child, I felt the terror imposed on my parents and family that included a constant threat to life. This was probably not much different in Rupert’s case. Rupert certainly suffered from the early death of his father, the political pressure of the Nazis and the Czechs, being expelled from his birth place, and then the annoyances of life under the communist doctrine in the German Democratic Republic (DDR).

Fig. 1.

Rupert (about 4) with his mother Anna and sister Roswitha.

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Fig. 2.

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Rupert (about 14) with other voluntary helpers in farming at Ru¨gen, DDR.

The DDR claimed to be the state of workers and farmers and only these groups of people had easy access to higher education. Children of other groups had to serve in the army, police, or other services before an academic career could be started. Consequently, Rupert was not allowed to study chemistry although he passed his final examination at high school with high grades in 1954. He worked at Leuna for a year and applied a second time but his application was again turned down. I can easily imagine how upset Rupert was, knowing his impatience against subjective restrictions in later life. Rupert took a rather dramatic step and left the DDR. He joined his grandmother and her daughter, Mitzi Resek, in Vienna. Mitzi’s husband Hans was a blacksmith. Being refugees, the Reseks had restricted financial means that were insufficient to support a student fully. With their help, however, Rupert was able to study chemistry but every second term he had to work to earn money. His favorite job was at the Department of Clinical Chemistry at the Hanusch Hospital in Vienna. He loved the Vienna Opera House and enjoyed many performances with cheap tickets, with which one could only stand behind the last row. I still remember my own exhaustion from similar visits of the Opera in Berlin, which I experienced at about the same time. Rupert acquired his love for Austrian wines during this stay with his grandmother and the Resek family. Hans Resek was an expert in Gru ¨ ner Veltliner and his influence came through even


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to the times when I met Rupert in the 1960s. Rupert’s wine parties with lots of this wine were well known and much liked. Rupert became an Austrian in other ways too. He met his wife, Liane, in Vienna. Liane is born in Vienna and her German has the typical Vienna sound, which is well known from many songs and the actor Hans Moser. She brought a daughter, Nora, to the young Timpl family. Their son Peter was born in 1963. Rupert naturalized as Austrian and when I met him later he was always very proud of his home country. One of his favorite phrases was that the main aim of all Austrians is to reach a very early retirement and then enjoy life. I do not know whether this is true, but in any case Rupert did not follow this philosophy at all. I should also mention that Rupert was very active during the time of the revolt in Hungary against the Soviet rule. He was member of a political group of students that helped Hungarian refugees to settle in Austria. Another important experience was a study period in Munich from 1958 to 1960. In the university’s domicile he met Helmut Langer, who became his closest friend throughout his life. Helmut is still full of memories of long joint excursions with a motorbike. Other close friends of Rupert from this time are Heinz Furthmayr, now retired professor of pathology at Stanford University, Udo Becker, Hans Nowak, and Klaus von der Mark, Professor of Medicine at the University in Erlangen.

Training in Chemistry and First Contacts with Biology We have seen that Rupert managed to study chemistry overcoming severe difficulties. He also became exposed to clinical chemistry by working in a hospital. At this time, chemistry and physics were good choices for students interested in quantitative molecular biology. Like Rupert, whom I did not know at this time, I also started to study chemistry at about the same time as he did, first in Berlin and, after the second term, in Munich. In Europe research had suffered very much from the war. Very many leading scientists had left Germany and Austria, were forced to emigrate, or were killed in concentration camps because they were Jews or did not follow the Nazi doctrine. When Rupert and I started our university training 10 years after the war some recovery was visible but at many places conditions were still very bad and research was frequently old-fashioned.

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Most of the exiting science news came from the United States. At this time great progress was made in biochemistry. American medical schools were leading, often with new directions started under the influence of European immigrants. As an exception from the rule already shortly after the war outstanding research was done by Fedor Lynen in Munich. He explored the fatty acid metabolism and established a biochemistry unit in the department of chemistry at which I received my basic training. In Vienna, Hans Tuppy started an innovative modern group and was appointed head of the Biochemistry Department of the Vienna Medical Faculty in 1963. In Graz at the Institute of Physical Chemistry, Otto Kratky was gaining international reputation with structural studies on biological macromolecules by small angle X-ray scattering. At the same institute Rupert worked for his diploma from 1961 to 1962. In 1966 Rupert earned his PhD from the University of Graz, officially supervised by O. Kratky but reporting his work at weekly intervals to H. Tuppy and G. Riethmu ¨ ller, who is now Professor of Immunology at the University of Munich. I guess, that the deal with Graz was due old-fashioned rules of the medical faculty, not allowing promotion of chemistry students. Rupert told me about the exiting literature seminars at Tuppy’s department dealing with the elucidation of the domain organization of immunoglobulins by Rodney Porter and Gerald Edelman. I recall a visit of these two later Nobel laureates during my post-doctoral time in Israel and my own excitement about their fantastic achievements. Rupert received a fellowship of the Austrian Research Council from 1966 to 1967 and was research assistant at the Department of Immunology from 1967 to 1969 together with Heinz Furthmayr. Decisive ideas for his later research work developed already earlier during his work at the Hanusch Hospital from 1961 to 1966, at which he spend a part-time position during his chemistry studies. There Carl Steffen, professor of pathology was assembling a group of connective tissue immunologists. In 1965, this small group, which at this time consisted of Rupert and another biochemist, Ivar Wolf, was joined by Heinz Furthmayr and Georg Wick, now professor in Innsbruck. Georg Wick is an early collaborator and friend of Rupert and he describes him as follows [1]: The group was headed by the young eager beaver Rupert, at this time still a graduate student who already had an impressive list of highly cited publications and – typically for him – also supervised


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three postdocs, me being one of them. This graduate student was short, lightweight, heavily smoking, not reluctant to drink a pint of beer, completely up to date in his field, and always open for new ideas, although Rupert himself was certainly the most important generator of such ideas in the whole group. The 16 first publications of Rupert mentioned by Georg Wick were all published before 1966. These papers deal with immunological assays, epitope determinations, and, most importantly for the future work, with the immunology of collagen.

The State of the Art of Matrix Research in the 1960s Before turning to Rupert’s achievements in matrix research, I should give a short account of the knowledge from which it started. The period of 1960–1970 was a great time for quantitative biochemistry on a molecular level. I remember my enthusiasm when I learned about the atomic details of the structure of proteins from the work of Max Perutz [2] and John Kendrew [3]. I had to review the structure of hemoglobin in the literature club of the Max-Planck-Institute of Protein Research in which I worked as a PhD student. Many methods such as protein X-ray crystallography had been developed and opened our eyes to completely new features. Protein sequencing by the Edmann method had just started. On the other hand, one has to be aware that many methods which are daily tools in today’s research were still missing. Cloning, sequencing on a DNA level, and even SDS-PAGE electrophoresis were unknown. It is also difficult to recall that computers were still in their infancy and computerbased libraries did not exist. I remember endless calculations of ultracentrifugal results with a mechanical calculator and my time-consuming attempts to keep up with the literature with the help of manually punched cards. In 1960 the term extracellular matrix was not common and instead most people talked about connective tissue. The name indicated a purely mechanical function. Typical connective tissues are bone, cartilage, tendon, skin, and blood vessels. The material of the connective tissue was seen as the ‘‘ground substance’’ between cells and some scientist wrote about the ‘‘cement’’ between cells. Not much was known about the composition of this cement.

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In the late 1960s, collagen, elastin, and mucopolysaccharides were known as components of the connective tissue. A popular textbook of this time describes the extracellular matrix as follows [4]: The mechanical and supportive functions of connective tissue are accomplished by extracellular, insoluble protein fibers embedded in a matrix termed the ground substance. There was no evidence for more than one collagen and only short collagen-derived peptides had been sequenced, which demonstrated the Gly-X-Y-repeat in which the amino acid residues in X and Y position are frequently proline or hydroxyproline. It was assumed that the collagen extracted from tissues is the collagen secreted from the cells. Nothing was known about possible precursor forms. However a soluble form of collagen had been extracted by acid buffers and was named procollagen, under the misapprehension that this was a biosynthetic precursor. Pioneering work showed that collagen was composed of three polypeptide chains [5]. The principal structural arrangement of the assembled triple helix was explored by fiber diffraction of tendon [6–8]. More accurate crystal structures were not known at this time. The assembly of ‘‘collagen,’’ which was later identified as the fiber-forming collagen type I, had been explored by electron microscopy and the principle mode of fibril formation was understood [9,10]. The process of covalent cross-linking of collagen into insoluble networks was of central interest, and was believed to be a major impact of aging. It was also known that connective tissue, collagen in particular, was involved in diseases such as rheumatism, arthritis, and heritable disorders, but no details were known. Elastin, which was described as the second major protein of connective tissue had been discovered in the ‘‘yellow ligamentum nuchae,’’ an elastic ligament connected to the spinal bones. Short regions of the unusual sequence of elastin were known. Its rubber-like properties and covalent cross-links were well studied and its contribution to the elastic properties of, for example, blood vessels was correctly appreciated [11]. The mucopolysaccharides attracted much interest but it was unknown that most of them are attached to proteins, which nowadays are known as proteoglycans. In view of the limited knowledge of matrix components many researchers favored a rather simplified model in which collagen fibers and elastin were


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embedded in a ground substance of mucopolysaccharides. Collagen and elastin were believed to provide the mechanical properties and the polysaccharides to act as a kind of lubricant. In this early literature little can be found about the interactions of the matrix with cells or of any guidance functions in tissue development and embryogenesis. Information on cellular receptor molecules for matrix proteins was completely missing. Interestingly, however, the glycoprotein fibronectin, which is one of the major players in cell–matrix interactions, was already intensely studied by a well-known biochemist of this time, John Edsall [12]. He was convinced that the high molecular weight ‘‘cold insoluble protein’’ (an alternative name for fibronectin) was very important but he only knew the protein in its plasma form. Its location in the extracellular matrix and association with cells was not discovered until later [13,14]. Following pioneering work by Robert Garrone, who found a collagen in sponges [15,16], developmental biologists recognized early on that collagens are phylogenetically very old. Today it is known that early evolutionary origin and conservation is a property of many extracellular matrix components. A surprising match of matrix components in flies, worms, and mammalian has been demonstrated, suggesting common functions in spite of large differences in organization and mechanical functions [17]. Along these lines, the zoologist Volker Schmid working on hydromedusae [18] began to believe that the abundant collagen and other matrix components of these animals might have a function in cell differentiation and trans-differentiation during regeneration. He told me that he mentioned this idea in a grant application in the 1960s. The concept was turned down by the reviewers, who believed in the restricted mechanical function of the connective tissue. In contrast, reviewers of today would severely criticize an application that did not include the major role of the extracellular matrix in cell differentiation.

From Immunology to the Complexity and Structure of the Extracellular Matrix As mentioned already in section Training in chemistry and first contacts with biology, Rupert started to investigate the immunological properties of collagen in 1962. His first papers on this topic

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originate from his stay at the Hanusch Krankenhaus in Vienna and then for a short period at the new Institute of Immunology at the University of Vienna, were published with Carl Steffen, Georg Wick, Heinz Furthmayr, and others. The work soon led him into the unexpected complexity of the collagen structure and the fact that there is more than one collagen. He also collected evidence for other matrix proteins and the matrix heterogeneity of different tissues. In 1969 he decided to join the leading group for matrix research, built at this time around Wolfgang Grassmann and Klaus Ku ¨ hn at the Max-Planck-Institut for Protein and Leather Research in Munich. This institute had been founded in Regensburg as one of the Kaiser-Wilhelm-Institutes devoted to applied research, namely leather and tanning. It was established as a Max-Planck Institute in Munich in 1965 and contained modern research groups such as peptide synthesis (headed by Erich Wu ¨ nsch), X-ray crystallography (headed by Wolfgang Hoppe), and electrophoresis (headed by Kurt Hannig). The institute was well equipped, for example, with an analytical ultracentrifuge at which I worked for my PhD. Rupert began with a career development position in the research group of Klaus Ku ¨ hn, who had joined the institute from Heidelberg. Before an electron microscope was available at the Max-Planck-Institut in Munich, Klaus Ku ¨ hn was performing outstanding electron microscopic studies on the organization of collagen fibers [10] in Heidelberg. Klaus Ku ¨ hn also started the sequencing of collagens by the Edmann degradation method. In this environment, Rupert found a fantastic platform to develop his many ideas. The close interplay between Rupert Timpl and Klaus Ku ¨ hn that developed at this time and extended for more than 40 years, turned out to be extremely fruitful. I met Rupert Timpl for the first time in 1969 at the MaxPlanck-Institute for Protein Research. Before he arrived I was also engaged with collagen but I was mainly interested in the folding of the collagen triple helix from its three unfolded chains. My PhD work was done under the joint supervision of Georg Maria Schwab, professor of Physical Chemist at Munich University, and Wolfgang Grassmann, the head of the Max-Planck Institute. In 1965 I returned to this institute from a post-doctoral time with Arieh Berger and Ephraim Katchalski at the Weizmann Institute of Science and later with Gerhard Schwarz and Manfred Eigen at the Max-Planck-Institute for Physical Chemistry.


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In 1967 I was promoted to Privatdozent (lecturer) and directed a small research group when Rupert joint the institute. At this time Chris Anfinsen had just published his famous finding that the three-dimensional structure of ribonuclease is determined by its primary sequence and is formed without additional information. I found that this also holds true for collagen, a finding which sounds very trivial today but excited us a lot at the time. Together with Klaus Ku ¨ hn we also found that renatured collagen can also reform collagen fibers. Klaus Ku ¨ hn and I however faced the problem that, in contast to ribonuclease, refolding was never complete, a point which would be resolved 10–15 years later in collaboration with Rupert Timpl. I do not remember our first contacts but I recall the immediate deep interest Rupert showed in my work. I mention this because it was my first encounter with a very typical feature of Rupert’s scientific interest. He was never turned back by unfamiliar approaches and complex techniques, in my case biophysical techniques such as ultracentrifugation, light scattering, and spectroscopy. Instead he was strongly attracted by a scientific idea and problem and new possibilities to explore them. Later I realized that this broad interest and willingness to accept very different approaches opened the way to integrate scientists of different backgrounds for a joint problem. Already at this time Rupert was full of ideas and started to stimulate my own work in a decisive way. We will see in the coming sections that this happened with many other scientists. At the top of his career Rupert was collaborating with a large group of scientists all over the world. Perhaps Georg Wick, Heinz Furthmayr, and I may be looked at as first members of what I may call the ‘‘Rupert Club.’’ My first joint work with Rupert was on the physical properties of the amino-terminal precursor specific portion of type I procollagen [19] and on conformationally distinct domains in the amino-terminal segment of type III procollagen and its rapid triple helix-coil transition [20]. This work was followed rapidly by two important publications on the folding of type III collagen [21,22]. At this time it was realized that the folding of collagens occurs at the level of much larger precursor forms, which contain N- and C-terminal domains. The C-terminal domains, called C-propeptides connect the three chains in a proper alignment (two a1-chains and one a2-chain in type I collagen, three identical chains in type III collagen). This critical step initiates nucleation

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of triple helix formation at the correct site and abolishes the formation of wrong refolding products with misaligned chains. Interestingly, in procollagen type III a disulfide knot is formed at the start of refolding which helps to connect and align the three chains. Type III collagen is therefore a convenient model for folding studies without the problems of partial folding which I faced during my PhD thesis (see above). Our work led to the discovery of cis-trans isomerization of peptide bonds as rate limiting steps in collagen folding and to clear proof that folding starts at the C-terminus and zippers through to the N-terminus. Before this work was started, I had moved in 1972 to Basel as Professor of Biophysical Chemistry at the newly established Biozentrum. Two highly gifted PhD students Peter Bruckner ¨chinger (now professor in Mu ¨ nster, Germany) and Hans Peter Ba (now Professor in Portland, Oregon) joined my group in Basel. We kept in close touch with Rupert, who was still in Munich, interrupted by a visiting professorships at Rutgers Medical School in the laboratory of Darwin Prockop, Piscataway, USA and a short but disappointing professorship at the Medical University of South Carolina. In 1972 the Max-Planck Institutes for Protein and Leather Research had been merged with the Institute for Biochemistry and Cell Chemistry to form a new, very modern Institute of Biochemistry in Martinsried near Munich. This institute contained many new departments and the one for Connective Tissue Research was headed by Klaus Ku ¨ hn. Here Rupert defined a sequential antigenetic determinant in the non-helical regions of collagen [23,24]. In my opinion this was the first sequential determinant identified in a protein and compares with the results by the group of M. Sela with synthetic determinants obtained at about the same time [25]. In our joint work with Rupert, the biophysical work was mainly ¨chinger in Basel but done by Peter Bruckner and Hans Peter Ba this was only made possible by the fragments of collagens that were prepared by Rupert and his coworkers in high purity and sufficient amounts for biophysical work. Here I am touching on another very important side of Rupert. He was extremely good and careful in what he called the ‘‘Handwerk,’’ namely the application of analytical and preparative tools. A slogan, which he often repeated was ‘‘Die meiste Wissenschaft ist Handwerk (most of science is manual skill).’’ This statement is a typical


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exaggeration of Rupert’s because his science was mainly based on ingenious concepts. However, the technical side was an important reason for his success. He adapted the most modern methods of biochemistry and later molecular biology, but he also maintained high standards of immunology. He prepared purified antibodies against all of the many proteins and protein fragments prepared in his lab and the radio-immuno assay remained a standard technique in his lab. Even at the top of his career he evaluated all these assays himself and kept accurate records of all the compounds and antibodies. A numbering system helped to trace differences in preparation and storage time, and helped to explain differences in experimental results. Generously he supplied other groups with these reagents. It might be interesting to investigate how much of the progress in extracellular matrix research is based on Rupert’s reagents, even in those cases in which Rupert was not directly involved.

The Basement Membrane Proteins Collagen IV and the Discovery of Laminin I think that it was in summer 1978 that George Martin from the National Institute of Health brought a transplantable mouse tumor, the EHS (Engelbreth-Holm-Swarm) sarcoma to Martinsried. This tumor produced an unidentified extracellular matrix material. It was very fortunate for matrix biology that Rupert and George Martin decided to analyze this matrix in great detail. They found out that the tumor produced a matrix essentially like that of basement membranes, but at much higher yields than could be purified from normal tissues. Basement membranes are welldefined protein layers, which underlie the epithelial cells in skin and many other tissues. Rupert and his group were able to isolate the basement membrane protein collagen type IV from the EHS tumor in much larger amounts than previously possible from placenta tissue [26]. In this way it was possible to explore the structure of this unusual collagen by careful investigations of various fragments or domains and electron microscopic visualization. The main collaborator of Rupert in establishing the now famous network model [27] was Klaus Ku ¨ hn but we also contributed biophysical data on some of the fragments. I consider the basement membrane collagen work to be a major advance in

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matrix research. It had a major impact on the understanding of basement membranes. For basement membranes, the intimate contact between cells and the matrix is visible already at a structural level. It was already known that basement membranes such as Reichert’s membrane play a role in embryogenesis and tissue development. This work may therefore be considered an important link from protein biochemistry to cell biology and developmental biology. A second step of even higher importance was the discovery and isolation of the major glycoprotein of basement membranes, which Rupert and George Martin named laminin [28]. This publication has been referenced several 1,000 times, mainly because of the impact of laminin in developmental biology, morphogenesis, neurobiology, and medicine. It was recently reprinted as a classics in a special issue of J. Biol. Chemistry (http://www.jbc.org). Today the laminin family has grown to nearly 20 members and the first laminin discovered in 1979 is now called laminin 1, or in the most modern nomenclature laminin 111 [29]. Of course all this remained unknown in 1979, but Rupert already had a very strong feeling that this unusually large protein might be of outstanding importance. I remember how excited he was shortly after its discovery. Perhaps this feeling was based on the new findings for another giant glycoprotein, fibronectin, the cold insoluble globulin of John Edsall [12], whose first function in cell biology had just been explored [14,30]. Rupert’s vision was that the extracellular matrix has a major influence on many facets of cellular behavior and that laminin and other still unknown proteins play a key role. Clearly, his thinking was not limited to a mechanical function of the connective tissue. Our first task was the elucidation of the structure and domain organization of the giant laminin molecule. Rupert gave full power in his own lab but typically he also engaged his friends. The first electron micrographs were obtained by Heinz Furthmayr at Yale University. Heinz applied rotary shadowing with heavy metals first developed for DNA and adapted by Dan Branton at Harvard to spectrin, to the thread-like proteins of laminin, fibronectin, collagen IV, and many others. Surprisingly, it was shown that laminin has the shape of a Greek cross with three short arms of about 35 nm length and a 77 nm long arm [31]. Rodlike regions and globular regions could be distinguished in the


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short arms and at the terminus of the long arm. I recall our excitement about the pictures and the many questions, which we tried to answer. How can a protein form a cross? What are the rod-like regions? Are the globular structures seen in the electron micrographs real or artifacts of the technique? The problem with rotary shadowing is that the decoration of the specimen with metal crystallites leads to blown-up diameters, thus only the length dimensions are correct. In view of the routine computer-aided image reconstruction methods of today it is amusing to remember my own approach at this time. I manually rebuilt Heinz’s many laminin micrographs by wire. The problem in averaging was the flexibility of the arms, which led to the molecules being imaged in many different shapes. I straightened my wire models and superimposed them all in the same conformation. In this way the real globular domains superimposed and the artificial did not. It is still difficult to believe that the model derived in this way was near to correctness, as proven by much later studies. From gel-chromatography and analytical ultracentrifugation we also knew that laminin consists of three different chains. The main problem remaining was of how these chains connected to form a cross. The preliminary model of laminin suggested that the protein consisted of many different domains. With the techniques of the time, Rupert approached this by fragmentation with different proteases [32]. The fragments derived by limited digestion with pepsin, elastase, cathepsin, and others proved to be very useful for the first functional studies. Their localization in the intact laminin molecule were derived from their electron microscopic shape, partial sequencing, and immunology. The affinity purified antibodies against these fragment were of utmost importance for functional work. To appreciate these achievements we should remember that the sequences of laminin were unknown and that cDNA sequencing and recombinant expression of domains became possible only many years later. There are many reviews on laminin and the full scientific story will not be repeated here. It should only be mentioned that the most important contribution to the understanding of the full domain organization was the cDNA derived sequences of the three chains of laminin 1 by M. Sasaki in Yoshi Yamada’s and George Martin’s lab [33–35]. Sequence data, electron microscopy,

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and other information complemented each other and led to a detailed model of laminin and the recognition of its protein domains, which included domains already seen in other proteins and a large number of laminin-specific domains [36]. This was the beginning of the general recognition that complex multifunctional proteins, including those of the extracellular matrix, consist of many domains that may have independent functions. Homologs of these domains are frequently repeated, either in the same molecule or in other proteins [37,38]. A current model of laminin 1 is shown in Figure 3 together with some functional data, which were obtained much later. The figure also indicates the very recently solved crystallographic structures of some of the domains. a6b1 bind to the LG-domains located at the C-terminus of the a1-chain at the end of the long arm. The inserts show atomic structures of selected regions (with kind permission by Sasaki et al. [39]).

Discovery of Laminin’s Cell Biological Activities As mentioned above, it was Rupert’s vision that laminin and other basement membrane proteins provide signals for cell differentiation thus guiding morphogenesis. Shortly after the discovery of laminin he therefore got in touch with scientists who employed relevant experimental test systems. The new members of the Rupert Club were the Finnish developmental biologist Peter Ekblom, at the Friedrich-Miescher-Laboratorium in Tu ¨ bingen, and David Edgar, a British neurobiologist who worked in the Max-Planck Institute for Psychiatry that was located close to Rupert’s laboratory. Peter Ekblom was an expert in kidney development. With Rupert’s antisera against laminin 1 he collected indirect evidence for a role of laminin in the formation of embryonic kidney epithelium by immuno-staining. This was published in 1980 [40]. With the help of various fragments and domains of laminin it was later shown that the activity is located in the carboxyterminal LG-domains of the laminin a-chain that are located at the tip of the long arm [41,42] (Figure 3). The fascinating story of the role of laminin and other matrix proteins in mesenchymal induction led to a large number of publications in Peter Ekblom’s lab.


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Fig. 3. Model of laminin 1. The three chains a1, b1, and g1 are linked by a three-stranded coiled-coil domain in the long arm of the cruciform protein. Nidogen/entactin is linked to an EGF-like domain of the g1-chain and integrin. The integrin a6b1 bind to the LG-domains located at the C-terminus of the a1-hain at the end of the long arm. The inserts show atomic structures of selected regions (with kind permission by Sasaki et al.[39]).

He died at a rather young age 2 years ago, but related work is being continued in many other laboratories. The second discovery, which made laminin famous in cell biology, was the discovery of its neurite outgrowth-inducing

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activity. The pioneering work was published in 1984 [43] and today neurobiological studies with laminins number many thousands. Rupert maintained a close and friendly relationship with Peter Ekblom, who became professor in Uppsala and Lund and David Edgar, who is now a Professor in Liverpool. Much additional work was done through close collaborations. In the present biography, which focuses on the history of Rupert’s research, it is impossible to cite all this work and I wish to direct the attention of those interested to modern research reviews.

Basement Membrane Proteoglycans Rupert systematically explored the EHS tumor matrix for more basement membrane proteins. The first proteoglycans were isolated by S. Fujiwara [44] and a low and high density proteoglycan were studied in collaboration with our lab in Basel. Mats Paulsson arrived from Dick Heinegard’s group in Lund to join Rupert’s lab in the beginning of 1980. He was an expert on cartilage and its proteoglycans and contributed know-how and new techniques to the laboratory. The low density proteoglycan of basement membranes was studied by him in collaboration with a group in the USA headed by Peter Yurchenco and with us in Basel [45]. This proteoglycan was later called perlecan. Perlecan is an abundant and important component of basement membranes and many research groups are still engaged in its exploration. The core protein is a huge multidomain protein with some similarities to laminin. Based on excellent experiments by Hanna Wiedemann (the electron microscopist in Klaus Ku ¨ hn’s and Rupert’s lab) [42], it was now possible to visualize individual sugar chains by the rotary shadowing technique. This rendered it possible to observe the three very long glycosaminoglycan chains of perlecan [45] and to derive detailed models of the cartilage proteoglycan, aggrecan [46]. Perlecan, laminin, and collagen IV are the major components of a commercial product, Matrigel, which is extensively used as a substratum or three-dimensional matrix for cell adhesion [47]. George Martin often demonstrated the astonishing self-organization of Matrigel to friends by a simple warming and cooling experiment. We could see how the solution solidified to a rigid gel, which could be reversibly destroyed and reformed by shaking. The little experiment was a nice demonstration of


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network formation by large multidomain proteins and proteoglycans of the basement membrane, and the extracellular matrix in general. Extensive studies of the calcium dependent self-assembly of laminin and other basement membrane components were started in Peter Yurchenco’s laboratory at this time. Today the function of the extracellular matrix in large networks or machines is well recognized [48].

Nidogen/Entactin, Collagen VI, and BM-40/SPARC In 1981 a very important additional basement membrane protein was isolated from a cell culture and named entactin [49]. An apparent molecular weight of 160 kD was reported and immunostaining showed its occurrence in kidney glomeruli. Three years later Rupert isolated a protein of 80 kD from the EHS tumor, established a multidomain model, demonstrated its occurrence in basement membranes and called it nidogen [50]. With time and the start of DNA sequencing it became clear that the two proteins were identical. The two names developed into a very controversial issue. Even today it is possible to define the relations of researchers to Rupert by observing which of the names they use. Researchers close to Rupert call it nidogen, the wider members of the Rupert Club nidogen/entactin (or the other way around), and people who oppose Rupert just call it entactin. It is a general phenomenon that deep emotions are attached to names of proteins. The name connects the protein to the name of the discoverer. Frequently names reflect different opinions on putative functions or depend on the species in which a protein was discovered. The many names used for the same protein always bothered me a lot because of my bad memory for names and also because of the confusion which was thus created. The peak of confusion is reached when fancy new names are introduced for fragments or domains of well-known proteins. To return to nidogen/entactin, an important finding in Rupert’s group was a specific and very tight complex of nidogen with laminin [51]. It forms a fourth short arm of laminin 1 and Rupert and I discussed the possibility of calling it a fourth chain of laminin. Fortunately this nomenclature was not invented because many laminins turned out not to contain nidogen/entactin. Also in Drosophila, which contains a number of laminins [52,53] and a

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putative nidogen/entactin no interaction between the two proteins was observed. Rupert and his group invested intensive research to elucidate the domain organization of nidogen. Together with Mon-Li Chu nidogen was sequenced by the cDNA method [54]. This work began in Darwin Prockop’s laboratory in which Rupert was a frequent guest. He (and I) were visiting professors at Darwin’s department at Thomas Jefferson University in Philadelphia in 1987 and owe him a lot of support and friendship. I remember Rupert sitting in the hallway of the department, the only place in which smoking was permitted, identifying clones of nidogen with the help of a coin. He claimed that the eagle side of the coin indicates that the clone was a true one. Fortunately Mon-Li addressed the problem in her serious and patient way and at the end of long joint efforts they would win the Max-Planck Research Award together in 1991. Very beautiful work was also carried out in an investigation of the localization and nature of the nidogen binding site in laminin [55]. This culminated in the first crystallographic structure of a laminin domain (see section Ringberg and other meetings). Rupert and his group also found that nidogen/entactin interacted with collagen IV and perlecan. Rupert therefore considered nidogen/entactin to be a central linker molecule in basement membranes. The function of nidogen/entactin is probably more sophisticated as shown by functional knock-outs of the nidogen binding site in laminin [56] and knock outs of the nidogens, which have grown to two members [57]. During the same period Rupert’s group collaborated with the Philadelphia group and our group in Basel to investigate the structure of collagen type VI. This collagen is again a complex multidomain protein with collagenous domains and a number of other domains that are also found in Von Willebrand factor and pancreatic trypsin inhibitor. The work accomplished was very fascinating and was published in many joint publications. Since collagen VI is not in direct contact with the basement membrane, I leave it with a single reference [58]. In his systematic search for new basement membrane proteins Rupert found a small protein BM-40 of molar mass 40 kD [59], which would occupy our efforts for quite a long time. It became apparent shortly after that the protein was identical with osteonectin/SPARC (secreted protein acidic and rich in cysteine) [60], a protein which was not considered to be an integral member


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of basement membranes and for which many other functions have been proposed. In 1987, I joined the laboratory of Brigid Hogan in London, who discovered SPARC together with Helen Sage. Helene Sage continues to devote a lot of her research to this protein in Seattle. Based on the sequence and the disulfide pattern of BM-40/SPARC we were able to establish a working model of its structure and calcium binding sites [61]. In particularly we recognized a single EF-hand motif but were unable to find its partner according to established EF-hand motifs. R. H. Kretsinger, who was at this time the pope of calcium binding EF-hands, wrote to me that a partner motif was needed for calcium binding, and suspected that my assignment of the first EF-hand was erroneous. This argument was supported by the fact that no EF-hand had been found in extracellular proteins before. All the high affinity EF-hand related calcium binding sites were believed to be located in cytosolic proteins such as calmodulins and many others. On the other hand, Patrik Maurer in my laboratory produced numerous experiments proving a high-affinity binding site for calcium. The puzzle would only be resolved by the crystal structure of SPARC/BM-40 (see section Domains and interacting proteins at atomic resolution).

Ringberg and Other Meetings The continuous exchange of ideas was very important for the reseach program headed by Rupert. Our research achievements were soon internationally recognized and we were frequently attending international meetings. The Gordon Research Conferences for Collagens and that for Basement Membranes were extremely fruitful. The Basement Membrane Conferences organized by Darwin Prockop and the Harden conference in Manchester also rank high in my memories. The most important meetings for us, however, were the meetings and conferences at Schloss Ringberg. These took place in December, a few days before Christmas each year. Because of the unusual time of the year, Klaus Ku ¨ hn and Rupert managed to reserve dates for this meetings at this otherwise very busy meeting center. The meetings were much liked by all members of Rupert’s group in Munich but also by the invited guests from outside. It is very

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typical of Rupert’s and Klaus’s philosophy of close collaboration that several people from other groups attended very regularly. These were Peter Bruckner, his wife Leena Tudermann, Klaus von der Mark, and myself. Thomas Krieg, Monique Aumilley and Mats Paulsson also attended Ringberg meetings for a long time after leaving Munich. Rupert also invited the technical staff to his Ringberg group meetings. These included Hanna Wiedemann, who was responsible for electron microscopy, Mischa Reiter, specialist for radio-immune assays, Vera van Delden, who was the soul of Rupert’s group carrying out most of the extractions and isolations. Schloss Ringberg is located near Tegernsee in Bavaria, about 1 hour drive from Munich. The castle has a strange history. Although it looks very ancient its founding stone was laid in 1912 by Herzog Luitpold in Bayern. Luitpold was a distant descendant of the kings of Bavaria, after the kingdom was long abolished. He studied History of art and painting at the Academy in Munich together with his friend, the painter Friedrich Attenhuber. The two friends designed the castle. Attenhuber mostly styled the interior, the furniture, lamps, and other household items and contributed a large collection of his pictures. The World War II interrupted all the activities, leaving the castle not yet ready for living in. Strangely, Herzog Luitpold continued the construction in 1953. The existing buildings were enlarged and new constructions were added but the castle was never made into a place useable as a home. Herr Ho¨rmann, the present manager of Schloss Ringberg, told us that Luitpold lived in a fancy hotel in Munich and inspected the building activities by occasional visits in a cab. Finally, the Herzog bequeathed it to the Max-PlanckSociety. After his death in 1973 it was converted to the present meeting center. We experienced fantastic meetings at Schloss Ringberg, which stimulated our science but also tightened many bonds of friendship. In addition to the 3 day meetings close to Christmas, Rupert organized several international meetings on matrix research at Ringberg. All of these meetings were of exceptionally high standard. We were linked by a joint idea and Rupert served as a mentor. He never dominated and was extremely fair in all collaborations. My group published about 50 publications with him and his group and I do not remember any bad feelings or quarrels about authorship. It is not possible to mention of all


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members of the Rupert Club but Figure 4 taken near to the lecture hall in Schloss Ringberg in May 1992, on Klaus Ku ¨ hn’s 65th birthday, may be representative. Meetings at Schloss Ringberg usually started with a bus that drove us out of Munich in the evening. At the bottom of the Ringberg hill snow chains had to be mounted. I remember a meeting at which I did not wait for the completion of this work and decided to walk up to the castle. I was already close to its entrance when I heard the bus approaching. I was alarmed by a strange noise, which was apparently caused by loose chains and sounded very dangerous. Suddenly there was silence. After a few minutes I watched my colleagues walking uphill with pale faces. I was told that the bus had lost a chain and only a tree stopped it from sliding down. On this day, the delicious cold dinner in the dining hall that includes an enormous painting of Herzog Luitpold hunting a deer by Attenhuber was enjoyed a little bit less than at other times. The best-liked room at Ringberg was the Hexenzimmer (witch room) that is decorated with wall carpets showing witches in all kind of activities, again styled by Attenhuber. We would sit around very large tables discussing science and non-science stimulated by beer and red wine. The wine at Ringberg was very good and obtained on a self-service basis, just placing a cross in a participant list for each bottle. Before departure everybody had to pay for his wine and beer. My bill was often very large, of course not due to my own consumption, but from my generosity in treating other people or due to crosses other people placed under my name. Rupert left the Hexenzimmer very late, usually around 1 am. Others left so late that I never witnessed their leaving. Despite this, nobody missed the lectures which started at 9 am. Rupert always sat in the last row, sometimes leaving for a short smoke. Klaus, by tradition, took care of the slide projector. Heinz Furthmayr caused a big sensation at one of the meetings by appearing with a computer and a well- prepared power point presentation. This indicated the beginning of the end for Klaus’s projectionist career. Even at the local group meetings the standard was very high and lecturers took great care to be well prepared. Rupert did not tolerate confusing lectures and was very critical of running overtime. Each minute overtime was charged by 100 Swiss Franks. This currency was apparently selected because Peter Bruckner from Basel invented this system. If Peter

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and the other speakers had paid up their punishments, all wine bills could have been settled, but this never happened. Domains and Interacting Proteins at Atomic Resolution When we started to explore laminin it was only a dream to see the structure of such a giant protein at atomic resolution. The hope


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was substantiated when the domain concept of laminin and many other proteins of this kind was recognized. Homologous domains of the same type were found to be repeated in the same protein and homologs were also found in other proteins. A typical example is the EGF-domain, which was first found in epidermal growth factor. The atomic structure of this growth factor was solved by NMR-spectroscopy [62] and so the global fold for many homologous EGF-domains in numerous proteins was known [38]. In addition, repeating structures such as the a-helical coiled-coil were recognized by sequence and spectroscopic data [63] and their structure could be predicted with fair accuracy. Many other important questions, such as the detailed individual structures and the connecting contact between domains, could be answered only by direct structural determination. For laminin the first direct structural determination was achieved by crystallography of the nidogen/entactin binding region of the g1-chain of laminin 1 [64]. The X-ray work was done by Jo¨rg Stetefeld, who worked in Robert Huber’s group and later moved to Basel. The work was based on the isolation of active peptides performed by Martin Gerl [55] and mutational studies by Ernst Po¨schl and Uli Mayer [65] in Rupert’s lab. A few years later Patrik Maurer in my laboratory succeeded in crystallizing BM-40/SPARC and Erhard Hohenester, a friend of Patrik’s, solved the structure. At this time Erhard was working in the Department of Structural Biology at

Fig. 4. Meeting at Schloss Ringberg in May 1992 at Klaus Ku¨hns 65th birthday. From left to right, 1st row: Shmuel Shoshan, John Scott, Monique Aumailley, Dieter Reinhard, Ju¨rgen Rauterberg, behind him half covered Rupert Timpl, Peter Ekblom, behind him Winfried Babel; 2nd row: Francoise Gaill, Roswitha Nischt, Karl Tryggvasson, Kari Kivirikko, Lisa Fessler, Karl Piez, Klaus Ku¨hn, Benoit de Crombrugghe, Klaus von der Mark; 4th row: Mon-Li Chu, Emmanuelle Tillet, Uli Mayer, Thomas Krieg, Robert Glanville, Elke Genersch, Andreas Kern, Roberto Perris, Helga von der Mark; 5th row: Darwin Prockop, Francesco Ramirez, Peter Mu¨ller, Ralph Golbick, Eva Schlosser, Helmut Ho¨rmann, Karlheinz Mann, Judith Brown, Herbert Germaier, Martin Gerl;6th row: Peter Fietzek, Martin Humphries, Mike Grant, Dick Heinegard, Ilse Oberbaümer, Annette Schmidtmann, Michael harnischmacher, Yoshi Yamada; 7th row: Raul Fleischmaier, Richard Mayne, Ju¨rgen Engel, Rainer Deutzmann, Martin Pfaff, Johannes Eble, Michaela Streubert, Walter Go¨ring, in front of him: George Martin; 8th row: John Fessler, XX, Ulrich Specks, Milan Adam, Ernst Po¨schel, Volker Gu¨nzler; top row: XX, Michel van der Rest, Peter Bruckner, Mats Paulsson.

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the Biozentrum in Basel. This work proved that high-affinity calcium binding EF-hands exist in extracellular proteins and resolved the puzzle of the partner motif in the EF-hand of BM-40/ SPARC mentioned in section Nidogen/entactin, collagen VI and BM-40/SPARC. Erhard discovered a very unusual partner motif, which could not have been distinguished by sequence predictions [66]. Erhard Hohenester continued crystallographic studies of ECM (extracellular matrix) domains in close collaboration with Rupert. Rupert was convinced of the importance of the determination of structures at atomic resolution and often said: ‘‘After the structure is known, biology will follow.’’ This indeed happened for the structure of the domains of extracellular matrix domains and the work gained broad international recognition. The first achievement was the determination of the LG-domains at the tip of the long arm of laminin. These are involved in the cell binding of laminin (see Figure 1; section Cellular receptors for matrix proteins). It will not be possible to describe this exciting work in any detail and only a review will be cited, which Erhard and I wrote together [67]. This review summarizes the knowledge of ECM-domain structures up until 2002, that was, in part, obtained by Erhard and Rupert or Jo¨rg Stetefeld and my group in Basel. The work included a study of a complex between interacting domains [68] and such interaction studies will be very helpful for future research.

Promotion to Scientific Member of the Max-Planck-Society and the arrival of Takako Sasaki In writing the last section I lost track of the historical sequence of events and Rupert’s personal life. I shall try to correct this in the present section. In 1992 he was elected by the President of the Max-Planck-Society as ‘‘Wissenschaftliches Mitglied’’ (scientific member) and director of the Department of Protein Chemistry at the Institute of Biochemistry in Martinsried. The former village of Martinsried is located in the southern part of Munich. The Max-Planck-Institute for Biochemistry and the Max-Planck-Institute for Psychiatry were the first scientific institutions which were built in Martinsried (see section From


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immunology to the complexity and structure of the extracellular matrix). Subsequently, major parts of the University of Munich have moved out to Martinsried and a large technology park was built there. Rupert and his wife Liane bought a small house in Gauting, which is located 10 kilometers south of Martinsried. Of their children only Peter still lived with them, but he was already very independent. Liane was taking care of elderly people in an institution supported by the church. At the time of his promotion, Rupert’s publication list numbered 388 papers. This and their high quality can only be explained by very hard work. His normal style of work was to arrive at the laboratory at noontime, to work until very late hours, and to leave home for writing publications until 3 o’clock at night. For most of his publications he composed a draft at an early state and the growing manuscript served as a guideline for any missing experiments. All his publications were written by hand and only later transferred to the computer by Frau Wesse, his secretary. Rupert read a lot and had an excellent memory. One of his routines was to spend 1 hour per day in the library reading and screening newly arrived journals. He continued this habit even until the time when the information service of the institute distributed excellent literature surveys and articles could be copied electronically. His knowledge was very broad and reached from classical biochemistry, immunology, molecular biology and genetics to medicine. It was fascinating how well he answered complicated questions and how many details he remembered by heart. His main driving force was his deep interest in matrix research combined with the will to find out how things happen. As far as I can judge, his collaborators liked him because of his competence and the relaxed and objective way in which he dealt with daily problems. He often made jokes, which were usually ironical and contained a second meaning. George Martin, a very close friend of Rupert, mentioned at the Rupert Memorial Meeting in 2004 that he ‘‘never understood Rupert’s jokes.’’ The issues Rupert was very serious about were soccer games. He played soccer with a Max-Planck-team every Friday afternoon and organized the soccer matches ‘‘America against the Rest of the World’’ at the Basement Membrane and the Collagen Gordon Conferences (Figure 5). Another tradition was his wine parties with Austrian white wine, which I mentioned already in section

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Fig. 5. Rupert (front row, second from right) organized a soccer game at the Gordon Conference for Collagen 1991.

Training in chemistry and first contacts with biology. On the other hand, work in Rupert’s lab was very intense. Mats Paulsson, who moved from Munich to Basel, mentioned that the Basel way of working was much more relaxed, a statement which conflicted with the opinion of other people about the working atmosphere in my lab. I frequently visited his lab and he often visited Basel. Coming to his place, I would find him in a small office, which he shared with three to four collaborators. Only after his promotion to scientific member did the room conditions improve a bit, and Rupert had his own office, filled with long piles of lab-reports and documents. Next to this office was a big laboratory room in which Vera van Delden was the queen. She usually prepared a strong coffee for us and I tried my Serbian on her to impress people. Vera is Croatian and I learned Serbian during the time with my parents as a boy in Nisch and Belgrade. Rupert and I would then enter a discussion of current and planned work. I remember Rupert in these discussions to be very intense and directed to the main point. The discussion would normally end with a long list of fragments and proteins, which Vera loaded into a Dewar vessel, to be carried back to Basel for measurements. My parents in law lived in a neighborhood close to Martinsried and Rupert would then drive


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me to them, or lend me his big Mercedes for an excursion with my aged mother in law on the next day. Rupert’s addiction to science did not have a positive effect on his relationship with Liane. She decided to settle in Vienna and to come to Gauting only for short visits. My wife Annelies expressed much understanding for Liane. She correctly blamed me to spend too little of my time for my family. I feel that this conflict is not uncommon to scientists. In 1992 Takako Sasaki, a post-doctoral fellow from Japan arrived. I remember meeting her the first time at the Ringberg meeting in 1993. I was chairing a session in which she gave her first lecture and she was very shy. Takako became one of the most successful and most permanent collaborators of Rupert, who co-authored with him about 80 publications. Gradually, a deep friendship developed between herself and Rupert. She was the most important person for him and even after his death Takako has a deep appreciation for Rupert.

Cellular Receptors for Matrix Proteins We now know that the numerous interactions between the extracellular matrix and cells are naturally mediated by a large repertoire of receptors. In the 1980s, the groups of Erki Ruoslahti [69]and Richard Hynes [70,71] defined the family of integrin cell surface receptors and it was soon recognized that this is the most important family of ECM receptors. The group of Erki Ruoslahti found the important RGD recognition site for integrins in ECM proteins. In the beginning they believed that RGD was an universal motif and called the integrins arginine-glycine-aspartic acid adhesion receptors. A prominent function of integrins is cell adhesion, but integrins should also be looked at as molecular machines with many functions including inside-out and outsidein signaling [48,71]. In Rupert’s laboratory, Monique Aumailley was the expert on cell adhesion. Monique came from a strong extracellular matrix group in Lyon and is now Professor in Cologne. I remember her plating all kinds of cells on laminincoated plastic plates. At this time, her technique to count and discriminate attached cells from loosely adsorbed cells by shape looked very subjective to a physical chemist like me. With tricks such as adding RGD-peptides as inhibitors and using cell with

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known receptor composition, Monique succeeded in obtaining very valuable information from these adhesion assays. For example she used the adhesiveness of different laminin fragments to localize sites of integrin binding in the laminin molecule [72,73]. It was also possible to find out which of the many different integrins were involved in laminin recognition. These results stimulated a number of the structural studies on laminin [74]. I do not remember the first contact of Rupert with the integrin expert Arnoud Sonnenberg, who was working at the Red Cross in Amsterdam. From about 1990, Arnoud was a prominent member of the Rupert Club and his joint efforts with the Munich group produced a large number of outstanding results. Again I have to restrict myself to a few early references [72,75,76]. Arnoud Sonnenberg also showed up at the Ringberg Meetings and, to me, his loud laughter is somehow part of Ringberg castle. He presented his seminars at an unmatchably high speed yet would still overrun his time. Applying to Arnoud the 100 SFr per minute overtime fee mentioned in section Ringberg and other meetings, would have left him without the funds to return to Holland.

Collagen XVIII and Endostatin Rupert’s interests and activities grew with the increased potential of his group but also because of the explosive burst of knowledge in the extracellular matrix field. I may mention a few of the new topics of investigation only by name without including any details. With Francois Gaill, Professor in Paris, Rupert’s group, ¨chinger in Portland, the my group in Basel, and Hans Peter Ba unusual length and mode of stabilization of collagens of annelids from hydrothermal vents was studied [77,78]. The novel basement membrane proteins fibulin-1 and fibulin-2 were discovered [79]. A strange ring-forming protein Mac-2 binding protein was investigated [80,81]. The transition of BM-40/SPARC from a latent to an active form by matrix metalloproteinases was studied in great detail mainly by Takako Sasaki in collaboration with Gillian Murphy, Professor in Cambridge. I feel sorry that I have to omit all these interesting stories, each of which could fill a separate review. I wish however to recall my memories about the research on endostatin. I was personally not involved in this research area but


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I know that it was a central topic in the thinking of Rupert. The motivation for this work came from a hypothesis of Judah Folkman at Harvard Medical School that endogenous inhibitors of angiogenesis might act as efficient antitumor drugs [82]. This was a general hypothesis and the Folkman group tested several different compounds. By screening conditioned tumor sera they found a compound which they called endostatin, for which they published amazingly promising results in the treatment of tumors in mice [83]. The collagen expert at Harvard, Bjo¨rn Olson, worked in Martinsried as a Humbodt Visiting Professor, soon after Folkman’s first publication. Rupert and he started a collaboration on the biochemistry of endostatin. Endostatin was identified as a fragment of collagen XVIII comprising its C-terminal non-collagenous (NC) domain. The occurrence of fragments of extracellular proteins in sera is nothing unusual. They are apparently formed by limited proteolytic degradation and their increased appearance is frequently a pathological sign of increased tissue degradation. Rupert and Takako Sasaki approached the problem by expression of the fragment in mammalian 293-cells. This expression system had been developed in Rupert’s lab and was applied successfully for the expression of native protein domains with all their disulfides properly connected. For extracellular proteins and domains the mammalian cell expression is superior to expression in E. coli, which often yields unfolded inactive peptides and needs special treatments for oxidative disulfide formation. [84]. Soon after Erhard Hohenester succeeded in solving the crystal structure of the mammalian-expressed recombinant endostatin [85,86]. When I studied chemistry I was told that crystallization is one of the best criteria for the purity of a compound. This should even be more true for a protein for which the atomic structure was solved from the crystals. It was therefore surprising that the endostatin expressed in mammalian cells did not show the high activities reported earlier for the fragment expressed in E. coli. I remember heated discussions about the relation between native state and activity between the physician Michael O’Reilly and the chemist Erhard Hohenester at an international Ringberg conference on endostatin organized by Rupert. Much hope was raised among cancer patients by a very positive article in New York Times [87] and by other premature reports.

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A close science colleague, whose child suffered from an incurable tumor and died a year after, asked Rupert for help, which however was not possible. This event touched me very deeply and stimulated a strong opposition against the political and commercial issues that had been connected with endostatin. These issues were broadly discussed in leading journals [88,89]. Subsequent clinical tests of endostatin were negative and it disappeared from the headlines [90].

The Last Years In summer 2000 I met Rupert at the European Connective Tissue Meeting in Patras. It was extremely hot and Greece suffered from terrible forest fires at this time. Takako, my wife, Rupert, and I went for a swim. I remember this evening because it was the first time that I realized that Rupert was in a dangerous health condition. I was just shocked by his extreme meagerness. He was always a lightweight but there at the beach it was different. He made the joke that a transplantation from my belly to his body would help us both. In contrast to, or perhaps because of, his light weight Rupert was rather sporty. I already mentioned his soccer activities and I also remember hikes at a vacation on the Black Forest in which he was always leading. Also in normal walking I often found it difficult to keep pace with him. His high mental activity was reflected in fast movements and physical agility. On the other hand he was not living a healthy life with his heavy smoking. There were jokes about him, that his health was supported by tar and nicotine, because he usually made a very active and healthy impression. In Patras however I was alarmed and Klaus Ku ¨ hn shared my concern. We both talked to Rupert and tried to convince him to go to medical examinations and to turn to a more healthy life. I know that many others did the same but as Takako expressed it Rupert was ‘‘stubborn’’ and gave us the impression that we were interfering too much with his personal decisions. He continued his scientific activities at full power and ignored all signs of warning. From December 18 to December 20, 2000 the group seminar at Schloss Ringberg took place as usual and a year later Rupert retired from the official duties as director of the department.


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¨ssler also lectured at the group meeting. In 1992 he Reinhard Fa had been attracted to the Max-Planck-Institute of Biochemistry by Rupert, because Rupert was fascinated by the new technique of gene knock-outs. Reinhard applied this technique to extracellular matrix components and became well known with a deletion of the gene coding for the beta-1 component of integrins. He worked very successfully with several proteins, was Professor in Lund for 3 years, and was then appointed scientific member and director of the Department for Molecular Medicine at the Max-PlanckInstitute for Biochemistry in 2001. ¨ssler’s department was related to the extraThe work in Fa cellular matrix and Rupert was able to continue his work in this department after retirement, supported by his grants and the Max-Planck-Society. He was full of ideas and followed several new projects with Takako and several students. Unfortunately his heath was causing increasing problems. Even in Basel I felt this, because a steadily increasing number of the parcels with material to be studied were packed and handled by Takako and not by Rupert as usual. On the phone he said ‘‘since I have a lower salary, I do less’’ but this joke did not make me laugh. Apparently, sometime in 2002, Rupert learned that he suffered from lung ephysema, which was progressing at a rather high rate. At this time he had an unusually large publication list with about 600 publications. His last big work was a major review for Nature Reviews in Molecular Biology on fibulins, which he wrote with Takako, Mon-Li Chu, and Gu ¨ nter Kostka [79]. This review only appeared shortly before his death and reads like a program for future research. He also started to write a publication on the Del-1 proteins, which we had studied together. I saw a draft of this paper with him in the hospital. Takako was engaged in a fruitful collaboration with Erhard Hohenester on Gas6-Axl signaling, which led to important results only published after Rupert’s death [91]. Takako helped Rupert not only scientifically. She was the person closest to Rupert. He stayed in her apartment located close to the institute in summer 2003. He refused to be hospitalized and stayed as long as possible with Takako. I was in close touch with her and sometimes I also talked with him by phone. In September 2003 he was finally hospitalized and I visited him in the hospital in Munich. There was a time of recovery and he was able to walk around and to talk to us in an almost normal way. He was aware

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that he had to adopt to a new way of living. We were not thinking about the possibility of a sudden death. Takako was invited to a conference in Japan and I advised to travel. I am still very sorry about this bad advice. In the hospital Rupert caught a pneumonia and with his partially disabled lungs he was unable to fight against the infection. He passed away on October 20, 2002. Reinhard’s wife, Elisabeth, who is a nurse, was with him. Takako returned as fast as possible but it was too late. The Memorial Symposium, a Summary and Outlook Takako and Reinhard organized a memorial symposium for Rupert at the Max-Planck-Institute of Biochemistry in April 2004. The list of the participants is given in Table 1. All the friends of Rupert were very grateful for this possibility to remember Rupert and the joint work. We were all aware that this meeting was the last meeting of what I called the Rupert Club. A year later, American colleagues also organized a memorial symposium for Rupert and several articles about Rupert and his work were published. Science moves fast, exciting new discoveries are made, and memories will fade (Figure 6). What should not be forgotten about Rupert and the explosion of knowledge during his lifetime’s work? His friends will not forget his personality. I already mentioned several features which I liked about Rupert. His openness to new approaches, his deep motivation to find things out, and his friendly way to stimulate other people to join him in exploring the extracellular matrix. I also mentioned his accuracy and fairness. He was very critical also concerning his own work and criticism to other scientists was never offensive, which does not exclude that some colleagues were hurt by any criticism. He never took science deadly seriously and was always prepared to make a joke. As I mentioned, these jokes were very typical of Rupert and it often needed some intuition to understand them. He was however very serious about the problems of others and gave excellent advice in case of personal problems. I also mentioned that he was a very hard worker but fortunately he was able to switch to other things completely. Examples are the parties, the soccer games, excursions and vacations, visits to musicals, playing of sophisticated computer


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TABLE 1 Participants at the Symposium ‘‘Extracellular Matrix: Assembly, Functions, and Role in Disease – A Tribute to Rupert Timpl,’’ April 25–27, 2004, Martinsried

Aeschlimann, D. (Cardiff) Ahmed, N. (Regensberg) Angres, B. (Reutlingen) Asberg, A. (Lund) Aumailley, M. (Ko¨ln) ¨chinger, H.P.(Portland) Ba Bader, B. (Martinsried) Baranowsky, A. (Ko¨ln) Bateman, J. (Melbourne) Baumeister, W. (Martinsried) Bode. W. (Martinsried) Bo¨se, K. (Ko¨ln) Brancaccio, A. (Roma) Breitreutz, D. (Heidelberg) Brenner, R. (Ulm) Bruckner, P. (Mu ¨ nster) Bruckner-Tuderman, L. (Freiburg) Chu, M.-L. (Philadelphia) Colige, A. (Liege) Crombrugghe, B. (Huston) David, G. (Leuven) Dreier, R. (Mu ¨ nster) Dziadek, M. (Aukland) Echtermeyer, F. (Mu ¨ nster) Ekblom, P. (Lund) Engel, J. (Basel) ¨ssler, E. (Martinsried) Fa ¨ssler, R. (Martinsried) Fa Fessler, J. (Los Angeles) Fessler, L. (Los Angeles) Fox, J.W. (Charlottesville) Furthmayr, H. (Stanford) Gail, F. (Paris) Go¨hring, W. (Martinsried) ¨ssel, S. (Regensberg) Gra Haehn, S. (Ko¨ln) Hahn, E. (Erlangen) Hahn, U. (Erlangen) Hansen, U. (Mu ¨ nster) Hartmann, U. (Ko¨ln) Hausser, H-J. (Ulm) Heckmann, L. (Ulm) Heep, D. (Ko¨ln)

Heinegard, D. (Lund) Herken, R. (Go¨ttingen) Herzog, C. (Mu ¨ nster) Hirako, Y. (Wu ¨ rzburg) Hohenester, E. (London) Holak, T. (Martinsried) Humphries, M. (Manchester) Kefalides, N. (Philadelphia) Kirchisner, H. (Mu ¨ nchen) Klein, G. (Tu ¨ bingen) Koch, M. (Ko¨ln) Kostka, G. (Martinsried) Kramer, J. (Chicago) Kreil, G. (Salzburg) Krieg, T. (Ko¨ln) Ku ¨ hn, B. (Mu ¨ nchen) Ku ¨ hn, K. (Martinsried) Kurkinen, M. (Detroit) Langer, H. (Mu ¨ nchen) Langer, I. (Mu ¨ nchen) Maier, S. (Ko¨ln) Mann, K. (Martinsried) Martin, G. (Bethesda) Mayer,U. (Manchester) Miosge N. (Go¨ttingen) Mokkapati, S. (Ko¨ln) Murphy, G. (Cambridge) Nischt, R. (Ko¨ln) Nowack, H. (Bad To¨lz) Nowack, W. (Bad To¨lz) Ohhashi, T. (Okayama) Olsen, B.R. (Boston) Paulsson, M. (Ko¨ln) Pihlajaniemi, T. (Oulu) Po¨schl, E. (Erlangen) Prockop, D. (New Orleans) Prockop, E. (New Orleans) Quondamatteo, F. (Go¨ttingen) Ramirez, F. (New York) Rauch, U. (Lund) Rauterberg, J. (Mu ¨ nster) Reinhardt, D. (Lu ¨ beck) Reiter, M. (Martinsried)

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266 TABLE 1. (Continued ) Ricard-Blum, S. (Grenoble) Risteli, J. (Oulu) Risteli, L. (Oulu) Rousselle, P. (Lyon) Ru ¨ gg, M. (Basel) Ruggiero, F. (Lyon) Sakai, L. (Portland) Sasaki, T. (Martinsried) Schaefer, L. (Mu ¨ nster) Schittny, J. (Bern) Schonherr, E. (Cardiff) Seidler, D. (Mu ¨ nster) Shimanovich, I. (Wu ¨ rzburg) Smyth, N. (Ko¨ln) Sonnenberg, A. (Amsterdam) Sorokin, L. (Lund) Talts, J. (Copenhagen)

Tro¨ger, J. (Martinsried) Tryggvason, K. (Stockholm) Uitto, J. (Philadelphia) van Delden, V. (Mu ¨ nchen) van der Rest, M. (Lyon) Vogel, G. (Aalen) von der Mark, H. (Erlangen) von der Mark, K. (Erlangen) Wendt, C. (Martinsried) Werb, Z. (San Francisco) Wesse, C. (Martinsried) Wewer, U. (Copenhagen) Wick, G. (Insbruck) Wiedeman, H. (Martinsried) Yamada, Y. (Bethesda) Yurchenco, P. (Piscataway) Zaucke, F. (Ko¨ln)

¨chinger, and philosophical discussions games with Hans Peter Ba at which my wife retired at midnight. In science his name will remain connected to many discoveries of novel matrix proteins, especially the laminins. To find an unknown protein was very difficult at the time Rupert and George Martin found the first laminin. It is much easier today by means of genome analysis and cloning. The major contribution of Rupert and what I called the Rupert Club was, however, the very detailed biochemical characterization of the newly identified proteins, both at structural and functional levels that reached to cellular and medical aspects. With this approach he stimulated the entire field. Naturally this biographic chapter reviews mainly Rupert’s contributions and even these not completely. Of course large number of other groups also contributed. A comparison of the literature on extracellular matrix on a broader level will prove the claim of the title, that there was an explosion in research in the matrix field in these years. Rupert was several times in the Science Index list of most cited authors and his work will still be read by many young scientists. His approach will certainly remain important in the future. Modern fields like genomics, proteomics, and gene manipulations are based on efficient sequencing, cloning techniques, and mass spectroscopy. These alone combined with bio-computing certainly


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Fig. 6. Rupert, how his friends remember him, with a friendly but slightly critical look over his glasses. The photograph and the flowers were arranged on Rupert’s now empty desk.

cannot solve all the questions mankind will ask of nature. I feel that it is essential to develop these techniques, but also to develop new structural, biochemical, and dynamic techniques [48]. We will also have to go through the painful detailed work of studying the individual proteins and their interactions in the future. A novel aspect of our understanding is the network structure of the matrix and the machine-like cooperation of several matrix components and cellular receptors in a joint function. The importance of networks was already seen by Rupert with the nidogen/entactin network. I feel that the old days with claims that protein X causes the effect Y have passed, because a network of many proteins is usually involved and these networks again are controlled by complex signaling chains. The importance of large systems has been generally recognized and led to the concepts of System Biology. I feel that the study of large machines as

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functional units and large networks as complete systems will be an important issue in future matrix research [48].

ACKNOWLEDGMENTS

Many thanks to Takako Sasaki (Portland, OR), Liesel Hofmann (Merseburg), Klaus Ku ¨ hn (Munich), Heinz Furthmayr (Stanford), Ulrike Mayer (Norwich), and Erhard Hohenester (London) for critical reading, suggestions, and photographaphs. Special thanks to Josephine Adams (Cleveland) for many improvements including the English.

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[16] Junqua, S., Robert, L., Garrone, R., Pavans de Ceccatty, M. and Vacelet, J. (1974) Biochemical and morphological studies on collagens of horny sponges. Ircinia filaments compared to spongines. Connect Tissue Res. 2, 193–203. [17] Hynes, R.O. and Zhao, Q. (2000) The evolution of cell adhesion. J. Cell. Biol. 150, F89–F96. [18] Schmid, V. and Bally, A. (1988) Species specificity in cell-substrate interactions in medusae. Dev. Biol. 129, 573–581. [19] Engel, J., Bruckner, P., Becker, U., Timpl, R. and Rutschmann, B. (1977) Physical properties of the amino-terminal precursor-specific portion of type I procollagen. Biochemistry 16, 4026–4033. ¨chinger, H.P., Timpl, R. and Engel, J. (1978) Three [20] Bruckner, P., Ba conformationally distinct domains in the amino-terminal segment of type III procollagen and its rapid triple helix leads to and comes from coil transition.. Eur. J. Biochem. 90, 595–603. ¨chinger, H.P., Bruckner, P., Timpl, R. and Engel, J. (1978) The role of [21] Ba cis-trans isomerization of peptide bonds in the coil leads to and comes from triple helix conversion of collagen. Eur. J. Biochem. 90, 605–613. ¨chinger, H.P., Bruckner, P., Timpl, R., Prockop, D.J. and Engel, J. [22] Ba (1980) Folding mechanism of the triple helix in type-III collagen and type-III pN-collagen. Role of disulfide bridges and peptide bond isomerization. Eur. J. Biochem. 106, 619–632. [23] Stoltz, M., Timpl, R., Furthmayr, H. and Kuhn, K. (1973) Structural and immunogenic properties of a major antigenic determinant in neutral saltextracted rat-skin collagen. Eur. J. Biochem. 37, 287–294. [24] Rohde, H., Nowack, H. and Timpl, R. (1978) Localization of antigenic activity and immunogenic capacity in different conformational domains of procollagen peptide. Eur. J. Immunol. 8, 141–143. [25] Sela, M., Arnon, R. and Jacob, C.O. (1986) Synthetic peptides with antigenic specificity for bacterial toxins. Ciba Found Symp. 119, 184–199. [26] Timpl, R., Bruckner, P. and Martin, G.R. (1977) Basement membrane collagen. Curr. Probl. Clin. Biochem. 8, 20–28. [27] Timpl, R., Wiedemann, H., van Delden, V., Furthmayr, H. and Kuhn, K. (1981) A network model for the organization of type IV collagen molecules in basement membranes. Eur. J. Biochem. 120, 203–211. [28] Timpl, R., Rohde, H., Robey, P.G., Rennard, S.I., Foidart, J.M. and Martin, G.R. (1979) Laminin – A glycoprotein from basement membranes. J. Biol. Chem. 254, 9933–9937. [29] Aumailley, M., Bruckner-Tuderman, L., Carter, W.G., Deutzmann, R., Edgar, D., Ekblom, P., Engel, J., Engvall, E., Hohenester, E., Jones, J.C., Kleinman, H.K., Marinkovich, M.P., Martin, G.R., Mayer, U., Meneguzzi, G., Miner, J.H., Miyazaki, K., Patarroyo, M., Paulsson, M., Quaranta, V., Sanes, J.R., Sasaki, T., Sekiguchi, K., Sorokin, L.M., Talts, J.F., Tryggvason, K., Uitto, J., Virtanen, I., von der Mark, K., Wewer, U.M., Yamada, Y. and Yurchenco, P.D. (2005) A simplified laminin nomenclature. Matrix Biol. 24, 326–332. [30] Wagner, D.D. and Hynes, R.O. (1979) Domain structure of fibronectin and its relation to function: Disulfides and sulfhydryl groups. J. Biol. Chem. 254, 6746–6754. [31] Engel, J., Odermatt, E., Engel, A., Madri, J.A., Furthmayr, H., Rohde, H. and Timpl, R. (1981) Shapes, domain organizations and flexibility of laminin and fibronectin, two multifunctional proteins of the extracellular matrix. J. Mol. Biol. 150, 97–120.

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Chapter 4

A Life with Yeast Molecular Biology HORST FELDMANN Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Molecular Biology. Schillerstrasse 44, D-80336 Mu¨nchen, Germany E-mail: [email protected]

Horst Wilhelm Albert Feldmann

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Abstract Born 1932, I was brought up in Stettin. I was trained as an organic chemist at Cologne University, before I entered the Institute of Genetics there in 1962 to work on tRNA structure and function. From 1967 to my retirement in 1997 I worked at the Adolf-Butenandt-Institute (Munich University) on several aspects of molecular biology of the budding yeast, for example tRNA genes, Ty elements, the yeast genome project, and programmed proteolysis. I include facets of my engagement in national and international organizations. Keywords: Yeast; tRNA; Ty; Yeast genome; Programmed proteolysis; FEBS; Spetses

Within the recent 3 years, several anniversaries have been celebrated – the 40th anniversaries of the European Molecular Biology Organization (EMBO) and the Federation of European Biochemical Societies (FEBS) both of which were founded in 1964 – and that of the Spetses Summer Schools on Molecular and Cellular Biology in 2006. In 2005, the Cologne Institute of Genetics had organized a workshop to remember the ‘‘Early History’’ of this institution, the first of its kind in Germany opened in 1961. Finally, in 2006 many of the participants in the International Yeast Sequencing Genome Project gathered in Brussels to memorize the deciphering of the first eukaryotic genome in 1996. These venues remind me that I myself by now have devoted nearly all of my scientific life to Molecular Biology, especially that of a small unicellular ‘‘model’’ organism, the yeast Saccharomyces cerevisiae. But it took my first 30 years to bring me to this attractive field. My Early Years and Education I was born in Stettin, on a date the impact of which should determine the further political development in Germany and with all its consequences imprint the future of the next decades worldwide. On March 13, 1932 – my birthday – Paul von Hindenburg was re-elected Reichspra¨sident (German President). I keep an edition of the Frankfurter Zeitung (Frankfort News) of this day reporting on the massive and impudent election campaign, Hitler, Goebbels, and the Nazis had set up to push Hitler into the

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position of Reichspra¨sident. This time the Nazis lost because the majority of the Germans highly respected Hindenburg and trusted that he was the one to save German unity and independence (actually, Hindenburg’s nomination had to be confirmed on April 10, 1932). But already several months later, Hindenburg lost any confidence in Heinrich Bru ¨ ning, than Chancellor of the Republic, who had tried to overcome inflation and growing underemployment by emergency decree. Thus, Bru ¨ ning’s demission marked the end of the Weimar Republic, and Hitler was appointed Reich Chancellor on January 30, 1933. My father, Wilhelm Feldmann, born in Siegen (Westfalia) was the eldest of five children. My grandparents run a bakery, and my father visited a technical school in Siegen to be trained as an engineer for countryside cultivation, water management as well as canal and river engineering. He found a position as a civil servant in a government facility in Stettin responsible for these affairs and married my mother, Hertha Mansen in 1931. My maternal grandfather’s family originated from Schleswig Holstein, and his genealogy is the only one in my family which I could trace back to the 17th century. As an employee of the German National Railway my grandfather Albert Mansen had been moved to Stettin, where he married Agnes Bohm, whose relatives lived in Stettin and Berlin. I still keep good reminiscences of Stettin as a wonderful town and would be able to draw a city map with street names and important buildings in German (such a map is no longer available) though I spent only 11 years of my youth there. My parents and I lived in an apartment of a tenement quarter built during the Gru¨nderzeit and in the early years of the 20th century, in the same style as those quarters that became characteristic for Berlin and other fast growing towns around Germany. Fortunately, our domicile was located not far from the center, and my grandfather very often took me for long walks downtown explaining to me the meaning and history of buildings, monuments, and other attractive places. My favorite place was the famous ‘‘Haken-Terrasse’’ (a terrace and park named after a Stettin mayor) at the banks of Oder river surrounding a huge building that harbored the departmental administration and the shipping museum. From here, one could overlook the whole area: the harbor with boats of all kinds and steamers moored to the quays, the busy traffic on the Oder and its side arms, and, on the

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opposite banks, the free port area governed by huge cranes, docks, and shipyards. At home I transformed these impressions into drawings of ships, the essentials of which I knew from memory already at the age of four, or I constructed cranes with my metal construction set. My parents argued that with these skills and interests I would become a shipbuilding engineer. The harbor – already busy during the time when Stettin was a member of the Hanseatic league – at the end of the 19th century had developed into the most important port of the Baltic Sea and German trading place with the Scandinavian and Baltic countries. This became only possible after the strong fortifications had been torn down, so that the town could expand. New modern quarters and huge parks were created, which together with its natural lovely hinterland made Stettin a ‘‘green town,’’ offering numerous possibilities for relaxing tours. During summer time, we used to undertake week-end trips to one of the many seaside resorts at the Baltic Sea, which were served by regular pleasure steamers from Stettin. Sometimes, we visited my aunt and uncle in Berlin, and so I saw the Olympics in 1936 for one day. In 1938, I entered primary school, where we started writing in Su¨tterlinschrift; which was changed to Latin lettering during my third year at school. But I still can read and write this Gothic type which already my parents had been taught at school. Surprisingly, it was the Nazis who introduced the change. Other of their measures turned out to be less harmless, and I was at an age to realize some of the threatening developments, such as the increasing prosecution of the Jewish population and foreigners. For our daily life, the most far-reaching effect was Hitler’s rigorous expansion policy: in September 1939 he broke the nonaggression pact and started war. Progressively, all goods and food became scarce, but the extending war also began to exert a subliminal influence on our lives as schoolboys: we were obliged to collect warm clothing or money for the Winterhilfswerk (winter relief organization), or to collect waste paper and other waste material, even gnawed bones that were used for the production of soap. We had to set up silk moth colonies and provide mulberry leaves to feed them. Though this was given the veneer of a playful competition, my parents and all the more my grand parents were alerted. When the local Hitlerjugend forced me to visit their ‘‘social evenings’’ held in a dump and dreary cellar – a measure


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that I hated from the very first moment – my mother succeeded that I was exempted from this dismal obligation. The consequences of the war became worse for us during 1942, the year I entered secondary school, because air raids had reached Stettin. Measures against the bomb attacks were noticeable long before. Cellars had been transformed into air raid shelters and public air bunkers had been put up in public parks. Anti-aircraft units were installed all around the town and we could follow their practice to fix air planes by spotlights during night. I vividly remember that one nice morning in September 1942 one of my classmates appeared with singed trousers telling us that their house had been bombed last night, everything had burnt down and that this was left as his sole property. The attacks increased in 1943: we had to spend nearly every night in the shelter of our house. The most frightening missiles were the air bombs: coming down, they produced a sharp whistle. One started counting to ten, and if one was still alive after, the bomb had struck elsewhere. Finally, the authorities decided to evacuate all children to the countryside. Since my father, due to his occupation, had been exempted from military service and instead had been moved to West Prussia, my mother insisted that the whole family should take up residence in Kulm, a small town at the Vistula. So we went there in autumn 1943 and after some gipsy life obtained a newly built apartment. It was a quiet and splendid time for me. I attended an inter-denominational school in Kulm, where boys and girls were co-educated together with young Germanized Poles, and found my first love, a tender girl with curled blond hair. This happy period ended abruptly in bitter cold January 1945. The Russian army was on the advance and we could already hear the near-by artillery fire. The Nazis, rigorously prepared to defend Kulm began, like all around Germany, to conscript all youngsters as well as any old men to the Volkssturm (German territorial army) endowing them with bazookas. My mother’s reaction was to pack a few small suitcases. (My father, for obvious reasons, had to stay behind.) I was to carry my satchel with some ‘‘important’’ belongings of my own, our silver stowed in a small suitcase in one hand and my violin given to me by my grandfather in the other hand. Where to turn? All nearby bridges across the Vistula had been conquered by the Russians and railway connections had been interrupted. Fortunately, the Vistula was completely covered with ice, so that our doctor was able to drive

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us by car across the frozen river, where in a small town further to the west we waited for the ‘‘last train’’ towards Danzig. When it arrived it was already full of refugees. By the crowd storming the wagons I was pushed under the slowly rolling train but my mother pulled me out safely, only my violin case was hit by one of the wheels and has retained a deep notch until today. From Danzig we could make our way to Stettin to meet our relatives, but as the town had been heavily bombed the city was nearly 90% destroyed, and we did not get permission to stay. Finally, my mother decided to turn northwest, to cross the Danish border and to reach Sønderborg, where my grandfather’s cousin lived. As Denmark was occupied by the German army, more than 200,000 refugees from the Eastern territories had been transported to Denmark. Many like us were allowed to stay in private quarters, until in May 1945 the Germans had to surrender to the British, an event that occurred without firing a shot. My grandparents, my mother, and I were then accommodated at the Sønderborg Masonic lodge which served as an internment camp. We lived together with some 40 people in the ceremonial hall, the walls painted black with a blue ceiling decorated with stars – but no daylight. Given this mystic atmosphere, the elderly ladies used to practice occultism behind black curtains. In early 1946, all German refugees were concentrated in larger camps and we were transferred to one near Sønderborg, a former large shack camp of the German marine. With some 40 people, we had to share a tiny hut heated by one cast-iron oven that was also used for cooking, no nearby showers or toilets were available. A young girl of 16 in the bed next to me died from tuberculosis. As I fell seriously ill, our family was moved to a smaller camp (the former German school in Broager), which was sort of return to more freedom. There were some 40 children who could enjoy a huge playground and go for swimming. I organized a children’s circus, staged two of Grimm’s tales and founded a harmonica ensemble. I found much leisure to do handicrafts and to read books I discovered in the attic, such as Eddington’s ‘‘Popular Astronomy,’’ teaching books in algebra and geometry, physics, and chemistry. I had an opportunity to learn French and English, an elderly woman teacher taking care of me. In February 1947, my father was able to return to his parent’s house in Siegen, which however had been nearly completely destroyed during the war. We were allowed to leave Denmark and


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to join him. We were fortunate to find one intact room in this house and, more importantly, an intact garden to grow all kinds of vegetables scarcely available otherwise (food ration cards were still in use). I managed to re-enter the local secondary school, and my grandfather taught me how to play the violin. My free time I spent with my friend Dieter Zimelka, who was handicapped by reduced verbal and physical capabilities due to spastic lesions, which he compensated by intelligence and will. We undertook cycle tours, collected plants for a herbarium, and were both interested in chemistry. After he had passed school with the best marks, he studied engineering at Aachen Technical University. We kept contact by correspondence and mutual visits until he died at the age of 47. In 1949, my father found a position in Du ¨ sseldorf and we moved into a new modest apartment there. Three years later, I finished school at the Max-Planck-Gymnasium in Du ¨ sseldorf and had to think about my future occupation. On the one hand, I was attracted by chemistry. During my time at school, I had been allowed to set up experiments for our lessons in physics and chemistry, and I even had performed some risky experiments at home. One day I stunned my mother, when I tried to produce bromine, which however ruined all nutrients in our kitchen: unfortunately, the bromine had crystallized in the cooling device and caused an explosion of the whole device. On the other hand, I developed a foible for architecture. From my father I had learned some skills in trigonometric techniques, civil engineering, and how to draw plans. Above all, I felt that architecture was an ideal subject for creativity. (Later, I experienced that chemistry could do the same!) Finally, it was decided that I should enrol for chemistry at Cologne University. The argument was that I could live at home and travel to Cologne every day by train; the main station was near our domicile. Unfortunately, chemistry as a subject was over-crowded. A major bottle-neck was to allocate so many newcomers (about three hundred candidates had accumulated among which were many late returnees from prisoners-ofwar camps) to a lab space to begin practical work. So I concentrated on lectures in chemistry, physics, and philosophy. Together with one of my new companions, Max-Dieter, I enrolled for mathematical courses which I followed for four terms. In my second year, I passed a test and was admitted to the first lab course starting with an endless number of inorganic analyses, for

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which we had to provide our personal equipment and all necessary chemicals ourselves, on top of the usual university charge. The good news was that once one had successfully finished a given program after passing an oral exam, there was a guarantee to take up the next course, which culminated in the analyses of an ‘‘exploded drugstore.’’ The ultimate goal – before we were admitted to the intermediate diploma – was to correctly work out the quantitative analysis of a piece of mineral within 2 days. The only unforeseen difficulty that arose for me and a few other candidates was that our assistant had overlooked that the institute was completely closed the first day (a local holiday), so that we had to find our way in and out through a window in the basement. Despite the very rigid schedule, I found time for some interesting ventures. After our third term, Max-Dieter and I participated in a vacation program across Italy organized by the Italian student association, my first ‘‘voluntary’’ trip to a foreign country. By train we traveled via Munich to Rome and continued to Naples. There our German group met with students from Italy, France, Finland, and England. In the evening we embarked on a small boat to sail to the volcanic island of Stromboli, where we were accommodated for 10 days in an open air camp near the picturesque, black shore. At this time, Stromboli was nearly empty – few inhabitants, no tourists. The painful aftermath of the big eruption 5 years earlier were still visible: destroyed and left houses, uncultivated gardens, and wild shrubs. A commemorative plaque on one of the houses documents that Roberto Rossellini spent some time on the island with Ingrid Bergman to produce his film Stromboli (1949). The most fascinating enterprise was a guided night-tour to the peak of the volcano where we enjoyed a marvellous sunrise. We continued by sailing towards Sicily touching most of the scenic Eolic islands and spent a week’s time in Messina and Taormina. In my fifth term, I was elected member of the student representatives (Cologne Student Council) and my job became to take care of ‘‘social and cultural affaires.’’ Among other activities, I was able to invite well-known literary cabarets, readings, the University of Michigan student choir, a Jamaican (!) steel band, and to arrange for an exhibition of Cologne student paintings. A most successful activity was to organize cheap bus tours to Paris. Transport plus a stay in a small hotel for 6 days


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was offered for 40 (!) Deutsche Mark per person. During my time, some 20 Cologne students got a chance to participate in a meeting with French students in northern France. We were put up in a small hotel located in the dunes of the Channel, enjoyed excellent meals (with four courses!) twice a day, met for political discussions, and were invited for sight-seeing and official receptions in Lille, Calais, Boulogne-sur-mer, and Dunquerque. An incredible story happened at my time as a member of the Student Council. The university financed a secretary to help in our common office. We hired a young lady saying she had escaped from Deutsche Demokratische Republik (DDR). After a year or so she left to take a governmental post in Bonn. It turned out, however, that she was a high-ranked spy who had used her initial job as a springboard to infiltrate the Ministry of Defence in Bonn. Like many students, I regularly took a job during the vacations. I worked in my father’s office, at the central chemical laboratory of Henkel Company in Du ¨ sseldorf or the chemical laboratory of Leybold (vacuum technologies) in Cologne. Henkel at that time was developing long-chain fatty alcohols esterified to sulfonic acid as new detergents for washing powder. They kept it secret, and only much later I realized what I had been working with. Several times I volunteered at the municipal gardening bureau in Neuss, where I was responsible for designing children’s playgrounds, and in the office of an architect in Cologne. In between I earned money by designing graphical advertisement for shops, for the Cologne tram, or privately. At one of such occasions as a working student I met Hildegard Beissel, who was enrolled as a student of economics at Cologne University, and we married after my diploma in 1960. Her brother Heribert Beissel, then a young conductor, arranged for a contact with the Bonn Theatre, and together with the Bonn ballet we staged three pieces, Mozart’s Bastien and Bastienne; Negro Spirituals; and Prokofieff’s Peter and the Wolfe. The material for the simplistic scenery I built in my 9 m2 Cologne mansard. After my intermediate diploma, I had to return to serious life in the lab and to follow the strictly regulated training as an organic chemist. At that time, the chemical institute was housed in an old hospital near the main buildings of Cologne University. Meanwhile, the area has been used to build new facilities, the new Institute of Genetics being one of the latest. During their diploma or even doctoral thesis work, the trainees had still to fully pay for

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any equipment and material. During the practical courses, bench space was limited to about 1.5 meter in width, and normally 40–50 people had to share a huge laboratory. Despite this high density, there was only limited contact or communication among the fellows. Actually, everyone buried himself in his own subject, mostly not realizing what his next neighbors were engaged in. The only common activity consisted in (rather boring) two-weekly seminars, where every fellow had to report news he found in periodicals he had been assigned to. The situation improved during my thesis work: we obtained more space, communication grew more intense, and resulted in a good co-operation between the lab fellows. I had already experienced that chemistry in some respect is a dangerous subject (not to mention the sometimes stinky air in the lab), and nearly every one of my colleagues had to pass through one or the other bad experience. I remember three personal accidents from this time, which, however, ended without too serious consequences. During our practical courses I was obliged to synthesize a mustard gas-like compound, which handled even under extreme precautions, caused a cauterization of the corneas of both my eyes; fortunately it was cured by special treatment and staying in complete darkness for 1 week. Two compounds I prepared for my thesis work invoked explosions upon gentle distillation, which ruined all glassware in the lab. My thesis work started in 1960 under the guidance of Professor Leonhard Birkofer, who at that time was the only organic chemist in Cologne since Professor Kurt Alder had deceased in 1958. His main interests concentrated on natural compounds, such as dyes of flowering plants like Petunia which he obtained from a cooperation with the institute of botany, as well as silicon-organic compounds. But the problem I had to investigate was the possibility of synthesizing asymmetric diamino acids which had never become known before. This turned out to be very tricky, because these compounds were extremely unstable. However, some of the precursors I had to prepare yielded novel N-heterocyclic compounds upon further reaction, and in the end I had collected and characterized some 20 of such derivatives [1,2]. In June 1962, I finished my PhD after having passed oral exams in three subjects, organic chemistry, physics, and physical chemistry, with the best marks, for which I was awarded a prize by our university. During this period of time, I gathered a first teaching experience as an


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assistant in chemical courses for medical students and candidates for a teaching post. Usually, in those years young chemists had no problem to find a suitable job, but after a number of interviews in leading German chemical companies, I decided not to take a position in industry, rather I was attracted by the possibility to do basic research. As no free post for a research assistant was open at the chemical institute, my boss recommended to apply as a post-doc at the newly founded Institute of Genetics in Cologne. In September 1962, I was engaged as a post-doctoral fellow by Hans-Georg Zachau, who headed the department for nucleic acid research, after I had convinced Max Delbru ¨ ck, appointed the first director of the institute, in an interview that I had an honest interest and a good qualification to do research in molecular biology. Undoubtedly, this was the decisive switch in my career. One has to recollect that around that time – and even for so many years to follow – no training in biochemistry, never mind molecular biology, was offered at German universities. During my studies of organic chemistry, protein chemistry was touched only peripherally, nucleic acid chemistry simply did not exist. So my first notion of this field stems from a fascinating lecture by late Fritz Cramer talking about methods of oligonucleotide synthesis, when applying for the vacant chair in Cologne. Of course it took some effort for a ‘‘beginner’’ to grasp the essentials of biochemistry, genetics, and molecular biology. But excellent books helped open this new world, for example, Biochemistry by Peter Karlson, Classical and Molecular Genetics by Carsten Bresch and Rudolf Hausmann (both from the Institute of Genetics); books on nucleic acids such as by A. Michelson or Lord Todd.

Institute of Genetics and tRNA How different became life in the Institute of Genetics! While in organic chemistry there were only two departments, each employing some three assistants and the same number of technicians, the Institute of Genetics accommodated five departments, each – on average – endowed with six scientific co-workers and roughly the same number of technicians. Conceived as an inter-disciplinary research institute, Genetics was open to collaborators from different fields, such as physics, chemistry, biology, or medicine, and most remarkably, to foreign co-workers. A completely new

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experience for me was to find a perfect ‘‘infrastructure’’: secretariat, workshop, library, cleaning kitchen, etc. A completely new experience for me was team-work, the superior maxim in every department of the Institute of Genetics. People collaborated and talked to each other very openly. Contacts between people from the single departments were guaranteed by weekly seminars and colloquia. In the seminars, all researchers and doctoral fellows had to elaborate on a given topic. One of the outstanding issues in discussions at that time (1962/1963) was the Genetic Code: triple, quadruple, comma-free or not? [3]. The colloquia were covered by invited speakers. For 1963 only, I have counted 68 renowned scientists from all over the world visiting the institute. When Max was around, he used to sit in the first row in order to ‘‘control’’ the speaker. If he felt something mysterious in this presentation, he turned to the audience: ‘‘Everybody got it?’’ If there was no clear-cut answer, the baffled speaker was urged: ‘‘Better say it again!’’ The relaxed atmosphere of the institute became manifest through the many parties, for which Max had a foible. A most spectacular venue was the farewell party for Max on July 19, 1963, when he had decided to return to CalTech at Pasadena. I have tried to document this venue by editing the original manuscript we used for the performances. In our sketch, Max was damned to be tied and boiled by the ‘‘wild Zachaus’’’ in a huge container, which we used for our large scale tRNA preparations. To mimic the boiling water, we had put some dry ice together with a little water onto the bottom. After a while Max sighed in great pain: ‘‘Can’t you at least remove the dry ice; my back is already burning?’’ Even after Max had left the institute this atmosphere was not lost, even during the ‘‘stormy time’’ the crew had to pass. Figure 1 shows Carnival decorations Rainer Thiebe and I fabricated to illustrate this phase of depression. It would, of course, be really tempting to tell more anecdotes about people and life in the institute during the first years. Clearly, the unconventional setup of the Institute of Genetics made it the birthplace for Molecular Biology in Germany. In 1962, I could engage myself in this field, experiencing and applying new techniques devoted to the analysis of transfer ribonucleic acids (tRNA), the subject Hans Zachau’s group (Figure 2) had decided to work on. My first task became what was called the ‘‘2u/3u


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Fig. 1. Carnival at the Institute of Genetics in Cologne, 1964. From left to right: Commander Max Delbru¨ck, Lieutenant Walter Harm, Vice-Commander Peter Starlinger, and Able-bodied Seaman Hans Zachau.

problem’’: it had been established that for protein synthesis the amino acids are hooked onto specific tRNAs via an amino ester bond to the ribose of the A residue of the ‘‘3u-CCA-end’’ and from there are transferred to the growing peptide chain, but it remained open whether the 2u- or the 3u-OH group of the ribose was involved. In a series of experiments, using chemical and NMR (nuclear magnetic resonance) spectroscopic methods, we compared a number of synthetic amino adenosyl esters with aminoacyl adenosine isolated from total tRNA that had been charged with amino acids by amnioacyl tRNA synthetases. While all synthetic compounds exhibited a ratio 30:70 for the 2u- versus the 3u-esters (probably due to acyl migration under the reaction conditions), at least 95% of the ‘‘natural’’ aminoacyl adenosine consisted of the 3u-ester. With this finding, we arrived at the conclusion that the 3u-linkage was the one active in aminoacyl tRNA [4–8]. From theoretical considerations, it had been argued that the 2u-hydroxyl of the terminal ribose should be the more reactive in the amino acylation reaction. Much later, this contradiction was solved by showing that two structurally different classes of aminoacyl tRNA synthetases do exist, class I

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Fig. 2. Zachau’s group (‘‘3rd Floor’’) at the Institute of Genetics in Cologne, 1962. From top to bottom and left to right: Susanne Notz, Hans Zachau, Fritz Melchers, Dieter Du¨tting, Horst Feldmann, Anita Mosch, Hugo Gottschling, Gisela Schultz, Paula Pru¨fert, Rainer Thiebe, Wolfgang Karau, Gudrun Patzelt, and Heidi Heusinger.

enzymes transferring the amino acid to the terminal 3u-hydroxyl, class II enzymes to the terminal 2u-hydroxyl prior to a rearrangement yielding the 3u-derivative, which in all cases is the active form of the charged tRNAs in protein synthesis. In 1963/1964, I became integrated into Hans Zachau’s main project, deciphering the primary structure of the major serine specific tRNAs from yeast. This subject again challenged the analytical skills of a chemist, but at the same time met my interest in molecular architecture. In fact, it was pure and hard chemistry at the beginning. We had to isolate the starting material from large quantities of brewer’s yeast. I guess during this period I myself worked up about a ton of yeast slurry I had to supply from


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¨lsch’’ brewery in Cologne. Each batch (about 100 kg ‘‘SESTER Ko yeast slurry) had to be stirred with 200 liters of a phenol/buffer mixture for several days, then the aqueous solution was decanted, the soluble RNA precipitated by adding 250 liters ethanol, and harvested by sedimentation in a huge centrifuge. The raw tRNA, resembling a brownish shoe polish, was purified on a DEAE cellulose column and yielded some 30 grams of white material [9]. Later, C.F. Boehringer and Soehne, Mannheim, set out to produce yeast tRNA on an industrial scale following this protocol, and we were lucky that they supplied enough of this material to us free of charge. The ensuing steps, namely the purification of the serine specific tRNA, consisted of a series of counter-current distributions in different solvent systems, whereby every fraction had to be tested for amino acid acceptor activity (i.e. in this case, enzymatic charging with radioactively labeled serine for measurement). We were fortunate that Zachau’s group received sufficient support to buy the most modern equipment available on the market: an automatic counter-current machine composed of 300 tubes with 20 ml capacity each manufactured by E.C. Apparatus Co., Swarthmore, Pa., and an automatic liquid scintillation counter developed and sold by Hewlett-Packard Company. In principle, these devices worked reliably, but any operator’s error could have fatal consequences. Once a tube of the countercurrent machine had been broken, a whole battery of 10 tubes had to be replaced, because no glassblower around was able to repair it, so we had to wait for an original set supplied by the company. A nasty but advisable procedure was to clean the apparatus after each use by washing it with sulfochromic acid to avoid potential contamination by ‘‘finger’’ ribonuclease. At the end of 1965, we finished sequencing of yeast serine tRNA [10–12], shortly after Robert W. Holley’s group in Ithaca had published the sequence of the first tRNA from yeast [13]. The analytical procedures involved complete and partial digestions with T1 and pancreatic ribonucleases, resolved on extremely thin DEAE (diethylaminoethyl) cellulose columns in 7 M urea, subsequent digestions with snake venom phosphodiesterase or micrococcal nuclease, followed by paper chromatography and spectrometric identification of the single constituents generated from the oligonucleotides by alkaline hydrolysis. In all, we collected several thousand UV-spectra, all recorded by hand.

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In fact, we had solved the primary structures of two very closely related molecules not completely separated by counter-current distribution, termed Ser I and Ser II. But when I analyzed each of the side fractions, it became clear that Ser I and Ser II, both present in about equal amounts in our preparations, differed in only three nucleotides (one in the TCC loop and two in the extra loop) [14]. Later, when the complete sequence of the genome had been determined (see below), no gene for Ser I could be identified in lab strain aS288C but 11 copies for Ser II. The ‘‘secret’’ of Ser I has never been solved. Probably, commercial brewer’s yeast we used (C836) consisted of two related species. Along with the sequence determination, I became involved in characterizing the structures of two ‘‘odd’’ nucleotides, N6-acetylcytidin and isopentenyl-adenosin (iPA), the latter of which we had found in serine tRNA at the 3u side of the anticodon [15]. A report on the serine tRNAs [16] was included in the Cold Spring Harbor Symposium on Quantitative Biology devoted to the ‘‘Genetic Code,’’ 1966. This offered me the splendid opportunity not only to follow the current achievements reported at this venue but to visit a number of laboratories in the US, at the University of Albany, at the Roswell Park Memorial Institute in Buffalo, at the University of Illinois in Urbana, at Oak Ridge Natl. Lab. in Tennessee, and at the University of Chicago. I also visited old friends from Genetics, Fritz Melchers at the Salk Intitute in La Jolla, San Diego, Max Delbru ¨ ck with his wife Manny and Charles David at CalTech in Pasadena, and Thomas Trautner at the UCSF, Berkeley. In order to take the cheapest way of flying to New York and back, I choose a prop-jet of Icelandic Airlines starting from Luxembourg, which offered a 24 hour stop-over in Reykjavik. There I met Johann Gudmundsson, who had obtained his diploma in chemistry during my time in Cologne and had taken a position in a fish cannery in Reykjavik. On the spot, he managed a sight-seeing tour in his veteran Ford car and showed me around a lot of the impressing country. This was ‘‘Iceland in 24 hours,’’ but the impressions from this intense trip should stay for the rest of my life. With so many contacts and after having done some work in my ‘‘new field,’’ it would have been easy for me to find a post-doctoral position in the United States, but just during my time in Genetics our two daughters had been born (Barbara in 1963 and Miriam in 1966), and so we preferred to stay in old Germany.


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Institute of Physiological Chemistry in Munich tRNA Biogenesis In early 1967, we had to say goodbye to Cologne and Genetics: Hans Zachau had been offered a chair at the Institute of Physiological Chemistry in Munich, and I was happy to get a tenure position there and to start my own laboratory. My first contact with yeast extended to a general topic of my research during the years to follow. What I had learned in Molecular Biology from the ‘‘early days’’ on, I tried to pass on to our students, and I am happy that even many of the medical students realized the importance of molecular biology for modern medicine. At first, I continued to work on the multiplicity of serine tRNAs, trying to isolate isoacceptors (e.g. the one specific for the AGU/ AGC codons), but this failed because of the minimal quantities of this species occurring in yeast [17]. (Later my lab at least succeeded in characterizing the three genes encoding this tRNA.) So I decided to work on the methionine specific yeast tRNAs, as we could be sure that there were two discernible activities, one for the initiation and one for the elongation of peptide chains [18]. After initial experiments, we decided to completely change our former sequencing strategy, now employing [32P]-labeled material and adopting the elegant ‘‘Sanger technique’’ [19]. I got accustomed to grow 24 liters batches of yeast cells fed with 200 mCi [32P] phosphate each time we needed fresh tRNA. All manipulations for harvesting the cells could be done during night time leaving no traces of radioactivity in the lab or elsewhere. Supernatants were put to a device in the basement and stored in big tanks for decontamination, solid waste was kept in big iron casks for more than 10 half-life times of [32P] so that virtually no radioactivity remained. A more serious issue was to convince our colleagues that, with appropriate precautions such as special safety hoods and a CO2 extinguisher, there was no problem performing high voltage electrophoresis in tanks filled with 20 liters kerosin for cooling. Fortunately, I could pay a short visit to the Sanger lab at MRC (Medical Research Council), Cambridge, where Bart Barrell showed me several tricks how to run the analyses and how to interpret the radioautographs obtained after 2D-electrophoresis. We published the primary structure of the yeast non-initiator methionine tRNA (tRNAMet 3 ) [20,21] after Tom [22]. RajBhandary’s lab had finished that of tRNAMet i

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Besides this structural work I became interested in the characterization of precursors to tRNA in yeast as a model system. tRNA precursors had already been studied in E. coli and phage T4 [23,24] and Sidney Altman and collaborators had detected that endonuclease P was necessary to produce mature tRNA from its precursors [25], but no details were known in eukaryotes [26]. We used gel electrophoresis of ‘‘soluble’’ RNA preparations of yeast cells pulse-labeled with high doses of [32P] phosphate and found that some distinct bands migrating slower than mature tRNA appeared. The smaller of these products contained minor amounts of the tetranucleotide TCCGp characteristic for tRNA but only traces of other minor nucleotides, while the larger of these products contained the tetranucleotide UUCGp but no minor nucleotides. Thus we concluded that these bands represented precursors to tRNA. This was confirmed by incubating material isolated from several of these gel bands in vitro in a yeast cell lysate, which yielded mature tRNA in kind of a two-step process [27]. Soon after, Goodman, Olson, and Hall reported on the first yeast tRNA gene to have an intervening sequence [28]. In conjunction with our efforts to identify the precursors to tRNA, we developed a two-dimensional gel electrophoretic system that enabled us to reproducibly map 40–50 individual tRNA species including isoacceptors from yeast, as well as individual precursors [29]. tRNAs were identified by (i) co-electrophoresis of purified [32P] tRNAs with non-labeled bulk tRNA; (ii) comparison of patterns derived from pure tRNAs with bulk tRNA; (iii) fingerprinting of spots from pure [32P] tRNA species; (iv) electrophoresis of bulk tRNA charged with one [3H]- or [14C]amino acid, whereby the aminoacyl tRNA was stabilized prior to electrophoresis by transforming the amino group into a 100-fold more stable OH-group. We realized that this technique of high resolution capacity – among other applications – was not only helpful to islolate specific yeast tRNAs but to identify the amino acid accepted by them, likewise also for determining the specific tRNAs contained in a tRNA population from any organism. One example is kindly mentioned by Guy Dirheimer, whom I met for the first time in the 60’s, in volume 44 of these Personal Recollections [30]. He sent Jean Weissenbach to my lab to learn the details of the procedure. They applied it to separate and isolate the mitochondrial tRNAs from yeast and started sequencing of several of those (e.g. [31]), a subject the Strasbourg lab


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very successfully continued until 1986, also paying attention to the non-canonical codon recognition and the biogenesis of minor nucleotides in yeast mitochondrial tRNAs [32,33]. The collaboration between Strasbourg and Munich was intensified by mutual visits of our whole groups during these years. Excellent opportunities for contacts with the many colleagues working in the tRNA field and exchange of new results and ideas were offered by the yearly tRNA workshops each one organized by a particular laboratory at most attractive places world-wide. Of those I keep a deep memory (and the Abstract booklets, too) are ¨ttingen (1971), Nof Ginossar the ones in Cambridge (1970), Go (Kibbuz in Israel), organized by Uriel Littauer in 1975, Sandbjerg (Alsen, Denmark; 1976 – because it took me back to the area where I had spent 2 years as a boy), Aarhus (1978), Strasbourg ˚ (1987). (1980), Tokyo (1983), Taos (New Mexico; 1985), and Umea Most impressive was the workshop in Japan held in Hakone, a resort area by the foot of Fujiyama at a hotel built in 1899 for a visit of the German Emperor. I extended the trip with my friend Wolfgang Wintermeyer to visit Tokyo, Kyoto and Nara, Singapore, Hong Kong, Bangkok, and Taipei, which was easy to arrange as we flew by Singapore Airlines who offered multiple stop-overs at low cost. A very attractive and scenic place was Taos, where the meeting in 1985 was held in a motel, not far from the Taos skiing area, which some of the participants enjoyed for one free day. The 70 fantastic slopes had been built by an Austrian fellow and given fairy tale’s names like Schneewittchen (Snow White), Rumpelstilzchen, etc. To reach the meeting, I had to rent an Ugly Duck in Albuquerque, but afterwards this car was good enough to carry me about 2,000 miles around Four Corner’s Monument. I enjoyed the picturesque landscape with its big miracles of nature among others Mesa Verde, Monument Valley, Painted Desert, Petrified Forest, and Grand Canyon. The two-dimensional gel electrophoresis system prompted Walter Kleinow from the Zoological Institute in Cologne, who worked on mitochondria of Locusta migratoria, to start collaboration with us. From the minute amounts of RNA he was able to prepare, we succeeded to first detect the unusually low (GþC) content for mitochondrial rRNA and tRNA and than to resolve B27 tRNA spots, which migrated faster than the cytosolic tRNAs indicating smaller sizes of the mitochondrial tRNAs [34]. That the

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population of 27 tRNAs was far below the minimal number of tRNAs to translate all sense codons according to the Wobble hypothesis (32 in cytoslic tRNA) had just been reported by Dirheimer and colleagues (e.g. [35]) from their investigations on yeast mitochondrial tRNAs. However, the smaller size and the low (GþC) of the locust mitochondrial tRNAs were unexpected and pointed to further peculiarities of these molecules. The low amounts of material available to do further studies on locust tRNAs tempted me in 1976 to ‘‘bit to the side’’ and to devote part of our lab activity (Figure 3) to the isolation and analysis of mtDNA and tRNA from rat liver mitochondria. Around this time, some data on these issues had been published but they were rather inconsistent. So I interested a new doctoral student, Ru ¨ diger GroXkopf, to take up this work in 1977/1978. In the beginning we had to isolate the starting material each time from sacrificed rats as our laboratory had no permission for cloning as yet. Also home-made restriction enzymes had to be employed and I am still grateful to the colleagues from Zachau’s group for their generous gifts of several enzymes. So, as a basis for

Fig. 3. My collaborators in 1977: Antonin Eigel, Christa BleifuX, Maria Wagner, Petra Mu¨ller, Gabriele Goertz, and Ru¨diger GroXkopf.


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further experiments we established a reliable restriction map of the mtDNA [36], and finally could take advantage of cloning. Using the Maxam-Gilbert technique [37], large portions of the mtDNA were sequenced and analyzed [38–40]. These publications document that we did not give up our efforts, though I became aware on a trip to Spetses in 1979 that Fred Sanger’s group in Cambridge was about to publish the full sequence of human mtDNA [41,42]. But we consoled ourselves that we were two people that had started in ignorance, and that they were by 14 people. We were satisfied, however, that the work of Dirheimer and his colleagues on yeast mt tRNA or ours on locust mt tRNA had been extended to human mitochondria. This knowledge on the human mitochondria later formed a basis to recognize the many diseases caused by mtDNA mutations.

Yeast tRNA Genes and Ty Elements Once we had learned that even eukaryotic tRNA genes occupy more genomic space than prescribed by the structural part, we became interested in the problem, how many genes do code for a particular tRNA species in yeast and how are these genetic entities arranged within the genome? Two organisms had been studied thus far in some detail: E. coli [43] and Xenopus laevis [44]. For E. coli, it had been established that tRNA genes sometimes are encountered as singular transcriptional units but that the majority of them were found to be arranged as multimeric transcriptional units, either in polycistronic entities, or interspersed into ribosomal RNA transcriptional units. For eukaryotes it was known that tRNA genes occur at a higher redundancy than in prokaryotes. Particularly in Xenopus oocytes gene redundancy can amount to several hundred copies per tRNA species. Moreover, it had been demonstrated that here the tRNA genes together with non-transcribed spacers form clusters of serially repeated sequences. Our initial experiments using tRNADNA hybridization indicated the occurrence of B10 gene copies for some of the major tRNA species from yeast tested [45], but I was mislead to interpret our results as several (isogenic or nonisogenic) copies being clustered like in Xenopus [44] or in Drosophila [46]. A more reliable statement was obtained by measuring the lengths of transcriptional units of tRNA genes by

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the UV-light technique [47]. Clearly, the transcriptional units were no longer than 200 bp for almost all of the 13 tRNA species tested and we concluded that these tRNA genes are scattered throughout the genome in singular entities. In 1978, we began to investigate the genomic organization of yeast tRNA genes at the molecular level. This became feasible only when we had adopted the genetic engineering techniques developed from the mid-1970s on. The beginning of genetic engineering undoubtedly was the successful approach of Paul Berg and his collaborators to show that recombinant DNA could be maintained in a host cell [48]. I vividly remember a long night session with full moon in the courtyard of a monastery at the Summer School 1971 held in Erice, where Berg, Sanger, and Tomkins chaired a discussion on the three paradigm shifts initiating a revolution in Molecular Biology: the discovery and use of restriction enzymes [49–51]; the utilization of recombinant DNA; and the necessity for developing methods allowing the determination of long DNA sequences, which had to follow principles different from the ones applied to the sequencing of RNA and became reality in the years to follow [52–54]. Though since 1972, several methods had been developed for cloning and characterization of recombinant DNA molecules, it was only after the Asilomar Conference on Recombinant DNA [55] that safe and simple procedures and bacterial vehicles could be propagated for extensive use of cloning recombinant DNA molecules. A big advantage of cloning vehicles based on phage lambda was the larger size of DNA sequences that could be accommodated. Those latter properties were shared by the cosmids, plasmid gene-cloning vectors packageable in phage lambda heads. These methods were applied to yeast genes, too. The transformation of yeast cells by replicating hybrid plasmids, however, was the first successful transformation of a eukaryotic cell and marked a break-through for yeast molecular biology [56,57]. Our first approach to clone various tRNA genes from yeast was only semi-selective and largely followed conventional cloning techniques, but our aim was clear: we wanted to study the structure and genomic environment of these genes and learn how they are expressed. Judith Olah coming as a post-doc was to initiate this work [58]. Unfortunately, in October 1979 an


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unforeseen incision happened: I suffered a stroke and became paralyzed on my left body side. This put me back for a very long time. Luckily for me, I did not loose speech or mental capacity. Though I recovered after some years of physical restriction, at the age of 47 I had no longer a chance to look for a position abroad. So, I modestly continued the work on tRNA genes with the invaluable help of a very nice lab crew [59–61]. Once we had collected data for several tRNA genes, we noticed that their flanking sequences revealed extended blocks of homology, including those for isoacceptors as well as those for non-related genes [61]. These similarities were largely detected by eye-inspection, as computer-assisted searches at that time were not yet available. The occurrence of repetitive elements in this context was a novel observation, since all sequences of yeast tRNA genes determined by our colleagues did not reach very far into the flanking regions. We speculated that the sequence elements might be of functional significance for the expression (like the conserved internal A and B boxes or the 3u-termination signal) or for the process of dispersion of the multiple tRNA gene copies over the yeast genome. In 1982, with more detailed sequence information, we obtained evidence [62] that most of the repetitive sequences represented specimen of the newly detected class of yeast Ty1 elements or their dispersed LTR remnants (solo delta’s) [63]. Shortly after their discovery, Ty1 elements had been shown to mediate DNA rearrangements (e.g. [64,65]), and, in accord with their capability of transposition, they could be moved to new chromosomal loci into pre-existing Ty1 elements by a gene conversion mechanism or be excised from a given chromosomal locus leaving behind only one of their deltas. In consecutive studies we were able to confirm that the 5u-flanking sequences of tRNA genes constituted preferred integration sites for Ty transposition, and that these were localized in region-specific distances upstream of the genes; in many cases, multiple integration and excision events were documented genome-wide [66–68]. The phenomenon of preferred integration of Ty elements upstream of tRNA genes was later inforced by other groups investigating Ty-host interactions (e.g. [69,70]). Although target site selection is still not well understood for this general class of elements, it is becoming clear that Ty elements target their integration to very specific regions of their host genomes, probably in order to prevent disturbances of cell

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integrity. Targets containing genes transcribed by RNA polymerase III (Pol III) were found up to several 100-fold more active as integration targets for Ty1 than ‘‘cold’’ sequences lacking such genes. High-frequency targeting was dependent on Pol III transcription, and the region specific integration was found to occur in an upstream window of B700 bp [71]. The pattern of insertion was non-random and not distributed equally throughout the genome, but periodic, with peaks separated by B80 bp. Recently, it has been demonstrated that ATP-dependent chromatin remodeling by Isw2p upstream of tRNA genes leads to changes in chromatin structure and Ty1 integration site selection, and that Bdp1p, a component of the RNA polymerase III transcription factor TFIIIB, is required for targeting of Isw2 complex to tRNA genes [72]. Over the years, details on the structural and functional organization of the yeast transposons were worked out by several groups. The Ty elements became useful models for the epitome of retrotransposition, and manifold aspects pertinent to this theme are still under study to present. First indications that Ty elements represent autonomous genetic entities which direct expression of endogenous genes was obtained from experiments in Kingsman’s laboratory [73]. Soon it was established that Ty1 followed a retrovirus-like strategy for the expression of a large fusion protein [74]. Concomitantly, a second class of variant Ty elements, Ty2, was shown to obey a similar sequence organization and expression strategy as the Ty1 class elements [75]. Other experiments confirmed that Ty elements transpose through an RNA intermediate [76]. The retrovirus-like gene organization in Ty1 became also evident from its complete nucleotide sequence [77,78]. In the Ty1/2 elements, two open reading frames, TYA and TYB, comprise sequences encoding the retrovirus-like gag and pol proteins, whereby a translational frameshift (in a þ1 mode) can occur in the region overlapping TYA and TYB [79] thus producing a gag-pol polyprotein. Finally, the minimal site for ribosomal frameshifting in Ty1/2 was determined to be a seven nucleotide sequence which induces tRNA slippage involving a minor tRNA species [80]. This finding rendered an explanation at the molecular level as to why the gag versus pol protein precursors were produced in a ratio of 20 to 1: translation of TYA was stopped at a usual stop codon in this minimal site, while


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read-through by frameshifting was limited by the availability of a rare tRNA. Further evidence for the similarity between the Ty elements and retroviruses was provided by the finding that the RNA transcript of Ty together with the smaller proteins processed out of the precursor proteins by the Ty protease [81] are associated with virus-like particles in yeast [82]. In contrast to retroviruses, however, these Ty virus-like particles (Ty-VLP) turned out not to be infectious and hence are trapped within its host. The detailed characterization of Ty3 (a gipsy type retrotransposon) revealed [83] that this element also transposes via VLPs as transposition-competent particles and exhibits translational frameshifting in a þ1 mode. Previous studies by the group of Susan Sandmeyer had shown that Ty3 (or its Long Terminal Direct Repeats (LTRs), sigma) insertions had occurred consistently in a 16–19 bp distance upstream of several tRNA genes. In this concert, Rolf Stucka identified Ty4 as a new type of yeast elements occurring in low-copy number, belonging to the class of copia elements and possessing a gene organization and expression strategy similar to Ty1/2; Ty4 also integrates into tRNA upstream regions [84,85]. However, we found that transposition capability was rather low [86]. From a collaboration with Cećile Neuve´glise and her colleagues on the genomic evolution of retrotransposons in Hemiascomycetous yeasts it appeared that Ty4 is an evolutionary recent element [87]. The last retrotransposon found in yeast, Ty5, revealed a number of features deviant from those of the other Ty elements: its preferred target sites were identified to be silent chromatin regions, such as origins of replication at the telomeres and silent mating type loci [88]. Targeting was found to be mediated by interactions between Ty5 integrase and silencing proteins, and it was argued that recognition of specific chromatin domains may be a general mechanism by which retrotransposons and retroviruses determine integration sites [89]. The second question, whether there was a transcriptional interference between Ty insertions and tRNA genes was answered positively by our first experiments using micro-injection of various such constructs into Xenopus oocytes [90]: particular segments revealed a stimulatory effect on tRNA gene transcription. As this constituted a heterologous system, we sought to

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prove our findings in the homologous system. In order to be able to identify and quantitate transcripts from an individual gene, Robert Krieg and Rolf Stucka synthesized a unique ‘‘artificial tRNA gene’’ (SYN2): it was tagged by an intron-like sequence that could not be spliced out from its long precursor but otherwise behaved like resident tRNA genes [91]. This gene combined with various Ty constructs and integrated as a single copy each into the yeast genome was used to monitor the transcriptional interference between Ty (and segments thereof) and a flanking tRNA gene as well as the chromatin conformation of the stable transcription complex and its flanking regions [91,92]. Figure 4 shows a photograph of my collaborators involved in this work at a birthday trip in 1988. We observed that there is a modest stimulatory effect (like in the majority of regulatory systems in yeast) of Ty or LTR insertions upstream of a tRNA gene on its expression in vivo. Transcriptional interference between Ty1 insertions and two POL III-transcribed genes was later also shown in the cases of

Fig. 4. People of my lab crew around 1988: Gertrud Mannhaupt, Hans Lochmu¨ller, Susanne Mitzel, Robert Krieg, Rolf Stucka, Christa Schwarzlose, and Uschi Obermeier.


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tagged SNR6 and SUP2 [93]; vice versa, RNA analysis indicated a modest tRNA position effect on Ty1 transcription at native chromosomal loci. Further, this study revealed that tRNA genes exert a modest inhibitory effect on adjacent pol II promoters, a result that was confirmed in other experiments [94]. The problem of correlating tRNA gene expression and chromatin structure is more complex. The data we and others (e.g. Refs [91–93], and references cited therein) obtained supported the following model: (i) tRNA genes counteract the formation of a canonical chromatin structure over a window reaching from B30 bp each upstream and downstream. In other words, actively transcribed tRNA genes have to be kept free of nucleosomes. (ii) The general pattern tRNA genes exhibit in DNaseI digestion experiments is a triplet of hypersensitive sites resulting from protection of sequences at the A and B box elements and accessibility upstream and downstream from the structural gene and between the A and B boxes, reflecting the binding of TFIIIC to the intragenic promoter and the tight binding of TFIIIB to the upstream transcription initiation site (B30 bp in length). (iii) Accessibility of this site by TFIIIB is crucial for active tRNA gene transcription, so that this region has to be kept in a nucleosome-free configuration. (iv) In DNaseI experiments the adjacent hypersensitive site(s) indicating canonical nucleosome spacing are located B170 bp and B340 bp upstream from the initiation start site of actively transcribed tRNA genes. The first upstream nucleosome in these instances is found positioned in a way as to form a boundary induced by the transcription complex. (v) A prerequisite for the induction of such a constellation is that the formation of the transcriptional complex outweighs the formation of nucleosomes, a situation that preveiled in competiton experiments. (vi) Whenever the sequences upstream of a tRNA gene are ‘‘favorable’’ to assist this positioning effect, transcription is enabled at a normal or even slightly elevated level. In ‘‘unfavorable’’ cases, however, nucleosomes can be formed over these sequences thus exerting a constraint for transcriptional initiation. We documented this correlation in a number of experiments using appropriate constructs and mapping hypersensitive sites and transcriptional efficacy concomitantly [91,92]. The highest transcriptional rates were always found in constructs, in which Ty elements, delta or tau sequences had been placed into ‘‘native’’ distances upstream of a tRNA gene.

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The Yeast Genome Project In the years between 1985 and 1988, our laboratory work slowly developed into a new phase. During their thesis work, Joachim Hauber and Peter Nelbo¨ck-Hochstetter had used high-molecular weight yeast DNA to clone fragments 35–40 kb in length into a cosmid vector (pY3030) that W. Piepersberg from the Munich Institute of Genetics had kindly provided to us. Both of them felt that it was timely to turn to a genomic scale, dissecting the organization of genomic entities along whole chromosomes. With the help of a technician Peter managed to establish a cosmid bank of 3,000 distinct clones, to prepare DNA from these by ‘‘mini-preps’’ and to fix it onto filters in a slot-blot apparatus within 4 weeks. This procedure was repeated a second time and we ended up with two ordered cosmid libraries in all representing the 12.8 Mb yeast genome at P ¼ 99.99%, that is 12 times the ¨ck genome equivalent. Together with Rolf Stucka, Peter Nelbo established a physical map of chromosome II by means of ‘‘chromosomal walking’’ and succeeded in localizing a variety of tRNA genes, Ty elements, and genes for diverse functions. Though these results were only published in form of doctoral theses, somehow we had sort of a favorable undercover press, which caused Andre´ Goffeau from Louvain-la-Neuve to ring me up in the lab 1 day in summer 1988. He asked me whether we would like to contribute to an assessment on ‘‘Sequencing of the Yeast Genome’’ he was preparing for the BRIDGE Programme of the European Communities [95]. We were enthusiastic about this initiative and, together with Yde Steensma from Leyden, produced an overview on how to take advantage of ordered cosmid libraries for such an ambitious project. It is a pity that this assessment to which a number of renowned colleagues participated, has never been made publicly available. It contains some ideas or predictions which never became reality, but many others that did. Andre´ tells more details about the difficulties he encountered when considering the launching of chromosome III sequencing [96], which for a number of reasons had been chosen to be the first chromosome to be tackled in 1989. After he had solved the bureaucratic obstacles, the enterprise run rather smoothly thanks to his untiring effort as well as that of Steve Oliver from Manchester as the ‘‘DNA coordinator’’ and the 35 European colleagues who participated in this project. Gertrud


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Mannhaupt, whom I could hire as a senior collaborator, together with Irene Vetter, took care of our share in chromosome III sequencing but Gertrud finally became the ‘‘good spirit’’ of all our further scientific enterprises. We were happy that we received EC support for our sequencing contributions, about 5 ECU per final base pair assembled as Andre´ had promised and accomplished. Moreover, the payment was never delayed as soon as the sequences had been proven and annotated. The collection and assembly of the sequence data had been put in the hands of MIPS (the Martinsried Institute for Protein Sequences), that had been developed by Werner Mewes at the Martinsried Max-PlanckInstitute for Biochemistry. He had set up the necessary informatic infrastructure which he – sometimes under considerable bureaucratic difficulties – was able to offer for the whole time of the yeast genome project. As I myself had fun in computing, we were in close and most friendly contact all these years, and still are after he moved to another university institution near Munich. Before the sequencing of chromosome III was started in 1989, Andre´ had initiated regular meetings of a ‘‘Steering Committee’’ that should supervise current and future activities. As soon as the first data had been collected, the sequencers and the committee met at regular intervals for progress reports and to exchange know-how and expertise. We followed the proposal of Andre´ and Steve to publish coherent regions from chromosome III, whenever the analyses had yielded useful information on new genes [97–99]. Before chromosome III had been finalized in 1992 [100], Andre´ and the ‘‘network’’ had to decide how to continue. As our initial complete chromosome II cosmid library [101] was not found suitable to serve as DNA material to start the project (the DNA was from an industrial strain), Rolf Stucka took the initiative to develop a new library from the lab strain aS228C, which had been given preference in the discussions of the Steering Committee. Thus we were happy to be awarded the contract for sequencing chromosome II (B820 kb) this time. Concomitantly, Bernard Dujon’s laboratory which had prepared an ordered yeast cosmid library by means of another vector [102], started sequencing chromosome XI (B670 Kb) and finished in 1994 [103]. Though we were paid as ‘‘DNA coordinator,’’ EC had reduced the subsidiary amount allocated for each final bp to 2 ECU. Luckily for us, the helper in the hour of pecuniary need was the German Ministry for Science and Technology (BMFT) which

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raised our income per bp by another 2 ECU. I might insert here that our lab largely had to rely on funds (for staff, equipment, and consumables) we had to invite from grant-giving institutions. While, for example, the Deutsche Forschungsgemeinschaft (DFG) in those years was not prepared to subsidize any sequencing project, the BMFT was open for this type of funding, because they could expect to gain from novel technical developments and to draw new insights from initiatives such as the yeast genome project. Indeed, it was obvious for anyone that the final goal of this project was much beyond establishing the complete sequence of a small eukaryote, namely to use this information for concurrent or subsequent functional analyses. As a fact, the wealth of information obtained in the yeast genome project turned out to be extremely useful as a reference against which sequences of human, animal or plant genes, and those of a multitude of unicellular organisms under study could be compared. Andre´ has given credit [96] to BMFT’s ‘‘outspoken support y (without which) y the EC would not have been engaged in sequencing the yeast genome.’’ While Gertrud Mannhaupt with three technicians took care of the sequencing and the analysis of particular regions of chromosome II [104–107] and later those from chromosome XV [108], Rolf Stucka was providing cosmid clones to the 18 European sequencing laboratories. We used to travel to the internal meetings with an 8 meter long detailed map of chromosome II to co-ordinate the program and to avoid too much overlapping during sequencing. It is remarkable that Rolf found time to follow his own projects, one of which was aimed at aspects of sugar metabolism – in a nice collaboration with Carlos Gancedo from Madrid [109–112]. Before the sequence of chromosome II had been solved [113], our lab concentrated on a new theme (see below). None the less, we eagerly followed the rapid developments in sequencing the many chromosomes that were left and brilliantly managed and brought to success by Andre´ Goffeau within the next 2 years [114–116]. As Andre´ Goffeau mentioned [96] he succeeded ‘‘to keep a collaborative spirit in the international yeast-sequencing community’’ by arranging the full participation of some American groups (particularly with the help of Mark Johnston) as well as that of Howard Bussey’s, Bart Barrell’s, and Peter Philippsen’s groups to the project. The initial aversion of the American scientific community to engage in an


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international network for sequencing the yeast genome was made clear to me during the 1990 Cold Spring Harbor Meeting of ‘‘Genome Mapping and Sequencing’’ to which I participated. Andre´ had asked me to present our intentions in a short ‘‘free’’ communication, but the organizers curtly refused. Only Dr Ito, a Japanese who worked at the Max-Planck-Institute for Genetics in Berlin and who chaired this session, was kind enough to allot 5 minutes for my presentation. The sequencing project was followed by several collaborative initiatives for functional analyses of the wealth of novel genes that had been detected. We participated in a German network chaired by Karl-Dieter Entian from Frankfort [117], and I was able to document our continuous affiliation with the yeast projects in separate chapter of three books [118–120].

Yeast 26S Proteasome and Triple A Proteins In connection with our work on Ty elements and the yeast genome sequencing project we became interested in yeast transcription factors. By looking into the TATA box binding factor, Rolf Stucka found that the protein sequence had a bipartite structural symmetry – probably the consequence of an ancestral gene duplication – that helped explain its saddle-like structure [121]. The theme ‘‘transcription factors’’ became a major issue when I got involved in setting up a special DFG program devoted to ‘‘Factors and Mechanisms of Gene Activation’’ (see below). In this context, we wanted to search for new ¨ck had just factors involved in Ty expression. Peter Nelbo published a paper [122] in which he reported on a putative novel protein interacting with the human immunodeficiency virus tat transactivator, called TBP-1 [122]. As HIV in many respects resembled Ty, we asked for a probe to search for similar factors in our yeast cosmid library. Already the first experiments Rolf Stucka undertook in 1992 were successful: a dozen different clones could be isolated and sequenced by our lab crew. To our surprise, the most prominent feature of the encoded proteins was a highly conserved domain containing nearly 200 amino acids, in one or two copies, encompassing two sequence elements characteristic of a novel type of putative ATPases. The first observation of this new category of proteins and its designation

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‘‘AAA proteins’’ stems from a paper of the laboratory of Wolf Kunau [123]. They noticed that the novel motif occurred in three groups of proteins fulfilling different cellular functions, such as Sec18p and NSF; Cdc48p, p97, and VCP; and TBP-1 (quotations, see Ref. [124]) and argued that ‘‘these proteins are members of a novel family of putative ATPases and may be descendants of one common ancestor.’’ Indeed, within a short time this family was found to be more widespread than initially thought and comprised a large variety of members in both eukaryotic and prokaryotic organisms. The first international conference on AAA proteins was held in Gif-sur Yvette in 1995 and the name for this family accepted by the community. When we published our results, further members of the AAA family had been characterized ([124]; and references cited herein). From comparisons we inferred that our collection specified four members of the yeast 26S proteasome, Yta1p, Yta2p, Yta3p, and Yta5p, whereby Sug1/Cim3 (as well as Cim5/ Yta3) had been discovered [125] during our work on this subject; the sixth yeast proteasomal AAA protein was recognized as Sug2 [126]. In order to keep a unified nomenclature, these AAA ATPases, meanwhile identified as subunits of the regulatory particle of the 26S proteasome, were called Rpt1 through Rpt6, whereas the non-AAA proteins were designated Rpn1–14 [127]. Likewise interesting became the Yta10, Yta11, and Yta12 proteins, which were highly similar in structure and also revealed the presence of the novel ATPase domain but bearing additional modules. Homologies with ftsH/hflB from E. coli were obvious as well as sequences typical for mitochondrial ‘‘matrix-targeting domains.’’ These and further notions led us to propose [124] ‘‘that Yta10, Yta11, and Yta12 may represent subunits of a proteasomelike complex in yeast mitochondria. This complex might be similar and evolutionarily related to the cytosolic 26S protease and thus constitute a mitochondrial proteolytic system in addition to the one that bears similarity to the Lon proteases y’’ Reimund Tauer engaged in this subject [128] and – in a fruitful cooperation with Thomas Langer and his group [129] working in Walter Neupert’s department – it was established that these three AAA proteins formed a novel ATP-dependent complex in the inner membrane of mitochondria with proteolytic and chaperone-like activities [130], whereby the proteolytic activity was found to be exerted by a Zn-dependent metallo-protease module linked to the


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AAA domain. Thomas Langer and his collaborators took care of the further characterization of this ‘‘mitochondrial quality control system’’ [131] and since worked out a wealth of important aspects in the biogenesis of mitochondria [132]. Together with Thomas, we organized the second international workshop on Triple A proteins in 1997 at Tutzing (upper Bavaria) supported by EMBO. The S. cerevisiae genes encoding the proteasomal entities are single copy throughout, and the majority of them are essential for cell viability. As I was convinced that the yeast ubiquitinproteasome system (similar to the ribosome) fulfils the requirements of a regulatory network, in which expression of the single genes is co-ordinated, we followed this issue. Again, it turned out a justified speculation. First, we detected a unique upstream nonamer box (GGTGGCAAA) which we called PACE (proteasome associated control element) to occur in the promoter regions of 28 out of the 31 proteasomal genes, including the RPT genes and most of the RPN (non-AAA) genes, and in B50 further yeast genes involved in the ubiquitin-proteasome pathway as well as in genes mediating diverse regulatory functions in yeast. We set out to identify the cognate DNA binding factor and were surprised to come across Rpn4, a subunit of the 19S regulatory cap. The structure and the role of Rpn4 to act as a transcriptional activator was substantialized in a number of experiments [133], for which we took advantage of a reporter system we had developed many years before [134]. Unfortunately, after this seminal contribution I had to quit my lab due to age. In the following years, it was demonstrated that Rpn4 links base excision repair with proteasomes [135]. Further ingenious studies by Alex Varshavsky’s lab revealed that Rpn4p is a ligand, substrate, and transcriptional regulator of the 26S proteasome and exerts a negative feedback control (e.g. [136–139]). RPN4 appears to be under control of several stress factors, such as Yap1p and Pdr1/3p [140]. On the other hand, Rpn4p strongly mediates the cell’s adaptation to arsenic-induced stress as revealed by expression profiling [141]. Filamentous-form growth is controlled by many modules in an integrated network, in which the proteasome system is probably integrated through Rpn4 [142]. Also in mammals and Drosophila (for review [143]), a similar system appears to be operative, but no molecular details are available as yet.

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Administration and Teaching Medical Students In 1967, when we had moved to Munich, for some period of time I was to take care of all kinds of administrative business: purchase and maintenance of equipment, accounting, etc. One task that met my interests was to keep liaison with the university building department, which had begun to raise an eight-storey new institute in our courtyard that should offer more space to the two new chairs of biochemistry (see Ref. [144]) and physiology. Theodor Bu ¨ cher, who initially was the only head of the institute of physiological chemistry, in his negotiations with the Bavarian Ministry of Science had accomplished that three new departments were created. Though we could improve some internal facilities during the construction of the new building (such as lab equipment, isotope labs) we were not able to prevent serious mistakes that are still persistent despite many necessary and costly repair over the years. For my part I was happy to move into a new lab and a well-equipped isotope lab in 1971. All the years I worked at the Institute of Physiological Chemistry in Munich, I had to look after the training of medical students at several levels. These teaching obligations and the administrative loads connected with them were greatly put on the shoulders of the ‘‘younger’’ staff, because most of the professors preferred to concentrate on giving the general lectures. One of my first activities was to organize a serious practical course in biochemistry, setting up reasonable experiments. The most intriguing problem was to develop a fixed schedule to hurry so many students through these venues within the minimal time of four terms: logistics had to consider subject and number of students as well as time, space, and staff available, in other words the time-table had to be strictly non-overlapping for each individual. Whoever has encountered such a demand, knows quite well that it can hardly be solved by computer programs. With my colleague Joachim Otto from the neighboring department we managed to develop a ‘‘master-plan’’ which was followed through 25 years. In 1974, the faculty entrusted me with the coordination of pre-clinical education and I had to chair the meetings of a committee consisting of representatives from every discipline, university administration, and examination board. In the beginning this task took much of my time, but in the end the meetings had to take place only twice a year.


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Analytica In 1977, I was elected member of the Scientific Committee for the bi-annual Conference of Bioanalytics held in Munich, connected to a world-wide respected trade fair in biochemical, biomedical, and bioanalytical instrumentation, the Analytica. I was then nominated to take care of the formalities connected to the award of a special prize Biochemical Analytics (later: Molecular Bioanalytics), and to act as its Secretary. The prize was inaugurated by a donation from Boehringer Mannheim GmbH and later continued by Roche Diagnostics GmbH. The statutes specify that the prize should be awarded for outstanding work in the field of molecular bioanalysis, that is for the development of novel methods and for new scientific contributions to the advancement of molecular and biochemical analysis in diagnostics and therapy. The awardees are nominated by a prize committee consisting of personalities representing the Gesellschaft fu¨r Biochemie und Molekularbiologie, the company, and the biochemical, biophysical and medical research, and the prize is handed over every 2 years at the Analytica Conference. For many years, when the German Society for Clinical Chemistry was the body scientifically responsible for all enterprise connected to the Analytica, the prize winners and some hundred guests were invited to Bayerischer Hof in Munich to celebrate the event (Figure 5). I am delighted that the list of the Prize winners (Table 1) names eminent researchers, who indeed made outstanding contributions to the fields specified above. A satisfaction for all those colleagues that participated in the selection of these persons was that in several cases this Prize was given to the awardees well in advance of the Nobel Prize. At the same time, it documents some of the highlights in the development of molecular biology.

FEBS I became aware of FEBS (Federation of European Biochemical Societies) through their early meetings held in Warsaw (1966), Oslo (1967), and Prague (1968). These meetings provided excellent opportunities for a ‘‘beginner’’ to follow novel

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Fig. 5.

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Analytica 1982: With my wife at the Analytica reception.

developments in biochemistry and molecular biology and to present his own results in short talks. In 1983, I was fortunate to be nominated by the Gesellschaft fu¨r Biologische Chemie (GBM) for membership in the Advanced Courses Committee (ACC) of FEBS. I took this post with pleasure, because I felt in absolute agreement with the main objective of FEBS that since its foundation in 1964 has been ‘‘to advance basic research and education in biochemistry, molecular and cellular biology, and molecular biophysics on a European level.’’ My predecessor as chairman of the ACC was Giorgio Bernardi, who succeeded in continuously raising the number of courses held per year from only a few in the beginning to more than a dozen, before he had to retire from this duty in 1986. I remember my first participation to an ACC meeting that was held on the occasion of a lecture course in Maria Alm (Austria) on the Biochemistry of Ageing organized by Fritz Cramer and Brian Clark in 1984. In 1986, FEBS Council appointed me to become Giorgio’s successor. I took up this office in 1987 and was reappointed for two further 3-year periods in 1990 and 1993. I was lucky to work with a Committee the members of which were enthusiastic in contacting colleagues from all over Europe,


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TABLE 1 Awardees of the Prize Biochemical Analytics/Molecular Bioanalytics

1980 1982 1984

Fred Sanger and Alain Coulson, Walter Gilbert and Alan Maxam Ce´sar Milstein and George Ko¨hler Ed Southern

1986

Brigitte Wittmann-Liebold, Leroy Hood and M.W. Hunkapiller

1988

Charles Cantor and David Schwartz Sir Alec Jeffreys Karin Mullis and H.A. Erlich

1990 1992

Jean Lawrence and David Ward

1995

Gregory Winter

1996

Mario Capecchi and Rudolf Jaenisch

1998

Arthur B. Pardee (DanaFarber Cancer Institute, Boston) and Peng Liang (Vanderbilt Cancer Center, Nashville Tennessee) Franz Hillenkamp (Institut fu ¨ r Medizinische Physik und Biophysik, Mu ¨ nster) and Michael Karas (Institut fu ¨ r Chemie, Frankfurt/Main)

2000

Development of modern techniques for sequencing DNA Development of monoclonal antibodies Development of the DNA hybridization technique: ‘‘Southern blot’’ Development of methods and instrumentation for sequencing proteins at a micro scale Development of pulsed field electrophoresis DNA fingerprinting Development of the PCR technique Development and applications of non-radioactive highly sensitive in situ hybridization techniques Isolation of high affinity human antibodies directly from large synthetic repertoires In recognition of their pioneering work on the specific integration of DNA in mammalian cells and for establishing transgenes as a basic tool for research in molecular biology and medicine Development and applications of Differential Display

Development of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Biopolymers, one of the essential technologies in genomics and proteomics

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who would be willing to run a FEBS Course. Though the funds for FEBS Courses were raised to 1 Mio Deutsche Mark per annum, we had to set certain limits for the amount of money given as a support to each course. So it was highly appreciated if organizers were able to invite co-sponsorship from other grant giving institutions. One particular advantage of running a FEBS Course, however, was that Youth Travel Grants were provided to assist attendance at these by younger scientists. As half of the FEBS Courses budget was designed for this purpose, up to 25% of the participants in a lecture course and all of the participants in a practical course could profit from this type of support. In accordance with FEBS’ general policy, fellowships were preferably awarded to young scientists from Eastern European countries, who otherwise would have had little chance to receive funds from their national institutions. Another aspect connected to this issue was that the ACC sought to invite colleagues from these countries to organize FEBS Courses at their home institutions, an encouragement that in fact paid out successfully. During my time as chairman, the ACC consisted of 10 members: eight colleagues from different Constituent Societies as well as the FEBS Secretary General and the FEBS Treasurer. This arrangement has been kept, but fortunately more colleagues from former Eastern countries became members of the ACC since. In all these years, the ACC received enough applications to sort out inappropriate ones. Priority was given to practical courses, because the committee felt that this type of venue would be of greatest benefit to young researchers who had no other opportunities to experience novel laboratory techniques or to learn techniques, which they wanted to apply in new projects. Thus the practical courses complete the intentions of the FEBS fellowships’ program. Indeed, some of the practical courses were so successful that the organizers and the ACC decided to repeat them, sometimes in subsequent years or in a series. I gratefully recollect that for one particular course the main organizer [Wilhelm Ansorge from EMBL (European Molecular Biology Laboratory)] repeatedly undertook to transfer all special equipment and instruments needed for this course to a place that had no supplies of this kind. The significance of the Advanced Courses Programme is also documented by the fact that students themselves, the Young Scientists movement, took the initiative to organize a successful series of courses entitled ‘‘Young Scientists’ view of molecular biology and biotechnology.’’


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Extraordinary venues I remember were two FEBS courses organized by Giorgio Bernardi, one in Cairo and one in Harare, Zimbabwe. He felt that students from African countries should profit from recent developments in Europe. In conjunction with these courses Giorgio arranged for private tours with some colleagues (I think he was fond of traveling). As I shared his view to spend a few extra days on ones own expenses to see a country whenever one had an opportunity to visit it, I undertook an unforgettable tour from Cairo to Upper Egypt with Marianne Grunberg-Manago. In Harare, Marianne, Brian Clark and his wife, the wife of Francois Gros, and I rented an old Peugot and went cross-country for 10 days. Personally, I am most grateful to FEBS that they have supported the Spetses Summer Schools on Molecular Biology (see below) from 1983 to present in co-sponsorship with NATO (North Atlantic Treaty Organization) and EMBO, and finally have decided to give full financial aid to these well-known venues together with EMBO. The years in FEBS were always exciting and enjoyable. I was glad to meet and to work with so many nice and enthusiastic colleagues from so many different countries, above all the members of the Executive (Figure 6) and the ACCs (Figure 7), but not to forget, the organizers of the FEBS Courses and the numerous student participants at courses which I had a chance to attend. I vividly remember the splendid and jovial atmosphere at the Committee meetings governed by hospitality and friendship and many exhilarating episodes that occurred at these occasions. At least I cannot repress one that happened when the ACC met in Amsterdam (Figure 7) and tell it with our host’s, Karel Wirtz from the University of Utrecht, own words [145]: ‘‘It also gave me a chance to make the committee members familiar with the capricious nature of Dutch wind and water. Having been asked to organize the meeting in Amsterdam a 60-feet sailing barge of the early 1900s was chartered (the whole enterprise turned out to be less expensive than a hotel). This ship offered a bunk for each member and a spacious room under deck where we could discuss and review the applications. Arrived on Friday late afternoon we sailed from Amsterdam harbour the next morning to cross the Ijsselmeer. During an 8-hour sailing trip we finished the agenda while a two-men crew made sure we reached the port of Hoorn at the north side of this large body of water. On Sunday morning we had to hoist the sails again to return to Amsterdam.

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Fig. 6. FEBS Executive Committee Meeting, Budapest 1990. Sitting: Prakash Datta and Doria Cavallini. Standing: Horst Kleinkauf, John Mowbray, Karl Decker, Guy Dirheimer, Vito Turk, Horst Feldmann, and Carlos Gancedo.

On our way back, the wind had picked up to force 7 while heavy showers tested our endurance, Horst being in the kitchen to prepare lunch, lost his balance to become encased knee deep in macaroni. Somehow he still managed to produce a tasty meal.’’ He forgot to mention that the ship just went about without a warning. It was a pleasant feeling to know that the ACC got in best hands in 1996 with Karel as my successor. In a way, I miss all these activities, but I am grateful that despite my retirement I still have an opportunity to keep contact with many friends from my time at FEBS. In 1995, I was awarded the FEBS Ferdinand Springer Lecture Tour that offered the splendid opportunity to visit a number of Institutes around Europe and to talk about my current scientific work. Since 1995 I am acting as an Editor to FEBS Letters, the journal founded in 1968 by Prakash Datta for rapid publication of relevant novel reports and reviews in biochemistry, biophysics, molecular biology, and related topics; Giorgio Semenza, then Managing Editor of FEBS Letters, brought me in. In 2003, FEBS gave me an opportunity to finish a book Forty Years of FEBS – 1964 to 2003 – a Memoir [145], which was


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Fig. 7. ACC Meeting, Amsterdam 1992. On a sailing tour on the IJsselmeer. From left to right: Julio Celis, Horst Feldmann, John Mowbray, Slobodan Barbaric, Vito Turk, Paulette Vignais, and Thanos Evangelopoulos.

presented at the 40th Anniversary of FEBS celebrated at the FEBS Congress 2004 in Warsaw. Spetses Summer Schools The Greek island of Spetses is the place I had a chance to visit more often than any other one in beautiful Greece; in fact, I can call it ‘‘my second home.’’ I immediately fell in love with the island when I was admitted as a participant to the 3rd NATO Advanced Study Institute held there in 1969. Actually, Marianne Grunberg-Manago had started this series of International Summer Schools on Molecular Biology in 1966 and through her untiring initiative [146] the Schools have been kept as a series of well-known annual lecture courses until to date. There was a college (Anargyrios and Korgialenios School) large enough to accommodate students and a hotel at a short distance from it both of them adjacent to a good beach. The island was small enough to facilitate contacts between students and professors and it was large enough to provide peace and quiet.

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Participants to the Spetses Summer School on Molecular Biology in

My active involvement in the Spetses Summer Schools dates back to the year 1970/1971. Hans Zachau was asked to organize the third School in order to bring in the Germans as a third party and to make the School an annual event, the directorship being rotated regularly between France, England, and Germany. (For some interval, the directorship has included Tom Caskey (Houston) and John Hershey (Davis) from the US to reach a 4-yearly rotation.) From then on I helped Hans Zachau as one of the German co-organizers, and after 1988 I took over as a German organizer (Figure 8). Initially, the Spetses Summer Schools were sponsored exclusively by NATO. But their strict rules soon caused the organizers to apply for further grants from EMBO (sponsor since 1972), and also from FEBS (sponsor since 1983), as to allow to invite and support lecturers and students from non-NATO countries. In recent years, the School relied on financial support from EMBO and FEBS only. It may well be that some colleagues were skeptical that Spetses had been established as sort of a ‘‘club’’ and should not be funded. But the facts show that over the years nearly 500 (different!) renowned lecturers came to the island to


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teach some 5,000 young pre- and post-doctoral researchers. Indeed, one can realize until to date that Spetses participants form a community, and I am still in contact with some of the students (particularly Russians) who attended the School several years back. To give an account on the Spetses’ activities, I compiled an overview at a website [147]. The topics dealt with in these venues reflect important moments in the development of molecular biology over the last 40 years. Thus, the intentions of these courses of familiarizing young researchers with novel insights and recent advances in this field completely met those of the grant-giving institutions. In 1996, I arranged for a 3-day Workshop to celebrate the 30th Anniversary of the Summer Schools at Spetses and was able to invite former organizers and lecturers of outstanding merit. As the authorities and the inhabitants of Spetses were proud and most grateful that they were selected to host an International Course of this calibre for so many years, all previous organizers were presented with a brass plaque by the Spetses Mayor to affirm their honorary citizenship. A similar venue was repeated successfully to celebrate the 40th Anniversary in 2006, organized by Brian Clark and myself. It was again a wonderful occasion that so many old companions were to meet many of whom had not seen each other for nearly 40 years. As I promised to Marianne, when she fell seriously sick, as long as I could to have an eye on Spetses and to engage in keeping the tradition of the School.

Gene Technology I have already briefly recapitulated the story of recombinant DNA. In 1971 Cetus and in 1976 Genentech companies were founded. The first pharmaceutical products based on recombinant DNA were somatostatin (1977); insulin (1978); growth hormone (1979); and interferon (1980) (e.g. [148]). Also Suisse scientists and industries engaged early in gene technology. In Germany, however, the application of gene technology for industrial purposes lacked behind considerably, though we had little problems to get permission for cloning in the lab after the German guidelines had been worked out and published. For industry, there were two reasons for reluctance: (i) at the beginning, the bosses leading big German chemical or

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pharmaceutical firms publicly stated ‘‘y there is no need to engage in this doubtful enterprise, if it turns out promising, we will buy the know-how y’’; (ii) later, the first attempts to set up an industrial production (e.g. for human insulin in recombinant bacteria) failed as German authorities voted down a bill. In all honesty I have to say that fortunately the situation in biotechnology has changed thanks to the engagement from politics, research and industry, so that these days biotechnology has a good and respected standing world-wide. As I felt (fortunately not being the only one) that it was timely around the early 1980s to familiarize at least those people interested in the ‘‘chances and risks’’ of gene technology with the new developments, I accepted several invitations to discuss the relevant items with chemists, geneticist, pharmacists, medical doctors or even ‘‘laymen,’’ at congresses or privately (e.g. [149–151]). In 1981, German industry no longer could deny that gene technology was attractive. But except a few smaller companies, who showed a growing interest in adapting novel techniques, there was no sincere attempt from ‘‘in-house’’ to train their employees. Rather came an impetus from the Gesellschaft Deutscher Chemiker, who asked me in 1982 to organize an advanced vocational training course on ‘‘Methods and Results of Gene Technology’’ for some 20 participants at our institute. I could solicit the help of some of my junior colleagues ¨nggi, Peter Philippsen, Rolf Streeck, and (Fritz Fittler, Urs Ha Wolfgang Wintermeyer), but generous funding allowed me to also invite foreign lecturers. This course was offered and successfully repeated 3 times in the years after, until 1985. A remarkable feature was that there was a growing interest of patent attorneys in these courses. A number of large and well respected offices had been established in Munich, whose clientele recruited from renowned biotechnical companies world-wide, the reason being that the European Patent Office had been installed in Munich in 1977. The contacts brought about by the courses stimulated several of our young doctoral students to start a career as patent attorneys as well (in all about 10). The courses on gene technology in Munich had raised the particular interest of Boehringer GmbH, who wanted to set up a similar advanced vocational training course for their staff, which we called ‘‘Novel Methods in Gene Technology.’’ With my good colleagues and friends Wolfram Ho¨rz and Gustav Klobeck both


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from our institute, we managed to familiarize the participants with the latest practical and theoretical developments in molecular biology and genetic engineering in 3-day courses. I remember we started this series in 1985 and repeated these annual venues at least 10 times, until 1995. These venues had arisen from the good relations we had with some influential colleagues from Boehringer Company. The enterprise was abruptly stopped when the firm was taken over by Roche to become ‘‘Roche Diagnostics’’ in 1998. A very pleasant cooperation with the German Society for üXlich, the director Animal Husbandry was launched by Horst Kra of the Munich Institute of Animal Breeding. As the members of Deutsche Gesellschaft fu¨r Zu¨chtungskunde (German Society for Animal Breeding) had a growing interest in learning the capabilities of recombinant DNA technology and had heard about our courses, they invited us to run a lecture course for them in 1986. It was really satisfying to see that our seeding message was to bear fruit in the years to follow. The animal breeders successfully combined micro-injection of recombinant genes with in vitro fertilization to pigs, cattle, sheep, and other domestic animals [152]. The DFG supported these activities by generous grants in a special program, whereby the applications of the single members had to be evaluated bi-annually and the forthcoming results to be presented in common sessions. It was always a pleasure for me to be accepted by these colleagues as an ‘‘honorary animal breeder.’’

Sonderforschungsbereich 190 The DFG supported the research of our group during my entire time. In 1989, members of the Munich scientific community interested in mechanisms of gene activation sought a closer contact with each other. We wanted to build a forum that should foster a practical cooperation and a direct exchange of results and ideas and should overcome the disadvantage that the respective institutes were scattered throughout Munich. This was exactly the concept of the Sonderforschungsbereiche (SFBs), quite a number of which existed all over Germany. Prerequisites for funding were to present a timely and attractive theme and the willingness of up to 20 groups to cooperate for better or worse. The application presented to DFG

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by Herbert Jaeckle, then the new head of the Munich Institute of Genetics, Wolfram Ho¨rz from our institute and myself was accepted and financing started in 1990. The responsibility for smooth rolling was laid in my hands as chairman of SFB190 in consecutive 3-year ¨rz was elected periods (from 1990 through 1998), and Wolfram Ho my successor for the fourth period (the maximal life-time for an SFB, until the end of 2001). An ongoing problem was to catch the attention of new groups whenever current members dropped out. Many of the group leaders left Munich as they had been offered chairs in other universities or directorships at foreign Max-PlanckInstitutes, and in order to keep a ‘‘critical mass’’ these groups had to be replaced by newcomers. But we succeeded to solve this problem as Munich was an attractive place which helped recruitment of capable colleagues.

Other Encounters Before I engaged in committees, boards, or in the organization of international meetings myself, I was attending several venues Hans Zachau had initiated and organized as chairman [144]. Together with members from the Russian Academy of Sciences he started the series of German–Russian Symposia, with the first bilateral conference held in Munich in 1976. The continuation of these venues for over 20 years opened the extravagant chance for me to visit several interesting places in Russia and the former Soviet Republic. On the Russian side, the symposia were organized by scientists from the Engelhardt Institute of Molecular Biology in Moscow. The meetings always started with a short stay in Moscow and from there the participants were taken by air plane to the various locations the organizers had chosen. Every time Tatiana Venkstern, a scientist from the Engelhardt Institute, was responsible to take care of us and to act as our motherly travel marshal. All these visits (Zagorsk, Erevan, and Armenia, 1981; Irkutsk and Lake Baikal, 1989; Suzdal, Vladimir, and Riga, 1993), but also the friendship with Tatiana, left unforgettable moments in my life. Through these tours, I realized how difficult life for the majority of the population was those years and what I could personally do to alleviate the frustrating situation our Russian colleagues had to struggle with. One late compensation consisted in the possibility to raise money from the


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German Volkswagen-Foundation to start a fruitful cooperation with Vadim Karpov’s group from the Engelhardt Institute in Moscow [133]. My engagements in immediate duties, in faculty or university affairs have been mentioned above. Commitments at the national level included services to the DFG (as a member of many panels of experts), the Alexander v. Humboldt-Foundation and the German Studienstiftung. I always kept good relations with EMBO after I had been elected EMBO member in 1979. My activity for the German Society for Biochemistry and Molecular Biology (GBM) largely concentrated on collaboration with them during my time as Secretary for the Analytica. But I am proud that the GBM logo I designed for them 30 years back is still in use. What concerns my international commitments, not mentioned above, I acted for some years (1993–1997) as an advisor for the German-Israeli Cooperation Programme in Biotechnology (DISNAT) maintained on the German side by the Ministry of Research and Technology. Very pleasant memories are connected to many events I was invited to by my French colleagues, better to say friends. They had noticed that I was able to follow lectures and discussions in their own language. This permitted me to serve as examiner for several the`se de troisie`me cycle and qualification pour l’enseignement supe´rieur at University Louis Pasteur in Strasbourg, or in evaluations of CNRS programs at Gif-sur-Yvette. When the French yeast community started their Geńolevures program, a project aimed at the ‘‘Genomic exploration of the Hemiascomycetous Yeasts,’’ I was happy to help Jean-Luc Souciet from Strasbourg who is acting as a coordinator of the Geńolevures project [153]. Since then I have been invited to their meetings in Paris or Strasbourg, which I consider a very selfless gesture supporting our long-lasting friendly relationships.

Epilogue Contrary to many colleagues whose personal recollections I read with great interest, I have to confess that I never enjoyed the close guidance by teachers belonging to particular academic schools. This does not exclude that being trained as a chemist I never gave up to look at the world in a sense of chemistry – at least with one

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eye. This perception was intensified by several chances to meet eminent scientists at the Lindau Nobel Prize Meetings during my time at university. My wife and I still keep good memories of the 4th Meeting of Awardees in chemistry in 1961. A treasure for me is the reprint of a remarkable essay by J.H. van’t Hoff explaining the value of ‘‘Imagination in Science’’ which my scientific ‘‘grandfather’’, Richard Kuhn, had edited and handed out to the students [154]. From my childhood on I was used to grasp and accept what I felt was interesting or suitable for me, be it knowledge, reflections, experiences, skills, or obligations. So I am deeply thankful to all people who have contributed to my development in one or the other way, in more or less intense connections. This does not imply that I was always prepared to adopt their opinions or way of action, particularly of those who in my eyes displayed attitudes unacceptable for me. Of course, my gratitude includes all my gifted and motivated co-workers, some of whom I cited by names above, but also those who have not been mentioned explicitly. Our lab always was an assembly of chemists, biologists, medical students, and technicians, individualists originating from regions all over Germany. More splashes of color were contributed by people coming from various nationalities for different but often overlapping periods of time. In this respect I remember: Hermann Falter, a BavarianCanadian postdoc; Erwin Meixner, an Austrian PhD student; Giuseppe Pirro (Pino), an Italian post-doc from Modena; Antonin Eigel, a Czech immigrant; Anni Fradin, a student from Bretagne who lost her supervisor in Gif-sur-Yvette and worked for her dissertation in my lab on tRNAMet and methylation in yeast; Noel Deering, an Irish technician; Rick Baker, an American post-doc coming with a Humboldt stipend; Pamela Smith, a British secretary fluent in German, Italian, and Spanish; and Vadim Karpov with Olga Preobrashinskaia and two students, all coming from the Moscow Engelhardt Institute to collaborate with us on Rpn4. The same gratitude applies to all colleagues with whom I shared my daily life during my time at the Munich institute, which was like a safe haven for me. They helped me in scientific as well as in personal affairs. I do not repeat that I enjoyed to get acquainted with so many people outside the institute, some of them becoming collaborators or real friends. Even if there will be little chance to


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meet one or the other of them again, they are alive in my memory. It is them who have to decide on the quality of my endeavor. Privately, my family was my best support and place of refuge, although they may not have realized this during the time I was busy or away from home. As said my wife gave birth to two daughters, and initially I was disappointed not to have a son. But I soon comprehended that daughters are much more devoted to a father than sons can be. Fortunately my wife took care of the children in any situation in life, which granted me a feeling of independence. Otherwise I feel lucky that they share one or the other of my interests: both of them loved and performed (classical) music and participated as supernumeraries in opera or ballett. Barbara before she took up and finished medicine was educated and got a diploma as a ballett dancer, Miriam is fond of fine arts, languages, and gardening. As a zoon politicon I never became a member of a political party, because such an interest had been spoiled for me radically by my experiences as a youngster. Not even could I accept to be monopolized by an interest group whose policy I did not agree with or to be sqeezed into a corset hierarchy; though in German university one has still to deal with this habitude, the most miserable outcome being a condescending attitude of some staff people towards their ‘‘subordinates.’’ Like many other things doing science is fun. Whenever somebody has developed a liking for science, I feel he will stick to it for ever. I found this notion expressed in all recollections of scientists I read thus far: pursuing scientific research gives satisfaction and pleasure. Indeed, this must be due to the fact that a scientist enjoys many privileges. As an occupation it bears its challenges, joys but also disappointments. I hope my recollections preferably mirror the pleasant aspects, because these are best to remember and to forget about the rest. Die Erinnerung ist das einzige Paradies aus dem wir nicht vertrieben werden ko¨nnen.

Jean Paul

ACKNOWLEDGMENT

I wish to thank Giorgio Semenza for critical reading of the manuscript.

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Chapter 5

A Botanist Going Astray: 77 Semesters of Studying Membrane Transport and Protein Glycosylation WIDMAR TANNER Cell Biology and Plant Physiology, University of Regensburg, D-93040 Regensburg, Germany Email: [email protected]

Abstract After studying at the Ludwig-Maximilian University in Munich and working for a PhD at Purdue University, USA, I started to do research in Munich on aspects of photophosphorylation and on the biosynthesis of raffinose type oligosaccharides. Later in Regensburg this led into detailed studies of an inducible active transport system of a eukaryotic cell (hexose/Hþ-symporter of Chlorella) as well as to the discovery of dolichol-phosphateactivated sugars and their role in glycoprotein biosynthesis. An excursion into the controversial field of plant transpiration and one into lateral plasma membrane compartmentation is included. Diversions from research by various administrative obligations are admitted. Keywords: Proton-symport; Protein glycosylation; Plant transpiration; Plasma membrane compartmentation; Saccharomyces cerevisiae

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WIDMAR TANNER

Die Menge fragt bei einer jeden bedeutenden Erscheinung, was sie nutze, und sie hat nicht Unrecht; denn sie kann bloX durch den Nutzen den Wert einer Sache gewahr werden. – Die wahren Weisen fragen, wie sich die Sache verhalte in sich selbst und zu andern Dingen, unbeku ¨ mmert um den Nutzen, d.h. um die Anwendung auf das Bekannte und zum Leben Notwendige, welche ganz andere Geister, scharfsinnige, lebenslustige, technisch geu ¨ bte und gewandte, schon finden werden. Die Afterweisen suchen von jeder neuen Entdeckung nur so geschwind als mo¨glich einigen Vorteil zu ziehen. J. W. von Goethe, ‘‘Naturwissenschaftliche Schriften,’’ 5. Band Le doute est de´sagreáble, mais la certitude est ridicule Voltaire

When I was asked by Giorgio Semenza, whether I would be willing to write down my personal recollections, naturally I first felt flattered, but was also full of doubts, whether my story would be of general interest. On the other hand, hardly a botanist has ever been invited to write in this series and since in classical biochemistry textbooks plants also do not exist – except for that brief chapter telling medical students that oxygen in the atmosphere comes from photosynthesizing plants – I decided that a voice from this special corner of the living world, cannot be harmful. It is true, however, that I have seldom been considered a botanist by my colleagues. I guess that is the price one has to pay to end up in this book.

Youth and Eight Semesters of Studies in Munich In 1938, the year before World War II started, I was born as the first of three children in a small town, called Wagstadt or in Czech, Bilovec. Whenever I had to fill out a form, I did not know what country I should officially name my home country. Whereas it was Czechoslovakia at the time of my birth, it became Sudetenland half a year later by a heavily disputed historical event and subsequently it changed – or is it more correct to say, the country was forced to change? – its name another 4 or 5 times. I finally decided that I was born in Moravia, a designation true during all these times. My father was an administrator in a factory that produced writing pens; my mother took care of household and children. Both my grandparents were farmers. My early memories are all about the farms, which we visited every week end. I especially remember my father’s bee-keeping and

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that he had to be called half a dozen times, before he left his bees and came for lunch. Later my father used to tell a story, which supposedly proves my early curiosity. Once, at the age of 4½, going for a walk with him, I said: ‘‘Daddy, I know now everything, except where the babies come from and how glass is made.’’ Whenever I told this story, unavoidably someone would say, and nowadays you still wonder about the glass. Although I was in school for a few months in Wagstadt, I continued in Prien at the ‘‘Chiemsee’’ in Bavaria, a small town to which we fled from the approaching Russians in January 1945. We succeeded to take one of the last trains via Vienna. However none of the many separately sent parcels and fairly heavy wooden boxes ever arrived. Thus, my family had to start a new life with their hand luggage only. Although the after-war times were very difficult for the parents, especially when you are a refugee, we children did not really experience this. The beautiful lake area in Upper-Bavaria, close to the Alps, eventually convinced me that we were not driven out of but rather into Paradise. And this was true, not even considering the fact that communists ruled for 40 years, the country which we had left. In 1948 I entered high school in Rosenheim, a town 25 km away, which meant an hour train ride every day. The last 2 years of high school as well as the ‘‘Abitur’’ I finished in Munich, at the ‘‘Klenze Oberrealschule.’’ My father had found a job in his profession at Siemens, finally 8 years after the war. In the winter term 1957/1958 I started to study Biology, Chemistry, and Geography at Ludwig-Maximilians-University in Munich to become a high school teacher. Among the various influences that may have led me to this decision, was a little book in the series ‘‘Understandable Science’’ published by Springer with the title Virus. The Story of the Borrowed Life by Wolfhard Weidel. I was lucky to still have heard Karl von Frisch lecturing in his last term. I remember well the lectures by E.O. Fischer and by Rolf Huisgen in Chemistry, by Leo Brauner, Hermann Merxmu ¨ ller, and Otto Kandler in Botany, by Hans-Jochen Autrum and Alfred Kaestner in Zoology, by Feodor Lynen in Biochemistry and the highly entertaining lectures of Konrad Lorenz, although it was hard to detect a red thread in between his stories. It was not considered anything special that all these famous scientists – four of them received the Nobel Prize a few years later – presented the general

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introductory lectures in their various fields (2–5 hours per week for 1 or even 2 semesters). It might have been a mistake in the 1960s to cancel the so-called ‘‘Ho¨rgeld’’ (‘‘Listening Money’’) of 3 Mark per hour per semester, which each student had to pay and which each professor obtained in addition to his salary. It was important to present interesting and stimulating lessons to have a large audience. In this time I realized that my main interest lay in physiology and in biological chemistry. The final decision to specialize in this direction came about, when working as a student in the laboratory of Meinhart Zenk in the Institute of Botany. He had just returned from USA, where he had obtained a Masters and a PhD degree with Carl Leopold at Purdue University in biochemically oriented Plant Physiology. In Munich, at that time, he probably was the only biologist who worked with radio isotopes. When his new Isotope Lab was inaugurated, even the Mayor of Munich, HansJoachim Vogel, was present. What a difference to nowadays, when every German politician would be hiding, if the topic were radioactivity. Zenk encouraged me to take Lynen’s ‘‘Biochemisches Grosspraktikum,’’ where I got acquainted under the guidance of Guido Hartmann with purifying enzymes, isolating coenzymes and metabolites as well as with the contents of the Biochemistry text book of Fruton and Simmons. I found all this very exciting, but it was far beyond the curriculum for a biology teacher. Most important for my future was Zenk’s suggestion that I should go to the States and work for a PhD degree. To go for such a goal would have been impossible for me in Germany, simply for financial reasons. With the help of Zenk, Harry Beevers at Purdue University, Indiana, offered me a research assistantship and I was determined to cross the Atlantic after finishing my 8th semester in Munich. I asked Barbara Schlotmann, a pretty girl from Westphalia, who studied English, History, and Geography, whether she would not mind coming along as well. She decided to specialize in American literature and to follow me. Since Zenk, however, indicated that it was absolutely impossible to live in the Midwest in one apartment with two names at the door, we ‘‘had’’ to marry. Both our parents were quite surprised and not too happy. Nevertheless, we are married now for 46 years and we are fond parents and grandparents of four children (Gregor, Robert, Burkhard, and Susanne) and seven grand children. In September of 1961, I crossed the Atlantic on the Hapag Lloyd ocean liner ‘‘Berlin’’ with the help of a Fulbright travel grant.


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Together with several hundred Fulbright students, the voyage turned into a 10 days party. All these young people were looking forward to the greatest adventure in their young lives: America. I will never forget the very morning when we passed the Statue of Liberty and the skyline of New York took slowly contour out of the mist. The subsequent 20 hours train ride to West Lafayette, Indiana, became only slightly shorter due to reading Time magazine’s title story on J.D. Salinger on the occasion of his publishing Franny and Zooey. His The Catcher in the Rye – which much later became a literature classics in German high schools – was the first book I greatly enjoyed reading in the States. Barbara followed at Christmas; her parents expected that it might take several months, as in Germany, to find an apartment; it did not even take 1 day. Karl Mu ¨ ller and Rainer Hertel, both previously students in Munich and also sent to the States by Zenk, had already organized one for me before I had even arrived.

My PhD Work at Purdue University Harry Beevers had elucidated the fat-sugar conversion in castor beans (Ricinus communis) via the glyoxylate cycle, a pathway he and Hans Kornberg had discovered in plants in 1957. With the successful detection of all the key enzymes and the detailed radioisotope study of David Canvin, the reaction sequence was firmly established [1]. The high efficiency of the conversion process, however, was surprising. Since the citric acid cycle and the glyoxylate cycle proceed side by side in the same tissue and since the acetate carbons coming from fat degradation were shown to be converted to almost 75% to sugar, the citric acid cycle obviously was efficiently by-passed in vivo. Harry suggested that I work on this problem. To make a 2½ year story short: towards the end of my PhD time, doing some electron microscopy in one of Henry Koffler’s labs under the guidance of Mathew Nadukavukaren, I noticed that the fat-sugar converting tissue, the endosperm cells of castor bean seeds, were full of an organelle, unseen before. I somewhat naively called it ‘‘unknown bodies,’’ a name that of course did not last for long: Breidenbach in 1967 obtained firm evidence that these single membrane bounded organelles housed the key enzymes of the glyoxylate cycle and were named from then on the glyoxysomes [2]. Besides enzymes

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of the glyoxylate cycle these organelles also contain the enzymes for the b-oxidation of fatty acids [3]. Thus, it had become clear that due to this metabolic compartmentation, the fat-sugar conversion proceeds with such high efficiency. Although I was able to publish three papers out of my PhD work [4–6], I naturally was disappointed to have missed the major points. On the other hand, this made it easy to switch fields completely, which in the long run turned out to have been of real advantage. The time we stayed in the States, however, had enriched our lives tremendously. Experiencing this great country and the wonderful friendship of so many people is something, we still live on today. To name a few with whom we became close friends: Larry Larson, Joe Geronimo, Walt Splittstoesser, O.G. and Lea Ward, David and Linda MacLennan, with whom we still have cordial contact. With late Ann Oaks we hiked down the Grand Canyon and up again on the next day, and we were gloating over the poor people, who went down on mules and were not able to sit at the supper table anymore in the Ranch, where one stays overnight. And not to forget Jean and Harry Beevers, with whom we were close all these years, and when Harry spent a sabbatical as a Humboldt awardee in my lab, we even did experiments together at the bench again. The data obtained – once published – brought up the whole botanical world against us (see section My Excursion into Real Botany).

Back in Munich: ‘‘Wissenschaftlicher Assistent’’ and Finding my Own Way Arriving in Munich, I simultaneously got two offers for an assistantship with a German professor. Karl Esser, one of the first chair holders at the newly founded University of Bochum, invited me to work with him on the regulation of tryptophan biosynthesis in Neurospora. Since the whole topic of regulating activity and amount of enzymes was probably the most exciting one in biochemistry at that time – comparable with the attraction signal transduction had not too long ago – the offer was a big temptation for me. Otto Kandler, on the other hand suggested that I work on some of the still open questions in photosynthesis. I recall that I saw me ‘‘working on the appendix of photosynthesis.’’ Turning down the potentially more attractive offer of Esser was not easy.


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However, both the problems Kandler made me to work on, their originality and in addition all the personal freedom he very generously allowed, soon developed into two, not at all overcrowded, new and promising areas, which kept me busy all my life. One of the major achievements of Otto Kandler had been, to study energy conservation related to photosynthesis. He presented very early in vivo evidence for light-dependant ATP formation and thus for photophosphorylation [7]. As Arnon later pointed out, Kandler’s only ‘‘shortcoming’’ was not to have gone one step further and work with isolated chloroplasts [8]. In the early 1960s it had become highly likely that photosynthetic CO2 fixation required two light reactions. The question therefore arose, which of the two light reactions is necessary for ATP formation or whether both of them are required. I was supposed to try to find an answer, using the method of measuring lightdependant glucose assimilation in the unicellular alga Chlorella. Under strictly anaerobic conditions, the light dependence of an energy requiring process was interpreted as measuring photophosphorylation. The results clearly demonstrated that the ATP formation measured under these conditions had a lower quantum requirement at 711 nm than at 660 nm [9,10] and therefore had to be caused by photosystem I. The work was carried out together with Eckhard Loos, who had visited Otto Warburg in Berlin for 2 weeks to get acquainted with the methods measuring quantum yields. Back in Munich he built an Ulbricht sphere of 80 cm diameter, we obtained a large surface bolometer and we were able to measure quantum efficiencies of photo reactions. We also showed that 80% of photo-assimilated 14C-glucose is incorporated into sucrose and starch; the rest appears in sugar phosphates, citric acid cycle intermediates, and amino acids; therefore it could safely be estimated that 1.5–2 ATP have at least to be supplied for each molecule of glucose assimilated. The quantum requirement was found to be 4.1 quanta/glucose taken up at 711 nm. These results were considered as evidence for the existence of cyclic photophosphorylation in vivo driven by photosystem I. During these measurements I noticed that photo-assimilation of glucose did not proceed from the very instant the light was switched on, but rather started with a lag of about 30 minutes. Considering the lag, the question arose, which enzyme had to be induced before the cells were able to assimilate glucose at a linear

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rate? With the exception of two enzymes, hexokinase and a potential hexose transport protein, all enzymes required for glucose assimilation were expected to be identical to those required for photosynthetic CO2 fixation and its conversion to various products. The activity of hexokinase did not change during the lag period, whereas the uptake activity for a glucose analog, 3-O-methyl-glucose, increased by a factor of more than one hundred. In addition, it was noticed that non-metabolizable glucose-analogs were accumulated by induced Chlorella cells several 100-fold [11–13]. Thus, a eukaryotic inducible membrane transport system had been discovered and could now be studied in parallel to similar bacterial systems, like the b-galactoside permease of E. coli. Over the years my group characterized the Chlorella hexose uptake system: we established for the first time that proton symport exists in eukaryotic cells, cloned the first gene for a plant transporter, functionally expressed the corresponding gene in yeast, purified the transport protein to homogeneity, and tested it in an in vitro liposomal system (see section An Inducible Active Transport System of a Eukaryotic Cell). Very recently I got interested in the role of lipids in membrane transport and in the related question of lateral membrane compartmentation. This is what my small group is presently working on. Most likely this will be my last field of interest. What it is all about, will be summarized later in these recollections (see section Over 40 Years of Membrane Transport Proteins: How about the Role of Lipids?). Before, however, I would like to point out the second research project Kandler in 1964 wanted me to join in, when I started to work with him in Munich. He and Margot Senser had noticed a number of new, highly radioactive spots on paper chromatograms from extracts of certain plants, after these had fixed 14CO2 photosynthetically. These spots were identified as raffinose type saccharides. Raffinose type sugars, galactosides of sucrose, are the most common oligosaccharides besides sucrose itself in the plant kingdom. In a number of plant families they are the most prominent sugars and function as long distance transport substrates that are translocated from photosynthesizing leaves to flowers, seeds, and roots. It was proposed by Kandler that the galactosyl moieties in the biosynthesis of raffinose sugars are transferred in an unusual way. Indeed, one of the spots mentioned above was identified as galactosyl-myo-inositol. This


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compound had originally been isolated from sugar molasses of sugar beets and called galactinol [14]. In photosynthesizing leaves it kinetically behaved as if it were the galactosyl donor for the oligosaccharides of the raffinose family [15]. We found and characterized the enzymes for the corresponding reactions and thus established a new role for myo-inositol: it serves as a cofactor in galactosyl transfer reactions in analogy to UDP-Gal [16–20]. My speculation at that time that inositol (or maybe phosphatidyl inositol) may also participate in the transfer of other monosaccharides, eventually led to the discovery of a lipid intermediate [21] – later identified as dolichol-phosphomannose (see below) – and started my investigations on protein glycosylation. How this finding that was published in BBRC (Biochemical and Biophysical Research Communications), was received by the scientific community, can be seen from a somewhat funny event. In the summer of 1970 an International Carbohydrate Congress took place in Paris, where I presented a brief talk – posters were not yet invented – about our galactinol results. During one of the plenary lectures at this meeting, visited by more than 500 people, the well-known Russian chemist Nikolay Kochetkov talked about saccharide biosynthesis and new types of glycosyl donors. He repeatedly referred to the potential importance of lipid activated sugars. Since it was not clear to me, whether he had any evidence for such compounds or whether they were pure speculation, I asked him in the discussion. Peaud-Lenoel, who was the chairman of the session, enthusiastically started to explain that there indeed is a report and he summarized for a couple of minutes all the evidence recently published. When I answered that I know about this paper, since it is my work, there was a big laughter in the audience. However, no further evidence from any eukaryotic organism was given. This event was the beginning of a friendship with Peaud-Lenoel and especially also with Nicolas Behrens, a coworker of Louis Leloir in Buenos Aires, who was in the audience as well. Nicolas had started with Leloir to also look for lipid intermediates in mammalian tissues, and they had a paper in press in PNAS (Proceedings of the National Academy of Sciences) about their first success [22]. In that paper, they presented evidence that chemically phosphorylated polyprenols can serve as a lipophilic sugar acceptor in their tissue extracts. That they tried it with polyprenols, was of course influenced by the results of Jack Strominger and Phil Robbins, who both found an undecaprenyl

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based lipid intermediate in bacteria [23,24]. During the Paris meeting, Nicolas decided to come to Munich and to check with me, whether phosphorylated prenols would also accept mannose form GDP-Man in the yeast system. Later in the fall within 1 week of Nicolas’ presence, two exciting things happened: first, we got the experiment to work [25] and second, even much more exciting, it was announced in this very week that Louis Leloir obtained the 1970 Nobel Prize for Chemistry for his work on sugar nucleotides. After Nicolas had tried to get in touch with his professor by phone for half a day, we finally could go on again with our experimental work. In the following year I got an invitation to tell my yeast story in Bariloche, Argentina, where Leloir’s Nobel Prize was celebrated. From Germany also Heinrich Kauss, who had described the first lipid intermediate from plant cells [26] and Feodor Lynen, were present. The role of lipid intermediates in glycoprotein biosynthesis became the second long-term interest of my laboratory (see section Glycosylation of Proteins). ¨ck In the very hot summer of 1969 the microbiologist August Bo and I, we passed in a tandem event the special procedure that we have in Germany, the ‘‘Habilitation.’’ At least in the past, a Habilitation was required to get the permission to present lectures at Universities (venia legendi). With Gustl a close friendship developed during my assistantship in Munich and even more so, when we organized the teaching, carried out common seminars, and took part in the ‘‘Monday evening football matches’’ of both our institutes at the new University of Regensburg, once we both had arrived there in the following years. In April 1970 I got an offer to take a chair of biology at the newly founded University of Regensburg, which I gladly accepted. In the first week of June I obtained my certificate from the rector Karl-Heinz Pollok; I mention the time interval of 7 weeks, because nowadays frequently more than 1 year of negotiations passes in Germany. I could take along to Regensburg both my scientific topics, which I had started in Munich. Kandler (Figure 1) was neither interested in working on membrane transport nor on protein glycosylation. He got deeply involved in bacterial systematics and was the first person to support Carl Woese’s concept of two prokaryotic kingdoms. Kandler had accumulated a large amount of data on the varying composition of bacterial cell


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Fig. 1. Otto Kandler, Professor of Botany (Plant Physiology) at the LudwigMaximilians University in Munich, 1976.

wall peptidoglycans and used it for bacterial systematic. He found some irritating exceptions, like the methanogenic bacteria, which perfectly fitted into Woese’s scheme. My teaching duties in Regensburg were quite heavy; however, I enjoyed teaching. For the first semester students, I started with 25 hours of Cytology and a part of General Botany and for the third semester students I read 45 hours of Plant Physiology. Both lectures took place in the winter term, when I had to present 8 hours lectures per week for 6 weeks and after each such week, I had the good feeling that I know what I got paid for. After a few years, when various additional obligations were on my shoulders, Gu ¨ nter Hauska – later also Iris Maldener – took over a part of the physiology lectures (the chapter movements of plants) and Ludwig Lehle – also partly Thomas Roitsch – the section on plant development and differentiation. All the practical courses had to be built up from scratch and we tried to establish a fairly large number of new and – as we hoped – better practical examples. Thus, I wanted that every student should know and understands about an enzyme that it shows saturation behavior and that it lowers the activation energy of a reaction. We determined a Km value, which is of course a task commonly set in courses, but we also determined starch hydrolysis with a-amylase and with HCl at three different temperatures.

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The result always comes out beautifully: with the enzyme the activation energy and the Q10 is considerably lower than that of the acid catalyzed reaction. All my life, I have disputed with transport physiologists that a low temperature dependence of a membrane transport reaction is no evidence for a non- or poorly catalyzed reaction, the opposite is true.1 They generally do not distinguish between diffusion in solution and permeation through a membrane! A special effort to improve teaching in Regensburg was made in 1990, when together with the faculty I started to establish a new curriculum for Biochemistry that allowed the students to obtain a Diploma in 8 semesters. Although the faculty of chemistry – not yet realizing the importance of biology also for their own field – was opposing this endeavor very severely, we succeeded. Since the experimental diploma work within this curriculum lasted 6–9 months and was included in the time of 8 semesters, our students (20 per year) indeed had the shortest studying times of Biochemistry students in Germany for almost 15 years (in the average 8.5–9 semesters). With the general change to Bachelor and Master programs in Europe, the faculty was forced by our politicians – who for many years were complaining about too long studying times in Germany – to again extend the minimal time for studying to 10 semesters.

The Time Spent in University Administration and in General Science Politics University/German Science Advisory Board/DFG In the fall of 1975 I got a phone call from the President of our University, Dieter Henrich, Professor in the Faculty of Law, asking me, whether I would be willing to be a candidate for one of his two, newly to be elected Vice-Presidents. With 37 years of age I mainly thought that I was much too young to already spend a lot of time with administrative duties. Since I had been elected Dean of the Faculty of Biology and Preclinical Medicine for 2 years, I considered to have accepted a load big enough. In addition 1 For example, we measured a Q10 of 2–3 for a well-catalyzed membrane transport reaction and around 8 for a poorly or non-catalyzed reaction [27].


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I remembered the remark of my Canadian colleague Jack Dainty, who once, when looking for a new Dean in his Department at Toronto University told me: ‘‘The person we are looking for, should be experienced in administration, but should hate it.’’ I certainly was not experienced; should disliking administration be sufficient to qualify? But after I had visited Henrich in his presidential office, with his charm he had talked me into it and my deanship was taken over by Karl-Heinz Wrobel, one of our anatomists. It was the first period the young University was supposed to be led by a President and two Vice-Presidents. The second candidate, Henrich had chosen, was supposed to be an eminent theologian. This definitely turned out to be true; it was Joseph Ratzinger. I remember only two occasions beyond every day’s business with him. One was, when the Bishop of Regensburg, Rudolf Graber, was supposed to get an honorary degree. It was the first Dr h.c. that was awarded in the history of our University. In the Senate I argued that it makes a poor impression for the public, if the first honorary degree of a young University were given to the local bishop, especially since the town of Regensburg within all of Germany had the flavor of a catholic stronghold. In addition, we had fixed in our University bylaws that an honorary degree should only be given for outstanding scientific achievements. Ratzinger got up and said: ‘‘Dear Mr. Tanner, you may not have noticed that an honorary degree is exclusively a matter of faculties, here the Faculty of Theology and therefore, this case does not have to be discussed in the Senate at all.’’ Rainer Jaenicke, at that time a member of the Senate as representative of our Faculty, bravely fought with me. Eventually, we considered it a minimal victory that the University President did not take part in the honorary degree ceremony arranged by the Faculty of Theology. Already after 1 year as Vice-President, Ratzinger was called upon to take the chair of the Arch-Bishop of Munich. He accepted the renowned office and after 1 year in Munich he was invited by ¨t’’ (‘‘the Friends of the the ‘‘Verein der Freunde der Universita University’’) to present a lecture in Regensburg. We met on this occasion and I was deeply impressed when he told me: ‘‘You cannot imagine, how I envy you that you still can do science.’’ During my third and last year as Vice-President of the University, the DFG (Deutsche Forschungs-Gemeinschaft) called upon me to join their Senate Committee for ‘‘Sonderforschungsbereiche,’’

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SFBs (Collaborative Research Centers). The person who called me used one of the many tricks that finally makes you give in. When I told him that I cannot do it, because in the moment I am Vice-President of our University, he mentioned that it is best to do several such jobs at the same time, since you cannot do anything reasonable anyway. This was my entry into the major funding agency of German Universities, the DFG, which I served in various positions for about 15 years. I enjoyed the work in the SFB committee, since I had to visit 10–12 universities per year, stay for about 2½ days and listen to the best of their science. Half the time the scientific programs were close to my own field, and for the other half one had to take part as a sort of a distant observer and which therefore could be in any scientific field but your own one. I still remember with pleasure that on such an occasion I learned all about plate tectonics from the geology experts of the Technical University at Karlsruhe. Thus, I learnt a lot, especially also, how an SFB has to be organized and run. In 1979 Manfred Sumper (Figure 2) had joined our Faculty as a biochemist, succeeding Hermann Eggerer, who had accepted an offer to go to Munich. When Manfred

Fig. 2. Manfred Sumper (left), Professor of Biochemistry, and Gu¨nter Hauska, Professor of Plant Biochemistry, both at the University of Regensburg. Picture taken during an excursion to the Monastery of Weltenburg during the First Glycobiology-Symposium of the SFB 43, 1982.


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suggested that we should try to run our own SFB, the idea was enthusiastically taken up and I guess, not the least because it seemed obvious, who should do the work. Those 15 years as speaker of the SFB 43 with the title ‘‘Biochemistry of cell surface and membrane components’’ was scientifically, but also due to the harmonious interactions with all the colleagues, a wonderful rich time. Members of the SFB – some at least for a certain period – were: Christian Bauer, Wolfgang Jelkmann, Rainer Jaenicke, Franz Xaver Schmid, Eckhard Loos, Gerhard Franz, Ru ¨ diger Schmitt, Gu ¨ nter Hauska, Wolfgang Lockau, Karl Stetter and Rainer Rachel, Wolfgang Buckel, Ludwig Lehle, Norbert Sauer, ¨ffler, Wolfgang Oertel, and last but Felix Wieland, Georg Lo certainly not least Manfred Sumper. And speaking of rich, we also felt well supported and enjoyed besides the scientific exchange and cooperation, the unbureaucratic way an SFB can be run. After 15 years a follow-up SFB with the title ‘‘Lower eukaryotes as model organisms’’ succeeded our first one and it was Manfred’s merit that this was achieved without one day of support lost in between the two SFB periods. The second SFB lasted for 9 years only and eventually collapsed due to too many retiring principal investigators. Among them were the geneticist Ru ¨ diger Schmitt and Karl Otto Stetter, a world-famous microbiologist and ‘‘inventor’’ of the hyperthermophilic bacteria and archaea. As speaker of an SFB one is, of course, not permitted to be a member of DFG’s committee for SFBs. Unfortunately my DFGless time was noticed by some ‘‘friends,’’ who proposed that I serve in a special German advisory institution called ‘‘Wissenschaftsrat’’ (a national ‘‘Science Advisory Committee’’), a group of about 30 scientists of all fields and the same number of politicians of the Federal Government plus of the 16 States. This National Advisory Board is supposed to help politicians decide in all questions related to science and research, whether carried out in universities or in university-independent institutions, except in Max-Planck Institutes. Thus for example any new university building or even costly repair of old ones is checked by a sub-group of the ‘‘Wissenschaftsrat.’’ Of all this kind of work that I had to do in my active time, I disliked this job the most. As a normal scientist you soon got the feeling, you serve to a large extent an alibi function. Although it was not uninteresting to get close to these very political and influential circles, altogether it was not my cup of tea and thus I did not want to

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be re-elected after 3 years. Looking back, I consider an advisory system, where the politicians responsible have a few top scientists as their immediate advisors as much more efficient. For very special and involved questions, a corresponding committee can be installed, but then you would have the real experts sitting at the table. After the ‘‘Wissenschaftsrat’’ there followed another 6-year period in the DFG Senate and 4 years as a DFG Vice-President under the Presidency of Wolfgang Fru ¨ hwald. As Vice-president I had to chair the DFG Senate Committee ‘‘Grundsatzfragen der Genforschung’’ (‘‘Principal Questions of Gene Research’’). These were the times of strict non-acceptance of ‘‘Gene-Food’’ – what stupidity already in this designation! – and of the revolution in the field of human embryonic stem cells (I still remember the scary remark of an American colleague: ‘‘Your German embryos are obviously too valuable, to be used in your country!’’ And this phoney situation indeed still holds today!). It was also the time of the 25th anniversary of the construction of the first transgenic organism in 1973 by Stanley Cohen and Herbert Boyer and for this reason I published a few articles [27b] in the FAZ (Frankfurter Allgemeine Zeitung, a leading German newspaper) with the aim to calm down the German public. The resonance from those that were already of my opinion was excellent, but most likely I hardly reached any of all the others. To chair this DFG Senate Committee was a challenge, but at least it meant to discuss serious science. I also launched a program to sequence the genomes of low eukaryotes that promised to be helpful model organisms. It was decided to start with Neurospora and Dyctyostelium and to do this as an international enterprise. Although to cooperate with Wolfgang Fru ¨ hwald and the other, partly overlapping seven Vice-Presidents (Wolfram Boeck, Rudolf Cohen, Gerhard Ertl, Lothar Gall, Karl-Friedrich Knoche, KarlHermann Meyer zum Bu ¨ schenfelde, Ju ¨ rgen Mlynek, Sigrid Peyerimhoff, Sigmar Wittig, Ru ¨ diger Wolfrum) was extremely pleasant, I eventually wanted to go back to my laboratory and my students. After successfully chairing the ‘‘Finding Commission’’ for Fru ¨ hwald’s successor, I finished my time in the DFG. The biochemist Ernst-Ludwig Winnacker followed the Germanist ¨rbel Wolfgang Fru ¨ hwald as DFG President; the Microbiologist Ba Friedrich succeeded me.


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The Dichotomy of Basic Science in Germany: Universities Versus Max-Planck Institutes Having been a member for 7 years in two of the main organizations responsible for science politics in Germany, the ‘‘Wissenschaftsrat’’ and the Presidency of the DFG, it is unavoidable that one frequently discusses very informally general aspects of science – that is German science of course. For example the question, why Germany is fallen so far behind the Americans, the English, and the Swiss – at least for the biological sciences, I dare to say this. Certainly, the loss of many outstanding talents during the Nazi Regime is one reason; however, should one not expect that this becomes negligible more than 60 years after the war? What then are main differences in the organization of science between Germany and the Anglo-Saxon countries? Unique in Germany certainly is the dichotomy of Institutions for high quality basic research: the Universities and the MaxPlanck Institutes (MPIs). Is it really an advantage that the presumably best scientists are uncoupled from teaching the young generation, among them those really important youngsters that enthusiastically absorb knowledge and information like a dry sponge absorbs water? It is not difficult to name a few additional points why this bifurcation of science may be of disadvantage: (1) Originally MPIs were supposed to be founded around very unique and extraordinary persons (Harnack Principle) and in these Institutes problems were supposed to be tackled that could not be or not well be handled in Universities. Today the MPIs are working – for example in the Life Science – with few exceptions on exactly the same problems that are investigated in University Institutes. This leads to harsh and in principle unfair competition between the young scientists of both places, combined with the effect that potential teaching excellence also of young MPI people does hardly get used. (2) The Max-Planck society not only takes away excellent professors again and again from well-organized and successfully run University Institutes – not to mention most Nobel Prize Winners, of course – but it also spoils young scientists with an enormous amount of supply money for research, an amount they will never be able to obtain, once they get

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offered the position of a University professor. It is never very motivating in life, if you have to step down. In addition scientists hired from MPIs are subsequently seldom enthused to teach basic classes and courses, as they never were involved in this before. (3) It is true that students doing their PhD at Max-Planck Institutes will be well trained and will have excellent working conditions and it is reasonable that the best students should get the best chances and enormous support. But does Max-Planck really get the best of the rising generation? Whereas a nationwide competition, for example in the United States, guarantees that Stanford and Harvard can select the top people of the best ones applying, such a system is not established in Germany. Thus the scientists at MPIs neither have the direct contact to the young people while studying, nor later the possibility to choose from a preselected group of excellent students. As a matter of fact, even in the right to award a PhD degree at all, they are not autonomous. The consequence of all this: Too often average people do their scientific work, especially for a PhD at MPIs. (4) It is not easy to understand that in various international ranking lists of universities of all countries no German University ranks among the first 50 (the best one at position 53), whereas for example two English and eight American ones rank among the best ten. (Reference: Academic Ranking of the World Universities, 2007, an activity of Shanghai Jiao Tong University.) Whatever one thinks about such rankings – and of course they are wrong – these data are of enormous influence concerning the decision of young people all over the world, where to go for their studies. Whenever such data are discussed, no one ever mentions that we have 78 MPIs, where we are ‘‘hiding’’ so-to-say our best potential university part! These problems are discussed once in a while among apprehensive and responsible scientists of universities, but the discussion is hardly ever made public. This is because for one, MPIs are more or less taboo – see the fact that the Wissenschaftsrat never deals with matters of the Max-Planck Society! – and secondly, critics would rapidly be put into the drawer labeled ‘‘envy complex’’; thus critical statements are at best a topic for someone retired.


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But to cite at least one renowned scientist, who dared to criticize the Max-Planck Society (previously the Kaiser-WilhelmGesellschaft) openly during his active time, Max Dellbru ¨ ck said in an interview: ‘‘In the Kaiser-Wilhelm-Gesellschaft the best people were taken away from teaching and they were made directors of these pure research institutions. One can view this as extremely successful (in the short run), but I view this as disastrous (in the long run), because one removed the best people from teaching, and the contact to students impoverished very much’’ [28a]. What should be done? First, no further MPIs should be founded; rather those that are far out of sight the Harnack Principle, should be closed. Secondly, successful MPIs with research activities paralleling those of universities should be integrated into the university system. Of course, this will have to happen in steps, with one step partly being made already: The founding of Max-Planck Research Schools, that is a close cooperation between Universities and MPIs for PhD programs. Once fully part of the University, with equal rights and duties, the individual Max-Planck Institutes may very well stay as special establishments with their own budget. They could be something like an elite institute with endowed chairs. It is obvious that several of the problems mentioned above could be minimized in this way and the scientific productivity – I am firmly convinced – would be improved in Germany. And last but not least, the esteem of the German University system in the world would rise very significantly (see also [28b]). Finally, I want to make one last remark concerning German Science politics. In the very moment we start a discussion (in the country of Wilhelm von Humboldt!), whether we should introduce a system of teaching professors versus research professors. Teaching professors might make sense for first year students. Basic knowledge could very well be taught by talented high school teachers and one of the main goals in this period is anyway to equilibrate different levels of high school knowledge. However, the very moment, when university teaching really starts, which is long before the start of graduate courses (‘‘Hauptstudium’’), teachers are needed who ask new questions, who search and do research. I cannot state it any better than Hans Krebs, what difference it makes from whom we learn. He said some 30 years ago: ‘‘Those who learn from somebody, who himself is in the

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process of learning, are drinking from the clear water of a running brooklet. Those who learn from somebody, who has already learnt everything, are drinking from the staying water of a green covered pond’’ [29]. Let’s hope that our Wissenschaftsrat takes notice of Hans Krebs and of those many others, who are of the same opinion; let’s hope that our universities, which were my home for exactly 50 years, will not loose even more attractivity.

My Excursion into Real Botany When in 1976 I visited Harry Beevers for the first time after he had moved from Purdue to Santa Cruz and told him of our new Hþ-symport results (see section An Inducible Active Transport System in a Eukaryotic Cell), we walked through the impressive Red Wood forests and philosophized about the height of the trees and the potential problem to move the water up to the top. I mentioned the ideas of the German botany school, which claim that the existence of transpiration is a real blessing for plants, since it takes care of all the long distance water and mineral transport and in this way the plants do not have to invest any energy for all this work. Harry looked somewhat bewildered and asked: ‘‘Do you really believe that?’’ We had extensive discussions from then on, but I was soon convinced that Harry with his doubts as well as a hand full of other scientists like the water expert Paul Kramer raised a valid point. Kramer stated ‘‘transpiration can be best regarded as an unavoidable evil’’ [30]. What Kramer meant with his statement is the following: Plants would require a skin that is impermeable for water vapor, but permeable for CO2, the daily bread for plants. But through the whole time of evolution no such skin was ‘‘invented.’’ Thus the best compromise for plants is to have a skin (cuticle), which is tight for both and in addition possesses small holes (stomata), which the plants at least can close, whenever water supply becomes a problem. I subsequently started to check all botany books I could get hold of and I found in all school books and most university texts my original statement made to Harry confirmed. I realized that there exists indeed a wide open question and potentially a widespread wrong conception. I also noticed that anyone you talk to, scientist or not, believes in the usefulness of transpiration for water and


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mineral transport up the plants and especially up high trees and in addition in the transpiration’s importance as a cooling device. I did, however not find a single experiment in the literature that would prove these points. Plausibility obviously is a convincing teacher! Thus, I persuaded Harry that it is important to check experimentally what is right or wrong and I invited him to spend a sabbatical year in Regensburg, where we could do experiments as well as discuss all day long and read all the old and new literature available. He and Jean arrived in the summer of 1987 in our medieval town, which they soon deeply loved. The experiment we planned had to be a very simple one, since Harry and I wanted to carry it out with our own hands (Figure 3). Corn plantlets (Zea mays), about 15 cm high were raised in hydroculture until flowering. The plants were in a growth chamber which was held as close as possible at 100% relative humidity (RH). The controls were grown at a RH of 60%. After 5 weeks the plants were 1.5 m high and flowering. No difference in fresh weight, dry weight, and mineral content of the two groups of

Fig. 3. Harry Beevers (right), Professor of Plant Physiology at University of California Santa Cruz, spending a sabbatical year in Regensburg, during which we try to measure effects of high relative humidity on the growth of maize plants. Helmut Zech, helping us preparing the large volumes of growth medium and taking care of the medium change at 7 o’clock in the morning in the case of the sun flower experiment (see the text).

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plants was observed. However, the difference in water lost by transpiration amounted only to a factor of 3. The reviewers of the paper we had submitted – all most likely lecturers, who had been teaching about the importance of transpiration for many student generations – did not like our results at all. Eventually the paper got accepted, but not as a full paper, instead as an ‘‘Opinion’’ [31]. The reviewers criticized that the difference of a factor 3 in the rate of transpiration is too little. If for example – they said – we had measured an enzymatic reaction at high substrate concentration (far in the Vmax region), a decrease in the amount of substrate by a factor 3 might not affect the velocity of the enzymatic reaction at all, which is a valid argument. However, we realized that it most likely is not possible to achieve a more convincing difference in transpiration, due to a phenomenon already recognized by Francis Darwin [32], the son of Charles Darwin. He pointed out that the temperature of a plant leaf in the light, due to the energy absorbed, will always be somewhat higher than the surrounding temperature, for example that of a growth chamber. The micro climate at the leaf surface as well as of the gaseous spaces within the leaf will not correspond to 100% RH and therefore water will be continuously lost by evaporation from a photosynthesizing plant. And concerning our experiment, the plants have of course to carry out photosynthesis to grow. Subsequently, we thought of a different experimental setup. This time we grew sun flowers in hydro-culture, but supplied them with minerals only during a 12-hour night period (the transpiration being very low in the dark), whereas during the 12-hour day, the plants were kept in deionized water – actually, we had to use a low concentration of CaCO3, because deionized water over an extended time period is toxic for plants. The control plants obtained the opposite regime. Now the difference in the amount of water lost during mineral uptake and long distance transport amounted to a factor of 12. Again the plants grew and flowered without a significant difference and this was true also for the dry and fresh weights and the mineral content of the leaves [33]. Control plants that obtained minerals day and night yielded 50% more in mass, which showed that the mineral supply under both regimes was indeed limiting. We submitted the manuscript to Science. Two reviewers found the results reported very interesting, one reviewer, however, insisted that still 10% transpiration occurred in our plants and we had not shown that


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plants can do without that. Of course I was upset. After having shown that 90% of transpiration is without a function to accommodate the plants’ mineral and water supply, and considering the fact that no experimental evidence for the opposite assumption is available, I wished it were possible to force this reviewer to prove his point. In our two papers [31,33] we also have discussed the mechanisms and transpiration-independent forces available that are able to explain the long distance transport even up the tallest trees; to enter into a detailed discussion of this would, however, go beyond the limits of these recollections. It should suffice that one of the positive Science reviewers was a physicist, who did not see any problems with our explanation. It has been a pity of course that we could not publish our results in the wide spread Science magazine. This may have been the last chance for mankind to learn that it is most likely a great advantage for an organism to be independent of the frequent changes in relative humidity. A Science publication would have reached most of the text book writers and might have had consequences, therefore. So however, any new textbook states something like ‘‘the ascent of xylem sap depends mainly on transpiration and the physical properties of water’’ (see Ref. [34]). To ‘‘depend,’’ however, means that without transpiration no xylem sap ( ¼ water plus minerals) would be moving. Many botany teachers even compare the transpirational transport in plants in its importance with the mammalian heart and its function [35,36]. But just think what happens, when a heart stops beating for a couple of minutes and compare this with a situation of several weeks of continuous rainfall and thus zero transpiration. The plant stays alive and will happily grow, although at a reduced rate due to a decreased rate of photosynthesis. Finally, I would like to briefly remark on the supposedly beneficial cooling effect of transpiration [37]. In most areas on this globe the main problem for plants is a shortage in water. For this very reason plants generally close their stomata at noon, when relative humidity is lowest due to high temperatures. In this way, the amount of water lost is lowest when it should be high according to the cooling theory. I usually say that the only plant where the loss of water for cooling would make sense, are the water-lilies (Nymphaeaceae), with their big leaves swimming directly on the water. It is true however that Otto Lange has observed deep rooting desert plants which are in contact with

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water reserves deep down and which indeed cool by transpiration, for example Citrullus colocynthis and Calotropis procera [38]. Discussing these results with Otto Lange – the only person, by the way, who ever observed this rare phenomenon – he fully agreed that even among desert plants, these few plants were exceptions. Tremendous heat and nevertheless sufficient water availability does not often happen on this globe. The many discussions about transpiration – I especially remember a very stimulating and helpful one with Hubert Ziegler of the Technical University of Munich – including the highly polarized ones Harry and I had with many colleagues (‘‘foes and friends’’) have been a lot of fun and I am very happy and thankful that we walked through the Californian Red Woods and started this discussion exactly 31 years ago. In April of 2004, I visited Harry a last time. In January that year he had celebrated his 80th birthday with his family and friends and they were singing old English songs. Harry loved to sing and was famous for even singing during his seminar talks, presenting for example the Leuconostoc method of degrading glucose carbon by carbon as a self-made song. When he learned that I would come for a visit, he was planning that we go for a walk again, this time along the coast. But his health situation did not allow this anymore. I visited him together with his wife Jean in the hospital. Although in very bad condition, his mind was awake. He showed me a picture of many of his co-workers taken at a special symposium honoring him in Riverside in 1991. He knew the names of everyone on the picture. Then he pointed at me and said: ‘‘only this guy I do not remember anymore.’’ I told him some recent results of Thomas Roitsch and myself concerning the action of cytokinin, a plant hormone that was discovered in the laboratory of Harry’s best friend, Folke Skoog. He did listen, but did not ask anything and Harry neither asking nor making comments – frequently funny ones – never normally happened. He died one week after my visit. Thomas Roitsch, whom I just mentioned, did his PhD with Ludwig Lehle and joined me as an assistant after a few years as postdoc with Eugene Nester in Seattle. When Thomas started in 1993 I suggested that he could work on a plant tissue culture and see, to what extent the many things we had learnt about the inducible membrane transport in Chlorella (see below), may also hold for higher plant cells [39]. In addition, a favorite idea of mine


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since many years had been, to find out, whether plant hormones, especially cytokinin, do affect transport processes. Finally, I convinced Thomas that he should also have an eye on cell wall invertases, which had been postulated to be important for unloading of phloem, that is the tubular cells that transport assimilates and distribute it in the whole plant [40,41]. From Wolfgang Barz in Mu ¨ nster we obtained a Chenopodium rubrum cell culture, which can be grown auto- and heterotrophically. With these cells Thomas studied the various questions. His most interesting observations were: (1) Cytokinins dramatically induce a specific cell wall invertase gene [42]; (2) the retardation of senescence caused by cytokinin – originally observed by Mothes – can be explained to a large extent by the increase in cell wall invertase activity [43]; (3) finally, a knockout tobacco strain, lacking a specific cell wall invertase, is unable to fill the seeds with starch. Thus he had obtained evidence for an essential role of sucrose hydrolysis and uptake of monosaccharides into the ripening seed [44]. Thomas Roitsch took a professorship in Pharmaceutical Biology at the University of Wu ¨ rzburg in 2001.

An Inducible Active Transport System in a Eukaryotic Cell Ewald Komor, one of my most brilliant students, joined my group in Munich in 1968 for his diploma work and came along to Regensburg, when the lab moved in 1971. We decided to characterize the newly discovered Chlorella hexose uptake system. One has to realize that membrane transport catalyzed by proteins was then not at all a generally accepted concept. Also Kandler was a supporter of diffusion through membranes as described by the classical lipid-filter theory and of the widely believed ‘‘acid trap’’ idea for substrate accumulation, but he did not mind that we follow the permease concept of the bacterial b-galactoside school [45,46]. A hypothesis heavily discussed at the time was phosphorylation of the transported substrate. The PTS-system had just been discovered (the phosphoenolpyruvate phosphotransferase system [47]). It took a while before it was clear that it was not involved in b-galactoside transport of E. coli and restricted to anaerobic and facultative aerobic bacteria. That is why

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Komor first checked the glucose analogs: 1-desoxy-glucose ( ¼ 1,5anhydro-glucose), 2-desoxy-glucose, 3-O-methyl-glucose, and 6-deoxy-glucose; since they all were transported competitively with glucose by Chlorella cells and also accumulated, it seemed highly unlikely that phosphorylation of the sugar was part of the transport step [48]. In the summer of 1970 Komor and I took part in a FEBSSummer School on Membrane Transport and Bioenergetics at the Lake Balaton in Hungary. Susan Hollan was one of the main organizers and an expert in studying membrane transport using the so-called ‘‘erythrocyte ghosts,’’ the emptied human blood cells. As far as I remember there were only microbiologists and people working with mammalian cells taking part in the course, besides us two botanists. When I mentioned this in a subordinate manner, like ‘‘yes, we also work on transport, but we are only botanists,’’ Susan Hollan immediately answered ‘‘what do you mean? You botanists started it all.’’ I guess it was then, when I lost my minority complex in the transport field. Yes, it is true that it was in botany that a number of major contributions of general biological importance were made, like discovering the ¨geli, Pfeffer) and – to name biological membrane (Dutrochet, Na another discovery – Mendel’s laws of inheritance. No reason to be too modest, in case you work with plants. At this Summer School one of the main, fairly new concepts that were heavily disputed was Peter Mitchell’s chemi-osmotic theory. Allan Hamilton as one of his few followers at that time most heartily defended his concepts. A young Englishman, Ian West, was also present and he told us that he had a paper in press in BBRC, in which he showed the first evidence [49] that the prediction, Mitchell had made 7 years earlier was most likely correct. Mitchell had speculated in 1963 [50] that a proton gradient may not only be a prerequisite for ATP synthesis but may also be the driving force for b-galactoside accumulation in bacteria. We had already tried, whether a sodium or potassium gradient might be responsible for active transport in Chlorella, in analogy to observations made with mammalian cells by Robert Crane [51]. This was not the case, however. Encouraged by the story Ian told us while booting together on Lake Balaton, Komor intensively tried to show that protons disappear from the medium and that this correlates with sugar uptake in Chlorella. He succeeded with a highly sensitive pH-meter, especially


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constructed by our electronic shop. It was not much bigger than a couple of match boxes, but 10 times more sensitive than the regular lab pH-meters. Figure 4A shows the original pH-trace Komor obtained [52]. After having made sure that the phenomenon was more than a plain correlation [53], the data where first presented at the 1974 plant transport meeting in Ju ¨ lich organized by Ulrich Zimmermann and Jack Dainty and subsequently in the same year at the 9th FEBS meeting in Budapest during a symposium organized by Arnost Kotyk. It took considerable time until Ian West’s data were accepted among the bacterial transport groups. The dominating figure in the b-galactoside transport field in all these years, Ron Kaback, had proposed a direct redox reaction of the transport protein coupled to respiratory electron transport and involving SH/S-S

Fig. 4. The crucial experiments demonstrating Hþ symport in eukaryotic cells. (a) The pH change after the addition of 6-deoxyglucose to transport-induced Chlorella cells (curve A); controls: the use of non-induced cells (B) or the addition of a sugar (a-methylglucoside), which is not transported (C). The original data of Komor’s FEBS Letters in 1973 [52]. (b) The experimental set up, how we measured sucrose-Hþ symport in the cotyledons of Ricinus communis. (c) The original pH trace obtained and published in Ref. [55].

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group interchange for energization of transport in E. coli. Publicly – as far as I am aware – Ron only accepted Mitchell’s mechanism at the 1976 FEBS Symposium on ‘‘Biochemistry of Membrane Transport’’ in Zu ¨ rich. Our data, on the other hand, did not get much credit from the botanical field either, since many people had problems, considering unicellular algae as real plants, although they were good enough for the elucidation of the path of carbon in plant photosynthesis in Calvin’s laboratory. It was for this reason that I recalled the plant of my PhD work, Ricinus communis, and the transport experiments that Paul Kriedemann had published together with Harry Beevers [54]. This brought us onto the right track for proton symport in ‘‘higher’’ plants. The cotyledons, two tender leaflets surrounded by the fat storing endosperm tissue, take up sucrose at a high rate, comparable to that of the small intestine in animals, however, without hydrolyzing sucrose and thus differing from Giorgio Semenza’s sucrase system in the animal field. The cotyledons take up sucrose against a large concentration gradient [54]. During germination the fat in the endosperm is broken down, by the glyoxylate cycle converted to sucrose, which is released into the free space between the endosperm and the leaflets. The tender leaflets function as a specific and active resorption organ with a very high sucrose uptake capacity. Thus in the summer of 1976 Ewald Komor and Marianne Rotter succeeded to follow sucrose uptake by Ricinus cotyledons and they were able to show that indeed protons are taken up together with the disaccharide. The set up of the experiment and the initial data are shown in Figure 4B and C. The radioactive sucrose was accumulated 45-fold at an outside concentration of 1 mM and more than 100-fold at 0.1 mM [55]. When Komor accepted a professorship at the University of Bayreuth in 1980, he took the Ricinus problem along, including the important question of phloem loading in general. Of course loading the phloem, the long distance transport system of plants, is the start of the transport path within any photosynthesizing leaf supplying all the non-green, heterotrophic tissues and has been an important problem in Plant Physiology since long. In Regensburg we kept working with Chlorella. They were easier to handle, as Derek Lamport used to say, when he had established a tissue culture of maple trees: its easier to work with pipettable plants – and there were enough questions open


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concerning active membrane transport in general, like for example: (1) Is sugar proton symport electrogenic and is the energy available sufficient to explain thermodynamically the accumulations of sugar analogs that were observed? (2) How do the transport kinetics change, when the transport cycle runs without protons? Can Vmax changes and/or Km changes be observed? (3) Is the system obligatory coupled to energy or can the transport protein also work as a facilitator? Later, when the methods of molecular biology became available, further questions could be studied. (4) Can the gene for the Chlorella hexose transport protein be cloned? (5) Can substrate binding sites of the transporter be experimentally defined, for example by mutagenesis? (6) Is it possible to purify the protein to homogeneity, incorporate it into liposomes and study it in vitro? Can it be crystallized? (7) How does the coupling to the proton gradient mechanically affect the transport protein and thus the transport process? These and related questions were studied in many laboratories, mainly however with bacteria and with mammalian cells. In Regensburg for almost 30 years quite a number of diploma and PhD students tried to answer these questions using Chlorella. Main contributions came from Ewald Komor, Eckhard Loos, Michael Decker, Norbert Sauer, Thomas Caspari, Andreas Will, Ingrid Robl, Renate Grassl, Ju ¨ rgen Stolz, and Miroslava Opekarova, a visiting guest scientist of the Czech Academy of Sciences in Prague. Komor, by the way, who loved transport kinetics [56], wanted to do a postdoc with the famous kinetics expert Arnost Kotyk at the Prague Academy, but for some obscure reasons – some bad feelings that just happened to have arisen once again between Czechoslovakia and Germany, possibly due to some statement of the Sudeten-German-Association – Ewald did not get a permit to entry the country. Instead he went to Bodmin and worked with Peter Mitchell for a year on mitochondria.

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Results from my laboratory that have been or could have been of general interest concerning membrane transport will be summarized in the following paragraphs. The Membrane Potential To determine the plasma membrane potential Dc as well as Dcchanges with time of small organisms like Chlorella have generally been a considerable problem. We followed the lipophilic cation procedure worked out by Liberman and Skulachev (see Ref. [57]). A membrane potential of 135 mV inside negative was determined for Chlorella with the tetraphenyl phosphonium cation (TPPþ). However the method was not suited for following rapid changes of Dc in Chlorella, because TPPþ entered the cells rather slowly. It took more than 30 minutes until the distribution equilibrium of TPPþ was reached and the equilibrium was required for calculating the potential. Komor had the brilliant idea that Dc should not only determine the equilibrium distribution, but also the velocity of entry of a lipophilic cation. He calibrated the rate of TPPþ entry versus the membrane potential. This allowed us to determine changes in Dc within seconds. With this method a transient depolarization of 70 mV was detected after the addition of glucose or a non-metabolizable glucose analog to the cells [58]. The repolarization to the starting potential is brought about by an increased activity of the plasma membrane Hþ-ATPase, and by an increased Kþ efflux as a consequence of the lowered Dc. At about the same time plasma membrane Hþ-ATPases were discovered and intensively studied in a number of fungal organisms and later also in plant cells [59]. For the glucose induced proton influx we determined a stoichiometry of close to one [53], which was in good agreement with an increased rate of oxygen uptake that was observed when the non-metabolizable sugar 6-deoxy-glucose was added to Chlorella cells: the easily measurable increase in oxygen uptake corresponded to slightly more than one ATP required for the uptake of one hexose [60]. This nicely paralleled the stoichiometry of the plasma membrane Hþ-ATPases determined in various cells; it was shown to be close to 1 proton/1 ATP [59]. Finally, from the DpH determined for Chlorella as 1.1 (acidic outside) and the membrane potential of 135 mV (inside negative), it was estimated that the proton motive force was sufficient to explain


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the more than 1,000-fold accumulation of glucose analogs that had been observed [58]. Transport Kinetics þ/ Protons The accumulation of glucose analogs in Chlorella as well as that of b-galactosides in E. coli is brought about by a steady state system, which means that once the accumulation plateau is reached, the rate of influx corresponds to that of efflux. (This differs from amino acid uptake systems, where a plateau is reached, when the influx, which slows down with increasing inside concentrations, becomes zero.) The simplest explanation for substrate accumulation in a steady state system is a difference in the substrate affinity of the transport protein, when its binding site faces the inside as compared to its facing the outside of the cell. To my knowledge, this has first been shown to be the case in E. coli [61]. Winkler and Wilson determined the substrate influx Km of the lacpermease as 0.3–0.5 mM for O-nitrophenyl-b-galactoside, whereas the half saturation value of efflux amounted to 20 mM. The ratio of Km (internal) to Km (external) should determine the accumulation ratio, in this case approximately 60-fold; the actual accumulation observed, however, was about 3 times higher. The difference could be due to the transport velocities, since the ratio of the velocity constants affect the accumulation ratio as well [56,62]. When we first determined the various constants for Chlorella, an accumulation ratio of W1,500 was calculated, which was close to the experimentally determined accumulation for 6-deoxyglucose [56]. This whole kind of analysis had rarely been done in the past. As a matter of fact, in the plant field I do not know of a second example. In the present time with its preponderance of molecular cloning, such whole cell physiology is completely outdated. It is obvious that the kinetic differences make the system an asymmetric one and this is the very reason for accumulative uptake. The corresponding differences must be brought about by the energy made available by the cell and used in the process. The next question arising was, therefore, how does Mitchell’s protonmotive force (pmf) affect the kinetic constants? Lowering the proton concentration in the medium clearly showed that the transport protein in Chlorella indeed decreased the affinity for

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the substrate. At an external pH of 6.0 the Km for 6-deoxyglucose is 0.3 mM, at a pH of 8.0 the measured Km (external) of 50 mM was found to be comparable to Km (internal) of 42 mM, that is to that for 6-deoxyglucose efflux [63]. Thus, as long as the proton ATPase of the plasma membrane pumps out sufficient protons to sustain the DpH, the system would actively transport. Since the energy, here ATP, is used for proton pumping and the sugar transport is powered only indirectly by the Hþ gradient built up, Erich Heinz in Frankfurt, if I remember correctly, introduced the logical nomenclature of ‘‘primary’’ and ‘‘secondary active’’ transport (Figure 5). In animal cells primary active transport is brought about by the sodium/potassium ATPase, originally discovered by Skou and secondary active transport in this case corresponds to the sodium gradient driven uptake of various substrates like sugars and amino acids [64]. As just described for Chlorella, the Km differences inside and outside the cells are caused by a difference in proton concentration. But how does the membrane potential Dc contribute to the kinetics of a secondary active transport system. This was not a question that could have been asked by Winkler and Wilson in 1966, several years before the chemi-osmotic theory was shown to hold for concentrative membrane transport. Expectations that the translocation constants are affected by lowering Dc turned out to be wrong, at least for glucose transport in Chlorella. Komor observed that at pH 6.0, the rate of uptake did not change by decreasing Dc by as much as 60 mV. Surprisingly, however, the pH value at which half maximal activity was reached was shifted half a pH unit into the more acidic region [65]. The simplest explanation for such behavior is that the crucial group that has to be protonated is located half way through the membrane and is accessible in a water-filled pore. The potential decrease of 60 mV (corresponding to 30 mV considering half the width of the membrane) would thus be compensated by a change in the bulk pH of half a pH unit. Mitchell had postulated a so-called ‘‘proton well’’ for this phenomenon. An alternative explanation is that the difference in Dc changes the pK of the protonable group. This will have to stay undecided until a 3D structure at atomic resolution might be available. In summary, the DpH was shown to be responsible for the difference in substrate affinity of the transport protein inside and outside the cell and Dc – at least according to one theory


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Fig. 5. Title page of ‘‘Biologie in unserer Zeit’’ (Issue of February 1985) accompanying my review for students and high school teachers entitled ‘‘Ionenstro¨me und Substratflu¨sse: Durch ihre Kopplung ko¨nnen Zellen Stoffe aktiv aufnehmen.’’

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supported by some evidence – seems to increase the pH difference at the actual proton binding site.

Can the Transport Protein also Act as a Facilitator? It had generally been assumed that proteins which facilitate membrane transport until the concentration equilibrium is reached, like the glucose transporter of erythrocytes, act strictly symmetrical. And even for active transporting systems it was assumed that in the absence of energy they behave symmetrical and, therefore, in principle like a facilitator. Indeed the E. coli lacpermease equilibrates accumulated lactose-analogs rapidly, when the energy supply is shut off by uncouplers. The hexose transport system of Chlorella surprisingly did not behave in that way. For this reason we published its first detailed characterization in 1972 as ‘‘Unusual features of the active hexose uptake system of Chlorella vulgaris’’ [27a]. It is still not fully understood, why Chlorella cells that have accumulated analogs several 100-fold, do not run empty, when uncouplers like FCCP are added. One observation, which may be related to the unorthodox behavior of the Chlorella transporter, was made with the antifungal toxin nystatin. In its presence the hexose transporter did behave like a facilitator. Accumulated sugars were rapidly released when FCCP and nystatin were present at the same time. However nystatin did not produce holes, as frequently stated and it only acts in metabolizing cells by the way [66,67]. Since nystatin specifically binds to 3u-b-hydroxy-sterols, it was postulated that the transporter protein binds to sterols and that nystatin interferes with this interaction. If the sterol–protein interaction were responsible for the non-reversibility, the whole phenomenon could be explained. Unfortunately this question has never been resolved and whenever I proposed that somebody should study it, the proposal did not find much resonance. Of the various problems one eventually has to leave as open questions, I consider this one the most interesting one. And indeed, when in later years the HUP protein was purified to homogeneity, it was found to contain 2–3 sterols bound per molecule of protein [68] and that in addition the protein is localized in a sterol-rich lateral plasma membrane compartment (see section on the role of lipids).


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Cloning the Gene Coding for the Hexose Uptake Protein (HUP1) When the possibilities of molecular cloning became available, the first membrane transporter genes were cloned from E. coli [69] and subsequently from human erythrocytes [70]. Gabriele ¨gl in my laboratory started to prepare a cDNA bank Schnelbo from induced and non-induced Chlorella cells and tried to find among the differentially expressed genes the transport protein. The project did gain considerably in speed, when Norbert Sauer returned from his postdoc position with Chris Lamb at the Salk Institute. In only a few months he was able to identify a protein with a hydropathy profile of a membrane protein, which was expressed in Chlorella cells only when pretreated with glucose and which was absent in a mutant not able to transport glucose. Although our HUP gene was the first transport protein cloned from plants and was cloned still the hard way, we were not able to publish it any better than in FEBS Letters [71] and in a meeting report [72]. But at least Norbert was more successful, when 1 year later he and his co-workers were able to place the homologous gene from Arabidopsis into the EMBO Journal [73]. And working with Arabidopsis became a good start for Norbert, who accepted a professorship in Erlangen in 1995, where he intensively worked on sugar transport genes in this and other ‘‘real’’ plants. A final proof that HUP1 codes for an actively transporting membrane protein was obtained by Thomas Caspari, who functionally expressed HUP1 in S. pombe. The protein in its new host obviously felt quite well: it showed the same Km and a comparable Vmax value as those in Chlorella [74]. The fact that yeast cells – S. pombe as well as S. cerevisiae – were excellently suited for functional expression of heterologous membrane proteins, as demonstrated for our HUP protein as well as by others for bacteriorhodopsin [75], was subsequently followed by many plant groups. At the ‘‘Xth International Plant Membrane Biology Workshop’’ with about 400 participants, more than a dozen new plant transport proteins were reported, all cloned by functional expression in yeast. We had organized this conference in Regensburg in 1995. Thanks are due even today to Veronika Mrosek and Doris Urbanek for their enormous organizational efforts, since this was still a completely self-made congress without the help of any

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commercial agency. The same was true for the even larger Congress of the German Botanical Society in the first week of October 1990. In 1790 the ‘‘Regensburgische Botanische Gesellschaft’’ was founded and as such it is the oldest, continuously existing Botanical Society in the world, with Goethe for example having been one of its members. Andreas Bresinsky, Professor for Systematic Botany and close colleague, was the Societies President in 1990 and because of the centennial we succeeded to bring the meeting to Regensburg. At this conference of the German Botanical Society, the first one after the reunification of East- and West-Germany, more than 700 participants were in Regensburg and about 40 from the previous DDR. Many of the older participants taking part for the first time again in a meeting of the Society since the Wall was built in Berlin in 1961. The actual day of reunification, October 3rd – now our national holiday – was in the middle of the meeting week. A larger group of participants – I remember Eike Libbert and his wife from Rostock – got together in the evening of October 2nd in the restaurant Orpheé. At midnight, when ‘‘Freude scho¨ner Go¨tterfunken’’ sounded from the loudspeakers, we all were embracing each other and we were not ashamed of our tears of joy. However back to the HUP1 protein that in principal was responsible for bringing the ‘‘Xth International Plant Membrane Biology Workshop’’ to Regensburg. Structure/Function Relationships in HUP1 Structure/function relationships of membrane transport proteins were excessively studied with the E. coli lac-permease, of which almost any one of the 560 amino acids has been exchanged with various other ones by quite a number of postdocs in Ron Kaback’s laboratory [76]. Especially the cystein scanning method was a great idea and a procedure that became widely used afterwards. It is needless to say that no granting agency would have paid a number of postdocs for similar work in Chlorella. Thus we had to think of a more modest approach that could be followed by one PhD student at most. Andreas Will undertook it to screen for random mutations in the HUP1 transport protein that showed reduced affinity for transport substrates. He used non-optimal PCR conditions that produced 1–2 random mutations per HUP1 gene, expressed the mutated genes in an S. pombe mutant not


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able to grow on glucose [77]. The transformed pombe cells were plated on low concentrations of the toxic sugar analog 2-deoxyglucose, while growing on gluconate as usable carbohydrate source. Under these conditions cells transformed with wild type HUP1 protein are intoxicated at a concentration of 105 M of 2-deoxy-glucose. Cells transformed with the mutated HUP1 that were able to stand even 104 M of the toxic sugar, were checked for transport Km values. Twelve different mutants were obtained that showed decreased substrate affinity [78]. They all clustered in four different transmembrane domains: I, V, VII, and XI. Using the simplest interpretation – not necessarily the correct one, of course – this was taken as evidence that the four membrane domains form the sugar specific transmembrane pore or at least part of it. We also pointed out [79] that it may not be accidental that the two helices in the first half of the HUP molecule and the two corresponding ones in the second half are affected (I and VII, V and XI), since it had been postulated that 12 transmembrane transporters evolutionary may have arisen by gene duplication [80]. The very same overall conclusion was drawn by Kaback, when he published his lac-permease crystal structure [81]. Based on a massive amount of additional data, the Kaback lab was even brave enough to propose a very detailed proton coupled lactose translocation mechanism [82]; however atomic resolution of the transporter will be required, before it can be decided, whether the exciting story of active membrane transport that went on for more than 30 years, is completely understood. In vitro Transport Tests and HUP1 Protein Purification Procedures to study membrane transport in vitro with plasma membrane vesicles became established in the 1970s by groups working with bacteria. Important technical knowledge came from colleagues in the mitochondrial and the chloroplast field. In Regensburg, we were happy therefore that Gu ¨ nter Hauska (Figure 2) with his great experience in bioenergetics in general and with liposomal work in particular, joined the Institute as professor in 1976. He had spent a postdoc’s period with Efraim Racker in Cornell and subsequently a number of years with Achim Trebst in Bochum. With minor modifications the vesicle methods could be applied for transport studies in plants and for preparing active plasma

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membrane vesicles of plant cells (e.g. Ref. [83]). The results obtained by various groups studying transport in vesicles, strongly supported the in vivo results for secondary active transport in all eukaryotic cells, but did not give rise to real new insights. On the other hand, pure transport proteins were rarely studied, not to speak about the lack of any crystals and 3D structures of transport proteins. To study co-transporting proteins purified to homogeneity, they had to be tested in proteoliposomes; these are liposomes that could be energized due to the additional incorporation of proton motive force generating proteins. Will Konings’ ascorbate/cytochrome c/cytochrome oxidase system ´ brought became a favorite one in the field [84]. Mirka Opekarova the method from Groningen via Prague to Regensburg [85,86]. Using it for studying the HUP1 protein, led to the conclusion that this protein alone is sufficient for concentrative sugar uptake [87] and in addition, it gave important hints, concerning the optimal lipid requirement of the protein [88]. We observed that depending on the detergent used, the HUP1 protein obligatorily required phosphatidyl choline (PC) for activity and stability. In addition, we could show that the nonyl-b-D-glucoside solubilized protein contained PC, phosphatidyl ethanolamine (PE) and ergosterol of two molecules each per molecule of protein [88]. The purification of the active HUP1 protein in large amounts was a heroic task. Ingrid Robl wanted to tackle it with the aim of crystallizing the protein together with a monoclonal antibody she had prepared against the transporter. She did indeed obtain 2D crystals, however the quality was too poor and thus we finally gave up. It still took 3 years before Ron Kaback and his crew obtained good ˚ resolution) of a lac-permease mutant specially crystals (3.6 A suited for crystallization [81] and another 2 years before the first transport protein – not taking in account MacKinnon’s ˚ ) by potassium-channel – was solved in atomic resolution (1.65 A Yamashita et al. [89]. The latter is the leucine transporter of the hyperthermophilic bacterium Aquifex aeolicus, and – what made the results especially exciting – a homolog of a family of neurotransmitter transporters. Most interestingly the results showed that transmembrane helices with short helix interruptions exist, giving rise to half transmembrane helices. This principle may represent an important principle of substrate binding and translocation. A. aeolicus had originally been


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isolated, by the way, by the microbiologists of the University of Regensburg [90]. How our work on membrane transport was continued, although with quite a change in direction, is discussed in the next section.

Over 40 Years of Membrane Transport Proteins: How about the Role of Lipids? In the famous Fox and Kennedy paper of 1965, the gene responsible for lactose transport activity in E. coli was identified as coding for the M protein [91]. This was the first transport protein seen as a band on a gel. Somehow this result supported the idea that membrane functions have to be considered almost synonymously with membrane proteins. The other 50% that constitute a biological membrane, the lipids, were thought to be a passive matrix, important for separating two spaces but otherwise solely forming the solvent for the biologically important membrane proteins. Lipid experts, however, like Joachim Seelig pointed out that several hundreds of different molecular lipid species exist in any membrane and if it were just for separating functions, the composition would be expected to be much simpler. When the signaling role of phospho-inositides was discovered [92], it became more than obvious how important specific lipids can be. But it was also suspected that less conspicuous roles may be played by membrane lipids. Thus for example, all membrane proteins that have been crystallized so far do contain specific lipids as building blocks and as such they play a structural, but may very well also play a functional role [93]. As mentioned above, we got increasingly interested in membrane lipids during our eventually unsuccessful trials to purify and crystallize the Chlorella glucose transporter. On the other hand, since the field of cloning and characterizing further transport proteins started to explode, it was time to get out of ´ of the Czech it. I was happy, therefore, when Mirka Opekarova Academy of Science in Prague and frequent guest scientist in Regensburg suggested that we should look in vivo – using corresponding yeast mutants – for the requirement of specific lipids for transport processes. We started with a mutant low in phosphatidyl ethanolamine, because Chen and Wilson had shown already in 1984 [94] that the E. coli lac-permease changes its

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characteristics in vesicles lacking this phospholipid: the transporter is not able anymore to transport against a concentration gradient on the account of pmf, but it is less affected in counterflow activity. Subsequently Dowhan and co-workers demonstrated that phosphatidyl ethanolamine serves a chaperone-like function for correct folding and membrane integration of the protein [95]. Our first experiments with an S. cerevisiae mutant low in PE showed a significantly decreased rate of arginine, uracil, and maltose uptake, all catalyzed by proton symporters, whereas glucose uptake – mediated by facilitators in yeast – was not affected [96,97]. For the Can1 protein, the arginine transporter, which we started to study in detail, we were able to show with a Can1-GFP fusion that the lack of PE affects the targeting of the protein to the plasma membrane. It got stuck in the Golgi [98]. However, the most surprising result during these investigations was the appearance of the Can1-GFP fusion protein in the plasma membrane. In the cross-section under the confocal microscope the membrane looked like drawn with a dashed or dotted line (Figure 6). Can1-GFP obviously is not homogeneously distributed

Fig. 6. It has been a great surprise to see that the arginine/Hþ symporter (here as Can1-GFP fusion protein) is not homogenously distributed in the plasma membrane of yeast cells. This became a fascinating new research area (see text). (A) Confocal cross-section; (B) confocal surface view; Bars 5 mm.


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in the membrane, but rather concentrated in lateral compartments consisting of 50–80 fairly round patches per cell, each with a diameter of about 300 nm [99,100]. This phenomenon became the starting point of a series of experiments and of an expedition into a completely new field. Rafts or Raft Clusters or a New Type of Plasma Membrane Micro-Domains A lateral compartmentation of membranes, especially of the plasma membrane had long been postulated, based on a variety of observations (reviewed in Refs [101–103]). Kai Simons coined the name rafts for one type of compartment, which became the most popular one. Rafts are postulated to constitute within the membrane specific small areas enriched in sterols and sphingolipids with long acyl chains and specific proteins are thought to be preferentially, often transiently, concentrated there. Are ‘‘our’’ membrane domains rafts? Since typical rafts of mammalian cells are much smaller, we suggested that they could be raft clusters. They behave like ‘‘DRMs’’ [104] – detergent resistant membranes, a phenomenon that developed into the most prominent criterion for rafts – however in yeast, all plasma membrane proteins behave like DRM proteins, independent of whether they are located in those patches or not [105]. Finally ‘‘our’’ Can1 patches are very stable: once formed they can be observed as a constant spot for a whole generation time [99], whereas mammalian rafts are highly dynamic arrangements. Thus, it is a riddle what the patchy compartment in yeast cells really is and what fixes it so stably in the plasma membrane. I have a small, highly motivated group, working on the problem in a humorous atmosphere – partly at the Czech Academy of Sciences in Prague; it is not surprising, therefore, that I enjoy this work even 2 years after my official retirement. The members of the group are: Mirka Opekarova, who started the whole project, Guido Grossmann, who did his diploma and now does his PhD work with me, Jan Malinsky, a physicist and expert in confocal microscopy, Ingrid Fuchs, our technician, and Ina Weig-Meckl, a diploma biologist, who enjoys part-time recovering from household and children. At times we were accompanied by two postdocs, Katka Malinska and Linda Novakova, and two diploma students, Wiebke Stahlschmidt and Martin Loibl. And with Ju ¨ rgen Stolz, now at the Technical

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University Munich, and Wolf Frommer at Carnegie Institute/ Stanford we have a fruitful exchange of ideas. What have we found so far? (1) The yeast plasma membrane is subdivided into at least two compartments: the patchy one, in which first Can1 was found to be concentrated and which is called MCC (membrane compartment of Can1), and a second one that looks like a reticulate structure encompassing all the patches; it houses Pma1, the proton ATPase, and is called MCP (membrane compartment of Pma1) [99,100]. (2) In MCC so far 19 different proteins have been detected together with Can1, as for example Fur4, Tat2, and Sur7. In the MCP compartment Pmp2 and most likely Mid2 co-localize with Pma1 [106]. In MCC also ergosterol seems to be enriched at least threefold [107]. (3) A number of membrane proteins are homogeneously present in the membrane and thus occur in both compartments, like Hxt1 and Gap1 [99,105]. (4) Although shape and position of the compartments are stable, FRAP (fluorescence recovery after photo bleaching) experiments showed that in the MCP compartment bleached Pma1-GFP equilibrates within minutes. Actin cables and microtubuli do not seem to be involved in compartment stability [100]. (5) The Chlorella hexose transport protein HUP1, heterologously expressed in yeast, co-localizes with the various yeast proteins that concentrate in MCC [108]. (6) The protein composition of MCC depends on the membrane potential Dc: depolarization leads to a spreading of the proton symporters Can1, Fur4, and Tat2 , not however of Sur7 [107]. In lipid biosynthesis mutants affected in the synthesis of ergosterol or sphingolipids also a homogeneous distribution of some of the patch-forming proteins is observed. The main problem for our future work – as well as for anyone else who may get interested in these phenomena – will be, of course, to find out the biological relevance of this lateral compartmentation of the plasma membrane. For mammalian cells strong evidence suggests that lipid rafts play a decisive role in signal transduction across the membrane ([109,110]; see also


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the reviews given above concerning lipid rafts). For S. cerevisiae we have no hint so far, except possibly that a new organelle has been discovered recently, and called eisosome, which is localized intracellularly in the immediate neighborhood of the MCC patches. Eisosomes have been postulated to play some role in endocytosis [111]. Unraveling this problem, might be a challenging near future with still a lot of science, provided the DFG will support the group and I remain in good condition to plan, think, read, discuss, and write grants.

Glycosylation of Proteins Biochemistry and Molecular Biology As briefly discussed in the section ‘‘Back in Munich and Finding my Own Way’’, I had studied sugar transfer reactions in plants and this by some detour gave rise to the discovery of lipid activated sugars in yeast. With a lipid intermediate, that is a mannosyl residue attached to a lipophilic component, a compound with a high group transfer potential (half time of hydrolysis in 0.1 N HCl at 181C of 10 minutes) in the luggage, we arrived at the new University of Regensburg in September 1971. The two obvious scientific questions then were: What is the natural intermediate chemically – is it really a polyprenol, as indicated by our preliminary experiments described above? [25] and second, to which acceptors do the mannosyl moieties get transferred from the lipid intermediate? Peter Jung was set to solve the first, Peter Babczinski the second question. A lipid extract of yeast cells was found to stimulate the incorporation of radioactivity from GDP-14C-Man into the chloroform/methanol/water fraction of isolated membranes. With this test Peter Jung first showed that the stimulating activity is highest in the non-saponifiable fraction of S. cerevisiae log-cells. We got the Merck Company to grow a fermenter with our S. cerevisiae strain in a defined medium. From 2½ kg of log-cells Peter was able to purify almost 2 mg of a phosphorylated lipid factor. This compound stimulated in his test; after dephosphorylation it was identified by mass spectral analysis (carried out by Hoffmann-La Roche, Basel) as a mixture of polyprenols 14–18 isoprenyl residues long. The hydroxylated

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terminal isoprene was saturated [112]. The family of polyprenols with saturated a-isoprenyl units had originally been described as natural products first isolated from kidney [113]. The authors named this group of compounds dolichols, because of the length of the molecules, from the Greek ‘‘dolichos’’ ¼ the Olympic long distance run. Peter Babczinski prepared 14C-mannosyl lipid, thin-layer purified it and incubated it with crude yeast membranes. He found incorporation into a glycoprotein fraction; unexpectedly however, more than 70% of the radioactivity was detected in oligosaccharides that were alkali-labile, which meant that they are O-linked to either serine or threonine. This protein modification was originally described from yeast by Sentandreu and Northcote [114] and was for a long time thought to be a fungal specificity (see however below). Peter also presented evidence that the oligosaccharides released by mild alkali were up to four mannoses long, but only the first mannose, the one bound to the protein, was transferred from the lipid donor. The extension was brought about directly via GDP-Man [115]. Later it became clear that the first mannose gets attached to proteins cotranslationally in the ER (endoplasmic reticulum), whereas further mannoses are added in the Golgi [116]. With this information the sequence of reactions of protein O-mannosylation was in principal understood. The reaction products were glycoproteins, almost exclusively components of the plasma membrane and of the yeast cell wall. Prominent members were for example the mating type specific agglutinins [117,118]. In the years from 1975 to 1985, the field of glycoproteins and protein glycosylation broadened considerably and since we had started the lipid activated sugar work, many people came and visited us over the years to learn some of the special tricks how to incubate lipophilic compounds in a watery reaction mixture, or how to prepare substrate amounts of dolichol from pig liver. Some visitors came for a few days or weeks, like Ralph Schwarz, Ernst Bause, Dianna Bowles, Frans Klis, and Markus Aebi, others stayed for a postdoc time like Graczyna Palamarczyk, Peter Orlean, Mike and Kathy Marriott, Jim Linden, Andre´ Hasilik, Francoise Quigley, Tilman Achstetter, and Chandra B. Sharma. The latter visited the lab altogether 10 times after his first stay from 1972 to 1974 [119–121]. Chandra had already been looking forward to come a 12th time for my ‘‘Abschiedsvorlesung’’ in the


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summer of 2007. Very sadly he died in the year before. Most of these visitors worked under the guidance of Ludwig Lehle, who had become the center of the protein glycosylation work in the Institute. Ludwig came along from Munich, where he did his PhD work with Kandler and me on the biosynthesis of raffinose in plant seeds [19,20,122]. In Regensburg he first switched to yeast mannosyl transferases that work with GDPman as mannosyl donor [123]. When it became clear that Dol-P-man in yeast acts also as mannosyl donor in protein N-glycosylation [119], Ludwig slowly took over this research area [124–126]. He eventually became a widely acknowledged expert in glycobiology and contributed significantly to the molecular biology of protein N-glycosylation and to the elucidation of CDGs (congenital disorders of glycosylation; see below) [127]. My group apart from Ludwig’s stayed with protein O-mannosylation, thought to be fungal specific and, therefore, a possible target for developing fungicidal compounds. Glaxo and later Oxford Glycoscience were interested in our efforts. The main goals now were to purify the Dol-P-man: protein O-mannosyl transferase, to try to clone the gene coding for it and see whether a deletion of the gene may be lethal for S. cerevisiae as well as for pathogenic yeasts. Fortunately, all the many methods required for molecular cloning and for gene knockout by homologous recombination became available through the efforts of many brilliant scientists during the 1980s. Thus the methods were available to tackle these kinds of problems. But it still turned out to be one of the hardest problems that got solved in my Institute. Toni Haselbeck started it and achieved a preliminary purification of the membrane bound enzyme [128]. The work was frustrating and Toni for some time was not sure, whether his decision to stay for a PhD thesis and not start as a high school teacher – he had passed the state exam for teachers – was the right one. Only after he had carried out a real breakthrough experiment, showing that dolichol carries mannose through an artificial liposomal membrane from the outside to the inside, something that had been postulated all the time to be the function of lipid intermediates, Toni relaxed and became a happy researcher [129] (Figure 7). After a postdoc time with Randy Schekman and some further years with me, he joined Boehringer Mannheim. The fact that Toni has been one of the very few in his new position, who had experience with glycoproteins, was very important for his future

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Toni Haselbeck (left) celebrating his ‘‘Doktorvaters’’ 50th birthday.

career. One of Boehringer’s main products became erythropoietin, which is a glycoprotein. And Toni stepped up to the top management of what is now Roche Pharma Research in Penzberg in Bavaria. The work of enzyme purification was continued by Sabine Strahl. She had conducted her diploma work under my and Toni’s guidance, in which she had worked out an enzyme test, using serine/threonine-containing peptides, which accept mannosyl residues from Dol-P-man. But nevertheless, I considered the purification of an extremely labile membrane protein, most likely present in minute amounts in the cells, a very risky project and tried to talk Sabine out of it. However she insisted to tackle it [130]. What Sabine required most in the subsequent 3–4 years was the ability to withstand an extremely high frustration level. She never ever gave up! In this respect she was a unique PhD student among the close to 50 or so, who I had the pleasure to work with. I found it always difficult but nevertheless important to console co-workers, after another experimental ‘‘down.’’ And it at least ended in a smile, when I told them: ‘‘You know, if it hardly ever works, you are doing research. If it works every day, you are most likely working in a post office.’’ After Sabine had reached a considerable enrichment of the enzyme activity, a 92 kD band appeared, but it was far from being


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pure. An antibody against this protein inhibited the enzymatic activity by 50%. From now on the protein was not any more purified by measuring the enzymatic activity, but by following the enrichment of immuno-positive material. As it turned out, this indeed was successful and led to the right sequence and the PMT1 gene (protein: mannosyl transferase) could be finally cloned [131] (Figure 8). The paper has the number 150 in my publication list and we devoted it to Meinhart Zenk on the occasion of his 60th birthday; he sent a box of several bottles of wonderful red wine in response. It soon turned out that there exist at least five more PMT genes in S. cerevisiae and that some of these had a very pronounced substrate specificity, concerning the proteins they mannosylate [132,133]. To our knowledge, this was a first indication that protein glycosyl-transferases not only have to recognize the immediate accepting site for the sugar, but also require additional information of the protein to be glycosylated. The latter is responsible for the phenomenon that only one or the other of various transferases is able to do the job. We have even observed

Fig. 8. The pathway and cellular localization of protein-O-mannosylation in Saccharomyces cerevisiae and next to it Sabine Strahl, who purified the enzyme, which catalyzes the initiating reaction (Protein O-Mannosyltransferase, Pmt) and cloned the corresponding gene.

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that in one and the same protein different domains get O-mannosylated by different Pmt proteins [134]. With the knowledge of the various PMT genes we were finally in a position to answer the most pertinent question: What happens to a yeast cell, if one or several of the PMT genes are deleted? Is O-mannosylation of proteins an essential reaction in the life of a yeast cell? With multiple knockouts Martina Gentzsch clearly demonstrated that protein O-mannosylation is an essential protein modification [132]. The reason for this lethality has been elucidated by Sabine Strahl, who has accepted a professorship in Heidelberg in 2004, and by David Levin at Johns Hopkins (see section Functional Aspects of Protein Glycosylation). Once the PMT genes were published, papers appeared that demonstrated that these genes were not restricted to fungi and that homologous proteins are present in higher organisms. The first one was the report of a Spanish group, Martin-Blanco and Garcia-Bellido [135], who found the gene corresponding to the yeast PMT4 in Drosophila. Especially interesting was their observation that a classical Drosophila mutant of the 1920s/ 1930s, the rt mutant (rt for rotated abdomen), had a mutated PMT gene. The histology of the mutant showed a defect in the orientation of the abdominal muscle cells and the authors speculated that mutations in PMT genes might be responsible for one of the many different types of inherited muscular dystrophies. This turned out to be correct (see below). The Spanish authors also noticed subsequently a PMT gene in the published human genome – named according to the nomenclature rules POMT1 – and Tobias Willer, a doctoral student of Sabine Strahl detected and characterized a second PMT gene of humans, the corresponding sets of genes in the mouse, and a second one also in Drosophila. In all these cases it turned out that the two copies of PMT genes of these organisms correspond to the yeast PMT2 and PMT4 gene [136]. Together with the laboratory of Jesus Cruces of Madrid we decided to produce an mPOMT1 knockout mouse. In Eckhard Wolf’s laboratory at the Gene Centre Munich the actual mouse part was handled, the corresponding embryonic stem cells were produced and most of the subsequent analyses were carried out by Tobias in our ‘‘Botanical’’ Institute. It turned out that the homozygous pomt1/pomt1 mouse is embryonic lethal; the embryos die between day 7 and 9 [137]. In the meantime


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paediatricians had been successful to relate severe neuromuscular disorders, frequently associated with defects in neuronal cell migration in children, with congenital defects in POMT1 and POMT2 genes [138,139]. The diseases are known as Muscle-EyeBrain disease (MEB disease) and as Walker-Warburg-Syndrome (WWS). Functional Aspects of Protein Glycosylation Whereas nowadays no doubt exists that protein glycosylation is of eminent biological importance – and to a large extent this knowledge is due to the great number of congenital diseases related to protein underglycosylation known today – for a long time the role of saccharides attached to proteins had remained an enigma. Halina Lis and Nathan Sharon had described an impressive set of examples showing how cell–cell interactions, mainly pathogen–cell interactions of higher organisms, depend on saccharides of glycoproteins [140]. But it was obvious of course that cells did not glycosylate their cell surface proteins to get successfully conquered by pathogens. There must exist cellular reasons for all these structures. In a lecture with the title ‘‘What do yeast cells glycosylate proteins for’’ – being unicellular! – I had postulated at the 44th Mosbach Meeting 1993 [141] that for N-glycosylation we have to distinguish between ER associated glycosylation reactions, which are extremely conserved in evolution from unicellular yeast to men and which, therefore, must play important intracellular roles, and the Golgi-type modifications, which differ widely from yeast to men and from species to species and are destined to play a major role in cell–cell recognition and interactions in multicellular organisms. This statement was based among others on our observation that for the only biological important cell–cell recognition event in the life of a yeast cell, the mating process, saccharides of glycoproteins do not play a role. Although it was well established that for agglutination of sexually differentiated a- and a-cells, two glycoproteins, a- and a-agglutinins were required [142], we could demonstrate that the interaction between the two agglutinins was a protein–protein interaction [143,144]. Potential intracellular functions that were known were the lysosomal targeting via the mannose-6-phosphate signal and the process of quality control for correct folding of secretory proteins, including the degradation of

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misfolded ones. Since frequently N-glycosylation mutants are osmo-labile, it seems that an important function for protein N-glycosylation in yeast is also the establishment of a stable cell wall, which consists to 40% of mannoproteins. A typical example for carbohydrate based cell–cell interactions in multicellular organisms – also in Lis and Sharon’s list of course – were the selectins in the process of lymphocyte homing. Although the consequences of protein underglycosylation are hardly ever understood on a molecular level, it is easy to imagine that protein-bound cell surface saccharides constitute important developmental signals. When they are missing, this may give rise to the very complex phenotypes of CDG patients. These types of congenital diseases has been first recognized by the Belgian paediatrician Jaak Jaeken in the late 1980s and a number of mainly European research groups were very successful in elucidating the detailed genetic defects within the complex biosynthetic path of protein N-glycosylation. The important role which the model organism yeast has played in this process has only recently been summarized. Specific yeast N-glycosylation mutants can be healed by the corresponding human genes; if not, this very gene is affected in the patient [127]. As already briefly mentioned, congenital diseases based on disorders in the syntheses of O-mannosylated proteins do also exist. For historical reasons they are not included so far in the list of CDGs, although they definitely are ‘‘congenital disorders of glycosylaton’’ and certainly should be and sooner or later will have to be classified within this group of diseases. The molecular mechanism causing the phenotypic expression of the disease in patients with defects in protein O-mannosylation is largely known. Due to the work of Ten Feizi in London and to Tamoa Endo in Tokyo it became clear in the late 1980s that in the mammalian brain and muscle tissue proteins with yeast-type O-mannose modification exist [145,146]. Endo identified the saccharides of a-dystroglycan, an extracellularly facing subunit of a plasma lemmal protein complex. This dystroglycan complex connects the intracellular cytoskeleton (via the protein dystrophin) with the extracellular matrix (ECM) [147]. In addition the Endo lab showed that the extracellular a-dystroglycan binds through its saccharides (Neur-Gal-GlcNAc-Man-Protein) to the ECM component laminin. A defect in the POMT genes produces sugar-free a-dystroglycan and thus muscle cells orient wrongly in


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their tissue context – as observed in the rt mutant of Drosophila (see above) – and give rise to a specific type of congenital muscular dystrophy. The prediction of our Spanish colleagues was right. The mental retardation frequently observed in the same patients indicates that neuronal cell migration during brain development also seems to be dependent on glycosylated a-dystroglycan. The question, why for yeast cells this protein modification is of life-necessity, was still unanswered, however. Several of the multiple PMT knockout yeast strains were highly osmo-labile [95] and, therefore, we were looking for essential, O-mannosylated cell wall proteins. Vladimir Mrsa, who joined me as a postdoc in 1992 with a DAAD stipend and characterized a cell wall endo-b-1,3glucanase, originally cloned by Franz Klebl [148,149]. Vlado visited Regensburg several times subsequently and became the cell wall expert of the institute. He first introduced an efficient and specific method to label cell wall proteins. With a nonpermeating biotinylation agent he biotinylated all external proteins, which were subsequently detected by avidine coupled tests [150]. He discovered more than a dozen new cell wall proteins, most of them heavily N- and O-mannosylated, but none of them, once deleted, was essential nor did the mutants show a strong phenotype [151–153]. Vlado also detected a new class of cell wall proteins, the so-called ‘‘Pir-proteins’’ (proteins with internal repeats) [150,153] and we – together with Rainer Deutzmann, our extremely helpful protein sequencing expert in the faculty – could show that they bound to the cell wall matrix by a completely new linkage type [154]. In the cell wall field we also had a very fruitful exchange of ideas with Vladimir Farkas’ laboratory in Bratislava. Vlado also stayed in Regensburg for a few months as well as Sergej Sestak, who was in the lab a whole postdoc term and together with Ilja Hagen, a doctoral student, they were fighting those difficult to handle cell wall proteins [155,156]. However the main problem, why protein O-mannosylation is so crucial for yeast remained obscure. It was only after a new signaling pathway, the so-called ‘‘cell wall-integrity pathway’’ was established [157,158] that the essential function of protein O-mannosylation could be pinned down. Two types of receptor proteins (the so-called ‘‘Mid-’’ and ‘‘Wsc-proteins’’) measure cell wall integrity by an unknown mechanism. Both receptors possess one transmembrane helix and a large ectodomain, which is highly O-mannosylated. Deletions of sensor

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genes is lethal and deletions of PMTs leads to unglycosylated receptor proteins, which get rapidly degraded [159,160]. Checking of cell wall integrity is a very important and essential feature within the life cycle of a yeast cell. This becomes clear, when one considers that each mother cell has to flexibilize its wall at a certain spot and instance to initiate the formation of a bud. This loosening of the cell wall obviously has to proceed hand in hand with measuring cell wall integrity and responding correspondingly if loosening gets too far. Indeed do pmt mutants die the very moment a tiny bud is being formed [160]. To summarize the PMT story: After 30 years of work on a serine/threonine protein modification by mannosyl residues, a feature originally thought to be fungi specific, and after unraveling its unusual path of biosynthesis, the same modification was found in mammalian cells. The mannosyl transferase responsible for this modification is present in several copies (up to seven) in fungal cells and in two copies in all higher organisms, except in plants and in Caenorhabditis elegans, where these transferases are missing completely. The loss of one copy in mice is embryonic lethal; mutations in these genes in humans lead to severe diseases, frequently to childhood death. The corresponding gene for this congenital disorder has been identified by medical doctors. However, only due to many years of work with yeast, this fantastic model organism that in addition cares for our eating and drinking pleasures, the medical doctors were able to name the reaction affected by the gene defect.

Balance Sheet and Outlook On June 20, 2007, I presented my farewell lecture to the Department. At the end of this lecture I tried to draw a brief quantitative balance of 35 years of teaching and doing research. I estimated that about 5,000 students could have heard – not necessarily have heard – my lectures for first year students. Approximately 200 diploma and 80 PhD theses have been written in the institution I headed, the Institute of Cell Biology and Plant Physiology. About half of these have been conducted under my personal guidance. At least 19 students and postdoctoral fellows working for some time at the Institute stayed in science and eventually have obtained a professorship either in Germany or at


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various places all over the world. Nine of them had originally made their PhD work in the Institute, they are: Ewald Komor, now in Bayreuth; Norbert Sauer, Erlangen; Sabine Strahl, Heidelberg; Peter Babczinski, Bayer/Du ¨ sseldorf; Martina Rickauer, Toulouse, France; Martina Gentzsch, Chapel Hill, USA; Ed Hurt of Hauska’s group, Heidelberg; Ludwig Lehle, Regensburg, and from his group Thomas Roitsch, who is now in Wu ¨ rzburg. Ten have been postdoctoral co-workers: Andrej Hasilik now in Marburg; Peter Orlean, Urbana, USA; Tilmann Achstetter, Bremen; Josef Lengeler, Osnabru ¨ ck; Grazina Palamarczik, Warsawa; Vladimir Mrsa, Zagreb; Jim Linden, Boulder, USA; Chandra Bhan Sharma, Roorkee, India; Lev Okorokov, Campos dos Goytacazes, Brazil and last but not least, Wolfgang Lockau of Hauska’s group who is now in Berlin. In my opinion there hardly exists a more beautiful and fulfilling occupation than being a scientist. One tries to solve interesting problems and communicates with basically the same type of people in the rest of the world. The possibility to choose any topic one is intrigued by and wants to work on, is limited only to the extent that a few referees find it also interesting and propose that it should be supported by the DFG. In the German system you may even be able to tackle a problem solely with institutional money, which you obtain for you and your team as university professor. This gives you a certain freedom and allows approaching even risky projects. I am grateful to the State, but also to the taxpayer, who in principle unknowingly and with no real understanding supported my work. As mentioned in the chapter concerning the role of membrane lipids and membrane compartmentation, towards the end of my time I will try to understand why the plasma membrane is laterally organized in domains and how this is brought about. Thus the only thing that has changed since my official retirement, besides leaving the teaching duties and the decreased size of my research group, is the fact that I go home at noon and enjoy a 1-hour nap. REFERENCES [1] [2]

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WIDMAR TANNER Ehness, R. and Roitsch, T. (1997) Co-ordinated induction of m-RNAs for extracellular invertases and a glucose transporter in Chenopodium rubrum by cytokinin. Plant J. 11, 539–548. Balibrea-Lara, M., Gonzalez Garcia, M.-C., Fatima, T., EhneX, R., Lee, T.K., Proels, R., Tanner, W. and Roitsch, T. (2004) Extracellular invertase is an essential component of cytokinin-mediated delay of senescence. Plant Cell 16, 1276–1287. Goetz, M., Godt, D.E., Guivarc’h, A., Kahann, U., Chriqui, D. and Roitsch, T. (2001) Induction of male sterility in plants by metabolic engineering of the carbohydrate supply. Proc. Natl. Acad. Sci. USA 98, 6522–6527. Cohen, G.N. and Monod, J. (1957) Bacterial permeases. Bacteriol. Rev. 21, 169–194. Kaback, H.R. (1970) Transport. Ann. Rev. Biochem. 39, 561–598. Kundig, W., Ghosh, S. and Roseman, S. (1964) Phosphate bound to histidine in a protein as an intermediate in a novel phospho-transferase system. Proc. Natl. Acad. Sci. USA 52, 1067–1074. Komor, E. and Tanner, W. (1971) Characterization of the active hexose transport system of Chlorella vulgaris. Biochim. Biophys. Acta 241, 170–179. West, I.C. (1970) Lactose transport coupled to proton movements in Escherichia coli. Biochem. Biophys. Res. Commun. 41, 655–661. Mitchell, P. (1963) Molecule, group and electron translocation through natural membrane. Biochem. Soc. Symp. 22, 142–168. Crane, R.K. (1962) Hypothesis for mechanism of intestinal active transport of sugars. Federation Proc. 21, 891–895. Komor, E. (1973) Proton-coupled hexose transport in Chlorella vulgaris. FEBS Lett. 38, 16–18. Komor, E. and Tanner, W. (1974) The hexose-proton symport system of Chlorella vulgaris. Specificity, stoichiometry and energetics of sugarinduced proton uptake. Eur. J. Biochem. 44, 219–223. Kriedemann, P.E. and Beevers, H. (1967) Sugar uptake and translocation in castor bean seedlings. I. Characteristics of transfer in intact and excised seedlings. Plant Physiol. 42, 161–173. Komor, E., Rotter, M. and Tanner, W. (1977) A proton-cotransport system in a higher plant: Sucrose transport in Ricinus communis. Plant Sci. Lett. 9, 153–163. Komor, E., Haass, D., Komor, B. and Tanner, W. (1973) The active hexose-uptake system of Chlorella vulgaris. Km-values for 6-deoxyglucose influx and efflux and their contribution to sugar accumulation. Eur. J. Biochem. 39, 193–200. Bakeeva, L.E., Grinius, L.L., Jasaitis, A.A., Kuliene, V.V., Levitsky, D.O., Liberman, E.A., Severina, I.I. and Skulachev, V.P. (1970) Conversion of biomembrane-produced energy into electric form II. Intact mitochondria. Biochim. Biophys. Acta 216, 13–21. Komor, E. and Tanner, W. (1976) The determination of the membrane potential of Chlorella vulgaris. Evidence for electrogenic sugar transport. Eur. J. Biochem. 70, 197–204. Serrano, R. (1985) In Plasma Membrane ATPase of Plants and Fungi. Baco Raton, Florida, CRC Press, Inc. Decker, M. and Tanner, W. (1972) Respiratory increase and active hexose uptake of Chlorella vulgaris. Biochim. Biophys. Acta 266, 666–669. Winkler, H.H. and Wilson, T.H. (1966) The role of energy coupling in the transport of b-galactosides by Escherichia coli. J. Biol. Chem. 241, 2200–2211.

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Strahl-Bolsinger, S., Immervoll, T., Deutzmann, R. and Tanner, W. (1993) PMT1, the gene for a key enzyme of protein O-glycosylation in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 90, 81164–88168. Gentzsch, M. and Tanner, W. (1996) The PMT gene family: Protein O-glycosylation in Saccharomyces cerevisiae is vital. EMBO J. 15, 5752–5759. Gentzsch, M. and Tanner, W. (1997) Protein-O-glycosylation in yeast: Protein specific mannosyltransferases. Glycobiology 7, 481–486. Ecker, M., Mrsˇa, V., Hagen, I., Deutzmann, R., Strahl, S. and Tanner, W. (2003) O-Mannosylation precedes and potentially controls the N-glycosylation of a yeast cell wall glycoprotein. EMBO Reports 4, 628–632. Martıń-Blanco, E. and Garcıá-Bellido, A. (1996) Mutations in the rotated abdomen locus affect muscle development and reveal an intrinsic asymmetry in Drosophila. Proc. Natl. Acad. Sci. USA 93, 6048–6052. Willer, T., Valero, M.C., Tanner, W., Cruces, J. and Strahl, S. (2003) Omannosyl glycans: From yeast to novel associations with human disease. Curr. Opin. Struct. Biol. 13, 621–630. Willer, T., Prados, B., Falcon-Perez, J.M., Renner-Mu ¨ ller, I., Przemeck, G.K.H., Lommel, M., Coloma, A., Valero, M.C.M., de Angelis, M.H., Tanner, W., Wolf, E., Strahl, S. and Cruces, J. (2004) Targeted disruption of the Walker-Warburg syndrome gene Pomt1 in mouse results in embryonic lethality. PNAS 101, 14126–14131. Beltran-Valero de Bernabe´, D., Currier, S., Steinbrecher, A., Celli, J., van Beusekom, E., van der Zwaag, B., Kayserili, H., Merlini, L., Chitayat, D., Dobyns, W.B., Cormand, B., Lehesjoki, E., Cruces, J., Voit, T., Walsh, C.A., van Bokhoven, H. and Brunner, H.G. (2002) Mutations in the O-mannosyltransferase gene POMT1 give rise to the severe neuronal migration disorder Walker-Warburg syndrome. Am. J. Hum. Genet. 71, 1033–1043. van Reeuwijk, J., Jannssen, M., van den Elzen, C., Beltram-Valero de Bernabe´, D., Sabatelli, P., Merlini, L., Boon, M., Scheffer, H., Brockington, M., Muntoni, F., Huynen, M.A., Verrips, A., Walsh, C.A., Barth, P.G., Brunner, H.G. and van Bokhoven, H. (2005) POMT2 mutations cause a-dystroglycan hypoglycosylation and Walker-Warburg syndrome. J. Med. Genet. 42, 907–912. Sharon, N. and Lis, H. (1993) Carbohydrates in cell recognition. Sci. Am. 268, 82–89. Tanner, W. (1994) Why does baker’s yeast glycosylate proteins. In Glycoand Cellbiology (Wieland, F. and Reutter, W., eds.), pp. 81–89, 44th Colloquium – Mosbach 1993, Springer-Verlag, Berlin, Heidelberg. Lipke, P. and Kurjan, J. (1992) Sexual agglutination in budding yeasts: Structure, function, and regulation of adhesion glycoproteins. Microbiol. Rev. 56, 152–179. Cappellaro, C., Hauser, K., Mrsa, V., Watzele, M., Watzele, G., Gruber, C. and Tanner, W. (1991) Saccharomyces cerevisiae a- and a-agglutinin. Characterization of their molecular interaction. EMBO J. 10, 4081–4088. Cappellaro, C., Baldermann, C., Rachel, R. and Tanner, W. (1994) Mating type-specific cell–cell recognition of Saccharomyces cerevisiae: Cell wall attachment and active sites of a- and a-agglutinin. EMBO J. 13, 4737–4744. Yuen, C.-T., Chai, W., Loveless, W., Lawson, A.M., Margolis, R.U. and Feizi, T. (1997) Brain contains HNK-1 immunoreactive O-glycans of

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WIDMAR TANNER the sulfoglucuranyl lactosamine series that terminate in 2-linked or 2,6-linked hexose (mannose). J. Biol. Chem. 272, 8924–8931. Chiba, A., Matsumura, K., Yamada, H., Inazu, T., Shimizu, T., Kusunoki, S., Kanazawa, I., Kobata, A. and Endo, T. (1997) Structures of sialylated O-linked oligosaccharides of bovine peripheral nerve a-dystroglycan. The role of a novel O-mannosyl-type oligosaccharide in the binding of a-dystroglycan with lamini. J. Biol. Chem. 272, 2156–2162. Evrasti, J.M. and Campbell, K.P. (1993) A role for the dystrophin– glycoprotein complex as a transmembrane linker between laminin and actin. J. Cell Biol. 122, 809–823. Klebl, F. and Tanner, W. (1989) Molecular cloning of a cell wall exo-b-1,3glucanase from Saccharomyces cerevisiae. J. Bacteriol. 11, 6259–6264. Mrsa, V., Klebl, F. and Tanner, W. (1993) Purification and characterization of the S. cerevisiae BGL2 gene product, a cell wall endo-b-1,3glucanase. J. Bacteriol. 175, 2102–2106. Mrsa, V., Seidl, T., Gentzsch, M. and Tanner, W. (1997) Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O-mannosylated proteins of Saccharomyces cerevisiae. Yeast 13, 1145–1154. Cappellaro, C., Mrsa, V. and Tanner, W. (1998) New potential cell wall glucanases of Saccharomyces cerevisiae and their involvement in mating. J. Bacteriol. 19, 5030–5037. Mrsa, V., Ecker, M., Strahl-Bolsinger, S., Nimtz, M., Lehle, L. and Tanner, W. (1999) Deletion of new covalently linked cell wall glycoproteins alters the electrophoretic mobility of phosphorylated wall components of Saccharomyces cerevisiae. J. Bacteriol. 181, 3076–3086. Mrsa, V. and Tanner, W. (1999) Role of NaOH-extractable cell wall proteins Ccw5p, Ccw6p, Ccw7p and Ccw8p (members of the pir protein family) in stability of the Saccharomyces cerevisiae cell wall. Yeast 15, 813–820. Ecker, M., Deutzmann, R., Lehle, L., Mrsa, V. and Tanner, W. (2006) Pir proteins of Saccharomyces cerevisiae are attached to b-1,3-glucan by a new protein-carbohydrate linkage. J. Biol. Chem. 281, 11523–11529. Hagen, I., Ecker, M., Lagorce, A., Francois, J.M., Sestak, S., Rachel, R., Grossmann, G., Hauser, N.C., Hoheisel, J.D., Tanner, W. and Strahl, S. (2004) Sed1p and Srl1p are required to compensate for cell wall instability in Saccharomyces cerevisiae mutants defective in multiple GPI-anchored mannoproteins. Mol. Microbiol. 52, 1413–1425. Sestak, S., Hagen, I., Tanner, W. and Strahl, S. (2004) Scw10p, a cell-wall glucanase/transglucosidase important for cell-wall stability in Saccharomyces cerevisiae. Microbiology 150, 3197–3208. Heinisch, J.J., Lorberg, A., Schmitz, H.P. and Jacoby, J.J. (1999) The protein kinase C-mediated pathway involved in the maintenance of cellular integrity in Saccharomyces cerevisiae. Mol. Microbiol. 32, 671–680. Levin, D.E. (2005) Cell wall integrity signalling in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 69, 262–291. Philip, B. and Levin, D.E. (2001) Wsc1 and Mid2 are cell surface sensors for cell wall integrity signalling that act through Rom2, a guanine nucleotide exchange factor for Rho1. Mol. Cell Biol. 21, 271–280. Lommel, M., Bagnat, M. and Strahl, S. (2004) Aberrant processing of the WSC family and Mid2p cell surface sensors results in cell death of Saccharomyces cerevisiae O-mannosylation mutants. Mol. Cell Biol. 24, 46–57.


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Chapter 6

Viktor Mutt: A Giant in the Field of Bioactive Peptides ¨ RNVALLa, BIRGITTA AGERBERTHa AND HANS JO MICHAEL ZASLOFFb a

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden Email: [email protected] b Department of Surgery and Pediatrics, Georgetown University School of Medicine, Washington DC 20007, USA

Abstract ¨rve, Estonia, Viktor Mutt was born on December 29, 1923, in Kidja and died on September 9, 1998, in Stockholm, Sweden. He came to Sweden via Finland as a refugee during the World War II and passed his thesis at Karolinska Institutet on the purification of secretin in 1959. So began a career that resulted in the discovery of over 50 naturally occurring biologically active peptides, including secretin, cholecystokinin, VIP, NPY, PYY, and many others. His scientific approach, which depended on the extraction of animal tissues on an industrial scale, permitted him to discover peptides present in low abundance, including hormone precursors and processing intermediates that could not have been elucidated by any other means with the technology available at the time. With him the era of large-scale purification from natural sources peaked and is now coming to an end, replaced by recombinant protein production. Viktor remained active to the end of his life, and only now, as this memorial is being written, have the many projects he directly initiated finally come to an end. His life was

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that of a devoted and successful scientist, a kind and benevolent man worthy to commemorate. Keywords: Secretin; Gastrointestinal peptides; Gut-brain-skin bioactive peptides; Large-scale native preparations; Central nervous system peptides; Antibacterial peptides; Innate immunity

Early Days ¨rve, Estonia, in 1923, passed his Viktor Mutt was born in Kidja initial education in Tartu, came as a refugee to Finland in 1943 and continued to Sweden in the autumn of 1944, where he started his career in the scientific world from a base at Karolinska Institutet. He died in Stockholm, Sweden, in 1998 at the age of 74 years. Two of us worked closely with him in different positions. It may seem strange to commemorate him at this time, years after his death, but his work did not end abruptly when he passed away; he left us with an active research program in place, with students, ongoing projects and huge amounts of starting material, intermediate fractions and purified peptides at different stages of analysis. Only now his last projects have been completed, his last ˚ ke Norberg and Essam Refai) received their PhDs students (A (with HJ) and employment in novel projects, and the whole era of peptide chemistry based on large-scale preparations from material of natural origin come to an end. The new ‘‘omic’’ era has begun, one of ‘‘peptidomics, proteomics, and metabolomics,’’ with nano/femto/attomole-scale analyses of natural products, DNA sequencing of genetic messages, and the recombinant production of proteins. Viktor saw the new field coming and both encouraged its arrival and made early contributions. For all these reasons we feel it is now appropriate to review the life of Viktor Mutt and his contributions to science. Viktor completed his ‘‘gymnasium’’ studies in Tartu in 1943 in the humanistic line, but the war had altered Estonia. It was annexed by the Soviet regime in 1940 but was under occupation by Germany between 1941 and 1944. In the autumn of 1943, Viktor fled to Finland, and after a year he continued as a refugee to Sweden. He never communicated much about his personal details surrounding those early days, but we know he worked at a farm outside Stockholm during his first year in Sweden. Through the knowledge of a nearby family, he was recommended to get in

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contact with Erik Jorpes, a famous scientist and professor in the Department of Physiological Chemistry at Karolinska Institutet, known to be engaged in helping refugees. Viktor followed the advice, and simply showed up one day at the Karolinska, knocked at Professor Jorpes’ door and asked if he could start working with him. Erik Jorpes was a highly regarded, internationally recognized physician and scientist whose research was based around preparations of bioactive material. Jorpes was the first to begin large-scale production of both insulin and heparin in Sweden. He ˚ land archipelago of the Baltic Sea, was born in Ko¨kar in the A ˚ bo in Finland and Stockholm in Sweden. halfway between A During the Finnish Civil War, Jorpes was on ‘‘the Red side,’’ the side that lost there. He fled to Russia where he practiced as a physician. In 1919, in the midst of the ensuing post-revolution turmoil he fled to Sweden. In Jorpes, Viktor found a mentor with whom he shared scientific devotion and hard work. In 1953, Viktor completed his Swedish ‘‘gymnasium’’ studies in the natural sciences. This time was tough for him, with work daytime and studies at night, but he managed and continued, passing his graduate thesis in Jorpes’s laboratory in 1959 [1]. Much later, and appearing only after Viktor’s death, he, together ¨ck, wrote a review of Erik Jorpes and his with Margareta Blomba life achievements, published [2] in the same series as this report. Viktor became an Assistant Professor (‘‘Docent’’) in 1960 with grant support from the Swedish Medical Research Council. In 1970 he became Associate Professor and in 1979, Professor of Biochemistry (Figure 1). During his early years at the Karolinska, Viktor married Birgitta Werner, who became a ‘‘Docent’’ in pediatrics, and the highly recognized founder of the toxicological emergency center at the Karolinska Hospital. Actually, she was supposedly the one who was on visit to Erik Jorpes and presumably opened the door when he initially came there to ask for a position in Jorpes’ laboratory (above). They had two children, Valter and Maria, who developed careers outside of chemical and medical fields. The home of the Mutt family was always hospitable, warm, and with excellent and tasty food prepared by Birgitta. We spent many memorable evenings there, together with scientific friends, guests, and visitors. The period starting from just before Viktor’s arrival into Sweden, and continuing during his early and middle research


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Fig. 1.

Viktor Mutt. (Photo: HJ, Bend 1980s.)

period, was an extraordinary time in molecular medical sciences and natural product chemistry at Karolinska. A critical mass of extraordinary scientists was assembling throughout the various departments. It was a period when world-famous discoveries seemed to be taking place every day and the Institute was crowded with internationally renowned scientists. Karolinska became a scientific Mecca for the analysis of biomolecules, much like Cambridge (cf. Ref. [3]) with its similar explosions of discoveries in protein and nucleotide chemistry! Ulf von Euler identified new hormones, Sune Bergstro¨m purified them, soon joined by a young Bengt Samuelsson who characterized them; Ragnar Granit unraveled neural signals; and Hugo Theorell purified and characterized enzymes. Each received the Nobel Prize in Physiology or Medicine. Pehr Edman was at Karolinska for a period, continued his work on the famous sequencing reaction [4]; Einar Stenhagen, Ragnar Ryhage, and Jan Sjo¨vall launched biomedical mass spectrometry and participated in the construction of the first commercial gas chromatograph-mass ¨ck began spectrometer (with LKB); Birger and Margareta Blomba


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the isolation of blood coagulation factor VIII, naturally occurring with von Willibrand factor [5]; Torvard Laurent initiated characterization of hyaluronic acid [6]; Peter Reichard characterized enzymes and coenzymes in deoxyribonucleotide synthesis, Torbjo¨rn Caspersson developed techniques to characterize chromosomes, Rolf Luft unraveled mitochondrial diseases, and as already stated, Erik Jorpes taught us about heparin, and Viktor Mutt opened the vast field of bioactive neuro-intestinal peptides. Many of them were members of the National Academy of Sciences of the USA (Figure 2). This was also a high time in the hospital clinics of Karolinska, with Herbert Olivecrona initiating neuro˚rdh modern anesthesia, and Lars Lexell surgery, Torsten Ga contributing to the refinement of neurosurgical procedures (his son later launching neurosurgical instruments in a world-known company, Elekta). During this time, essentially just over one generation, professors at Karolinska received the five Nobel Prizes mentioned, but could have received more! This must have

Fig. 2. Celebration in the laboratory when the message about Viktor’s election into the National Academy of Sciences of the USA had arrived. Long-term scientific collaborators shown are Mats Carlquist (2nd from left), Kay Tatemoto (4th from left), Arvid Ahlroth (4th from right, originally from the pharmaceutical company Astra) and BA (3rd from right). Remaining persons around Viktor are laboratory personnel, from left: Ulf, Jan Wiberg, Vera Andreasson, Ewa Papinski, and Louise Melin. (Photo: Essam Refai.)


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been an exciting period of time for Viktor to have been a part of, and is one of the highlights thus far in the history of Karolinska, soon to celebrate its 200 year anniversary, in 2010.

Secretin Viktor’s initial scientific work was published in 1949 [7]. He then turned to purification of the gastrointestinal hormone secretin [8], a work which culminated in his thesis [1]. Secretin was a substance first detected as a biological activity by Bayliss and Starling back at the start of the 20th century [9]. They extracted dog jejunal mucosa with dilute acid, injected the preparation intravenously into a dog, and observed pancreatic secretion. They called the substance secretin and 2 years later called this class of messengers ‘‘hormones’’ from the Greek o´rm′ – ‘‘to set in motion.’’ For many years the chemical nature of secretin was a mystery; it was to be Viktor who purified it [1] and showed it to be a 27-residue peptide [10,11]. Secretin stimulates release of bicarbonate from the pancreas. When Viktor began his studies, very little was known about the nature of gastrointestinal hormones. In textbooks of those days, such as Fruton and Simmonds’ classic General Biochemistry [12], gastrointestinal hormones were allotted one quarter of a page of text, versus 16 pages for the hormones of the pituitary, other glands, pancreatic islets, testes, and ovaries. That textbook mentioned secretin and referred to ‘‘pancreo-zymin, cholecystokinin, and enterogastrone,’’ noted that ‘‘none of these hormones have been purified extensively’’ and that ‘‘their chemical nature is not established.’’ To give a sense of the magnitude of Viktor’s contributions to the field, one should appreciate that within 10 years, he and his colleagues had purified these hormones and had demonstrated that pancreozymin was not a separate entity, but simply an activity of cholecystokinin [13]. One scientist, with a devoted team, had opened the field of gastrointestinal hormones. Viktor intuitively understood the physical properties of biological materials. First and foremost, he realized, along with Erik Jorpes, that the proteolytic activity in intestinal tissue extracts had to be tamed in some fashion to recover an intact protein. They solved this problem by ‘‘simply’’ boiling the crude preparation and


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by developing peptide-selective precipitation/fractionation steps using salt and ethanol. In fact, Viktor’s preparations, although large in scale and from the small intestine – amongst the richest sources of proteases of any organ – were so protease-free that he could use them even for detection of labile hormone precursor forms [14] before they were characterized by cDNA sequencing. Secondly, he recognized the need for a suitable, quantitative assay to detect and quantify the amount of the intestinal peptides present in extracts, and so developed a highly reproducible secretion assay using a cannulated cat. Finally, he was amongst the first to utilize solid phase peptide synthesis to validate the structure of peptides purified from natural sources, and to explore through the synthesis of analogs, how activity varied with the structure [15,16]. Viktor’s thesis on the purification of secretin was published in Arkiv fo¨r Kemi [1], in those days a leading journal. The starting material was pig small intestine on the ton scale (Figure 3), that was boiled rapidly in large water-vapor-heated tanks, minced, and extracted in acetic acid. The peptide-rich fraction was concentrated on alginic acid, which was collected and rinsed with ethanol. Finally, the polypeptides were eluted and precipitated with salt to end up with a new, intermediate starting material, ‘‘concentrate of thermostable intestinal peptides’’ (CTIP). Using this material as the starting point for subsequent chromatographic separations, he obtained pure secretin. Viktor’s purification of secretin constituted a milestone. He had not only isolated the ‘‘hormone’’ detected by Bayliss and Starling and shown it to be a peptide, he established the basic methodology that would be applied repeatedly by Viktor and collaborators over the subsequent decades to isolate and characterize over 50 different intestinal peptides. Many of these ‘‘intestinal’’ peptides were subsequently found to be present in both brain and skin, resulting in the explosive growth of research in neuropeptide biology. Following the discovery that intestinal peptides could also be isolated from the central nervous system, Viktor and his colleagues initiated efforts to better understand their role in brain function. Using highly purified or synthetic peptides, antibodies were produced; the antibodies were then used to create detailed micro-anatomical distribution maps of specific peptides in the CNS by immunohistochemical techniques, a field

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Fig. 3. Porcine intestinal starting material, arriving deep-frozen and being handled for some decades by ‘‘Janne’’ (right) and ‘‘Johnny’’ (left), Jan Wiberg and Johnny So¨derlund, here in their early years. (Photographer unknown.)

¨kfelt and collaborators pioneered by Viktor’s colleague Tomas Ho ˚ [17]. Before that Nils-Ake Hillarp had, together with his student Bengt Falck in Lund, discovered the now classical histological formaldehyde fluorescence method which permitted visualization of catecholamines and serotonin directly in the microscope [18]. With this method, Kjell Fuxe and Annica Dahlstro¨m mapped these monoamine systems in the rat brain [19]. Together, these two detection methods using antibodies and formaldehyde, respectively, transformed the Department of Histology at Karolinska Institutet and made it into the Department of Neuroscience, showing the world that peptides and adrenergic compounds coexist in the brain [20], together regulating our behavior.


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Fig. 4. Large-scale extraction and filter tanks for the early preparative steps. (Photographer unknown, probably from Karolinska Campus Photo Center.)

Viktor’s laboratory extracted pig small intestines on such a scale, and produced so much secretin of such a high quality that he attracted the attention of pharmaceutical companies. Viktor entered into collaborations with several Swedish pharmaceutical companies, Vitrum, Kabi, and Ferring, in exploring the introduction of intestinal peptides into medical practice. Viktor’s laboratory became a ‘‘factory,’’ occupying a special building at Karolinska Institutet (Figure 4), and financing much of his research activity. This peptide production continued for decades, and Viktor’s laboratory became the world’s principal source of secretin for medical use.

The Post-Secretin Period Following the isolation of secretin, Viktor and his collaborators continued to purify and characterize peptides from the ‘‘CTIP’’ material. Cholecystokinin was a substance contained within the CTIP fraction that, as implied by its name, stimulated the release of bile from the gall bladder. By measuring the potency of various

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fractions to stimulate gall bladder contraction in guinea-pigs [21], Viktor isolated the responsible activity and discovered it to be a peptide, CCK. Surprisingly, the pure peptide also potently stimulated the release of digestive enzymes from the pancreas. It thus became clear, as noted above, that CCK was itself responsible for the hormonal activity ascribed to a presumed substance called ‘‘pancreozymin.’’ He also identified the amide part of this hormone [22]. The search for ‘‘enterogastrone,’’ based on an assay that measured the activity of the purified CTIP fractions to inhibit the secretion of acid by the gastric mucosa of dogs, led to the discovery of the peptide responsible, GIP, or gastric inhibitory peptide [23]. At about the same time, Viktor and colleagues, using as an assay the activity of various fractions to cause an increase in peripheral blood flow and arterial hypotension in dogs, chased VIP, or vasoactive intestinal peptide from its hiding place in the CTIP fraction [24,25]. The isolation and identification of somatostatin-28 [26] and GRP, gastrinreleasing peptide [27] followed in 1979. Multiple forms of somatostatin and CCK were subsequently identified, differing in length and potency, and indicated early on to Viktor and colleagues the existence of post-translational processing in hormone maturations. Viktor realized that bioactive peptides might exist for which biological assays could not be predicted or anticipated. Because several of the newly discovered peptides possessed a chemical modification, an amide, at the carboxyl-terminus, Viktor and Kazuhiko Tatemoto, developed a chemical method which could identify peptides bearing a C-terminal amide [28,29]. Using both chemical and biological assays, a rush of new discoveries emerged from Viktor’s laboratory: peptides PHI (peptide with N-terminal histidine and C-terminal isoleucine amide) and PYY (with tyrosine and tyrosine amide, respectively) came in 1980 [29], of peptide NPY (with proline and tyrosine amide at the two positions) in 1982 [30], of galanin (peptide with glycine and alanine amide, respectively) [31] and cardio-dilatin (an exception, with known function already at the purification stage) in 1983 [32], of neuropeptide K in 1985 [33], and of pancreastatin in 1986 [34]. We have only ‘‘scratched the surface’’ of Viktor’s achievements in this summary. In addition to the work mentioned, he discovered many other peptides, such as PEC-60 in 1989 [35],


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endosulfine [36] in 1996, and dopuin [37] and daintain [38] in 1997, the latter two named by Chen, his Chinese collaborator, to reflect the peptide activities in Chinese. Daintain turned out to be identical to macrophage allograft inflammatory factor-1 [39,40]. Viktor also improved previous purifications [41] and discovered additional peptides as well as sub-forms and species variants of several of the parent peptides mentioned above [42,43]. The discovery of each new peptide almost always provoked an ‘‘algorithmic’’ sequence of subsequent studies within Viktor’s group or with his many collaborators: The new peptide would be sequenced; variants would be created to establish sequence– activity relationships; the tissue distribution would be determined and immunohistochemical studies applied to establish its micro-anatomical location within tissues; pharmacological properties would be discovered and physiological functions hypothesized. Possible therapeutic applications were frequently proposed, based on pharmacological studies of the native peptide and its synthetic analogs. Viktor would also embark on comparisons of the peptide in various species, to glean further insight into critical sequence determinants. His work brought together experts in a diverse group of specialties; Viktor clearly understood the value of advancing science through extensive collaboration, creating the multidisciplinary teams so fashionable today. A major contributor to the field of biologically active peptides, and a contemporary of Viktor, was the great Italian pharmacologist Vittorio Erspamer. While Viktor explored the search for new mammalian hormones, Erspamer chose tissues from nonmammalian species, especially the skin of frogs and toads. Viktor wrote a tribute to Erspamer, ‘‘An appreciation of the work of Vittorio Erspamer’’ [44]. In this, he wrote: ‘‘It is evident that in initiating investigations on a wide scale of the occurrence of pharmacologically active substances in non-mammalian species, Vittorio Erspamer has done in our time what two of his countrymen, Christopher Columbus from Genoa and Amerigo Vespucci from Florence, did some 500 years ago – discovered a continent to explore.’’ Viktor, like Erspamer, guided the ships of science to new subjects and novel compounds. In the 1980s a new class of biologically active peptides – antimicrobial peptides – were discovered in several laboratories, including those of Hans Boman who discovered cecropins in the silk moth pupae [45], and MZ, who discovered the peptide

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magainin in the skin of the African Clawed frog [46]. Cecropin was also detected in the CTIP material [47] but turned out to be one of several peptides derived from intestinal worms [48]. Other ‘‘alien’’ peptides, derived from plant food, were also detected in the intestinal peptide preparations [49]. Viktor and his collaborators (including HJ and BA) also discovered genuinely mammalian antimicrobial peptides, PR-39 [50] and NK-lysin [51], peptides that are now recognized to represent critical components of the mammalian innate immune system. Together the separate discoveries of antimicrobial peptides contributed to the new research field of innate immunity, where pattern recognition receptors (PRRs) sense the presence of microbes [52]. A flood of new antimicrobial peptides from many species of animals were discovered in laboratories worldwide, followed by the recognition of the existence of PRRs (which regulate the expression of antimicrobial peptides in many animals) highlighted by the discovery of Toll-like receptors [53], linking research groups engaged in peptide chemistry, microbiology, immunology, and drug development in a worldwide interdisciplinary research community. As should be obvious from this summary of Viktor’s work, his discoveries of so many of the now well-known bioactive peptides positioned him as a true pioneer in the founding of three currently ‘‘hot’’ scientific fields: gastrointestinal hormones, peptide neurotransmitters, and antimicrobial peptides. The impact of Viktor’s work in medicine might not be appreciated by those who fail to realize that peptides central to fields such as psychiatry and disorders of behavior and innate immunity were discovered by Viktor and his colleagues.

Viktor: Teacher, Scientist, and Friend Viktor was a scientist who had the curiosity and knowledge to participate in the diverse interdisciplinary scientific milieu that was his world. He read the scientific literature voraciously and had a memory that would permit us to accurately describe him as a ‘‘living encyclopedia.’’ He even assigned a former laboratory technician (Karin Diel, later Termenius, initially working with Pehr Edman) for many years, more or less full-time, to assist him in searching literature at the Karolinska library. He would send


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notes to his friends and collaborators on scientific news he had seen in the literature and wanted us all to read. His capacities in literature knowledge and general judgments did not go unnoticed by the Nobel System at Karolinska Institutet. Viktor was (1977–1989) a member of the Nobel Committee at Karolinska and in 1970–1989 a member of the Nobel Assembly (before 1977, the Nobel Faculty). In 1983, he became a member of the Royal Swedish Academy of Sciences, and remained active there until the very end. It was after having attended one of the meetings at the Royal Academy that he suddenly died. Perhaps, it is appropriate, at this point, to highlight some of Viktor’s personal characteristics. Until his last days, Viktor could be found working in the laboratory at the bench, or in his office reading and writing. Despite his worldwide fame, he was a shy and humble man. He was very generous in offering advice, sharing ideas, and providing reagents. Viktor and Birgitta had many close friends and were recognized as part of the social ‘‘glue’’ of the Karolinska community, providing help to colleagues in matters relating to their personal lives. Viktor’s office reflected his character. It was one of the smallest offices at Karolinska, if not actually the smallest, furnished with only a small writing table, a few simple wooden chairs, and some bookshelves for filing reprints. With difficulty, he could squeeze one or two visitors into the room; he would usually offer the visitors coffee (from a thermos suddenly pulled off of a shelf) and a little cake. After Birgitta retired from her position as physician, she became Viktor’s secretary, managed his budget, and all his ‘‘right-hand’’ duties. Viktor was always busy, always going somewhere, sometimes almost running to scientific lectures. Nevertheless, he always had time for you (Figure 5). Retirement did not slow down either Birgitta or Viktor. Viktor continued to work, without any change in pace, keeping his laboratory intact until the very end, launching new purification programs and drafting new research manuscripts. Viktor was a popular teacher renowned for his inspiring lectures. He presented his lectures with great animation and often at a fast pace. Stories about his teaching style during the biochemistry classes for the medical students inform how he sometimes would write chemical formulas of peptides on the blackboard with the chalk in his right hand and the eraser in his left hand to make room immediately for more sequences.

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Fig. 5. Viktor during an interview. (Photographer unknown, probably from Karolinska Campus Photo Center.)

Nevertheless, he always made time for the individual student and was recognized by the medical students as a patient and kind teacher. Of particular interest with respect to Viktor’s career, and concerning the history of molecular sciences and of universities in general, are several characteristics of his approach to research. Historically, the extraction of naturally occurring substances and their characterization were central to the early development of the biological and microbiological sciences. Academic centers that were engaged in these areas of research created ‘‘extraction centers’’ capable of working at the ‘‘industrial scale.’’ At the Karolinska, both the Departments of Biochemistry and


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Bacteriology established large scale production centers, with enormous incubators, extraction facilities, and purification equipment. It is not surprising that several pharmaceutical companies, now quite large, started as small companies in the shadows of these facilities around Karolinska, Uppsala, and later further university centers in Sweden. The Karolinska Institutet created its first such large-scale facility in the late 1920s under the direction of Erik Jorpes who purified insulin. Jorpes had visited the laboratories of Banting and Best in Toronto and brought their insulin purification technology to Sweden. The Jorpes facility eventually supported the large-scale production of heparin and subsequently was adopted by Viktor for the purpose of isolating gastrointestinal peptides. The Karolinska extraction facilities required ‘‘industrial size’’ equipment and both Erik Jorpes and Viktor had to convince the Karolinska administration to permit them to physically alter the configurations of the buildings in order to accommodate the ‘‘oversized’’ equipment, including some very large chromatography columns (Figure 6), extraction facilities (Figure 4), huge lifting devices for handling raw material, and big centrifuges. Many stories from this period are still told and illuminate the atmosphere of the early days. For example, an unbalanced rotor in one of Hugo Theorell’s huge centrifuges was said to have become unbalanced, rotating out through the wall of the building. Another often told story describes Viktor’s huge Sephadex column requiring technicians equipped with buckets to serve as fraction collectors. In 2005, when the old Biochemistry department had already been transformed into the Department of Medical Biochemistry and Biophysics, Viktor’s production facility was disassembled. New technologies now permitted the dramatic miniaturization of classical methodologies. Micro-scale analytical technology allowed for characterization and sequencing of minute amounts of lowabundance proteins, no longer requiring large quantities of starting material. And through application of DNA methodology proteins could be produced recombinantly in virtually unlimited quantities. One should appreciate that the ‘‘old’’ extraction methods, now considered ‘‘old-fashioned’’ by some, were surprisingly effective. In Viktor’s hands, industrial-scale methodologies, utilizing a ton or so of pig intestines, yielded labile intermediates and lowabundance peptides that could not have been discovered at the time by any other route [14]. Similarly, proinsulin was detected in


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Fig. 6. Human-sized Sephadex column. When in use, being handled by handheld buckets as fraction collector, but here stored by Viktor after a time of preparation. (Photographer unknown, probably from Karolinska Campus Photo Center.)

its pro-form by large-scale purification of pancreatic extracts in 1967 by Steiner and coworkers [54], long before the age of modern molecular biology. We can only wonder how many naturally occurring substances might be discoverable through continued large-scale purification of natural sources.

Conclusions Viktor was a pioneer. When he and his colleagues began their search for bioactive components, little was known of the nature of


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the substances; neither was it obvious to anyone how to attack the problem. Viktor isolated novel gastrointestinal hormones, opened new avenues of scientific inquiry, and freely distributed pure hormones around the world. His efforts made it possible to unravel complex physiological processes, the production of immunological reagents, and clarification of the role of peptide messengers in the gastrointestinal tract, the peripheral and central nervous systems, and the immune networks. Viktor’s pioneering research initiated new medical insights and spawned novel fields of pharmacology. Pure hormones led to the discovery of their receptors, resulting in deeper understanding of human physiology. It is fitting, as we have now entered the era flooded with unprecedented amounts of genetic data, and as we witness the passing of Viktor’s approach to peptide discovery, that we remember his early days, his life, and his achievements.

ACKNOWLEDGMENTS

¨kfelt and Jan We are grateful to Maria Mutt, and to Tomas Ho Sjo¨vall (both professors at Karolinska) for help with some details of our recollections.

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Chapter 7

Sailing Side by Side BRIGITTE M. JOCKUSCHa and HARALD JOCKUSCHb a

Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092 Braunschweig, Germany E-mail: [email protected] b Developmental Biology and Molecular Pathology, University of Bielefeld, D-33501 Bielefeld, Germany E-mail: [email protected], [email protected]

BMJ & HJ at the annual ball of the Technical University of Braunschweig, 1999

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B. M. JOCKUSCH AND H. JOCKUSCH

Abstract The authors describe their lives as a scientist couple, extending over more than 40 years. They worked in related, but not identical fields of molecular cell biology and biomedicine, in many different places, and raised two sons. Although this life demanded frequent compromises, they consider this as a gain rather than a loss and conclude that they were granted a privileged life. Keywords: Cell adhesion; Cytoskeleton; Histogenesis; Host cell–pathogen interaction; Mould; Mouse genetics; Neuromuscular diseases; Protein folding; RNA viruses; Tumor biology

Il moto e` causa d’ogni vita Motion is the cause of all life Leonardo da Vinci (1452–1519)

BMJ and HJ: About this Chapter Giorgio Semenza (editor of this series) and Rainer Jaenicke (former co-editor) invited us to contribute our personal recollections. We were more than hesitant, but Rainer Jaenicke eloquently convinced us to accept. We hope that we have captured the moods and the problems of (largely) the second half of the last century, a time of transition in many aspects of life – technologies of communication, the role of the universities, women in advanced scientific positions, to name just a few relevant for this chapter. We have usually worked on different scientific projects, but the foundation of a family was a joint enterprise, which meant that at all stations (and there were many) of our careers we had to aim for positions either at the same place or within a reasonable distance. Often our personal points of view on how to proceed were quite different and compromises had to be found. Regarding the presentation of our report, we followed the example of ´ra ńy (Vol. 41 of this series) and chose the Michael and Kate Ba form of a duet. Scientifically, we were not in the same boat as ´ra ńys but sailed alongside – by now for 44 years. were the Ba Another peculiarity, proposed by Rainer Jaenicke, is that we include some examples of drawings and paintings by one of

SAILING SIDE BY SIDE

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us (HJ). Again, this is not entirely new in the series, as there are most elegantly drawn caricatures by Kaj Linderstro¨m-Lang (Vol. 44 of the series).

HJ: Growing Up in the Orbit of Chemistry I was born in March 1939 in Frankfurt (Main) as the younger of two brothers and named Harald Helmut. My father, Helmut Jockusch, from Bielefeld, a town in Westphalia, had studied chemistry at several German universities; he later moved to Berlin where he carried out his PhD thesis with the physicochemist Max Bodenstein. At Berlin University he met his later wife, Gerda Seifert, daughter of a hydraulic and shipbuilding engineer, who studied biology and chemistry for the qualification of high school teacher. Before my later parents married, my mother had won a stipend for a college year in the United States. Supported by the German Academic Exchange Service (Deutscher Akademischer Austauschdienst, DAAD – still in existence) she spent the academic year 1928/1929 in Columbia, Missouri, and at Bryn Mawr College in Philadelphia. During this year she made a number of American friends for life. Some years later, my father got hold of a position as an assistant at the University of Marburg, with professor Meerwein. Salaries for these positions must have been extremely poor by present day standards. In these years, my elder brother was born. My father was looking for a position in industry, and got two offers: one from Leuna Bitterfeld in the industrial region of Saxony, the other from Farbwerke Hoechst in the west of Frankfurt. As I was told, the salary in Bitterfeld was higher, but my mother decided ¨chst because the environment was much nicer. for Frankfurt-Ho For my growing up this was a fundamental and fortunate decision: Bitterfeld ended up in the Russian occupation zone, and later in the German Democratic Republic, Frankfurt in Hessen was located in the American Zone, and later became the business and financial center (although not the capital) of the German Federal Replublic. My mother’s decision, then based on personal preferences, for the West and against the East, was irreversible at least from 1961 until 1989, from the erection of the wall to the reunification of Germany. We lived 5 miles north of the Farbwerke Hoechst (in the name of the company ‘‘oe’’ was used

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instead of ‘‘o¨’’) and depending on the weather we observed grey and yellow clouds of smoke leaving the chimneys, and often smelled the acrid by-products of the trade on which so many people in the region lived. From age 5 onwards I had two main interests: drawing and animals. Like many children, I was fascinated by the looks and behavior of insects, especially ants and beetles. I had the urge to draw what I saw around me and before my inner eye: leaves, beetles, fish, mice, and witches – but there were no neat white sheets of paper available to do so. My parents gave me all kinds of leftover papers, like ‘‘Feldpost’’ (letter sheets for army postal service) and Nazi proclamations, with some free space at the margins or on the backside (Figure 1). Early post-war years were times of overcrowded flats, due to the massive influx of refugees into the western parts of Germany. Among the 13 people living in our flat were my mother’s parents who had arrived from Berlin in the last months of the war after their living quarters in Berlin had been destroyed by bombs. Our small town, some 10 miles distant from Frankfurt city had only occasionally been hit by bombs, but after the war we were reduced to rather primitive standards of life, and had to grow our own potatoes and vegetables, and collect wood for heating. Like everybody else I was severely undernourished but appreciated the adventures of self-subsidy. And there were aftermaths of the war: ‘‘Bombensplitter,’’ remnants of bombshells, were collector’s items among children – as were fuses; my brother was nearly killed when investigating one at the age of 10. The ‘‘quality of life,’’ a concept not yet en vogue in those days, was so low that it could only improve. In the Frankfurt region the help of the Americans was greatly appreciated: Soldiers gave oranges and candy to the kids, official CARE packets with milk powder, instant soup (not yet known to us at that time) and other food were of great help, especially for families with kids. In addition, our family got ‘‘private CARE packets’’ from my mother’s old friends, from Boston and California. Eventually, life improved. My maternal grandfather was the earliest admirer of my drawings, especially of my cartoons. He died when I was 11 and left a little cardboard box in which he had collected my sketches and cartoons. There were not only positive experiences with my drawings. In the first or second year of elementary school, I was 7, we had to draw animals at home. The next day


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Fig. 1. Early drawings by HJ. Upper part: Plant galls (dog-rose, oak, beech) and witch, pencil, 1945. Lower part: Male staghorn beetle, India ink, 1951. By now staghorn beetles are nearly extinct.

I presented a drawing with three geese, and to my surprise the teacher scolded me and called me a swindler: ‘‘These drawings are not yours, they were done by one of your parents or by your elder brother.’’ Although I was usually close and shy at school, this massive insult in front of my class mates enraged me. I asked for a piece of chalk and chance to draw on the blackboard. Within 3 minutes I had the geese sketched out and abashed the teacher. A few years later, when I was 10 or 11, I had a similar experience with biology at high school, at age 11: The teacher

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asked how anemones survived the winter, and I answered: ‘‘by their rhizome.’’ The teacher got furious that I didn’t use the German word ‘‘Wurzelstock’’ (rootstock). It wasn’t my intention to show off with the scientific term ‘‘rhizome,’’ I had simply read this expression in a book on wild flowers that was on our bookshelf at home. At least at this school it was considered bad manners to break ranks. Such experiences made me cautious and I would not talk at school unsolicited about my private progress in drawing or knowledge of plant and animal life. This was discovered by my biology teacher and he would scold for ‘‘holding back’’ my knowledge. There was a piano in the family, a standard bourgeois piece of equipment, and when I was about 10, it was considered that I should learn to play, especially since my elder brother made already very good progress with this instrument. The piano teacher, testing my abilities, told my mother that I would probably be able to play an instrument, but she would have to teach a recalcitrant child. Thus I settled with my parents that drawing and painting would be good enough a cultural activity and I was exempt from piano playing. The big difference was, of course, that I could decide by myself when to paint and when not; but drawing and painting is a lonely job. From my childhood onwards, though, I discovered ‘‘my music,’’ that turned out to be the opus by Johann Sebastian Bach, Claude Debussy, Igor Strawinsky, and Carl Orff. I believe that many young children have a similar preference. Only later, in puberty, one discovers Mahler and Tschaikowski, and enjoys the emotional surges of their music. After 1948, when the worst times were over, my family resumed pre-war activities and traveled again for vacation. In the summer of 1951, we took an overnight train to Hamburg, and from there to the Island of Sylt, at the border to Denmark. Aged 12, strongly influenced by the English painter William Turner, whose paintings I had seen at an exhibition, I carried out water colors of birds in their natural environment, the open shore or ponds between the dunes. In contrast to many of my later paintings, these are still liked by many people. The scientists at the Marine Biological Station in List on the Island of Sylt – the place where I stayed more than half a century ago – assure me that these serene sceneries still exist in the northernmost part of the island (www.hal-jos.org/animals, plants, and their environments).


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BMJ: The Roots: Memories from an Uneventful Childhood I was born in Berlin, shortly after the beginning of World War II, as Brigitte Schenk, the first child of a civil servant working in the State Bank of Germany (Deutsche Reichsbank). At these times, civil servants had to move wherever their employer, the State, was sending them to. Hence, when I was still an infant, my family moved to Troppau, Czechoslovakia, because my father was sent there after the incorporation of this region into the ‘‘Reich.’’ Later on, my family moved to Marktredwitz, a small town in the region of Oberfranken, Bavaria. In 1942, my father was drafted to the army, and my mother stayed in Marktredwitz, tending to my younger brother and myself. As Oberfranken was a remote, rather rural area, we were not afflicted by war activities, until spring 1945, when many members of my parent’s families came to our place as refugees from the east (Silesia), and food became very scarce. In May 1945, World War II ended, and I, as a 5-year-old child, was confronted for the first time in my life with darkskinned people speaking a language unknown to me (American soldiers), offering fruit unknown to me (oranges and bananas). Although Oberfranken, like Frankfurt, was within the American Zone of Occupation, there was not much help in obtaining basic food from the American army, and we lived for several seasons from dried mushrooms and turnip leaves. It was only in 1946, when typhoid fever hit the community that there were also CARE packets directed to Marktredwitz. In September 1945, I started elementary school, but teaching lessons were scanty, due to lack of books, paper and, in winter, coal or wood for heating. In 1947, my father returned home, after he had spent some time as a prisoner of war of the British army. In 1949, the Republic of Germany had established a new State bank (Landeszentralbank), and my father was offered a position in Landshut, an attractive small town close to Munich in Bavaria. The medieval nucleus of the city was completely conserved, as, like Oberfranken, this part of rural Bavaria had not suffered from war calamities. However, when my family moved there, we realized that although the gothic skyline of Landshut was unharmed, its social peace was greatly upset. Thousands of refugees were flooding the town. The endogenous population, very conservative, Roman catholic and speaking a heavy Bavarian dialect was confronted with their demands for shelter and

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employment, with Protestantism and various dialects and idioms from the Eastern parts of the former German Reich. Although, strictly speaking, I was not a refugee child, I was placed in the same category as these displaced people: I was not of Bavarian extraction, not catholic, and I did not speak the local dialect. At these times, the conservative life style of rural Bavaria included rather old-fashioned ideas about female education. Thus, there was no admission of girls to the public high schools in Landshut. Consequently, my parents sent me to a private convent school, for girls only. This was run by nuns of the Cistercienser convent who offered their pupils an excellent education in languages and music, but only a selected and filtered approach to history, politics, and science. However, I was lucky in that during my later years at that school several young nuns appeared who had just finished their education in mathematics and physics at the University of Munich and were eager to transmit their knowledge to interested pupils. I was deeply impressed by the motivation of these persons, as well as by a music teacher who made it possible for me to try the harpsichord and even sometimes the organ in the magnificent 18th century convent church. There was one key experience which I never forget and which coined my musical taste for the rest of my life: when I was 11 years old, I was given the first sheet music by Johann Sebastian Bach. Outside school, my musical education was completed by piano lessons and by joining a small madrigal choir specializing in medieval and pre-classical music. Thus, when I finished high school in 1958, I was quite undecided where I wanted to turn to: music, in particular to a professional training in old key instruments like harpsichords and virginals, or to science, but for either topic, I wanted an academic education.

HJ: Too Many Years of Lessons and Lectures In the early post-war years I entered elementary school later, but finished high school younger than my classmates, on my 19th ¨chst, there was no coeducation birthday. In Frankfurt/Main-Ho but separate high schools for girls and for boys. The boys’ gymnasium was named after Gottfried Wilhelm Leibniz who had invented calculus at the same time as Isaac Newton. However, this gymnasium offered a balanced education in both, liberal arts


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and science. Due to change in education politics in the state of Hessen, for the last 3 years at high school, we had to choose between either a science and mathematics or a language branch. I decided for the former, but did not see the reason for this specialization – after all, my father was a chemist but could read classic Greek in the original, as typical for higher education in his generation. I was never particularly fond of school, but some teachers impressed me: A math and physics teacher who became lyrical when talking about the particle-wave dualism; a biology teacher who was an expert on crayfish but told us about the DNA double helix already in 1954; a music teacher who made us feel what music is about from Gregorian to (then) contemporary composers like Hindemith; a young protestant minister who had been commander of a submarine but very rarely talked about it. When the end of high school approached with the final high school exam, the ‘‘Abitur,’’ the choice of a professional training was due. When official advisors came to our school and asked about our future plans, I answered ‘‘I want to study biology.’’ My advisor said: ‘‘So you want to become a teacher?’’ – ‘‘No!’’ – ‘‘OK, then you will be unemployed.’’ This reflected the advisors’ state of information in 1958, while at the same time the chemical industry in Germany had already started to employ microbiologists. My final grades in math and the arts were better than those in biology, so why not become a physicist, an engineer, or even an architect or artist? I considered a profession in the graphic arts but figured that in order to earn my living, I would have to consent to people’s taste and not be free in my artistic production. Unfortunately, nobody told me that one could be quite successful in biology when trained in physics. Some colleagues of my father recommended that I should study ‘‘hard core chemistry.’’ However, finally I stuck to my original plan and studied biology. For financial reasons – tuition was free for inhabitants of the Federal State of Hesse – I enrolled at the University of my hometown Frankfurt. What did I face at the relatively young, then 44-year-old Johann-Wolfgang-Goethe University? On the whole, I was disillusioned. I had not been very fond of high school and was looking forward to an atmosphere of intellectual challenge, individuality and creativity at the university. These expectations were not fulfilled. There was a rigid schedule of lectures in

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mathematics, physics, chemistry, and biology; the latter was classical zoology and botany at that time. In between lectures, bells would ring in the hallways just as at school, and the smell, a concoction of floor wax and sweat, was not much different from that in the school building. Yet, lectures in mathematics, physics, and physicochemistry were at least entertaining. The lab course in inorganic analyses was held in the basement of the chemical institute: water pipes were running along the ceiling, and since they were cold, all kinds of aggressive vapors condensed on their surface and corroded the cast iron so that a rusty liquid was dripping from the ceiling. This was more or less the status at many German universities in post-war years: The ‘‘Wirtschaftswunder,’’ the economic miracle, had not yet reached the universities; however, at least in Frankfurt, the chemical industry supported the chemistry department with donations of materials like ether. In this and subsequent chemistry lab courses I got the definitive feeling that I lacked a most important property any chemist should have: patience. My interest in biology was mainly in agriculture, applied microbiology, and genetics but also in theoretical biology, and these were not taught to beginners. The only book I found to read on theoretical biology was by Ludwig von Bertalanffy, ‘‘Theoretische Biologie’’ from 1932. I liked the book because it elegantly bridged thermodynamics and – as far as I know – introduced or at least popularized the important concept of organisms as open systems far from the thermodynamic equilibrium. Of course, it did not cover molecular biology and this is probably the reason why it is forgotten by now. In later years of my studies, my favorite book was Erwin Schro¨dinger’s ‘‘What is life’’ which is still quite popular. In those years, 1958–1960, I was considerably distracted from my studies at the university because I still lived at my parents’ home – this was inexpensive and convenient – and had taken up to draw, paint, and do metal work. In the alluvial clay at the site of our family home I had discovered inclusions of gritty iron oxide, ranging in color from ochre yellow to dark red (called ‘‘Englisch Rot’’ by German Artists). Chemically, these were the same pigments as sold in artists’ supply stores. To these I added dark green chromium (III) oxide that I bought for a few Pfennigs at the store of the chemical institute (this extremely stable pigment is ¨lner Gru also called ‘‘Ko ¨ n’’ or ‘‘Adenauer’s Gru ¨ n,’’ because Konrad Adenauer, the Lord Mayor of Cologne (later the first


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chancellor of the Federal Republic of Germany), had all the Rhine bridges in Cologne painted with chromium green. While listening to Igor Stravinsky’s ‘‘Sacre du Printemps,’’ I carved a dancing man into a wooden board – the back panel of an old piece of furniture – and painted it in black, white (gypsum? titanium oxide?), and ochre, red, and green metal oxides [1]. In some paintings, I combined metal and paint on wooden boards. The sun in ‘‘Jacob and Esau’’ is of molten lead poured onto a charred board (www.hal-jos.org/paintings). Later during my stay at Frankfurt University, there was a tendency for modernization in teaching biology: a Chair of Microbiology was established in the Faculty and this attracted Hartmut Hoffmann-Berling to come for flying visits from the Max-Planck Institute for Medical Research in Heidelberg to report on the latest progress in the mechanisms of protein synthesis. In between his arrival and the start of his lecture he would slip into the department library and pick up the latest news from the journal issues on the shelves. His style of teaching was clearly very different from the routine lectures of most established professors. And there was another encounter that influenced my scientific education: due to my interest in physicochemistry and the personal relationship of my parents with the professor of physicochemistry and colloid chemist Joachim Stauff, I met his collaborator Rainer Jaenicke, 9 years older than me and thus young enough to give me advice on my studies. At this university, as in most German Universities of that time, there was no program for a diploma in biology and one had no serious examinations to take prior to the doctoral exam, unless one was enrolled in the program for high school teachers. During those and subsequent years I received the most influential practical training in industry, not at the university. I twice spent several weeks as a temporary worker in Farbwerke Hoechst. One summer, I worked in an outstation, involved in field and greenhouse experiments on fertilizers, insecticides, and the possible influence of factory emissions on lettuce and vegetables. My second job was in the laboratory for biochemistry and microbiology. Here, I had to carry out manometric measurements on cell respiration with the Warburg apparatus, to test the cell toxicity of newly synthesized compounds. This was certainly a good experience not only with regard to applied biology but also with respect to getting insight into the world of workmen. I was

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comforted to notice that the times were clearly over when industrial chemists would say, ‘‘Biologists are only good for the counting of dead flies.’’ To sum up: My intellectual progress during my formal school and university training could have been achieved in less time. For the generation of my grandchildren, this situation has improved: children will enter elementary school when 6 rather than 7 years old, and the final high school exam can now be taken after 12 rather than 13 years of total schooling. Furthermore, there is a strong tendency to tighten university studies.

BMJ: Education in Science I enrolled at the University of Munich for the winter term 1958/ 1959. My parents were surprised that her daughter aimed at an academic education, but did not object. My father, however, insisted that I should enrol in the program for high school teachers, to be on the safe side for future employment, as it seemed unthinkable to him at that time that I could ever succeed with a scientific career in industry or even head for a professorship at a university. According to the legal situation in 1958, I still was not of age, and thus I agreed to my father’s proposal. Consequently, I enrolled in three subjects: biology, chemistry, and geography, to qualify for a high school exam of the Bavarian Ministry of Culture. In addition, I tried to continue to study and practise music. However, when listening to the biology lectures in my first term at the University, I was so fascinated by this field that I decided to devote my time almost entirely to this topic, framed by the relevant chemistry and physics courses, and I put practising music, playing the harpsichord and singing in choirs, aside as a hobby. As of today, I believe that this was a wise decision, and given the chance, I would decide the same way. My first terms in Munich were governed by undergraduate courses in biology, chemistry, physics, geography, and geology. I still lived in Landshut with my parents, which meant that I had to get up at 5 o’clock in the morning, take a bus to the railway station, travel by train for about 1 hour to Munich, and walk to the lecture hall in the old Chemistry Institute of the University, to attend the classes starting at 8 o’clock, like Egon Wiberg’s ‘‘Inorganic Chemistry’’ or Rolf Huisgen’s ‘‘Organic chemistry.’’


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The lecture halls were situated in the Chemistry and Zoology Institutes close to the main railway station of Munich, both of them pre-war buildings that had still not been renovated. These auditoria were much too small for the number of students, so sometimes one was lucky even to find space to sit on the steps of the staircase. The rush for a seat was further complicated by the fact that the caretakers of the building opened the doors only 5 minutes before the lectures started. So, in winter, the students had to wait for some time in the cold outside. All these hardships notwithstanding, I enjoyed attending these lectures and cannot remember having missed a single one, but after four terms with such a life style, I came down with severe stomach problems. Hence, on medical advice, I moved from Landshut to Munich, but never turned into a fan of this city. Finding an affordable place to live was extremely difficult. Landladies were quite unfriendly, and as there was still no subway system in Munich, one spent many hours at drafty stations, waiting for street cars. There was a tuition to pay for attending classes as well as for taking exams, and we were to buy all the equipment needed for chemistry and biology courses. The laboratories destined for courses in analytical chemistry were a nightmare: too many students crammed into drafty rooms that were poorly or not heated in winter, with furniture composed of rotten wood, cheap plastic, and rusty ironware. I supplemented the small budget I received from my parents, which had to cover university fees, accommodation and living as well as the purchase of books, concert and theatre tickets, by various engagements to earn money. Thus, I sold fruit and groceries at shops, supervised small children in a vacation program of the city of Munich for underprivileged children, cleaned offices late at night, and tried my luck as a waitress. However, I never had to interrupt my studies for earning money. For future high school teachers, there were also compulsory seminars in pedagogies and philosophy, which I enrolled in and finished in due time by passing the relevant exams. As an advanced student, I also earned some money by assisting the professors in teaching lower grade students in biology courses like anatomy and genetics. I enjoyed all of these activities, which gave me glimpses of various aspects of life in the big city of Munich. However, none of them deviated me from my goal: to understand as many aspects of the living matter as ever possible. A lecture series on ‘‘general biology’’ by Hansjochem Autrum, head of the

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Zoological Institute of the University, wherein he described new insights in muscle structure and function as published by the MRC in Cambridge, UK, was instrumental in attracting me to this topic. At that time, the biology program for high school teachers focused on zoology and botany. The Botanical Institute and the Botanical Garden were quite remote from the city center, while zoology, chemistry, physics, and geology buildings were downtown. Hence, it was tedious to reach the botany classes, since, as I already mentioned, Munich still had no subway system. As it was also recommended for students to change to another University at least once during academic training, I decided to go to a different University for my compulsory training in botany. Based on a recommendation from one of the professors in the Munich Botanical Institute, I enrolled for the summer term 1962 at the University of Tu ¨ bingen. I was surprised to learn how easy life was in this small town as compared to Munich: everything concerning studies, living and evening activities was within walking distance, native people were friendly and used to students, and one got acquainted with the fellow students in very short time. It was in the botany course I had booked that I met a student from the Frankfurt region, Harald Jockusch, who seemed to share my taste in music, history, politics, and biology. This impression turned into certainty when we found the opportunity to talk to each other at several botany excursions in the lovely surroundings of ¨bische Alb’’ as well as at the ‘‘Federsee,’’ Tu ¨ bingen, at the ‘‘Schwa a small lake encircled by reeds and remnants of prehistoric settlements. Harald admired my painted toenails displayed in sandals and fed me the cherries sent to him by his mother, and I discovered that I liked his courtship. I was particularly attracted by his skills in drawing, although he sometimes used this gift to tease me with his own ‘‘improvements’’ of my clumsy sketches of the botanical specimens and tissue sections. In the last weeks of my stay, we got quite close. Hence, departing back to Munich was not so easy for me. However, as I had already invested much time and money in the program required of becoming a high school teacher in the State of Bavaria, I returned as planned. Back in Munich, I was attracted to the ongoing research in the Zoological Institute of the University, which primarily focused on insects. Karl Ritter von Frisch, emeritus since 1958, was still frequently present in the institute, and we, the students, were


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fascinated to watch him when he came in, swiftly walking, in his ‘‘Lederhosen’’ and equipped with an old-fashioned ear trumpet. The old bee hives that he had used for his pioneering work on the social behavior of bees (awarded with the Nobel Prize in 1973), were still there in the garden of the institute, and were used by one of his followers, Martin Lindauer. Shared interests in behavioral science was also the link to the close-by Max-Planck-Institute of Behavioural Biology in Seewiesen, where Konrad Lorenz (awarded the Nobel Prize together with von Frisch) acted as director. Sometimes, when we could find someone owning a car, we drove there to attend a seminar by one of their famous guest speakers. I still remember the first occasion I met Konrad Lorenz: he was late for some reason, and thus the seminar had not yet started. The speaker and the entire audience (with the students sitting in the back, on the floor) waited in silence for several minutes. Suddenly, the wooden panels in the wall behind me opened with a bang, and there was the famous host, entering the seminar room like the giant Leviathan, with ‘‘Lederhosen,’’ suspenders and a fuming pipe! Some of the scientists from this Max-Planck Institute also participated in teaching at the Zoology Institute, and in one of the lab courses, üs Eibl-Eibelsfeld frequently present, telling tales we had Irena about his adventures when studying gestures and facial mimics of natives in New Guinea or other exotic places in the South Pacific. Under the directorship of Hansjochem Autrum, the Zoological Institute concentrated on two major research topics. One was behavior of insects and of small mammals like tupaias. The second was the physiology of vision. Hansjochem Autrum and his group were pioneers in analyzing the compound eyes of flies and locusts with electrophysiological methods, and there were other groups in the Institute investigating the vision of squid and octopus. I was inclined to learn more about insects. Consequently, I wrote the small thesis required for the school teacher exam on the feeding behavior of the larvae of hover flies. I finished the written and oral exams for biology and chemistry in 1963, and the ones required in geography (including geology) in 1964. Thus, I could have left the university then and get employed as a high school teacher anywhere in Bavaria. However, life as a schoolmistress did not appeal to me, and so I decided otherwise.

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HJ: Everybody Should have been to Tu ¨ bingen By 1960 I decided I should move to a ‘‘real university town’’ – Frankfurt is a city of trades – and preferably one in which biology professors had a good reputation. Also, the presence of MaxPlanck Institutes (MPIs) with well renowned research in modern fields of biology was an important aspect. Since I was a bit tired of the big city of Frankfurt, my choice was Tu ¨ bingen, a town with a nearly 500-years-old university tradition. I liked Tu ¨ bingen from the beginning because, in contrast to the situation in Frankfurt (at least for the students in science), there was a lively student life around the university, and one made personal contacts most easily. An important argument for the University of Tu ¨ bingen was the reputation of plant physiology there. With this in mind I attended courses in botany but I was disappointed that Erwin Bu ¨ nning, professor of plant physiology and internationally renowned for his work on the biological clock, did not personally show up in the laboratory course. Of course, it was not unusual that a professor left the practical teaching entirely to his collaborators even if his name was linked to this course in the university calendar. What I found out later, too late for me: those who wanted personal contacts with Erwin Bu ¨ nning had to participate in his famous excursions to Lappland! Karl Grell, professor of zoology, an expert protozoologist, also taught genetics, whereas microbiology was treated as a minor subject of botany. I had come to Tu ¨ bingen to learn more about plant and animal physiology, and perhaps some virology and molecular genetics which I knew were the research subjects of the Max-Planck Institutes for Biology (halfway up the hill) and Virology (on top of the hill). At the University of Tu ¨ bingen, a group of bright students were convinced that by spending too much time with the old fashioned aspects of biology which, like in Frankfurt, dominated the curricula of the University of Tu ¨ bingen, they might miss the exciting developments in modern cell and molecular biology. Independent of any professorial supervision, they founded their own journal club, with meetings in the attic of the Botanical Institute, a century old building in the Botanical Garden. I was encouraged to join. At late hours, we discussed the most exciting news in physiology and cell biology. What we could read in


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‘‘Scientific American’’ had not yet made its way into German university lecture hall. The ‘‘founding members’’ of this group proposed to produce a German translation of ‘‘Scientific American’’ and to sell it to fellow students. Of course, as a business idea of a few students without a penny of seed capital this was fairly naı¨ve, but many years later the German equivalent of ‘‘Scientific American’’ appeared, titled ‘‘Spektrum der Wissenschaft.’’ In January 1962, while carrying out an experiment in the physiology lab, I got a telegram from my mother, informing me that my father had suddenly died, at age 56. He had suffered from tuberculosis and its sequels since he was a young man, but his untimely death was totally unexpected and dampened my mood for several months. At the same time this loss made me realize that I was finally grown up and totally responsible for my further career – if there was any. My father’s employer, Farbwerke Hoechst, generously sponsored my further studies until the end of my doctoral thesis. I continued my training in organic and physical chemistry, and was interested in too many different fields of biology. But, like in Frankfurt, the botany courses were in no way exciting, so my attention was drawn to a female student from Munich, Brigitte Schenk, who spent the summer term of 1962 in Tu ¨ bingen, as she had been told in Munich that Tu ¨ bingen was a hot spot for modern plant biology (which is certainly true nowadays). By the standards of Tu ¨ bingen biology students, this graceful girl was of lady-like, urban elegance. Her appearance certainly helped to lighten up my mood and I started to tease her – and finally got to know her quite well. But then she had to return to Munich to take up her thesis work at the Zoological Institute of the University. While I was a student at Tu ¨ bingen I participated in two excursions to the seaside. The first, late in September in 1962, took us to the Mediterranean Sea. Under the supervision of Professor Grell, we worked in the marine biology station at Ville Franche sur Mer, close to the Spanish border. In a most relaxed atmosphere we studied marine protozoa, a field Karl Grell was an expert in, and invertebrate metazoa. The second marine biology excursion took place in early spring 1963 to the island of Helgoland in the German North Sea, to study algae. Although Brigitte Schenk was back at Munich at this time, I arranged that she was listed as a Tu ¨ bingen student and could come along. It turned out that she had been several times to the Mediterranean,

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but never north of Frankfurt. As a scientific experience and an advertisement for the German north, this excursion was certainly no success. The winter had been exceptionally cold with ice on the North Sea, and the ice sheets in the surf had scraped off all the kelps and algae from the Helgoland rock shore – so their reproduction, the main topic of the course, could not be studied. In addition, there was permanent fog during the entire stay and the foghorn was hooting all the time in a nerve-wrecking volume and frequency. In this spooky setting, Brigitte felt extremely uncomfortable – even scared. For the following decades, she turned down all my invitations to join me in the student excursions to the Marine Station of Helgoland, which I, together with my collaborator Peter Heimann, arranged every second year from Bielefeld. These excursions, however, dealt with the reproduction of marine invertebrate animals and were therefore performed later in the year, in June or July. During these 20 years we witnessed the spectacular development of a gannet colony on the rock island of Helgoland and observed the huge grey seals closer and closer, in addition to the large numbers of harbor seals, on the sandy beach of the adjacent ‘‘dune island.’’ I became emotionally attached to the marine life of Helgoland, and for several years was a member of the board evaluating the research and teaching activities of the Marine Biology Stations on the islands of Sylt and Helgoland, which had become outstations of the Alfred-Wegener-Institute for Polar and Marine Research at Bremerhaven, on the mainland. In the 1960s, Tu ¨ bingen University modernized its program in what would now be called ‘‘life sciences.’’ Chairs of microbiology and genetics were established, and a curriculum of biochemistry was developed – the first at a German university. The curriculum in biochemistry, with the possibility to obtain a diploma on this subject would have been attractive to me. But I felt it was too late for me to make this turn, because I planned to start my thesis work as soon as possible. Like in Frankfurt, a diploma exam was not required for becoming a PhD student. Already towards the end of my Frankfurt studies, I had heard lectures by Alfred Gierer, from the Max-Planck Institute of Virus Research, and by Georg Melchers, director at the Max-Planck Institute for Biology, both at Tu ¨ bingen. They were guest lecturers of the Institute of Microbiology, as their topic was the genetics of tobacco mosaic virus (TMV), a field necessarily transforming into molecular


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genetics. These two scientists were extremely different in their appearance and style! Melchers tall, with a reddish face and white hair, presenting the first steps towards molecular plant pathology in colorful slides and baroque sentences with a loud voice. Gierer, in the habitus of a shy student with a soft voice, introduced the genome of TMV with a straight line of chalk on the blackboard and from this modest drawing developed the mutagenesis experiments on TMV. I had liked both presentations, and they greatly influenced my thoughts towards a choice for my doctoral thesis. However, I decided to participate in the seminars of the Max-Planck Institutes, before deciding on a thesis project. There was a seminar on the latest developments in virus research, with Gerhard Schramm and Hans Friedrich-Freksa representing the elder generation, and Alfred Gierer, Walter Vielmetter, and Heinz Schuster as rising stars. The hot topics were nucleic acid chemistry and bacteriophage genetics as routes towards the understanding of the molecular nature of the gene, gene regulation, and the genetic code. Schramm usually argued as a hardcore chemist. It was reported that he had commented on Jacob and Monod’s Lac repressor ‘‘Before I don’t have it in a bottle, I don’t believe it.’’ The animal virus group headed by ¨fer did not participate regularly in this seminar. Werner Scha But there were other offers at the ‘‘Max Planck Hill’’: In the newly founded MPI for Biological Cybernetics I participated in a ´ had joined Werner Reichardt’s ¨ Varju student seminar. Desco group after some time with Max Delbru ¨ ck and through him I learnt about Delbru ¨ ck’s experiments on the light perception of the mould Phycomyces. As always, my problem was that all of that interested me: plant pathology, molecular genetics, and, lately, biological cybernetics. In addition, I had slipped by chance into a then rather new field: science journalism. In 1960 or so I had written to the science editor, a chemist named Kurt Rudzinski, of the Frankfurter Allgemeine Zeitung (FAZ) and complained that this highly respected newspaper had published several articles on medical subjects that were studded with mistakes whenever topics of molecular genetics were involved. Rudzinski’s reaction was: ‘‘Why don’t you write these articles for us?’’ Thus, during my later years as a student, I earned some additional money by writing for the FAZ and later for the weekly newspaper DIE ZEIT, where the mathematician Thomas von Randow was acting as a science editor. At that time,

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it was still quite unusual for German newspapers to reserve one entire page per week for science reports. To my fellow students I explained the deal between the editor and contributor: for ‘‘DNS’’ (DNA) one gets 15 Pfennigs (less than 5 pence at that time), for üre’’, one Deutschmark (then a quarter ‘‘Desoxyribonucleinsa dollar), because the word fills a whole newspaper line. My most satisfactory experience as a moonlighting science journalist was the participation in the XIth International Congress of Genetics in The Hague, Netherlands, in 1963. It was an exciting congress, because of the latest news on the genetic code, and I wrote a long article reporting on it for DIE ZEIT, the honorarium for which was enough to cover my (modest) travel expenses.

HJ: Ending Halfway up the Hill Although I had heard many exciting news from the fields of molecular genetics and biological cybernetics, and received offers to do my thesis in these fields, I did not end up on top of the hill where the MPIs for Virus Research and for Biological Cybernetics were located, but halfway up, at the MPI for Biology, Abteilung Melchers, because I stubbornly stuck to my original plan to work in plant pathology. I had gathered all my courage and visited Professor Melchers, then 56, and asked him whether I could become a PhD student, ‘‘Doktorand,’’ at his institute. He answered: ‘‘‘Doctorandus’ in Latin designates somebody who may or will receive a doctoral degree. In this Institute there are no ‘Doktoranden,’ because nobody can say, let alone promise, whether a given person will receive a doctoral degree after 3 years work or so. Apart from that, we have very little space, but you may work here. It is best that you first talk to my collaborators, Drs Mundry and Wittmann.’’ Mundry, who had just returned from the California Institute of Technology, was very eloquent; Wittmann appeared as the hard worker who didn’t care for long discussions. I decided, I would work all by myself, as I already had a plan what to investigate: The genetics and biochemistry of virus–host interactions in the TMV-tobacco system. My plan was based on some findings by a Hungarian group of phytopathologists that the enzyme polyphenol oxidase or catecholase was induced in the course of host defence against TMV (hypersensitive reaction), mounted by certain tobacco cultivars. I reasoned


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that the genome of TMV must be simple because of its small size (6 kb of single-stranded RNA), and a single Mendelian gene, designated N (from ‘‘necrosis,’’ small patch of dead leaf tissue) was responsible for the hypersensitive reaction of the host. ‘‘Now, this is certainly not the hottest issue’’ Melchers commented on my proposal – the hottest issue then was the genetic code worked on by Heinz–Gu ¨ nter Wittmann, who, together with his wife Brigitte Wittmann-Liebold, analyzed the amino acid replacements in coat proteins of nitric acid induced TMV mutants. Finally, Melchers declared ‘‘You may work on your chosen topic in this institute, but prior to that you should get some professional training in biochemistry; why don’t you enrol for a lab course in Feodor Lynen’s institute in Munich.’’ ‘‘Oh no’’ I thought ‘‘still not finished with being trained.’’ But then, thanks to help from Brigitte Schenk who was busy preparing for her teacher’s exams in Munich, I managed to enrol at Munich University, and to find a place to live in Munich for the summer term 1963.

BMJ: A Munich-Tu ¨ bingen Connection The ties to Harald Jockusch in Tu ¨ bingen had never ceded. Soon after my return to Munich, there were letters, drawings, small presents, and eventually we were traveling between both cities. We exchanged opinions on how to continue our studies, but also soon on how to share more than that. So, I was pleased when Harald decided to spend one term in Munich to take a lab course in the Biochemistry Institute of Feodor Lynen (I booked the same course two terms later). In addition, I took up classical genetics, and although I found (and still find) this topic also quite interesting, I have to admit that my interest was strongly enforced by the knowledge that Harald was specializing in this field, working on a PhD thesis in the Max-Planck Institute for Biology. He owned a very old, but sufficiently reliable VW beetle ¨bische Alb,’’ the mountain with which he crossed the ‘‘Schwa range between Tu ¨ bingen and Munich. I traveled by train, whenever time and money permitted, and I can still to this day recite the many stops in the small towns lining the railroad track: Geislingen, Plochingen, Go¨ppingen, Metzingen, Reutlingen and, finally, Tu ¨ bingen.

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It was on one of those weekend visits to Tu ¨ bingen that I encountered his mentor and supervisor, Georg Melchers. Harald met me at the station downtown Tu ¨ bingen, and on the way uphill to the Max-Planck Institute, he informed me that prior to enjoying the weekend, I might help him to inoculate tobacco plants in the greenhouse with TMV. This was not the first time he asked for my assistance in this task, so I did not argue further but agreed. As it was a Saturday afternoon, there were no other persons in the greenhouse. Harald told me to wait for him there while he was looking for some tools in one of the service rooms close by. Suddenly, while I was inspecting the plants, both wings of the door flung wide open and there was Georg Melchers, more than 6 feet tall, in a white coat, his formidable jaw pointing rather menacingly towards me. The following remarks, prompted by my shy answers to his pressing questions, are still vividly present in my memories: ‘‘Who are you? A friend of Harald Jockusch? Congratulations, you picked a good one. Where do you come from? Munich? What a horrible city. What is your profession? You are a PhD student, working in a zoological institute on insects? Well, I hope you do not contaminate our tobacco plants with bugs!’’ Even this encounter did not squash my interest in Harald and the scientific atmosphere in Tu ¨ bingen, with its many different Max-Planck Institutes. Harald was awarded his PhD degree from the University of Tu ¨ bingen at the end of 1966, and we married in spring 1967, quite traditionally in Landshut, my home town.

HJ: In the Dungeons: From Phytopathology to Protein Stability I started my thesis work in the fall of 1963. I shared a tiny laboratory in the basement of the Max-Planck Institute with Lieselotte Rentschler, who worked with Heinz-Gu ¨ nter Wittmann on amino acid sequences of the coat proteins of ‘‘wild strains’’ of TMV. Our shared bench was about 1.2 meters in length and so high that Lieselotte had to use a stool when vacuum drying her tryptic peptides. Like in the chemistry lab at Frankfurt University there were all kinds of pipes running under the ceiling and in wintertime the room was moist and cold. Georg Melchers, who was very eager to head not only a productive but also a neat and beautiful institute – surrounded by blossoming


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roses all summer long – was aware of the ugliness of our working place, and correspondingly apologetic. We did not mind and appreciated to work in a forgotten corner with few distractions and disturbances. I started my work on polyphenoloxidase (PPO) and confirmed the dramatic increase of its activity accompanying the hypersensitive reaction (HR) in certain TMV-tobacco combinations – but I found a similar increase in leaves after cropping the stem of an uninfected plant; thus PPO induction appeared as an unspecific reaction to any insult to the plant, not as a specific answer to virus infection [2]. The tobacco cultivar that reacts hypersensitive to all TMV strains carries the dominant gene N, tobacco cultivars that lack a hypersensitivity gene allow unrestricted spreading of the TMV infection and are used for virus multiplication. In the older literature, the HR was reported to be temperature sensitive in reversible fashion (an effect used by the group of Barbara Baker at Berkeley to clone the N gene [3]); and dependent on gene dosage (see Ref. [2]. If one elevates the incubation temperature from 201C to 301C, the lesions become larger and blurred and finally the infection becomes systemic as in the absence of N. As the host defence breaks down, the production of virus increases steeply. The extractable infectious virus per lesion can thus be used as an inverse measure of the efficiency of the HR [2]. I realized that the gene dosage and temperature dependence of the HR might provide a handle to get at the ‘‘Pudels Kern,’’ the gist of the matter, of the HR. When I applied this test to the combination of TMV wild strains with tobacco homozygous for the N gene, the yield of infectivity per excised lesion steeply increased between 261C and 291C and, expectedly, a lot more infectivity was extractable after incubation at high than at low temperature. A nitrous acid mutant, Ni118, showed similar symptoms as the wild type virus with increased temperature, but the extractable infectivity dropped to near zero around 301C. Thus, although the symptoms indicated that the TMV infection had spread in the leaf, practically no virus particles had been produced [2]. Obviously, a temperature sensitivity of the virus mutant Ni118 HR was superimposed on the temperaturesensitivity of the plant defence mechanism (Figure 2). This conclusion was confirmed by repeating the temperature experiment on a tobacco cultivar that did not carry a hypersensitivity gene – the local lesion counts of this decisive experiment were

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Fig. 2. The discovery of temperature sensitivity in a TMV mutant. Breakdown of tobacco host defence with increasing temperature, resulting in increased yield of TMV per local lesion in wild strains vulgare (circles) and Mild (squares), but not with mutant Ni118 (triangles), despite similar symptoms (triangles and circles were exchanged in the published legend). From Ref. [2], with permission of the copyright owner, Wiley-Blackwell.

actually carried out by my colleague Lieselotte Rentschler, while I was in Frankfurt visiting my mother. At that time, conditional lethal mutants of T4 phage were a topic in the journal club of the Virus Research Institute. One class of conditional lethals, the temperature-sensitive (ts) mutants had become an important tool to define gene functions in phage


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morphogenesis [4]. Apparently, I was the first to observe temperature sensitivity in a plant virus mutant. I decided to abandon my original thesis subject, the biochemical mechanism of HR, and started to work on temperature sensitivity of TMV mutants. My results, obtained under controlled laboratory conditions, explained the variable yields of some long known TMV mutants in the green house as a trivial consequence of the variable weather conditions. Georg Melchers, always interested in, and excited about, differences between TMV strains or mutants, urged me to test more and more mutants whereas I would have been satisfied with analyzing the principles of temperature sensitivity with just two or three. The nitrous acid induced mutants had been produced to get information on the nature of codon triplets, via amino acid replacements detected in their coat proteins. This was ongoing work by Heinz-Gu ¨ nter Wittmann, his wife, Brigitte Wittmann-Liebold, and their collaborators. Thus, a number of amino acid replacements, which, according to my analysis, rendered the virus mutant ts, had not yet been published. Since other (then unknown) genes of induced TMV mutants might have been mutagenized as well, it became necessary to show whether the temperature sensitivity of ts TMV mutants in the plant cell, in vivo, was due to a destabilized coat protein. Thus I set out to test coat proteins of tr and ts TMV mutants by an in vitro assay. The function of TMV coat protein is, in a self-assembly process, to incorporate its RNA into the wall of a hollow cylinder with a helical arrangement of protein subunits (see contribution by M. Lauffer in Vol. 41 of this series). This function can be simulated, in the absence of RNA, by lowering the pH, for example from 7 to 5: the formation of ordered cylindrical (but not helical), soluble aggregates causes the clear solution to become opalescent. Opalescence, analytical ultracentrifugation and electron microscopy indicate whether the coat protein is functional. I carried out a first experiment with alkali-dissociated wild type and Ni118 TMV by dialysis from pH 7 to pH 5, in parallel at 201C and at 301C. The result was evident to the naked eye: whereas the other solutions became opalescent, the protein of Ni118 at 301C had turned into an ugly white curdle. According to this in vitro assay, nearly all of the ts mutants turned out to have a ts coat protein. Since TMV coat protein can be prepared in large quantities and – using free-flow electrophoresis to get rid of the RNA fragments – at high purity, the further subject of my thesis

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focused on protein denaturation. In fall of 1964, I sketched out a short communication on my findings; it was to describe the first case in which temperature sensitivity was related to known amino acid replacements: ‘‘In vivo und in vitro Verhalten temperatursensitiver Mutanten des Tabakmosaikvirus’’ (in vivo and in vitro behavior of temperature sensitive mutants of TMV). In the draft, I had proposed myself and H.-G. Wittmann as the authors. For reasons that were not clear to me, Melchers proposed I should be the sole author. The short communication appeared (in German) late in 1964, in ‘‘Zeitschrift fu ¨ r Vererbungslehre’’ [5], the oldest genetics journal of the world, with a name that was unknown to, and unpronounceable for, most scientists abroad. Protein denaturation at that time was not a big issue in lectures and textbooks, and there was nobody in the institute who knew much about it. However, I read helpful articles by Kauzmann and bought the newly (1965) published book A Physicochemical Approach to the Denaturation of Proteins, by M. Joly [6]. Browsing through the older literature I found Kunitz’ classical kinetic studies on the thermal denaturation of the trypsin inhibitor (Kunitz 1947, cited in Ref. [6]). Based on my readings I designed a kinetic assay for the denaturation of mutant TMV coat proteins. The experiments took place in the cut off lower half of a demolished plastic bucket, connected to a suction pumping thermostat. The small protein quantities, withdrawn at different times and centrifuged, were measured, after alkaline hydrolysis, by the sensitive ninhydrin reaction [7]. Ninhydrin reagent was always available in large amounts in the Wittmann laboratory. To determine protein concentration (after hydrolysis) with the sensitive ninhydrin reagent has been rediscovered in recent years, as has the usefulness of free-flow electrophoresis. During my thesis work I continued to get trained in various seminars and journal clubs; with only three (later four) PhD students, there was no journal club in the Melchers department. Wolfhard Weidel, director of the sister department within the MPI for Biology, was a leading scientist in the field of structure and synthesis of the bacterial cell wall and author of the first German popular book on modern biology Virus oder die Geschichte vom geborgten Leben (Virus or the Story of Borrowed Life). He was a brilliant writer and a merciless critic in his journal club, in which two PhD students of Melchers, Lieselotte Rentschler, and myself were guests. All modern developments


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of microbiology and molecular genetics were discussed, and there was an intellectual challenge that I appreciated. Because I admired Weidel, the following experience got stuck in my memory. We were discussing the enzymatic mechanism of DNA replication. I saw the replication fork and the asymmetric enzyme molecule in front of my inner eye and dared (though only a guest of the seminar) the remark: ‘‘If replication was catalyzed by only one enzyme with one specificity, then one strand must be synthesized discontinuously, in fragments and later linked.’’ Weidel just grunted ‘‘Nonsense’’ – of course, some years later, the Okazaki fragments were experimentally demonstrated and became textbook knowledge. Lieselotte Rentschler and I were shocked when Weidel unexpectedly died at age 47, in 1964. It was probably in the same year that one day the lab door opened and Georg Melchers appeared with a guest. He said to this gentleman: ‘‘This is Harald Jockusch. I will leave you here and he will explain to you what he is doing.’’ The visitor was Max Delbru ¨ ck. We had some problems in communicating, perhaps because the story of the temperature sensitive TMV coat protein was just too simple. A debut during my PhD time was the active participation in the 2nd International Biophysics Congress in Vienna, September 1966. I presented a short communication on the destabilization of TMV coat proteins by single amino acid replacements. This was my first performance at a congress, and Aaron Klug, then best known for his work on the X-ray analysis of TMV structure, was my chairman. He was very polite and friendly but didn’t seem very interested in my topic. In 1966 Georg Melchers became 60. There was a celebration in the Max-Planck Haus, the guesthouse of the MPIs on top of the hill. I think it was on that occasion that I delivered a short talk standing on my head. Heinz-Gu ¨ nter Wittmann asked: ‘‘Why is Jockusch standing on his head?’’ – Dadaism was not his cup of tea. I presented a slide, drawn directly with India ink on a tiny piece of cellophane ‘‘The vultures feeding on the remnants of TMV’’ – the vultures were, of course, the TMV researchers (Figure 3). The implicit prediction ‘‘TMV is dead’’ turned out to be wrong: Research on plant host defence (my first research topic) and on cell-to-cell movement of TMV turned out to be interesting topics in the years to follow. More than 30 years later, I myself took up TMV research again.

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Fig. 3.


Vignette: Vultures picking on carcass of TMV (HJ, 1966).

In 1966 I took my oral doctoral exam, in the classical form of a rigorosum, that is an examination in three subjects, in my case genetics (major), microbiology, and organic chemistry, with no relation to my thesis work. I had pressed Melchers for his consent that I could finish these last steps required for my PhD degree in 1966, and he finally agreed to the date: December 22, just before Christmas. This was my first academic exam and I passed with the best grade, perhaps due to the Christmas mood of the examiners. Later, absolutely nobody cared for that grade – as a postdoc I wasn’t even asked for any document of my doctoral degree when I was employed by the University of Wisconsin at Madison. As usual in those days, there was a big celebration of my exam, that started with me, dressed in a black suit, being picked up at the old University main building, downtown Tu ¨ bingen, and brought, enclosed in a huge ‘‘dialysis bag’’ (a plastic bag as used by dry cleaners) uphill to the Max-Planck Institute. For the night I had prepared a party based on the Brecht/Weill ‘‘Dreigroschenoper’’ (‘‘Three penny opera’’) – Melchers appeared as police inspector Brown with a bowler hat! Improvised and organized parties – like the famous carnival parties, arranged by the MPI for Virus Research, were typical for the academic life in Tu ¨ bingen at that time. After a miraculously short time period, my thesis appeared in print, again in ‘‘Zeitschrift fu ¨ r Vererbungslehre,’’ again in


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German, in two parts with a total of 43 pages [8,9]. What I didn’t know at that time: this documentation of 3 years of successful research was not only at a disadvantage by being published in German, but also had appeared in the last issue of the very last volume of the journal under its German name. From 1967 onwards the name was changed to Molecular and General Genetics (MGG, now Molecular Genetics and Genomics) and the oldest genetics journal was doomed to disappear in oblivion, in the outermost corner of the libraries, under the letter ‘‘Z,’’ far from ‘‘M.’’ And even worse, this last issue never made it to ‘‘Pubmed’’ and therefore the four papers (two of which were mine) in that issue disappeared in a black hole, at least for the search machines. Fortunately, a preliminary version of my findings had appeared in the September issue of Biophysical and Biochemical Research Communications (BBRC), [7] and had received an impressive response in terms of ‘‘reprint requests’’ – I suspect because ‘‘TMV’’ was not in the title, only ‘‘mutant proteins.’’ In those times, one received reprint requests on postcards with very pretty stamps, especially from Eastern European countries, and one made friends by giving these to collectors among technicians or people in the workshop of the institute. During this time, I revived my relations to Rainer Jaenicke in Frankfurt to carry out measurements on reversible denaturation of TMV proteins in solution using circular dichroism, a technique not available at the MPIs at Tu ¨ bingen. Rainer Jaenicke was (and still is) always in a hurry. In his lab he would suddenly excuse himself, kick the pipette drawer closed with his knee, grab his flute and rush to a concert to play his part. From these days of collaboration, a life-long friendship resulted. With Heinz-Gu ¨ nter Wittmann I collaborated on a nitrous acid mutagenesis experiment, to isolate TMV mutants temperature sensitive in a non-coat protein gene. In contrast to those of coat protein mutants the lesions of mutants affected in other genes were expected not to expand at non-permissive temperature. One such non-coat protein or class II mutant, designated Ni2519, was further characterized. The coat protein was thermostable and no amino acid exchange was detected. At non-permissive temperature no infectivity spread in the leaf tissue of either the necrotic or the systemic host [10,11]. Seventeen years after the isolation of TMV Ni2519, Jo Butler and Tony Hunter at the MRC Cambridge reported mutations of Ni2519 based on RNA sequencing: one,

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as predicted, in the gene for the movement protein, the other, unexpected, affecting the thermostability of the RNA secondary structure responsible for the assembly of the virus particle. The bacteriophage work on conditional mutants had influenced me continuously, because of its conclusiveness and elegance [4]. A disadvantage of TMV as compared to bacteriophages is its extremely low plating efficiency: less than 104 of the virus particles give rise to a local lesion, whereas nearly a 100% of phage particles produce a plaque. With sufficient dilution of the virus, however, a local lesion like a phage plaque represents a virus clone and can be used for the isolation of mutants. Because of the low plating efficiency of TMV and the fact that only the epidermal layer of the multilayered leaf tissue is infected during inoculation, most cells are infected secondarily, after spreading of the virus through cytoplasmatic connections between cells, the plasmodesmata. Thus I thought it would not be worth to embark on complementation assays that had been so important to define the more than hundred genes of T4 phage. Later, a Russian group showed complementation between a TMV coat protein mutant and Ni2519, the mutant affected in the movement protein. On the whole, the project on temperature-sensitive TMV mutants gave me great satisfaction. It had developed from a chance observation into a discovery. A simple in vitro experiment, involving just one protein in an aqueous buffer, explained what was going on in vivo, in the living cell, in an environment crammed with thousands of proteins and other macromolecules. The project had subsequently involved the use of a wide range of then up-to-date techniques, like electron microscopy, analytical ultracentrifugation, kinetics, and equilibrium measurements using CD. It gave me the opportunity for theoretical considerations based on activation energies (deduced from the temperature dependence of denaturation rates), roles of side chain hydrophobicities and charges, and of the change in rotational freedom when proline residues had been replaced. Furthermore, the importance of these effects in relation to the position of the amino acid replacement within the 158 amino acid polypeptide chain became evident and could be related to what was then known about the folding of the coat protein subunit [9,11,12] (Figure 4). One of the differences between the Melchers lab halfway up the hill and the Virus Research Institute on top of the hill was


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Fig. 4. The mechanism of temperature sensitivity in TMV mutants. Upper part: The polypeptide sequence of TMV vulgare (standard wild type) coat protein (CP) is shown as a horizontal line. Numbers give amino acid positions (total length 158 residues).The vertical axis indicates temperatures between 251C and 401C, showing the lowered denaturation temperatures resulting from the amino acid replacements indicated. From Refs [11,12], with permission of the copyright owner Springer, Heidelberg. Center part: Temperature resistant (tr) CP of TMV vulgare (wild type); from left to right: Amino acid positions 19, aspartic acid, and 20, proline; rods resulting from reaggregating CP at 201C and 301C (electron microscopy after negative staining). Lower part: Temperature sensitive (ts) CP of TMV mutant Ni118; amino acid replacement proline to leucine at position 20 (P20L); result of aggregation experiments at 201C and 301C, leading to amorphous aggregates at 301C (cf. Ref. [9]); diameter of rod shaped aggregates: 18 nm; ball-and-stick models of amino acid residues illustrate the increase in degrees of rotational freedom by the P20L replacement. Layout of this figure from Ref. [197], with permission of the copyright holder, Wiley-VCH.

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that we used mostly physicochemical and standard chemical techniques, like analytical ultracentrifugation, Tiselius electrophoresis, peptide analysis, etc. whereas uphill, radiotracer experiments were routinely performed, to study the synthesis of nucleic acids and proteins in virus infected cells. By 1967 Heinrich Matthaei had returned from the US: Together with Marshall Nirenberg he had performed in vitro synthesis of polypeptides using synthetic polyribonucleotides and thus cracked the first code words – a most exciting finding at that time. Matthaei was appointed director at the MPI for Experimental Medicine at Go¨ttingen. His department now offered a lab course on cell-free protein synthesis and I successfully applied for participation.

BMJ: From Ant Lions to Pond Snails After having finished my academic education as a high school teacher with the ‘‘Staatsexamen’’ in biology, chemistry, and geography, I continued with a doctoral thesis at the Zoology Institute of the University of Munich. I was still determined to work on insect behavior and thus asked Werner Jacobs, professor for systematics in the institute, for a PhD project. His proposal involved studying the behavior of ant lions, the larvae of a dragon-fly like insect. The approximately 1 cm long ant lions are equipped with a pair of needle sharp jaws and very conspicuous, black eyes. They build small funnels in sandy soil, bury themselves at the bottom and wait for passing ants to cross the rim of the funnel. Then, they throw sand at them, so the victims tumble down to the center of the funnel and are caught and eaten by this ferocious predator. The PhD project discussed with Werner Jacobs involved to find out how ant lions oriented themselves towards light, when sitting in the center of their traps. The ant lions I examined were collected all over Germany, and even Harald took pity on me and provided some from the vicinity of Tu ¨ bingen. After having watched the animals I kept captive in small vessels in the laboratory for a few weeks, I came to the conclusion that they do not care for the direction of the light. Hence, I changed gears, and contacted the groups in the same institute who studied vision using electrophysiology. I got invaluable help from the groups of Dietrich Burkardt and


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Kurt Hamdorf, borrowed some outdated but still useful equipment from them and started to examine the ability of the ant lion eyes (ocelli) in light perception and color discrimination. Thus, I abandoned the studies of insect behavior but still remained in the Zoological Institute of Hansjochem Autrum to work on a PhD thesis. To my surprise, I found that the eyes of ant lions, morphologically rather simple structures and much less sophisticated than the compound eyes of the adult insects, were quite competent in color perception. To complement my scientific education, I attended a lab course in electrophysiology headed by Dietrich Burkhard, and lectures in physics which I enjoyed and which were very beneficial for my thesis work now involving electrophysiology. In addition, I planned on taking genetics and biochemistry for minors, and thus took lectures in the genetics department which was also located in the Zoological Institute, and in biochemistry, given by Feodor Lynen. Already honored with the Nobel Prize for his achievements in unraveling the fatty acid cycle (1964), he irritated his audience by writing extremely rapidly the most complicated biochemical reactions on the blackboard, and then changing the side groups of molecules with two or three swiftly sponge wipes, while the students tried frantically to copy all these permutations into their note books. The atmosphere in the Zoological Institute was not very pleasant, with Hansjochem Autrum being a highly irascible, extremely autocratic ‘‘boss,’’ whom the students tried to avoid meeting in the building whenever possible. Social contacts during my time there were rare, and again I experienced the disadvantage of living in a big city. When working late in the Institute, it was always risky for unaccompanied girls to go home, especially since the Zoological Institute was within the red light district set up close to the main railway station at that time. So, once I had reached my modest quarters, I was certainly very reluctant to leave again for parties or other social encounters. Furthermore, my thesis project held another negative surprise for me: I came to the rather disappointing conclusion that ant lions, although equipped with quite competent eyes, do not at all need vision for their life subsidy. They catch and feed on their prey equally well when blindfolded. If nothing else, this certainly taught me a lesson in how to approach biological questions prior to doing experimental work.

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Taken together, I did not enjoy my time as a PhD student. While working on my thesis, I learned some interesting techniques but, due to the course of my thesis work and lack of a competent supervisor, as well as the difficulties with living in such a big city, I was happy when I received my PhD degree in 1967 from the University of Munich, with biology as a major, biochemistry and genetics as minors. Fortunately, my data on vision of ant lions proved quite interesting to some insect physiologists, and thus I managed to publish my thesis work in a renowned physiology journal [13]. Subsequently, I looked for a postdoctoral position in Tu ¨ bingen, to join my husband. I got employed as a postdoctoral fellow in the group of Gerhard Czihak at the Max-Planck Institute for Marine Biology, which had moved from Wilhelmshaven, at the north shore of Germany, to Tu ¨ bingen and was headed by Heinz Bauer, the discoverer of the polytene chromosomes of dipteric larvae. I was supposed to work on cell division and spindle orientation in snail embryos, with their spiral arrangement of the mitotic spindle. When Gerhard Czihak proposed this project to me, I found it quite fascinating, as I wanted to move into cell biology, and spindle orientation was, and still is, a topic quite attractive to me. However, it is only today that we know some of proteins involved in anchoring the spindle poles to specific regions at the inner face of the cell membrane. At the time I was supposed to work in this field, it was completely new, and I soon found out that I was doomed to fail for many reasons. Soon after I arrived, Gerhard Czihak left to work at the Marine Biology Station in Neapel, and I was left without supervision or advice. In the institute, there was virtually no equipment for characterizing cells or fractions thereof with biochemical methods, not even an ice bucket. Thus, not to loose too much time, I wrote a small review article on protein synthesis during cell division in pond snail embryos [11,14] and made use of the only good piece of equipment I had access to: a very good light microscope. This turned out to be useful in cooperation with Harald, in which we documented the disastrous effects of some TMV strains with altered coat proteins, the ‘‘yellow’’ TMV mutants, on the host cell chloroplasts [11]. However, I was glad that this employment ended when we moved to the United States already 10 months later, for 2 years, 1968–1970.


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HJ: Crossing the Atlantic: Too Small a Step? I could have continued my research on ts mutants of TMV without any pressure and publish some more papers on the topic, but, like many postdocs at that time, was considering a move to the United States. Larry Bockstahler, an American postdoc at the Melchers institute, had carried out his thesis with Paul Kaesberg, professor of Biophysics at the University of Wisconsin in Madison, Wisconsin. Upon Larry Bockstahler’s recommendation, Paul Kaesberg offered me a postdoctoral position in his lab. At that time, most young German scientists tried to get a postdoctoral position at places in California or the East Coast of the US. Despite the fact that this offer came from the less glorious Midwest, I was flattered and responded positively. When these plans substantiated, Brigitte had successfully applied for a DFG stipend to work for the McArdle Laboratory for Cancer Research. We finally sold (!) our 15-yearold VW beetle and bought a new one with US specifications. We booked on a freighter for June 1968. When we left the Channel a heavy swell of the open Atlantic caused the vessel to roll so that I had great troubles with my mechanical typewriter to finish a manuscript for the journal ‘‘Die Naturwissenschaften,’’ as the carriage went back and forth on its own. The paper was an overview on the role of mutations for the physical state of TMV coat protein, this time written in English – in fact, a version of a previous manuscript entitled ‘‘TMV mutants and the five levels of protein structure’’ which audaciously I had submitted to Science. I had been returned rather quickly with the remark that such a paper would only make sense with Georg Melchers as a co-author. But it was not Georg Melchers’ style to put his name on a manuscript written by a member of his institute.

BMJ and HJ: From the East Coast to the Midwest, by Beetle We arrived at New York City after an 11-day boat ride, with Brigitte having been heavily sea sick during the entire journey. After the rough Atlantic, it was an overwhelming experience to glide smoothly under the Verrasano Bridge into the cargo port,

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very early in the morning. Immigration officers boarded, examined visa and passports, and we had to present our chest X-rays, as proof that we did not suffer from tuberculosis. Finally, our VW beetle had to be sanitized – as we discovered later, this included the removal of the radio. These procedures took hours, and when we finally were permitted to enter the country, we drove to the Bronx, where we stayed for a few days with German postdoc friends. We were very short in cash, as in those days four Deutschmarks were converted to one dollar, and we were insufficiently insured, but were traveling in good spirits, from New York City to Madison, with a stop for a few days in Pittsburgh, at the house of Rainer Jaenicke and his family. We enjoyed their hospitality very much. Rainer Jaenicke was working on the physicochemistry of TMV, as a guest scientist of Max Lauffer (see Vol. 41 of this series) at the University of Pittsburgh. When we approached Madison, we gaped at the enormous Capitol, the second largest one in the US, but were also struck by the beauty of its location – on an isthmus between four lakes. During our journey and for some weeks after our arrival, we lived on ‘‘Kentucky Fried Chicken’’ and hamburgers from ‘‘McDonalds,’’ as it took another month before we received Harald’s first American salary. We had been lucky in being assigned to a pretty one-bedroom flat in ‘‘University Houses,’’ not too far from the university campus. Most of the residents were either Europeans or Japanese. We furnished the flat quite nicely with second-hand items from ‘‘VincentSt-Paul’s.’’ In summer, the Madison area with its lakes was rather humid and full of mosquitoes, but we enjoyed cycling to the labs in the morning, and fishing for our dinner on the way back at University Bay, where we kept a mooring for a small, half-rotten rowboat, a ‘‘hand-me-down’’ among German postdocs. We even took a sailing course as offered by the University and were proud to pass the final test required for a certificate – on one of the few days governed by a dead calm! In winter, life was more grim: the temperature dropped to below –201C, and the arctic wind attacked us cruelly when we walked from the parking lot to our labs, with Harald carrying the battery from our VW beetle into the institute, and Brigitte’s legs turning into icicles, since at that time, females did not wear trousers but skirts when working in US medical institutes or hospitals. But skating across the University Bay, from November to March, with fantastic views across the


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glittering lakes and snow-covered shores compensated for the inconveniences of Wisconsin winter. The scientific life on the campus was quite lively. There were brilliant groups like those of Har Gobind Khorana, Masayasu Nomura, and Gary Borisy; Khorana received a Nobel Prize in 1968. There were highly stimulating seminars in an international atmosphere. As many European postdocs, we were struck by the casual and friendly reception we received from the American members of both our laboratories. We enjoyed Thanksgiving and Christmas parties at people’s homes, barbecues in the lovely parks bordering the lakes of the city of Madison, and weekend trips to natural resort areas of northern Wisconsin. We vividly remember the special parties at Paul Kaesberg’s house; in one spare room, he had installed a record player and a slide projector, which continuously inundated the worshipping party guests with the music and texts by Simon and Garfunkel. The members of the groups we were working with were quite different. In the group of Paul Kaesberg, Harald met American and English postdocs quite interested in politics, while in the group of Harold P. Rusch, Brigitte encountered several German postdocs, and some from the US Midwest who were very friendly and firm in sports activities, but very reluctant to discuss politics. In this group, there was no critical comment about the President, Richard Nixon. Madison also offered good music and theatre activities, and we particularly remember an excellent performance of Stravinsky’s opera-oratorio ‘‘Oedipus Rex.’’ In a warm night in August 1968, when driving home from a movie theatre and listening to the radio, we heard shocking news from Europe: Russian troops had invaded Prague and crushed the promising political movement of the ‘‘Prague Spring.’’ Harald had discovered, in an artists’ supply shop in Madison, the newly developed acrylic paints and found them ideal for moonlighting painters with little time. A few months after the end of the Prague Spring a small painting documented our feelings (Figure 5, upper part). A large number of students on the Madison campus were quite actively involved in politics. The most important issue was, of course, the ongoing Vietnam War and the drafting of students. There were rather inconspicuous shacks on the campus erected by ‘‘Army ROTC’’ (reserve officer’s training corps) in which, as we were told, students were registered for the army, on a voluntary basis. This infuriated the pacifist students

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Fig. 5. The world 1968–1970, as seen from Madison, Wisconsin, USA. Upper: The end of the Prague Spring, August 1968 (Acrylics on canvas, 48 cm 64 cm, HJ, 1969). Lower: USA MOON 2 – Turmoil (Acrylics on canvas, 72 cm 134 cm, HJ, 1969).

and there were attempts to burn these buildings. These activities escalated, and in 1969, when the US government sent more troops to Vietnam, the National Guard was called in. In another nice summer evening downtown, while we were enjoying our soft ice cream cones, we were caught in a battle on the campus, between students and the National Guard, which used pepper gas and clubs to attack and disperse the demonstrators. Brigitte insisted on finishing her delicious ice cream cone as we tried to return to


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our car, despite the heavy clouds of pepper gas. She was sick the next morning. In June 1969 we were, like everybody else, excited about the landing on the moon. We did not own a television set, but it happened that at the day of the landing, the tenants of the flat below ours were on holiday and had asked us to water their plants in their absence. So, we had a key to their flat, in which there was a large, rather old-fashioned TV. In the evening, we rushed down there and fiddled frantically with all the knobs on this instrument. Only due to the fact that the landing was delayed for more than an hour, we mastered the adjustment in time to watch the key moment: Neil Armstrong stepping onto the dusty surface of the moon. This concoction of impressions and moods, the ending hippie period, the battles within the country and the moon landing inspired Harald to paint another (Figure 5, lower part) of a total of four acrylics.

HJ: ‘‘Harold from Biophysics’’ We appreciated the liberal attitude on the Madison campus, and became attached to the town and the university, not only because of the blue lakes and the clear light, but also because of the beautiful landscaping and buildings on the campus, and the fact that everything was in walking distance. In summertime, I preferred to have my ‘‘BLT’’ (bacon, lettuce, and tomato) or ‘‘Reuben’’ (sauerkraut and corned beef) sandwich together with Brigitte, so I called her lab to find out whether she could get off her work for a 20-minute lunch break in the sunshine. To the technicians who usually picked up the phone, I would introduce myself as ‘‘Harald, calling from Biophysics.’’ Thus, in the McArdle Lab I became known as ‘‘Harald from Biophysics.’’ Soon after my arrival, I had finished my manuscript on ‘‘Stability and genetic variation of a structural protein’’ (coat protein of TMV) and sent it to Springer Publishers in Heidelberg for publication in ‘‘Naturwissenschaften.’’ In the 1930s that journal had enjoyed a great reputation as most important articles (frequently very short ones) by German physicists and chemists had been published there. This fame, however, had deteriorated during world war times. Once my paper had appeared [11,12], would I be able to see my article somewhere on the campus of

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the University of Wisconsin? It took me a while to find out that the Department of Zoology was the only institution on the campus that still held a subscription of ‘‘Naturwissenschaften.’’ I had to climb a ladder to the attic and discovered that even the latest issues were immediately deposited up here – unread – and eventually bundled with cord. In Paul Kaesberg’s lab it took me a while to adapt to both, a totally different working style and to the slang spoken by most of my colleagues – it was so different from the English we had learnt at high school! As a German I was expected to be a tough and efficient worker – I wasn’t sure whether I should be flattered by that expectation; wasn’t that appreciation of German virtues somewhat outdated? The lab worked on ‘‘spherical’’ RNA viruses, the icosahedral shape of which had been discovered by Paul Kaesberg, both plant viruses (e.g. brome grass mosaic virus) and RNA bacteriophages (R17 and QX). I chose to work on bacteriophage because I figured I would learn more than by sticking to plant viruses. Paul Kaesberg suggested that I should continue with a new gene product, an extremely basic polypeptide, that had been discovered by his former co-worker Yamazaki [15], using R17 RNA directed cell-free protein synthesis. Due to the Matthaei course in Go¨ttingen, I was well prepared to immediately start on in vitro protein synthesis. However, I was unable to reproduce the ‘‘Yamazaki protein.’’ In those days, the RNA phage QX had become very prominent because of the RNA replicating enzyme activity it induced in the infected E. coli cell. This ‘‘replicase’’ was certainly more interesting than the major product, coat protein. Therefore I now performed in vitro protein synthesis with the aim of characterizing polypeptides coded for by QX RNA, especially the R polypeptide responsible for the replicase activity. I had a prominent competitor: at about the same time Severo Ochoa’s lab performed in vitro translation experiments on QX. Fortunately, I was not scooped and could publish the QX specific polypeptide pattern in Virology, together with my British postdoc colleague Andy Ball and Paul Kaesberg [16]. The replicase protein was easy to follow, because it is the only QX gene product that contains histidine. In 1969 I participated in the Cold Spring Harbour Meeting on Protein Synthesis. It was not as spectacular as the 1966 meeting on the genetic code must have been (where H.-G. Wittmann had presented the Tu ¨ bingen results on TMV mutants)


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but I loved the place, the shore of the Long Island Sound with limulus crawling through the mud and the famous lobster dinner. Many years later I returned to the Cold Spring Harbour Laboratory as a mouse geneticist. The postdoc period of 2 years passed very quickly, especially since we, like most German postdocs, took some weeks off for vacation, to see all the famous places like Yosemite and Yellowstone Parks, California, the Indian reservations in the Southwest, the Mississippi, New Orleans, the Caribic Shore – in other words, the usual ‘‘German postdoc tours,’’ which was the other aspect of ‘‘German efficiency.’’ Our first trip, already in 1968, brought us via California, including a visit at the Salk Institute, La Jolla, to Tucson, Arizona. There, my American senior competitors in the field of TMV mutants, Al Siegel and Milton Zaitlin, had invited me to give a talk. Both were very nice hosts with a good sense of humor. We had a party in Milton Zaitlin’s house and every so often our host would jump up and grab his rifle to chase the rabbits feeding on the precious plants in his desert garden. One of the guests said to me ‘‘I noticed, you got this funny slide with the vultures feeding on TMV from Jo Butler’’ – ‘‘What do you mean by: from Jo Butler?’’ – ‘‘Jo Butler showed the slide in his talk here not too long ago’’ – obviously my fun slide (Figure 3) had traveled faster to the US than I had! Ever since this first encounter with Tucson and the Southwest, we loved this area and returned to this region later on several times. To sum up, my scientific yield of those 2 postdoc years was modest but I had learnt and seen a lot and had met many interesting people.

BMJ: An Acellular Model of the Cancer Cell In the laboratory of Harold P. Rusch, I wanted to work on proteins related to mitosis, as I intended to continue what I had started with the pond snail embryos at Tu ¨ bingen, but hopefully with better chances for success. The Rusch group investigated protein and RNA synthesis during the various stages of the life cycle of the slime mould Physarum polycephalum. The acellular, plasmodial stage of Physarum can be grown to large yellow ‘‘pancakes,’’ wherein up to 108 nuclei assemble and separate their chromosomes synchronously, within approximately 60 minutes,

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on intranuclear spindles. As the Physarum plasmodium lacks a G1 phase, it was considered as a model for tumor cells, and the McArdle Laboratory for Cancer Research generously funded this research. At that time, this institute housed several excellent scientists, like Charles Heidelberger, Howard Temin, Waclav Szybalsky, and Bill Dove. In the elevator, as well as in the weekly seminars arranged by several groups, one met many interesting senior scientists, postdoctoral fellows and PhD students from various backgrounds and countries. Of course, English was the Lingua Franca, and the countenance of our American or British colleagues was sometimes severely challenged in seminar discussions among the European, non-English participants, like the following: ‘‘How many mouses did you use for this experiment?’’ ‘‘I used one mice!’’ Dr Rusch was very liberal in defining the projects in his group, and agreed to my ideas on identifying mitotic proteins. From my experience in Tu ¨ bingen, I was doubtful whether I could identify factors relevant for regulation of mitosis and the cell cycle (such protein factors were much later discovered in the laboratories of Paul Nurse and Timothy Hunt), but I decided to analyze the catalog of structural proteins involved in chromosomal separation. My plan was to precipitate tubulin and putative binding proteins from nuclei isolated in different stages of mitotic division. At that time, no reliable antibodies against tubulin were available, but there were several microtubule-binding drugs on the market that were used as mitotic inhibitors in cancer chemotherapy. Hence, I wrote to the company of Eli Lilly and asked for a sample of the drug vinblastine, which was supposed to act specifically on tubulin. I received a generous sample and indeed, it could be used to precipitate proteins from nuclear fractions. My first SDS gels showed a profile of various bands, in addition to tubulin, but one of them consistently migrated with actin! It turned out that vinblastine was not as specific for tubulin as previously thought, it precipitated a good number of acidic proteins due to electrostatic interactions, among them tubulin as well as actin. Due to several criteria, I was convinced that this was not a cytoplasmic contamination, but that actin had a role in nuclear processes, at least in this organism with an intranuclear spindle. When we left the US after 2 years, I had completed two publications on nuclear actin and its synthesis in Physarum [17,18] and was determined to devote my further scientific life to


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actin and its presumptive roles in the nucleus and the cytoplasm of non-muscle cells.

HJ and BMJ: Founding a Family in Good Old Tu ¨ bingen In June 1970 we returned to Germany, this time by passenger boat. We went back to Tu ¨ bingen, as Georg Melchers had both of us generously offered to work in his institute, and we could return to the same flat we had left in 1968, with our furniture and books. During our absence, this two-bedroom flat, on the grounds of the Melchers Institute, had served to accommodate numerous scientific guests of all the Max Planck Institutes in Tu ¨ bingen. On several occasions, during scientific conferences or meetings in the US, we were approached by people whom we had never met before but told us that they had stayed for some time in our flat and had vivid memories of the great parties they had enjoyed there. The only argument we had with Georg Melchers when discussing our return concerned the import our American cat. As Melchers was very fond of the birds breeding on the institute grounds and thus hated cats, he claimed that the first thing we would face after our return was the cat grilled at one of the famous institute’s barbecues! We did not believe him and our tomcat enjoyed the Max-Planck grounds for the next few years to come. With the beginning of 1971, Brigitte became pregnant. At that time, in Germany one still could not hope to meet a generous understanding from one’s boss for this condition, and, after all, Georg Melchers had invested a position, lab space, and money in her. Care centers for small children were virtually nonexistent, kindergarten slots were scarce and available only for children older than 3 years. Moreover, the general feeling in the ‘‘educated society’’ in Germany was still that mothers had to stay home and tend to their kids – unless they had to contribute to the financial situation of the family. Mothers working ‘‘just for the fun of it’’ were not highly respected, and it was prophesied that young children would suffer if their mothers would not be permanently present. It is quite amusing that today, 35 years later, the same absurd discussion celebrates revival in Germany. So we were quite nervous, and it was with millions of butterflies in the stomach that Brigitte approached Georg Melchers with the news

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of her pregnancy. He listened to her, rose from his chair and said with a bow: ‘‘What wonderful news! So I will be not only Harald’s ‘Doktorvater,’ but also soon a ‘Doktorgrossvater!’’’ For him, there was no question that Brigitte would continue to work in science, no matter what happened. To this very day, we are grateful to him for this reaction and his positive attitude to a scientist couple raising children. Luckily, Brigitte’s pregnancy did in no way interfere with her scientific activities. Our first son, Arne, was born on a Sunday, just like his father! The first babysitter we employed was not totally satisfactory, but when Arne was about 12 months, we were extremely fortunate to find an English girl trained as a nanny, who gradually turned into a friend and an important and beloved member of the growing family. She was still with us when Wolf, our second son was born in 1974, and moved with us, when we left Tu ¨ bingen again.

HJ: From Virus to Mould In 1970, Georg Melchers was 64, but vigorous as always. In previous years, he was more a science manager, whose aim was to found institutions in which modern plant science got its place and to defend – even within the Max-Planck Society – scientists against administrative restrictions, and to further the career of young scientists. But now he swiftly developed new scientific interests, or revived old ones, related to the applications of modern plant cell biology to problems of plant breeding. He was quite successful in this field and was elected a Foreign Member of the American Academy of Sciences. But still, he left it entirely to us to choose our research projects at his institute. As a capstone of my tapering journalistic activities I wrote a popular book ‘‘Die entzauberten Kristalle. Geschichte, Methoden und Ergebnisse der Molekularbiologie’’ (The Demystified Crystals. History, Methods and Results of Molecular Biology). I had received the request to write a popular book on molecular biology from a publisher specialized in economy, by the end of our stay in the US. Because I was always keen on new experiences, I airily agreed, but, of course, it turned out that writing a book on a moonlighting basis is a lot more strenuous than writing a newspaper article. I had started the project in Madison, continued


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a little bit on the boat back, and, from fall 1970 on, tried to finish the manuscript in Tu ¨ bingen. When Brigitte became pregnant I got quite hectic: Would it be possible to finish the manuscript before the child was born? I lost the race, but still finished the book because our first son, Arne Andreas, turned out to be quite cooperative. The book appeared only in 1973, had a misleading cover designed by an arts professor and in many libraries ended up in the section mineralogy. The Czech paperback edition was half as expensive and had an informative and beautiful cover. At least in Germany, it wasn’t considered respectable among scientists to write newspaper articles and popular books. Wolfhard Weidel’s book on virus was a lonely forerunner of today’s flourishing ‘‘popular understanding of science’’ (‘‘PUS’’) activities. For a very long time this book remained my last publication written in German. Now, a third of a century later, the German language in all fields of daily life is percolated with English words. My explanation, which certainly holds for the business and advertising world, is the high fraction of threeletter, one-syllable words in English: top, fit, job, hit, pet, etc., the German translations of which are usually lengthier. Despite this fact, a sentence I used to explain the triplet code and the mechanism of frame shift mutations: ‘‘DIE RNS IST AUF DEM WEG VOM GEN ZUR TAT’’ (the RNA is on its way from gene to action) cannot easily be replaced by a meaningful English sentence consisting only of three-letter words. In the Czech edition the translator managed, by asking the question (in Czech) ‘‘Who knows the code for the gene?’’ As for my research in the Melchers institute, there were three possible routes which I tried to follow up in parallel: To tackle some old TMV problems with new techniques, continue research on QX, and to start new projects in cell or developmental biology. In German speaking countries, after getting a PhD degree and having developed one’s own field of research, ‘‘Habilitation’’ would be the next step in an academic standard career – a career that I had not intended originally. Apart from a few exceptions the Habilitation used to be a prerequisite to successfully apply for a professorship. The candidate has to convince the faculty and the relevant committees in writing and orally that he/she is a promising scientist and academic teacher. Georg Melchers said: ‘‘This old-fashioned ritual is all complete nonsense (he used the rather harsh German word: ‘Quatsch’) but I advise you to go

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through it.’’ I did so although it wasn’t quite easy to pass a faculty rite as an employee of a Max-Planck Institute. The advantage was that I could now have my own diploma and PhD students, and thus could work on different projects at the same time. Progress with TMV was not too exciting. However, I did make progress on the replicase protein of QX, a project that I had taken along from the Kaesberg group. With the technician Ingrid Hindennach I characterized, by radioactive labeling of in vitro synthesized protein the peptide map of the 550 amino acid long replicase polypeptide, and, later on, with the PhD student Monika Happe (who was trained in the newly established biochemistry curriculum of the University of Tu ¨ bingen) we synthesized active QX replicase in vitro. This was not trivial, as the enzyme consists of a complex of one virus-specific subunit and three host subunits (provided by the E. coli supernatant); in fact, enzyme activity, of a complex sedimenting at the expected rate (7S), appeared only after ammonium sulfate precipitation. This work was published in a recently sprouted offshoot of Nature, called Nature New Biology [19] and in European Journal of Biochemistry [20]. We thought in vitro synthesis of active QX replicase was a neat complementation to the in vitro replication of QX RNA using replicase purified from infected E. coli cells. However, there was very little response to our publication. Not enough, after a rather short lifetime Nature New Biology was terminated! This reminded me of the case of ‘‘Zeitschrift fu ¨ r Vererbungslehre’’ and I started to feel like an angel of death for journals! I was quite disillusioned and got the definite feeling that the times of this kind of virus research were over. I started to think about how to get out of the virus field and into the analysis of developmental and morphogenetic processes. Of course, it would have been much more fruitful to think about a new field before going to the States as a postdoc. But if I were to leave the virus field, what project could I start in my tiny group of three or four people, based on my background in microbiology and genetics? After some reading, I figured a hormone controlled interactive morphogenetic system might be an interesting topic. The system I had found attractive from the literature was the mating reaction between plus and minus strains of the bread mould Mucor mucedo, a mould related to Phycomyces, which had been chosen by Max Delbru ¨ ck to study cellular light reception. ´ when ¨ Varju I had heard of Max Delbru ¨ ck’s projects from Dezso


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I participated in a journal club on biological cybernetics. Together with my PhD student Tilman Wurtz, I mutagenized Mucor with NMG and got a beautiful collection of mating-deficient mutants. The mating hormone was known, it is the carotenoid trisporic acid (TA). We were particularly interested in TA response mutants, both with absolute and with temperature sensitive deficiency. But other types of mutations were also found, for example with defects in later morphogenetic steps of the specialization of hyphal tips into mating organs that fuse to produce the zygospore. When we published a first paper to Developmental Biology [21] we got very positive comments from Max Delbru ¨ ck. Thus encouraged, we continued to produce and characterize mating mutants [22], with the final aim to get our hands at the TA receptor. It took a while before the severe disadvantage of this ‘‘model system’’ became apparent. In contrast to other moulds like Neurospora, Mucor (like Phycomyces) was not well suited for formal genetics. In the zygospore, there is no straight forward relation between the genotpyes of the approximately one thousand nuclei and the underlying meiotic events. Therefore, gene mapping and complementation assays are not easy to perform and methods of genetic manipulation were yet not available. Later, I found that it is much easier (although much more expensive) to perform mouse genetics than Mucor genetics. Looking back, I can say that Mucor still offers good chances for the analysis of hormone-dependent sexual interactions and morphogenesis in a lower eukaryote. Nowadays, many of its drawbacks for classical genetics can probably be overcome with modern methods of genetic manipulations.

BMJ: Would a Slime Mould Thrive at Tu ¨ bingen? As Georg Melchers imposed no restrictions whatsoever on my research plans, I decided to continue on what I had started in the US. Physarum plasmodia still fascinated me with their periodic cytoplasmic shuttle streaming and their synchronous intranuclear mitotic divisions. While in McArdle Physarum was considered a model organism for cancer cells, in Tu ¨ bingen it was classified as a rather unusual plant. Once I felt confident that I could continue to work on Physarum actin in Melchers’ institute, I asked the DFG again for support. Doing this, I followed Georg

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Melchers’ advice. ‘‘I am facing retirement and thus you will leave this Institute in short time. To be prepared for the future, you should achieve two things: First, strengthen your ties with the DFG as the largest and most renowned funding body in Germany. Second, like Harald you should aim for the teaching right at the University of Tu ¨ bingen (Habilitation).’’ Indeed, both these suggestions turned out to be very valuable for my future scientific career. I obtained another DFG grant with positions for a technician and a postdoctoral fellow, and thus began to establish a small group, by accepting several students from the Tu ¨ bingen University to work on their Diploma thesis, in addition to the postdoc and a technician. Then, my small group tried to identify and analyze structural proteins involved in various aspects of the intranuclear division and cytoplasmic streaming. We characterized nuclear myosin, and in collaboration with Harald and his group we compared nuclear actin of Physarum with rabbit actin. In addition, there were collaborations with several guests. I particularly remember studies we performed on the structure of Physarum chromatin with Ian Walker from the Oxford University who spent a Sabbatical with me [23], and a postdoctoral fellow, Alan Wheals, who taught me the secrets of Physarum amoeba mating types. Sadashi Hatano from Nagoya University visited us and taught me how to purify Physarum myosin with excellent ATPase activity, provided one was willing to spend endless hours in the cold room. We studied its capacity to form contractile elements with rabbit skeletal muscle actin, thus confirming some aspects on the evolutionary conservation of cytoskeletal proteins. Hatano, quite different from the formal Japanese whom I had expected, turned out to be a gifted babysitter when the nanny was not available! Within 4 years, we could present a number of well acknowledged papers, including the identification of nuclear actin by fingerprinting as the result of another collaboration with Harald [24], and on nuclear myosin [25,26]. Due to our excellent nanny and with Harald as a cooperative father, I could afford to attend conferences abroad and could also concentrate on the other aspect of my German Academic career: I engaged in teaching undergraduates at Tu ¨ bingen University and, after submitting the required papers, was awarded with the Habilitation (teaching rights) in 1972.


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¨ dtele hinaus’’ – Leaving Tu HJ: ‘‘y zum Sta ¨ bingen for Good Early in 1972, Georg Melchers became 66 and I turned 33. Numbers with identical digits are called ‘‘Schnapszahl’’ in German and I arranged a little birthday party for the lab under the title ‘‘Schnapszahl and half the age of the boss.’’ Melchers, usually a person of good humor (unless enraged by something that had gone wrong in the greenhouse) was not at all amused by that little joke and we soon found out why: the central administration of the Max-Planck society in Munich had notified him that he must now seriously consider his retirement. This meant that he had to give up his thriving research on plant breeding by cell fusion and other modern manipulations, including the cooperation with Japanese groups, and that he had to dismiss his scientific collaborators, because the department as a whole would be closed. In other words: we had to look around for another place to continue our scientific career. For this we had time till the end of 1974. I for my part, was determined to change my field of research, to leave virology for good and switch to cell and developmental biology as applicable to biomedicine. We considered two places: the Mill Hill Institute of the Medical Research Council in London and the Biochemistry Department at the Biocenter of the University of Basel. After having visited the Mill Hill laboratory, we realized that it would be hard to raise small children in London with both of us working in competitive fields of science. Thus we decided for Basel. At the biocenter, I talked to the head of the biochemistry department, Max Burger, and he was quite pleased to add two scientists clearly beyond the postdoctoral state to his group. Preparing for my new field of research in Max Burger’s group, I participated in an EMBO lab course on cell culture techniques, which was organized by the laboratory of Max Burger. During my second period at Tu ¨ bingen, I achieved the qualification as a university lecturer, in addition to doing research at the Max-Planck Institute consequently, I had a teaching obligation and participated as a lecturer in a microbiology lab course. While the teachers at University focused on bacteriology and antibiotics, I taught plant viruses and bacteriophages. The students had great fun simulating experiments that had recently led to the discovery

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of new bacteriophages like RNA phages and filamentous DNA phages. To this end, we took samples from the water of a muddy little stream running through the old town of Tu ¨ bingen and was heavily contaminated with sewage. We separated fractions on sucrose gradients with sedimentation markers and tested them for plaque formation on an E. coli lawn. Thus, we obtained a bacteriophage profile. The plant virus part involved a number of biological and biophysical techniques that can be applied to TMV. Many years later, this course was revived for undergraduate students at the University of Bielefeld. TMV also proved as a valuable means for teaching at the University of Regensburg, in the institute of Rainer Jaenicke.

HJ: Changing Fields Again What was my motivation to join the Burger group at the Biozentrum of the University of Basel? When I had studied the literature for a possible biochemical access to developmental biology, I had come across the work of Moscona and others who worked on cell–cell adhesion. I was attracted by the fact that these researchers had introduced unconventional mechanical tests like clumping of cells as a function of shear force, and Max Burger had used these techniques to show that lectins like wheat germ agglutinin could differentiate between tumor and normal cells, and also to characterize adhesion molecules in sponge cell aggregation. However, when I joined the Burger group, I discovered that cell–cell adhesion was not the main focus anymore, and Max Burger was now mainly interested in cellsurface properties of tumor cells. Hence, I figured I should work out my own program which would combine cell biology, embryology, and genetics of the neuromuscular system, if possible, in relation to human hereditary diseases. In those days, when nematode and zebrafish genetics were not yet popular, this implied working on the mouse. Fortunately, a small mouse room was available and I could start to get founder mice for the mutants I found interesting. I started with Dystrophia muscularis (dy, now known to affect the gene for laminin S) and motor endplate disease (med). I was impressed by Alan Peterson’s chimera experiments [27], designed to find out whether dy primarily affects muscle, or the nervous system, or both. These


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beautiful experiments, which, from today’s point of view rather introduce complications than answer questions, were a good opportunity for me to get into experimental mouse embryology. On the other hand, this route of research would totally isolate me from the rest of the Burger group. Mouse embryology and genetics involves the killing of mice. As a microbial geneticist I never had to kill a mammal. After more than 30 years I remember the eyes of the first mouse turning milky when I killed it. On the whole, I was more successful with tissue culture experiments than with embryo manipulations and had turned into an expert for the preparation of coupled nerve–muscle cultures from primary myoblasts of neonatal rats or mice and slices of embryonic spinal cords [28]. Using co-cultures we could demonstrate trophic interactions between nerve tissue and muscle cells, for example stimulation of the synthesis of choline acetyl transferase, the marker enzyme of motor neurons [29]. However, in using a cell culture approach for the analysis of mutations affecting the neuromuscular system, there are two problems. First, if the gene is not yet identified and a mutation is lethal or renders, in the homozygous state, the mice unable to reproduce, one has to produce mutants by heterozygous matings and is faced with the problem of identifying the Mendelian quarter of the homozygous, affected embryos. Second, like in most human hereditary neuromuscular diseases, mutant phenotypes in the mouse often develop postnatally in a progressive fashion. Since even the most mature muscle cultures reflect at best a neonatal stage, it cannot be expected that disease symptoms show up in culture. Radically changing one’s field of research can be refreshing but has a severe disadvantage: one looses all scientific friends and has to struggle before one gets into the network of the new field. This was the reason for me not to return to the United States for 15 years. On the other hand, with quite modest contributions to conferences I got immediately a lot more attention than with my previous virus work, and I enjoyed going to the relevant meetings. Whether in Harvell, England, or at the famous Jackson Laboratory at Bar Harbor: the mouse genetics meetings always had a wonderful personal atmosphere. In England, I met people like Mary Lyon and Tony Searle, both famous in mouse genetics and most friendly, and chatted with them on a hike through the Vale of the White Horse. There were not so many people at these

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meetings, maybe 50–150. Mouse geneticists and embryologists, ‘‘mousers,’’ in the 1970s and 1980s exchanged their findings without fear of getting scooped. ‘‘Mousers’’ would reveal their findings in an informal journal called Mouse News Letter (later named Mouse Genome, now extinct) and did not ask for an ‘‘impact factor.’’ Whenever I mentioned Mouse News Letter in a talk in front of a non-mouser audience, people would laugh, probably because they imagined mice reading ‘‘their’’ newsletter.

BMJ and HJ: Science in Switzerland, Home in Germany We left Tu ¨ bingen in January 1975. Our first son was then 3 years and the second one was only a few months old. Our American cat, the English nanny and Brigitte’s German (male) technician moved with us. As we could not get a working permit for the English nanny from the Basel authorities, we decided to live on the German side in a small village, Inzlingen, close to the Swiss border. The administration of the Biozentrum was relieved that we did not insist on living in Basel, as there was a quota for foreigners working in Switzerland. So, we commuted and crossed the border every day, Harald sometimes using a small motorcycle. We loved Basel as a city (and still do), but from the beginning we knew that we could not stay there permanently. In addition to living in such an attractive area, there were also some disadvantages: Personal connections to Basel citizens were rare, and due to us not living within the city, we did not find many friends there. Crossing the border every evening was nerve wrecking because of heavy traffic of German commuters with jobs in the Basel pharmaceutical industry. After some months, the English nanny decided to return to Tu ¨ bingen, but was kind enough to arrange for a successor, to take care of ‘‘her children.’’ So, we imported another girl from England who also was quite reliable and lived with us for several years.

BMJ: From Slime Mould to Vertebrate Cells Harald had been offered an ‘‘Assistant Professorship’’ by Max Burger, which implied that he had teaching obligations in


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Biochemistry. I managed again to obtain funding by the DFG, this time for my own salary in the form of a fellowship, but in 1976 Max Burger offered me also a position in his group, and I, like Harald, became an Assistant Professor of the University of Basel. Like Georg Melchers, Max Burger accepted the fact that I was focusing on the actomyosin system in Physarum, and first I continued this work, together with my technician who had moved with us from Tu ¨ bingen and was paid out of my DFG grant. However, eventually I got tired of the many problems one had to face when working on protein chemistry with Physarum. Its given name ‘‘slime mould’’ truly reflected the most severe disadvantage: The plasmodia were full of slimy carbohydrates and it was tedious to obtain nuclear or cytoplasmic fractions without such a contamination. In Max Burger’s lab, I realized how much ‘‘cleaner’’ and easier to work with vertebrate tissue culture cells were. In 1975, I attended the ‘‘26th Mosbacher Kolloquium’’ of the German Society of Biochemistry (now Society for Biochemistry and Molecular Biology, GBM), titled ‘‘Molecular Basis of Motility.’’ I was struck by the fact that the attending members of this society were all male, all clad in grey suits, but was very impressed by the presentation of Susan Lowey, the guest speaker presenting elegant work on muscle function, which integrated biochemistry, biophysics, and structural biology. I would have considered it as a joke if someone would have told me during that meeting that 19 years later I would also organize one of these famous ‘‘Mosbacher Colloquia.’’ Impressed by the Mosbach Conference, I started to work on the actin-based motility of non-muscle vertebrate cells in Basel. I wanted to localize actin, myosin, and other actin-binding proteins in the different compartments of cultured vertebrate cells, and thus I began to raise specific antibodies against these socalled ‘‘contractile proteins.’’ This was a difficult task, as many of these proteins are highly conserved in evolution, and thus mice or rabbits did not easily produce high-affinity antibodies against the vertebrate or even invertebrate counterparts. At that time, there were only few reports on the presence, distribution and function of actomyosin fibers in non-muscle cells, and when we succeeded to raise specific, well characterized antibodies against actin [30] and several of the actin-binding proteins, a wide field of cell biological questions opened for research. The scientific

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community in the Biozentrum was quite interested in these topics, and I could discuss my projects and problems with Max Burger, Gu ¨ nther Gerisch, Eduard Kellenberger, and Ueli Aebi. I also liked the international, easy-going atmosphere in Max Burger’s group where many American postdocs and guests worked at that time. When my German technician left, I found an American girl, Kristin Kelley, as a substitute who was also quite motivated and a great help. Among the more important results we achieved was the localization of alpha-actinin at neurosecretory vesicles [31], and the identification of actin as a cofactor of RNA polymerase 2 [32]. Both papers were the harvest of collaborations with other groups in Switzerland (Giulio Gabbiani, Geneva, and Richard Braun, Bern), and both were well accepted and are still acknowledged by the cell biology community. Today, there is no doubt that secretory vesicles are transported along cytoskeletal elements, and actin has been identified as a cofactor for all three DNA-dependent RNA polymerases. So, my work was going well, my family was thriving and everything seemed perfect – except we knew that we could not hope for a permanent position in Switzerland and thus prepared to move again in 1978.

HJ: Down the Rhine: From Basel to Heidelberg To introduce myself to the German community in neurobiology I published (without Max Burger) a somewhat speculative overview on genetic analysis of neuromuscular interactions in vertebrates – despite my negative experiences with my 1968 article on the stability of mutant TMV coat proteins – in the good (good?) old journal ‘‘Die Naturwissenschaften,’’ and, even worse, in German [33]. In this I stressed the role of tissue interactions and the analytical approach by chimera experiments to distinguish myogenic from neurogenic and humoral mechanisms of hereditary neuromuscular diseases, with mouse mutants as models. Thus, as a converted former virus geneticist, I was distinctly ‘‘non-molecular’’ and did not refer to molecular cloning techniques, which, as it turned out, were to determine the future of human genetics and the elucidation of human hereditary diseases.


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Melitta Schachner, a scientist a few years my junior, had started her career in bacteriophage research in Munich and subsequently switched to neurobiology during her postdoc time at Harvard Medical School. Now she had been offered the newly established chair of neurobiology at the University of Heidelberg. In the academic system of those times the position of an associate professor usually came with the chair of the full professor. I contacted Melitta Schachner at a meeting on the Development of the Nervous System at St Odile in the Vosges Mountains in the Alsace (France), a place not very far from Basel. Melitta Schachner had been invited because of her work on immunological aspects of neuronal development, carried out in Richard Sidman’s group at Harvard Medical School. We discussed possible research projects at the planned institute at Heidelberg: Her topic was the cell and molecular biology of CNS development, especially of the cerebellum, whereas mine would be neuromuscular interactions. The biological basis for both projects would be a mouse colony with the relevant mutants. Melitta Schachner was quite positive about my application as in those days there were not too many people in the University scene with experience in mouse genetics, embryology, and cell culture. In German university institutions within biological faculties, neurobiological research was typically performed on insects. I applied for the position and after the formal procedures in the relevant comittees, I was offered the position of an associate professor in neurobiology at the University of Heidelberg. Thus I got my first permanent position, at age 38. But what about Brigitte?

HJ and BMJ: On the Move Again We had an agreement: both of us would apply for academic positions in any European country, but we would not consider the Americas, Asia, or Australia, since we wanted to educate our children in Europe. As our scientific fields were partially overlapping, but not identical, we always aimed at two positions in close vicinity. Hence, if one of us would be offered a position with tenure or tenure track, the other would try to adapt somehow. So, when Harald came with the good news that his application for a professorship at the University of Heidelberg

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had been successful and he was offered the job, Brigitte started to search for an adequate opportunity. When she had also found an adequate offer (see below), the family packed up again, and we moved to the tiny town of Neckargemu ¨ nd, upstream from Heidelberg in the Neckar valley. Harald had found a small, rather run-down house for us there, which we bought. Unfortunately, it turned out that it was in a rather desperate condition, and renovation was much more expensive and took much longer than we had anticipated. When the moving date approached in January 1978, we had to direct the van to a much too small three bedroom flat on a very steep hill, covered with black ice. In this rather inadequate substitute, we lived with the children, the nanny and the cat, about one hundred boxes and hundreds of books. It was quite an experience, but turned out not to be the last one of its kind in our life. A few weeks later, we moved into our modest but charming house, perched on a hill side, with a small, very steep garden. In the mornings, Harald took the road following the Neckar valley to the university campus, and Brigitte dashed through the forest with her VW beetle to her new working place in the hills above Heidelberg. We left our children with the English, and later on with an Italian nanny, who took the children first to kindergarten and later to school, and cared for them during the day. To compensate for the inconveniences our children sometimes experienced with two scientists as parents, we usually took them on Saturdays to Heidelberg, for lunch at McDonalds. This was considered a special treat.

BMJ: A Step up the Hill: EMBL at Heidelberg My application for a position at the Anatomical Institute of the Heidelberg University was not successful – at that time, medical faculties in Germany, in contrast to their counterparts in the US, were still quite reluctant to open their field to cell biology. Thus, I decided to inquire at the European Molecular Biology Laboratory (EMBL) for openings. The EMBL had recently been set up by EMBO (European Molecular Biology Organization), a congregation of several European countries and Israel, to support the modern branches of biology as well as the development of new instruments for science in Europe, with the aim to counteract the overpowering role of the USA in these fields. At the time I learnt


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about EMBL’s existence, it was not yet well known, but there were three Finns who had set out from their home country to this new place and were determined to mould it into a first class institution in cell and molecular biology: Kai Simons, Ari Helenius (his brother-in-law), and Henrik Garoff. All three of them worked on Semliki Forest Virus and were interested in host cell reactions during virus entry, transport and the formation of virus progeny. When I talked first to Kai Simons, he indicated that there might be some interest for a cell biologist working on the cytoskeleton, and he encouraged me to talk to John Kendrew, the first director of EMBL. I immediately got an appointment with Sir John in his new office. During the interview, he quietly listened to me and looked carefully at my credentials and publication list. Finally, he said in his low voice: ‘‘Come back tomorrow.’’ The next day, I went back up the hill, very uncertain about the outcome of this encounter. Sir John looked at me, smiled and said in his well polished, upper class Oxford English: ‘‘I will offer you a position as a group leader, on one condition: we have to teach you some proper English herey .’’ Of course I promised to get rid of my Midwestern American slang as soon as possible! So, I started my life at EMBL early in 1978, on a 3-year contract as a group leader, in a twin group with Graham Warren. Graham had been trained in biochemistry at Cambridge University and worked on vesicular transport through the Golgi compartment. We shared a laboratory and both were able to establish small groups. However, the idea of a close collaboration between the two of us, which had been the concept of Kai Simons, the senior group leader of the Cell Biology Department, did not really take off. Our interests in cell biological questions and our taste how to tackle them were rather divergent. During my time at EMBL, about a third of the scientists working there were British, and they dominated the scientific life in the Cell Biology Department. They came in late in the morning but usually stayed late in the evenings, including weekends and holidays, and for someone like me, having always to find a compromise between the time for work in the laboratory and for my family with small children, this was quite difficult. There were only three female group leaders at that time and all three were German: Christiane Nu ¨ sslein-Vollhard, Chica Schaller, and myself. I was the only one with children, and it was clearly my

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private job to cope with this situation, but I never complained. As a consequence, I never made it to the ‘‘inner circle’’ where decisions on EMBL policy and directions in science were made, usually in the evenings.Today, I believe that my male colleagues simply were not aware of the problems I encountered, and I probably should have been more outspoken. However, the international atmosphere at this great European institute, with a large budget and many excellent guests was greatly stimulating, and thus my research was going quite well. In different collaborative studies, I focused on the role of the structural protein alpha-actinin in lymphocyte receptor clustering [34] and as a structural component of the differentiating [35] and mature [36] skeletal muscle. Together with Frans Jennekens, a pediatrician from Utrecht, we also identified alpha-actinin as the major component in nemaline rods, pathological structures seen in the skeletal muscle of hereditary, congenital nemaline myopathy [37]. Several other studies focused on the changes of the localization of this and other actin-binding proteins during early events of cellular transformation [38–41]. In a collaboration with Gerhard Isenberg from a Max-Planck Institute in Munich, we also discovered that vinculin, another component of anchorage structures in muscle and non-muscle cells, bound directly to actin, thus contributing to the bundling of actin filaments close to the membrane [42–44] a finding which was dismissed for years by several American groups who could not repeat it. Finally, our results were acknowledged in my presence at a Cold Spring Harbour Meeting. The solution to this discrepancy was that vinculin can adopt an open as well as a closed configuration – and while we apparently had been working with the open form, the laboratories in the US had always the closed one in their test tubes. Furthermore, and in continuation with my former interests in the nuclear cytoskeleton, we demonstrated that non-muscle actin was able to shuttle between the cytoplasmic and the nuclear compartment. This was the result of a collaboration with Jean and Joe Sanger from the Pennsylvania University who spent a Sabbatical with me in Heidelberg [45] and turned into close friends. Occasionally, I made an excursion to cytoskeletal systems other than the actin network. With Harald, I investigated the organization of microtubules and microfilaments in the growth cone of cultured neurons [46], and together with Gareth Griffiths, the gifted electron microscopist from Wales who was


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assigned to my group at that time, and Graham Warren, we studied the transport of clathrin-coated vesicles towards the acrosome in developing sperm cells [47]. I always found such short side steps from the actin filament to other fibrillar cytoskeletal systems like microtubules and even to the clathrincoated vesicle system quite refreshing and kept this habit also alive in my later scientific periods. All our own achievements in the field of the cytoskeleton were based on ‘‘home made’’ good antibodies. They were used for localization at the light and ultrastructural level, but proved also very useful for functional studies on cells microinjected with them and for biochemical characterization of the proteins in question. So it was during my Heidelberg times when I developed a strategy which should prove useful during all my later scientific life: a combination of studying the biochemical properties of structural proteins in the test tube with parallel studies of the same components in cells. In 1978, I visited Sadashi Hatano in Nagoya, and attended the 6th International Congress in Biophysics in Kyoto. The president of this congress was Fumio Oosawa, the leading figure in research on contractile proteins in Japan, and therefore there were many sessions on biophysical properties of actin and myosin. In 1980, I attended the International Congress in Cell Biology in Berlin, organized by Werner Franke, and this event marked the foundation of the ‘‘European Cytoskeletal Club’’ (later on recoined in ‘‘European Cytoskeletal Forum’’). The idea of such an organization was propagated by Giulio Gabbiani, Geneva, and the founding members included Werner Franke (DKFZ Heidelberg), Klaus Weber (MPI Go¨ttingen), and myself. Subsequently, this organization grew steadily, presented itself with many excellent meetings in various European countries and Israel, and attracted a large body of people working in this field. It is still thriving. While my scientific life at EMBL was quite satisfactory, I spent only a small amount of my time teaching at the University. Harald, on a university position, had a much heavier teaching load, and the conditions for his research were not very satisfactory. Thus, when the University of Bielefeld finally offered him a chair for Developmental Biology (a position for which he had applied when we were still in Basel!), we were quite pleased. Furthermore, my own contract at the EMBL was not tenure

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track, and I could not hope to stay there for good. Hence, we decided to move again.

HJ: Brain and Muscle When I took up my work at the Neurobiology Institute, money and space were limited, and the teaching load was heavy. We started out in a prefabricated building made of aluminium sheets, one of the ‘‘Krebszellen’’ (cancer cells) as they were called because they had been erected to house the German Cancer Center before it got its definitive building. My office was on the upper floor, as was Melitta Schachner’s office, her labs, the small seminar room and the secretariat. My lab, however, was in the basement together with the darkroom and other jointly used facilities. The ceilings of the basement rooms were so low that the enlarger for photo prints, had to be placed directly on the floor, to accommodate its column. The fluorescence microscope was set up in the broom closet of the cleaning lady. The door of this cabinet had been removed, to gain space for a chair to sit down. With the smelly floor wax in front of me and with receiving every so often pushes in my back from people moving through the narrow corridor, it was difficult and certainly no pleasure to evaluate fluorescently stained tissue sections or cell cultures, and take photographs with long exposure times. Thus I often preferred to drive up to the EMBL, which, in comparison to the institute downtown, appeared like a resort hotel. Additionally, the fluorescence microscope to which I had access through Brigitte was of much better quality and the food in the EMBL cafeteria was excellent. With grant money of the DFG I had built up a small group consisting of a technician, a postdoc, and two students, covering, at a modest level, biochemical techniques, cell culture, embryology, and electrophysiology. I myself did the mouse breeding and genetics, embryology and tissue culture; molecular genetics was missing. I continued with cultures of embryonic spinal cord explants, isolated or in conjunction with muscle fibers, and studied the distribution of cytoskeletal proteins, for example in growth cones using Brigitte’s antibodies [46], or the blocking capacity of antibodies against surface glycoproteins (with Melitta Schachner [48]). The neurobiologist Georg W. Kreutzberg invited me to attend the 1979 Neurochemistry Congress in Jerusalem,


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and this was my first of several journeys to Israel. During this conference I met Gerhard Isenberg, an expert on the actin cytoskeleton like Brigitte, with whom I explored the historical sites of Jerusalem whenever time permitted. The members of the Institute for Neurobiology had to teach advanced courses in cell biology, developmental biology, and neurobiology. These courses had to be developed from scratch which was a stimulating challenge. But there were also lab courses in physiology with large numbers of medical students. In those days these involved the killing of frogs, then imported from Hungary, to teach basic electrophysiology using nervemuscle and heart preparations for extracellular recording. As far as I know, the use of animals in these beginners’ courses has in the meantime been replaced by computer programs simulating the behavior of excitable nerve fibers and muscle cells. One day, in 1981, I got a call from the University of Bielefeld: was I still interested in a professorship for developmental biology, now that I was professor at Heidelberg? It was the Dean of the Faculty of Biology of the University of Bielefeld who asked me that question. In fact, I had applied for that position some time earlier but wasn’t on top of the list. The University of Bielefeld (in the state of North Rhine Westphalia) had only been founded in 1969 and was totally unknown to most scientists in Southern Germany. A colleague in Heidelberg asked me ‘‘Why would you go to the tundra?’’ By this he not only referred to the ‘‘Siberian’’ chilly climate of Westphalia as compared to the mild climate of the Heidelberg region, but also implied a barren academic scene in comparison to the blossoming scientific landscape of Heidelberg. In fact Heidelberg had developed into a hot spot of molecular biology, cell biology, and medical research. At the same time, Heidelberg University could boast with century-old traditions. Besides the university there were several institutions devoted to up-to-date research in physics, the life sciences, and medicine. It was not easy to weigh the pros and contras: Brigitte would loose her excellent position at an international top institute (a position which, however, was not tenured) and her connections to the German Cancer Center; I would loose contacts to medical institutions, of which neurology and human genetics were the most important to me. But then, at least for me, there were strong arguments in favor of Bielefeld. In contrast to most other people in Southern Germany I was familiar with this town (because my

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father’s parents had lived there). I knew that it was not an exciting but a rather pleasant place, certainly not bad to bring up children. I was offered plenty of lab space as well as open positions for scientists and technicians, and a secretary – in short the possibilities to build up a research unit. Of course, there was also Julius Caesar’s argument to rather be the first in the village than the second in Rome. So we left Heidelberg.

BMJ and HJ: Settling the Family up North When we told the children that we would move again, they were quite upset. However, their worries subsided when we answered with ‘‘yes’’ to two questions: ‘‘Is there a McDonald in Bielefeld, like in Heidelberg?’’ and ‘‘Will you promise that we will not have to move again?’’ Brigitte’s positive answer to the first question was a straightforward lie – we knew that there was no McDonald. Fortunate for our reputation as parents, McDonald opened its first diner in Bielefeld just when we arrived, in September 1981. As for the second question, we kept our word: our children did not have move until they were grown up and could decide on their own. We stayed together until Arne, the older boy had already finished high school and Wolf, the younger, had only one more year to go. At home, we first passed rather choppy waters, but later on things smoothened. Like in Heidelberg, we first had to experience that the house we were to move into was not finished, and we had to settle in a rented bungalow. The move was done all in one night, in September 1981: a moving van 13 meters long, containing in addition to our private belongings the setup for electrophysiology that had been financed by the DFG and the students’ bicycles. Brigitte’s father, the kids and the English nanny, who had returned to us, were riding in one car, Harald drove our station wagon with the nanny’s brother and three hundred mice in about one hundred cages in the back. The house had not enough bedrooms to accommodate so many people, so we built a wall from the mover’s boxes, as an extra small room to accommodate the nanny and her brother. Of course, several times we were faced with the situation that an item we desperately needed was buried in one of these boxes, quite close to the floor!


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Somehow we survived the winter, and in spring of 1982 we finally moved into our own house. From there, it was only a 7-minute walk to the western end of the gigantic university building where the offices and laboratories of the Faculty for Biology were situated. The prosaic glass-and-concrete university building was called ‘‘thinking factory’’ by the liberal arts people. To walk its entire length, with shops and lecture halls flanking the main hall on the ground floor, took another 7 minutes. Harald was to work in that building for 23 years, in this town where his grandfather and father had grown up, and where our sons now spent their childhood and adolescence. As we had hoped for, both were soon integrated well in school and got new friends easily. After 3 years, our nanny finally left for good to return to England, and we were very fortunate to find an extremely nice and efficient housekeeper from a nearby village who stayed with us for the next 20 years. Bielefeld offered many interesting things for growing boys, like football and tennis grounds and an excellent music school. Both our sons learned to play an instrument, Arne the oboe, Wolf the transverse flute. Brigitte took up some music again on a small harpsichord (spinet) that Harald had given her as a present for Arne’s birth. The three of us even managed to perform in the Bielefeld Music School. Thus, it seems the sons inherited some of their mother’s interest in and talent for music, but, alas, none of them was as talented in painting or drawing. We had sent Arne to a conservative Bielefeld high school with Latin as the first foreign language, the same school his paternal great grandfather and grandfather had attended (as had Karl Lohmann, discoverer of ATP). When Wolf, being 3 years younger and a great admirer of his elder brother, was asked whether he would also want to join the same school, he said: ‘‘Yes. Arne is there, and there is a vending machine for hot chocolate.’’ So, this topic was settled. The school period was uneventful, and both boys were not too unhappy with their teachers. At home, we had lab parties and the house was big enough to accommodate guests of the institute. For years, there was a fixed ritual every night with bedside stories after dinner, and sometimes, the scientist guests would participate in this rather private affair. There were picnic excursions to the nearby Teutoburger Wald on weekends, and summer vacations. Twice we took the boys to the US, with stays in New York, Boston, and Woods Hole, and Wolf spent 6 months with the Sangers, to attend

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a high school close to Philadelphia. Both our boys finished high school in Bielefeld.

HJ: ‘‘Where is Bielefeld?’’ – From Mouse to Man Back to our professional life: on the one hand, it was fun to build the institute from scratch, on the other it was not easy to transfer collaborators from Heidelberg to Bielefeld. Using color photographs of the beautiful Westphalian landscape (the ‘‘tundra’’) in the light of late summer, I had convinced two diploma students to join me and to start their PhD theses with me at Bielefeld University. I took up mouse genetics by converting one half of the secretary’s position into that of an animal caretaker and changed an instrument room into a mouse room. Later I adapted a climate chamber, which was supposed to house experimental plants, into a room for nude mice that I needed for transplantation experiments. Although all of this was improvised, I could now start with more extensive programs on neuromuscular differentiation and mouse models for neuromuscular disease. Personally, we had a new experience: Whenever we attended a conference, people would stare at our name badges and ask ‘‘Where is Bielefeld?’’ – of course, nobody had ever asked ‘‘Where is Heidelberg?’’ I finally took to the habit of drawing a tiny map on my badge to show that this place, then totally unknown to cell biologists abroad, is about halfway between Cologne and Hanover. To people from Southern Germany I had to explain that it was neither situated in the industrial Ruhr region nor in the flat lowlands of Northern Germany. Later we sometimes met Englishmen who said ‘‘Bielefeld? Of course we know that place: we had a great time there’’ – these were young men who had served in the (so-called) ‘‘Rhine Army’’ (Bielefeld is located 250 km east of the Rhine), and we did not ask what exactly had happened during those ‘‘great times.’’ Obviously, it was now our task to put Bielefeld on the map in our research fields, cell and developmental biology. At a rather informal mammalian genetics meeting in London, in 1978, the biochemist David Watts from Guy’s Hospital in London showed a movie of a mouse suffering from a hereditary neuromuscular disease termed ‘‘arrested development of righting response’’ (gene symbol adr, phenotype ADR). The name was


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supposed to describe the behavior of the (homozygous) mutant in comparison to the wild type: When placed on its back a wild type mouse turns to its feet faster than one can take a photograph, whereas the ADR mouse would stretch and freeze in its position for a few seconds. David and Rosemary Watts had studied the enzymes in ADR muscle and found drastic changes in the levels of many enzyme activities in comparison to wild type control muscle. How could one explain that so many changes were caused by a single gene mutation? I asked David Watts whether I could have a founder pair of this mutant and he kindly suggested I should visit him at Guy’s Hospital right after the meeting and get the mice. The first attempt to get the stock carrying the adr gene to Bielefeld ended in a most unpleasant encounter at Heathrow Airport. These were the times when security checks had just started, in Britain because of the terrorist activities of the IRA. My mouse family from Guy’s Hospital was in a biscuit tin in a shopping bag. But I was forced to have this X-rayed and everybody could see the spines of the animals moving around. ‘‘Oh fish’’ a lady shouted. This caused a great turmoil and I was accused of trying to illegally introduce mice into Britain – a deadly sin! I tried to explain that I wished to export mice, only to be asked by the enraged lady officer ‘‘Don’t you have mice in Germany?’’ I nearly missed my plane and, despite all my arguments and offers to pay any airfreight charges, the mice were confiscated and probably destroyed. I finally received breeders carrying the adr mutation in 1982, when a postdoc from Britain brought them to the University of Bielefeld, without any problems, in his moving van. It should be mentioned that the adr mutation had arisen in, and was kept on, the A2G inbred background. A2G mice are graceful white mice with a silky fur. I did not know that this strain was famous for its inborn resistance towards influenza virus infection (see contribution by Lindenmann, Vol. 44 of this series). The chair I had been offered was originally designated in a somewhat old-fashioned manner ‘‘Entwicklungsphysiologie’’ (Physiology of development). Anticipating that I would use quite a bit of biochemistry and a lot of genetics, I chose a more general name ‘‘Entwicklungsbiologie’’ (Developmental Biology); finally, when I realized that we would work on disease models, I extended the name to ‘‘Developmental Biology and Molecular Pathology.’’ In the first Bielefeld years, my research drifted towards muscle

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research and only later, neurodegeneration became a prime topic again. In the following report on these years, I will stress what cannot be read in the publications, namely the anecdotal aspects, errors, failures, and thoughts. Ingo Stuhlfauth, a postdoc who had come along from Heidelberg, was very fond of 2D gel-electrophoretic separations of polypeptides (the O’Farrel technique, nowadays the first step of ‘‘proteomics’’) – the larger the gel slabs, the better. Although this was not the most brilliant approach, we thought we might analyze, with this technique, the muscles of all the neuromuscular mouse mutants available in our lab. On 2D gel electrophoresis, muscle yields a beautifully characteristic and reproducible reference pattern dominated by the limited number of abundant myofibrillar (‘‘contractile’’) proteins. We found that the polypeptide pattern of muscle from motor endplate diseased mice, which lived for only 2 weeks, was not significantly different from the controls. In contrast, in muscle of ADR mice, which suffered only from occasional cramp-like attacks and lived several months, it was immediately evident that a low molecular mass polypeptide was missing or drastically reduced. It did not take long to find out that this was parvalbumin, a highly soluble calcium binding protein abundant in wild type mouse muscle. Final proof came from an immunoblot with a polyclonal antibody against parvalbumin, which we obtained from Claus Heizmann in Switzerland [49]. Claus Heizmann was in search of a function of his pet protein parvalbumin and was happy that in the ADR mouse (genotype adr/adr) the lack of the calcium buffering protein seemed to cause a problem with muscle relaxation. This appeared plausible, as contraction and relaxation are regulated by the cytosolic Ca2þ concentration, and parvalbumin was known to be a cytosolic Ca2þ buffer. The simple assumption that in the homozygous state, the adr mutation might directly cause a parvalbumin deficiency became unlikely when we found that the adr gene was located on mouse Chr 6, and the parvalbumin gene Pva on Chr 15. At that time, chromosomal mapping was considered boring and useless by many scientists. The case of parvalbumin reduction in ADR muscle showed that genetic mapping data can be very useful to once and forever exclude false hypotheses based on physiological or biochemical findings. A normal concentration of parvalbumin in the Punkinje cells in the brain of ADR mice, as shown by immuno histochemistry, was


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an additional argument against a primary defect in the Pva gene as a cause of the ADR disease. What was the physiological nature of the after-contractions of ADR muscle? Was it an inability to immediately relax, as Heizmann liked to think? This was unlikely because in the soleus muscle, the force amplitude of the after-contraction was often higher than that of the foregoing, scheduled contraction [50]. Gerhard Mehrke, a PhD student who had moved with us from Heidelberg, studied the patterns of action potentials in ADR myotubes (immature muscle fibers) in cell culture but did not find any difference between ADR and wild type. To end his desperate search, I suggested: ‘‘Why don’t you have a look at mature muscle taken from the animal?’’ And indeed: within 1 hour of intracellular recording, he obtained a ‘‘run’’ of spontaneous high frequency action potentials, as typical for the muscle disease myotonia. We should have done this experiment before sending the parvalbumin manuscript to PNAS! Finally, the PhD student Heinrich Brinkmeier (now professor of physiology in Greifswald at the shore of the Baltic Sea) showed that the hyperexcitability of ADR muscle fibers was due to a drastically reduced chloride conductance [51] (Figure 6A). Using a drug that suppresses sodium channels and thereby symptomatically cures myotonia resulted in a restoration of almost normal levels of parvalbumin in genetically myotonic muscle [52]. From these findings it followed that the reduction of parvalbumin concentration in muscle was a consequence, rather than the cause, of the physiological abnormality of ADR muscle. Not only can myotonia be elicited by drugs that block chloride channels ‘‘from the outside,’’ the activity of chloride channels is also subject of regulation ‘‘from the inside’’: Heinrich Brinkmeier in my group discovered that activators of protein kinase C acutely suppress chloride conductance and thus cause myotonia in isolated muscle preparations [53]. The group of Richard Sidman at Harvard University had described a mouse mutant suffering from muscle stiffness. They had named the mutation myotonia (mto), because electromyography (extracellular recording with a needle stuck into muscle) had shown ‘‘myotonic runs.’’ I got hold of that mutant and we showed that the mutation mto was allelic to adr [54]. Of course, mto was a more appropriate name, but adr was older so mto was (provisionally) designated as an allele of adr. When I visited the

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Sidman laboratory in Boston, Paul Neumann, an expert on neuromuscular diseases, and Richard Sidman were a bit irritated that the American mutant had to adopt the gene designation of the British one, but I could not help it. Whatever the name, it was now clear that the ADR and MTO mice were valid phenotypic models (‘‘phenocopies’’) for human myotonia. Where they also genetic models (‘‘genocopies’’)? Our mapping experiments and the allelism to mto placed the adr locus on Chr 6, within a group of loci which in humans would map to the terminal long arm of Chr 7. It was extremely encouraging that a Canadian group had used the information on mouse myotonia and mapped the human myotonia gene to that very chromosomal location [55]. This indicated that the ADR mouse would not only be a phenocopy but very likely also a genocopy of human myotonia. If this were true, identifying the gene in the mouse would also identify the (or a) myotonia gene in humans. In the neurological literature a dominant, ‘‘Thomsen,’’ and a recessive, ‘‘Becker,’’ form of inherited myotonia were known. Thomsen myotonia has a peculiar history: It was described in 1876 by the Northern German physician A.J. Thomsen, who himself suffered from the disease. In his report he came to the conclusion that myotonia is due to an abnormality of the nervous system.

Fig. 6. From physiology to molecular genetics, from mouse to man. (A) Wild type (WT) control to the left, myotonic ADR mutant to the right. Pathophysiology of the ADR mouse from top to bottom: Cramp-like stiffness after sudden movement; after-contraction of muscle upon tetanic stimulation (indicated by bold horizontal bar); action potentials of muscle fiber in response to a single stimulus: the wild type fiber responds with a single spike, the ADR fiber fires a high frequency series, a ‘‘myotonic run,’’ the cause of after-contractions. This hyper-excitability is caused by a drastically lowered chloride conductance GCl. From Ref. [57], with permission of the copyright holder, Walter de Gruyter. (B) Schematic representation of the protein domains of the muscular chloride channel with mutations leading to myotonia in man and mouse. Vertical rectangles indicate alpha helices, of which B to Q are within the sarcolemmal lipid double layer, but not all of them span the whole membrane thickness. This topology is based on the X-ray crystal structure of a bacterial chloride channel, as published by Dutzler and MacKinnon. Two mutations identified in myotonic mice are indicated by a ‘‘mouse.’’ Amino acid residues are given in one letter code, X ¼ stop codon, ‘‘ins’’ ¼ insertion leading to abnormal transcript splicing in the ADR mouse – this was the first identified myotonia mutation [60]. Adapted from Thomas Jentsch, with permission.

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Electrophysiological analyses on both human muscle and muscle from another animal model, the myotonic goat, had revealed that a lowered chloride conductance of the sarcolemma (the plasma membrane of muscle fiber) was the cause of the hyperexcitability of myotonic muscle. Drugs that block chloride channels can reversibly produce the symptoms of myotonia. These physiological results, though important, did not permit to decide whether myotonia was a primary muscle disease, a myopathy, or whether it was caused by an abnormality of the nervous system, that is ‘‘neurogenic’’ as Thomsen had thought, or by some humoral disturbance. We could decide between these three possibilities by cross-transplantation of genetically myotonic muscle on a wild type recipient and vice versa: The transplanted muscle which had gone through severe degeneration and regeneration including reinnervation had retained the phenotype of its origin, that is it behaved according to its own genotype, not to that of its host. Thus the expression of the myotonic phenotype was muscle-autonomous [56], myotonia was a myopathy. What was the molecular nature of the myotonia gene? Humoral and neurogenic mechanisms were excluded by the transplantation experiments, and it was likely that the myotonia gene was expressed in muscle, yet not necessarily exclusively. Since sarcolemmal chloride conductance was reduced in myotonic mice, goats, and human patients suffering from myotonia, a muscleexpressed chloride channel would have been a prime candidate, but other possibilities like changes in the lipid composition of the sarcolemma could not be excluded [57]. In the meantime, Thomas Jentsch of the Center of Molecular Neurobiology at Hamburg (ZMNH, now Max Delbru ¨ ck Center, Berlin) had published an article in Nature on the expression cloning (using a functional assay in the Xenopus oocyte) of a chloride channel in the electroplax of the electric ray Torpedo [58]. The electric organ of this electric fish is a transformed muscle from which the contractile apparatus has been ‘‘squeezed out,’’ leaving only stacks of membranes (enclosing thin sheets of cytoplasm) that act like a capacitor. This specialization implies a dramatic relative increase of mRNAs coding for membrane proteins including ion channels, and this had enabled the Jentsch group to identify a chloride channel (later on designated ClC-0) that turned out to be a member of a whole family of


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sequence-related chloride channels. Having heard of our myotonic mouse mutant, Thomas Jentsch showed up at a workshop of our Sonderforschungsbereich (Special Collaborative Program) SFB 223 in 1990 and asked me whether we should not collaborate on mouse myotonia. At that time, the Jentsch group, especially Klaus Steinmeyer, had gone a step further, and had identified, by homology cloning, a chloride channel in rat skeletal muscle, termed ClC-1 [59]. The mRNA of this channel was exclusively found in mature skeletal muscle. There were visits of the Jentsch group in Bielefeld and the resulting collaboration, during the last months of 1990 and the first of 1991, was incredibly swift and successful. There was a changed restriction pattern in the gene Clc1 in the ADR mouse and this molecular marker mapped to the same location on Chr 6 as did the myotonia disease gene. The most striking result was obtained by RNA (‘‘Northern’’) blotting: The Clc-1 mRNA from ADR muscle gave a totally disrupted pattern as compared to the wild type: Instead of one RNA species of 4 kb there were four sizes, two smaller and two larger than the wild type 4 kb. Such a dramatic disruption, presumably due to a faulty splicing process, made it very likely that these transcripts were non-functional. The Jentsch group sequenced the abnormal transcripts and showed that the aberrant splicing pattern was due to the insertion of a transposable Etn element in the adr allele [60]. We had been lucky to work with the ‘‘British’’ mouse myotonia, the adr allele, because the ‘‘US’’ allele, mto, had a single base exchange not visible by Northern blotting. Sequence analysis, however, showed that this single base change had introduced a stop codon resulting in a chain termination of the ClC-1 polypeptide at position 47 out of 993 [61]. Expectedly the ClC-1 protein was later on localized to the sarcolemma. In muscle of myotonic MTO (erroneously designated ADR in the publication [62] and ADR mice no ClC-1 antigen was detected in the sarcolemma. Taken together, these results unambiguously showed that the myotonia gene of the mouse is identical to the gene coding for the sarcolemmal chloride channel, adr/mto ¼ Clc1. I was relieved that we had not come up some lipid defect to explain the lowered sarcolemmal chloride conductance. Despite my brief training in Feodor Lynen’s institute at Munich I abhorred lipid metabolism as a research topic. Ion channels, on the other hand, are directly

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linked to electrophysiology, a field for which we were well prepared. On the single channel level, the electrophysiological characterization of the sarcolemmal chloride channel turned out to be difficult [63], but the relation between chloride channel expression, sarcolemmal chloride conductance, and hyperexcitability during development of the mouse was clarified [64,65]. Subsequently, the research projects of the Jentsch group and ours diverged: The Bielefeld group concentrated on developmental and regulatory aspects of the muscular chloride channel, whereas the Jentsch group, in collaboration with a human genetics group at Marburg (Manuela Koch and K.H. Grzeschik), hunted for mutations in the ClC-1 gene (designated CLCN1) of human myotonic patients. The map position of the human myotonia gene had already indicated that it was homologous to the mouse Clc1 gene. Within a few months a paper appeared in Science describing all kinds of mutations in the human Thomsen/ Becker gene CLCN1, point mutations leading to amino acid replacements, to stop codons, microdeletions, and insertions. Fortunately, it was possible to analyze a descendent of Dr Thomsen; the authentic dominant Thomsen mutation turned out to cause a proline to leucine exchange, P480L (reminding me of TMV-coat protein mutants) (Figure 6B) [66]. The Bielefeld group further analyzed the exon–intron structure structure of the mouse chloride channel 1 gene and compared it to the homologous human sequences: The primate coding sequence was found to have an insertion of nine nucleotides coding fore the amino acid sequence ‘‘APE’’ (alanine-proline-glutamic acid) [67]. Now, that the mechanism of myotonia is unraveled, one might ask: what were all the changes in enzyme levels that David and Rosemary Watts had observed? Obviously, these were secondary changes, long-term effects of the altered excitation pattern of myotonic muscle. This shifts the muscle fiber phenotype from fast glycolytic to fast oxidative fiber type, implying changes not only in the concentration of parvalbumin and in the phosphorylation state of myosin light chains (Figure 7, upper part) but also in a number of cytoplasmic and mitochondrial enzymes as well as isoforms of myosin heavy chains (Figure 7, lower part). This phenomenon, ‘‘the plasticity of muscle fibers’’ [68], was the research field of Dirk Pette at Konstanz University who used chronic stimulation of muscle to achieve a shift in fiber type, according to the pattern of excitation. As a referee of our SFB he


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Fig. 7. Secondary effects of ADR myotonia. Upper part: 2D patterns of low mass muscle proteins. TM, tropomyosins; LC, myosin light chains; PV, parvalbumin. First dimension (horizontal) isoelectric focusing, basic left, acidic right; second dimension SDS electrophoresis. (A) Myotonic muscle with a very low level of PV, and equal levels of phosphorylated (black arrowhead) and non-phosphorylated (empty arrowhead) LC2. (B) Wild type control, showing a high concentration of PV and most of the LC2 phosphorylated. From Ref. [52], with permission of the copyright holder, Wiley-Blackwell. Lower part: Scheme of the regulation of gene expression in muscle by the excitation pattern, as dependent on the muscular chloride channel gene, Ccl1. The excitation pattern in myotonic muscle transforms glycolytic fast fibers into oxidative fast fibers. This implies changes in the enzymes of energy metabolism, of myosin heavy chain (MyHC) isoforms, parvalbumin level, and the level of Clc-1 mRNA itself. From Ref. [72], with permission of the copyright holder, Elsevier.

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used to say: ‘‘The ADR mouse is a wonderful model of our stimulation experiments,’’ but my version was ‘‘Dirk Pette’s stimulation experiments provide a nice model of myotonic muscle.’’ Not only does myotonia influence, via the excitation pattern, mRNA levels of myogenic factors and, probably indirectly, levels of all proteins characteristic for muscle fiber type [50,52,69,70], but also the level of ClC-1 mRNA and thereby by the function of its own protein product [71], leading to a closed loop of relation between excitability and gene expression in muscle [72] (Figure 7, lower part). Interestingly, there is a post-transcriptional dosage compensation, ensuring that the sarcolemmal chloride conductance is equal (and typical for the fiber type) whether there is a full dosage of ClC-1 mRNA (as in the homozygous wild type) or half the concentration as in heterozygous þ/adr animals [73]. In conclusion, the myotonia story was one of the few examples where a human disease was clarified on the basis of a mouse model, which turned out to be a genocopy and a phenocopy. However, myotonia is a relatively rare disease which is not life threatening, and it can be treated with drugs that suppress the hyperexitability of the sarcolemma (via suppression of sodium channel activity). As a disease model, the ADR mouse made it to the southernmost university in Europe: Diana Conte Camerino at the pharmacology department of the University of Bari, Italy, used it, in collaboration with organic chemists, to test all kinds of organic compounds on their ability to relieve muscle from myotonic symptoms [74]. All the work on neuromuscular diseases carried out at Bielefeld University had been supported by the DFG. Thus I found it appropriate (already prior to the identification of the gene) to write an article in the popular DFG journal ‘‘Forschung’’ (Research); for this I provided a montage showing a mouse at the moment of a myotonic cramp, the force trace with typical after-contractions and a trace of intracellular recording from a muscle fiber showing a ‘‘myotonic run’’ and the figures for sarcolemmal chloride conductance (like in Figure 6A); but the DFG would not print it, being afraid that it might provoke a reaction from animal-rights activists. Rather, they preferred a totally uninformative photograph of cultured myotubes. Myotonia cannot be studied in myotubes, but cell cultures were politically correct. Likewise, for the cover of the issue of Nature containing


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our adr ¼ Clc1 paper I had suggested a photograph of a cramping ADR mouse in front of the sequence showing the reason for the cramp: the insertional mutation in the Clc1 gene. Although graphically attractive (in my opinion) this was not selected for the cover, and I presume for the same reason as my figure for the DFG journal was unacceptable.

BMJ: Making the Best of it: Bielefeld University In the beginning, my scientific life at the University was not easy. I was spoiled from my time at EMBL, and I found it quite difficult to adjust to my new situation. In Bielefeld, the University was the only academic institution around, so I had to find a job there. Harald sacrificed one position as a ‘‘Wissenschaftlicher Mitarbeiter’’ (scientific coworker) of his institute for me, which by standing and salary was not more than a postdoctoral position, and the contract was also restricted to 5 years. This was of course in no way comparable to what I was used to. In addition, Harald provided me with one of his positions for a technician, sufficient lab space and unlimited use of all his equipment for my projects, but I had no financial means from the Faculty to employ postdocs or PhD students. Being employed on a subaltern, temporary position, I was not entitled to order anything, and was reprimanded by the dean of the Faculty of Biology that I was also not entitled to invite guests for seminars. Even worse was that I was not at all well accepted by the Faculty – most of the professors made it very clear to me that I was not appointed like Harald but was simply the ‘‘wife who moved along.’’ It did not seem to matter what my former contribution to science and teaching, or my standing in the international community of cell biology was, and whether now I could add something useful to these topics within the Faculty. Looking back, I believe that those faculty members who gave me a hard time were not really hostile, but were simply helpless in how to handle this situation, as it was unprecedented. In the meantime, times have changed and today, I would probably encounter much more understanding for my position. One solution for the problems of couples working in the same faculty is certainly the possibility of ‘‘tenure track’’ positions for the party not officially appointed. These should turn into ‘‘tenure’’

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after a positive external evaluation. When we moved to Bielefeld, such positions were not available, and lastly, this was one reason for me to move to Braunschweig later on and commute to be able to see my husband at least on weekends. I started to work in Bielefeld anyway. Due to generous support by the DFG, the Stiftung Volkswagen, the Stiftung Boehringer Ingelheim, and later the ‘‘Fonds der Chemischen Industrie,’’ I obtained sufficient means to establish a small group, mainly with Diploma and PhD students. Fortunately, some of the connections I had established when at EMBL still existed. For example, our studies with Frans Jennekens Utrecht on the pathological conditions of Nemaline Myopathy which we had started at the EMBL continued in Bielefeld, and when I visited a group of clinicians years later at Helsinki, I was pleased to learn that they used the information we had gained on that disease in Heidelberg [37] and Bielefeld [75,76] as a reference for diagnosis. In studies on the dynamics of actin filament networks, which I had started üli from with the group of Gisela Haemmerli and Peter Stra Zu ¨ rich when I was still at EMBL, we established that vinculin participates in connecting the submembraneous actin filament network to the cell membrane in the dynamic lamellipodium of locomoting human tumor cells [77,78]. The Sangers, our friends from the Philadelphia Muscle Institute, came to visit us several times in 1982 and together we studied composition and organization of actin filaments in different types of non-muscle cells [79,80]. We established an ‘‘antibody factory’’ again and this time we not only raised antibodies in rabbits but rather focused on the production of monoclonal mouse antibodies. We used our precious tools for microinjection and various immuno histochemistry techniques to continue what I had started previously: to characterize the domain composition and functional roles of various cytoskeletal proteins. For the generation of monoclonal antibodies, I got invaluable advice from Gu ¨ nter Gerisch, MPI Munich, whom I knew well from our overlapping stays in Tu ¨ bingen and Basel, and with whom I could also exchange opinions on the role of several cytoskeletal elements in cell motility of his ‘‘pet,’’ the Dictyostelium amoeba, and vertebrate cells. At the University of Bielefeld, my position required a lot of teaching to biology students, and I was not too happy to spend my


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days instructing undergraduate students in light microscopy, in overcrowded classes, with old and insufficient equipment. However, when I had adapted to these conditions and got acquainted with graduate students in lectures and seminars, I learned to cherish one of the positive aspects of science at Bielefeld University: there were many good and highly motivated students to choose from, while in Heidelberg, one always had to face heavy competition for such students with the different groups from the University, Max-Planck Institutes and the German Cancer Research Center. With a group of motivated students and a battery of antibodies disrupting vital cellular functions when microinjected into cells, new collaborations were now easily established, with many groups in Germany, the US, or the UK. In the field of actin-binding proteins, our work resulted in two highlights: in a collaboration with the groups of Ulrich Scheer and Werner Franke (Heidelberg) we demonstrated that microinjection of one of our antibodies against actin inhibited very effectively the activity of RNA polymerase 2 at the lamp brush chromosomes of Xenopus laevis nuclei [81]. This study nicely complemented my earlier work on nuclear actin as a cofactor of this enzyme. Furthermore, in another collaboration with Gerhard Isenberg we found that microinjection of an actin-capping protein into the cytoplasm of cultured mammalian cells disrupted their actin cytoskeleton. The corresponding publication [82] was the first of a long series of studies by many groups investigating the role of cytoskeletal proteins when microinjected into the living cell. At that time, microinjection in my group was usually performed by a very talented PhD student, Annette Fu ¨ chtbauer. Her skills in microinjecting antibodies or other proteins into living cells were nonsurplussed. She managed to manually inject large numbers of cells within a very short time, so one could even obtain sufficient material for subsequent biochemical studies. Our catalog of specific, high affinity antibodies and Annette’s skills in microinjection also permitted us to study the effect of selectively disrupting the interaction between different cytoskeletal proteins. Thus, in a collaborative project with the group of Eva and Eckhard Mandelkow from Heidelberg, we studied the consequences of disrupting the tubulin–tubulin interactions within the microtubular system of cultured cells on cytoarchitecture by injecting high affinity antibodies against beta-tubulin [83]. Together with Annette, I also began to analyze

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focal adhesions, the attachment sites of tissue-forming cells (in more detail see Refs [84,85]). In these structures, matrix proteins are clustered and make contact with the substratum or neighboring cells on the extracellular side, while the ends of actin filaments are anchored to the intracellular face. The search for transmembrane proteins linking these two populations of structural proteins kept the cell biological community busy for decades, until the integrins were discovered as the missing links, with binding motifs to extracellular matrix proteins and to actin ligands at the inner surface of the cell. The year 1985 was of great improvement in Harald’s and my conditions for research. The DFG had called for people willing to engage in one of its large funding programs, the ‘‘Sonderforschungsbereiche’’ (SFBs, Special Collaborative Programs). Applications required the involvement of the Bielefeld University as a responsible body, and the integrated approach of at least 15 different projects under a common research theme, with subdivisions. The grant application itself required writing a voluminous book of several hundred pages, demonstrating previous achievements of all project leaders and outlining the future research plan in great detail. Positions for PhD students, technicians, postdocs as well as money for equipment and consumables could be asked for. Harald, myself, and Harald Tschesche, the head of the biochemistry department, decided to apply for such a funding package, which, when granted, should relieve our financial worries for up to 15 years, and we had the full consent and encouragement of the Rector of the University, Karl Peter Grotemeyer. I was elected as the designated speaker of this enterprise coined ‘‘Pathomechanisms of Cellular Interactions.’’ On the site visit, which followed our application, we could convince the referees of the quality and novelty of our program, based on an intimate cooperation between groups from the Biological, Chemical and Biotechnological Faculties of the Bielefeld University, and two groups from the Medical Faculty of the University of Mu ¨ nster. We were granted most of what we had asked for. I was particularly happy: one especially positive result was that we were granted a specific ‘‘service project’’ to raise antibodies for all the groups within the SFB who were to benefit from custom-made tools for their own research. This implied that I finally had a position for my own technician working with me on antibody production and characterization,


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and indeed, in the future, I was lucky to find a string of talented and highly motivated young ladies for this position. Most enjoyable and satisfactory for me personally was the conclusion of the referees that my position as a scientific co-worker on a temporal contract in Harald’s group was not adequate for the speaker of such a program, and Rector Grotemeyer managed to obtain a position for me as Associate Professor from the Ministry of Science of North Rhine-Westphalia. I was appointed in 1986. Finally, my efforts at and for the University were greatly acknowledged, and I am still grateful to the late Karl Peter Grotemeyer for supporting me in every possible way during my stay in Bielefeld. Based on the Rector’s high estimate of my achievements under adverse conditions, the City of Bielefeld, in an attempt to strengthen the ties to its rather new University, awarded me its ‘‘Kulturpreis’’ (award of culture) in 1987. In 1985, I organized the third Meeting of the European Cytoskeletal Forum in Bielefeld, together with Harald, who also provided the relevant graphics: the pattern of rafters in the coatof-arms of the City of Bielefeld appeared as the cytoskeleton of a fibroblast on the conference booklet (Figure 8). We were especially pleased to welcome a large number of participants from Eastern Europe, for whom we had obtained visa. Also in 1985, I was elected Vice President of the German Society for Cell Biology, and later on served as a member on its Advisory Board. In 1989, we organized the Annual Meeting of this society in Bielefeld (Figure 9). This was a great success, with more than 800 people attending the conference which comprised symposia, poster presentations, job market, and industry exhibition, sponsored by the participating companies. The large University hall, with many auditoria extending from it, served this enterprise very well.

HJ: Cytoskeletal Diseases of Muscle In addition to hereditary muscle diseases caused by abnormalities in ion channels (for which the ugly name ‘‘channelopathies’’ came into use) there are others due to defects in structural proteins, especially components of the sarcomere, the unit of myofibrils in skeletal and cardiac muscle, and of structural proteins responsible for anchoring the sarcomeres to the surrounding

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Fig. 8. Third Meeting of the European Cytoskeletal Club. Program Cover, a cell with a cytoskeleton formed by rafters from the coat of arms of the City of Bielefeld, which is located in the cell nucleus. Note cytoskeletal elements in the nucleus! (HJ, 1985).

cytoskeleton, to the sarcolemma and, via transmembrane proteins, to the extracellular matrix (ECM). In 1987 there was a breakthrough in the genetic analysis of muscle diseases: The groups of Louis Kunkel (grandson of a scientist well known for his research on TMV!) at Harvard


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Fig. 9. Conference of the German Society for Cell Biology, Bielefeld, 1989. Poster, HJ, 1988. A large fibroblastic cell, again with the Bielefeld rafters forming the cytoskeleton. The figures symbolize the titles of the sessions, for example cyclist ¼ cell cycle; Rodin’s thinker (in front of the Bielefeld art museum) ¼ neurons; bus ¼ transport; crab ¼ cancer; smoking weaver (a monument in the old town of Bielefeld) ¼ inflammation; football hitting cell surface ¼ peptide hormone (Wolf-Georg Forssmann, who presented his work on peptide hormones, is an enthusiastic football player).

University and of Ronald G. Worton at Toronto had succeeded in cloning the human X-linked Duchenne Muscular Dystrophy (DMD) gene. The product of the largest human gene is a large cytoskeletal protein (but not the largest protein, which is titin, see below), which was termed ‘‘dystrophin.’’ In contrast to the relatively mild disease myotonia, DMD is a nightmare. It drastically reduces the life span of affected boys, who carry the mutation on their single X chromosome. Female carriers, with one

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normal and one affected X chromosome, display only very mild symptoms. These are due to the inactivation in each body cell of one of the two X chromosomes, resulting in a mosaic condition in females. Due to cell fusion during the formation of muscle fibers and the dominance of the wild type cell nuclei, the dystrophic phenotype is largely suppressed. Tragedy starts when mothers transmit their mutated X chromosome to a boy. In a textbook on neurology by Victor Dubowitz there is a photograph of three brothers afflicted by DMD, demonstrating the progression of muscle wasting with age. One can imagine the suffering of the youngest boy, seeing his elder brothers deteriorate and die, realizing that his was the same fate. The probability of giving birth to three affected boys is 1/8. Today at least the second and third case can be avoided by conventional prenatal or preimplantation diagnosis in conjunction with abortion or in vitro fertilization and selective reimplantation of the embryo, respectively. In 1984 Graham Bulfield of the Roslin Institute near Edinburgh discovered an X-linked muscular dystrophy in the mouse, which later turned out to have a nonsense mutation in the dystrophin gene. Thus, this MDX mouse is a genocopy of human Duchenne dystrophy. But MDX male mice reproduced and had a normal life expectancy. The same was true for homozygous females (DMD females in humans are extremely rare), so that there was no problem in establishing a breeding colony of MDX mice. Although juvenile muscle of MDX mice undergo a severe crisis with muscle fiber degeneration and regeneration, the adult mouse looked more ‘‘athletic’’ than control mice of the same strain, and its life span was not reduced. Could it be that very simple parameters play a role here, like absolute life span and absolute body mass? It seems that the pool of stem cells responsible for muscle repair, the satellite cells, is sufficient for the short life (about 2 years) of a mouse. Despite the fact that the MDX mouse, at the level of the whole organism, is not a phenocopy of DMD, it became one of the most widely used mouse models for disease. The rationale for using the MDX mouse was that it is a valid model at the cellular and the tissue level, where one can study the primary effects of dystrophin deficiency. Furthermore, in humans there are milder forms of muscular dystrophies caused by mutations in the dystrophin gene; they are collectively designated Becker Muscular Dystrophies (after the same Peter Emil Becker who had discovered the recessive form of myotonia).


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The advantage of a mouse model for progressive dystrophy is that one has a continuous supply of tissues and cells for years, all affected by the same mutation. Furthermore, all kinds of therapy, pharmacological, surgical (e.g. cell and tissue grafting) as well as gene therapy can be tried. Despite research going on in many laboratories, the mechanism by which dystrophin deficiency leads to the death of muscle fibers remained enigmatic. Could this ‘‘mechanism’’ be mechanics? The dystrophin molecule seemed to have some function in stabilizing the connection between the intracellular cytoskeleton of the muscle fiber and the ECM. It was reported that in human dystrophic muscle, fibers with large diameters were more susceptible to damage than those with small diameters. This was reminiscent of the engineer’s calculation that relates maximal tolerance to pressure to the diameter of a cylindric steam kettle (at given strength of its wall). I proposed to Andre Menke, then a diploma student in my lab, to test the mechanical stability of dystrophin deficient muscle fibers. Two types of muscle cells were used: isolated mature fibers from mouse toe muscles (as these are very small, they can be isolated intact by enzymatic digestion) and cultured myotubes that were prepared from satellite cells. The idea was that in both, dissociated muscle fibers recovered in culture and myotubes, which were surrounded by a minimal ECM (which can be demonstrated by staining for laminin), dystrophin contributed to their osmotic stability by connecting the cytoskeleton to the ECM. The inexpensive method for testing cellular stability was to apply osmotic stress by suddenly lowering the osmolality of the medium and score for burst cells. Andre Menke found that dystrophin-deficient muscle cells were more susceptible to osmotic stress than wild type cells [86]. In his PhD thesis, Andre Menke extended the findings on osmotic lability to human myotubes grown from biopsies, using influx of horseradish peroxidase to monitor membrane rupture [87]. The membrane biophysicist Otto Hutter and his group at Glasgow University, with whom I discussed these results, had measured membrane stability with a suction pipette and found only minimal differences between myotubes with and without dystrophin. However, the relation of these measurements to whole cell behavior remained unclear. It was obvious to check, by performing histology on a continuously used skeletal muscle, the diaphragm, of the MDX mouse, whether mechanical stress

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in situ would produce severe fiber degeneration; the student whom I had asked to look for this effect had private problems and did not find anything, although the effects are dramatic. A group in Philadelphia used forced excentric contractions on MDX and control mouse muscle and showed convincingly, and in contrast to a British group, that dystrophin deficient muscle is damaged to a higher degree than wild type control muscle. We continued our research on the molecular biology and biochemistry of MDX mouse during more recent years in collaboration with groups at the Weizmann Institute in Rehovot, Israel [88,89], a group in Estonia, and in Maynooth, Ireland [90]. Unfortunately, despite worldwide extensive experimental efforts, using cell transplantation and somatic gene transfer, there is still no cure for boys affected with DMD. However, it is now possible in families at risk to determine by prenatal diagnosis whether the embryo carries a known dystrophin mutation. Another mouse mutant that we kept was called ‘‘Muscular dystrophy with myositis’’ (phenotype MDM, gene mdm). Names of mouse mutants are often misnomers – like ‘‘arrested development of righting response’’ for myotonia. In MDM muscle, electron microscopy performed by Peter Heimann in our lab showed an increased number of satellite cells but no sign of inflammation, that is there was no myositis. One day, Klaus Weber from the MPI for Biophysical Chemistry at Go¨ttingen called me, suggesting that the MDM mouse might be affected in the titin gene – a remarkable suggestion, considering that he once had said to me: ‘‘Muscle is boring.’’ Titin is a giant protein, in fact with 3,000 amino acids the longest known polypeptide. In the sarcomere, it stretches from the Z-line to the M-line, that is the middle of the sarcomere. This means that its length must follow the length changes of the sarcomere during contraction and relaxation. Much research work has been invested into this giant molecule which is now viewed as a molecular spring, a safeguard against rupture of the sarcomere, and a stretch sensor and signaling molecule. We chromosomally mapped the titin gene in collaboration with the group of Siegfried Labeit, then at Heidelberg. The genes for titin and nebulin (another giant polypeptide of the myofibril) and the mdm gene were all located in the same region of Chr 2 of the mouse [91]. Siegfried Labeit, a restless worker, had become a renowned expert on titin, and he arranged for a European Community Project on titin. In this


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international project, the Bielefeld group was to do comparative analyses on titin genes in different vertebrate species and to follow up Klaus Weber’s titin hypothesis for the mdm mutation. First we planned to compare, by Southern blotting, other rodents with the mouse. To this end, Christian Witt, a diploma student, bought a Chinese hamster in a pet shop, but his girlfriend threatened to leave him should he sacrifice this sweet animal. To avoid trouble, I proposed to use, in place of animal tissue as a source of DNA, a permanent cell line, baby hamster kidney cells, BHK21. The BHK21 cell line can be induced by media conditions or by transfection with myogenic transcription factors to transform into a muscle-like cells containing myofibrils with Z-lines and correctly located titin filaments. We got a BHK21 clone from a colleague at Bielefeld University. The cDNA probe Christian Witt used first was from a mouse sequence coding for a segment of titin that was close to the anchoring point of the titin filament to the Z-line. Although mouse and hamster are sufficiently related, he did not get any hybridization signal and it turned out that by chance we had received a particular clone of BHK21, later designated BHK21-BI, which had lost a segment of its titin gene [92]. It was surprising that the loss of that gene region, probably due to some genomic mishap, had become homozygous. Whereas this condition would have been a lethal for the animal (see below), the integrity of titin, a muscle specific protein, was unimportant for these cells growing in culture. A control line freshly obtained from the American Type Culture Collection still had the complete titin gene. Upon induction of a muscle-like phenotype, the sub line BHK21-BI, with a mutated titin gene, turned out to be unable to form myofibrils [93]. Thus, by serendipity we had found a means to prove that titin is essential for myofibril assembly. Christian Witt performed his PhD thesis on titin in Labeit’s group and later on turned into a renowned titin expert as well. What about the MDM mouse? Gregory Cox at the Jackson Laboratory had a good idea and enough breeding capacity for high resolution chromosomal mapping to show that the mdm mutation indeed affected the titin gene, as had been suggested by Klaus Weber. The good idea was to make use of the observation that cardiac muscle in the MDM mouse is normal, implying that a skeletal muscle specific exon might be deficient in the MDM mouse. With this assumption the search for a mutation could be

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concentrated on a small fraction of the gigantic length of titin coding sequence. Cox and his co-workers were able to show that a binding site for the protease calpain-3 in a skeletal muscle specific exon was disrupted by the mdm mutation. [94]. Calpain-3 is a relevant ligand because mutations in its gene lead to limb-girdle muscular dystrophy type 2A in humans. Desmin is a muscle specific protein of the intermediate filament system, which in skeletal muscle fibers wraps around the Z-disks and is thought to align the myofibrils in register. It is one of the earliest structural markers which can be identified during the embryonic development of cardiac and skeletal muscle. A knockout of the desmin gene had been reported to block the development of skeletal muscle by an American laboratory, and to severely reduce contractile strength by Denise Paulin’s laboratory in Paris [95]. However, homozygote knockout, desmin-deficient mice that are born and survive invalidated the claim by the American group. The French knockout strain had the convenient addition of the knock-in of a ‘‘nuclear LacZ’’ gene, coding for bacterial beta-galactosidase with a mammalian nuclear import signal attached [96]. We were kindly provided with a stock of these desmin-deficient, beta-galactosidase transgenic mice. We thought for a few weeks that something was wrong with our PCR genotyping because all mice including those putatively homozygous for the knockout, moved around normally, without any sign of weakness. But finally it dawned on us that there was something wrong with the force measurements reported in the paper by the French group [95]. Although there were degenerative processes going on in the desmin-less skeletal muscle, we found that the contractile strength of non-degenerated desminless muscle fibers was close to normal. This shows how important it is to distinguish between acute, primary consequences of a mutation and long-term effects due to secondary degenerative processes. Unexpectedly, we found that the desmin deficiency in the mouse causes a defect in excitation–contraction coupling that was only evident during extended high frequency stimulation [97]. We attempted to publish these findings in renowned physiology journals but were denied acceptance, as we contradicted published work! As mice heterozygous for the desmin knockout are normal in muscle development and function, the nuclear LacZ gene provides a nice marker for cellular origin. We made use of this, in conjunction with a complementary jellyfish


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fluorescent green transgenic marker to study muscle stem cell migration after muscle transplantation [98]. From 1985 to 2000 most of the diploma and PhD students in my group were working on ion channels of muscle or on structural proteins of the contractile apparatus and their role in hereditary muscle diseases, whereas only a few worked on neurological mouse diseases [99] and formal [100] and molecular [101] genetics. Thus we were considered a ‘‘muscle group’’ by most of our colleagues. A linguistic remark: The word ‘‘muscle’’ Lat. musculus) means ‘‘little mouse.’’ Most Indo-Germanic languages use words for ‘‘mouse’’ derived from the same linguistic root (Latin mus; Vignette Figure 10). In French, Italian, and Spanish this rule was broken as a result of some zoological confusion, involving shrew, mole (?), and rat. Due to the work on muscle, the Bielefeld group became part of the international (informal) muscle network. Our group participated in the European meetings in muscle research, which started out under the name of ‘‘European Muscle Club.’’ This name was later changed into ‘‘European Muscle Conference’’ because the original name had caused some irritations with the internal revenue officers when participants wanted to deduce their travel expenses from taxation. At these conferences it was easy to find new and meet old friends, and I became a member of the board of the European Muscle Club, with Marcus Schaub, a renowned Swiss muscle biochemist and a person with a good sense of humor, presiding. Finally, it was our turn to arrange a European Muscle Conference. ‘‘European’’ was not defined as a geographical but

Fig. 10.

Vignette: Muscle means little mouse (HJ, 1990).

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rather as a cultural term, and thus it included Israel. Together with my colleague Horst Hinssen, an expert on smooth muscle, I organized a European Muscle Conference at Bielefeld University in 1992 (Figure 11). It comprised, in addition to the traditional topics of muscle structure, biochemistry, and molecular biology ‘‘Development’’ and ‘‘Ion Channels’’ as main symposia. Poster and program cover were designed by one of the organizers (HJ), and there was a fun performance on ‘‘Muscle Art,’’ in which scientist painters could show their products – with quite surprising results! (unfortunately the pun in the title of my ¨nnlicher Muskel vermessen’’ cannot be muscle painting ‘‘Ma translated into English; I donated the painting to Anna Wobus at Gatersleben). At the end of the intermissions I would call the participants to the lecture hall with a small but loud trumpet, instead of the usual and often inefficient clapping. There were many active participants from Eastern Germany, from Poland, and several from Russia. I had invited a historian of Bielefeld University to give a lecture on the fall of the iron curtain. Almost everybody in the audience acknowledged this lecture – with the exception of a few of the Russian participants in grey suits. With the advent of myogenic transcription factors skeletal muscle became an exciting subject for molecular cell biology, and EMBO workshops on muscle research were arranged, alternating with Keystone Conferences on the molecular biology of muscle. Of the EMBO conferences I found the one at Ein Gedi, a Kibutz at the West Bank of the Dead Sea especially impressive: The scientific program at the meeting was excellent and the view south from the large terrace is of biblical grandeur. One could observe ibexes grazing along their trails, hyraxes climbing in the shrubs, and workshop participants submersed in steaming hot sulfur brine or covered with the black mud from the Dead Sea. On the whole, my initial isolation as a newcomer in the field of neuromuscular development and its molecular pathology was quickly overcome, and this led to many contacts including collaborations, with colleagues in Russia, Poland, Israel and, of course, the United States. Whenever in the United States, I would visit the Sangers in Philadelphia and give a seminar there at the Muscle Institute of the University of Pennsylvania. During these years I was nominated a member of the scientific ¨mpfung der board of the ‘‘Deutsche Gesellschaft zur Beka Muskelkrankheiten,’’ DGBM (German Society for Fighting


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Fig. 11. XXI European Muscle Congress, Bielefeld, 1992. Program cover ¼ poster, HJ, 1992. Depicted is the university building ¼ myofibril, in front of the Teutoburger Wald ¼ endplate region of muscle fiber surface, with synapse and extracellular matrix; the church towers are patch clamp pipettes attached to a small muscle fiber (cloud) with membrane blebs caused by hypo-osmotic shock; to the left of the myofibril, is a trace of single channel recording. The sun is the seal of the University of Bielefeld.

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Muscle Diseases, later named ‘‘Gesellschaft fu ¨ r Muskelkranke,’’ DGM). This German society although boasting a long tradition, has never been as powerful as the American ‘‘Muscle Dystrophy Association’’ (MDA), and was soon overtaken in importance by the ‘‘Association Francaise contre les Myopathies’’ (AFM) and The´le´thon in France. I was invited to both their headquarters, of MDA in Tucson, Arizona (1996), and of the AFM in Evry near Paris (2006). On the whole, the American and the French societies acknowledged the use of animal models and the significance of basic research much more than the German Society for Muscle Diseases, in which the importance of research and fund raising for research was steadily declining, while it turned into a patient’s advisory organization. Because of our work in muscle regeneration, muscle reconstruction, and the migration of muscle stem cells I became a member of the Stem Cell Network North Rhine Westphalia, which was founded to counteract the legal problems of this research in Germany and promote the collaboration of Universities, Max-Planck Institutes, and Medical Institutes in this region. In 1993, Anna Starzinski-Powitz, Alfred Maelicke, and myself organized a nationwide research program of the DFG devoted to muscle research, of which I was the first speaker. It was nice to meet the most active research groups in the field from all over Germany, but I probably was not the most talented person when it came to organization or politics in science. This, however, did not deter Karl Peter Grotemeyer, mathematician and rector of the University of Bielefeld since its beginnings, to suggest me as candidate for the Prorector of Research and the Promotion of Young Scientists of the University of Bielefeld. After a bit of political skirmish I was elected and remained in that position until Grotemeyer retired. The tasks were interesting and not too time consuming. Wearing a tie at the sessions was obligatory, but this has changed with the successors of Karl Peter Grotemeyer.

BMJ: The last Bielefeld Years: Research and Science Politics The projects within the Special Collaborative Program (SFB) opened new venues for my own research. In a joint project with


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the group of Harald Tschesche, we studied the involvement of actin filament synthesis in granula secretion from leukocytes [102], and we analyzed the distribution and reorganization of several actin-binding proteins during platelet activation, with Jan Sixma from Utrecht and J. Hartwig from Harvard, USA [103]. During these times, observations by several groups suggested that within cells, cytoskeletal elements did not operate independently from each other but served as an integrated ‘‘super system’’ to execute the many different tasks connected with cell motility, like locomotion, adhesion, and intracellular transport of organelles. Thus, we, as many other groups of that time took up several studies to analyze certain aspects of such concerted actions of different cytoskeletal elements. In another joint SFB project involving the groups of Ju ¨ rgen Frey from the Biochemistry Department of the University of Bielefeld and Horst Robenek from the University of Mu ¨ nster, we followed uptake and fate of IgG receptors via clathrin-coated vesicles [104]. At that time, we had already shown, together with Milan Nermut from London, that focal adhesions are the hot spots of clustering and vivid uptake of such vesicles [105]. A few years later, we demonstrated that a well-known chaperone, hsp70, was intimately associated with clathrin-coated vesicles [106]. Subsequently, again with the group of Horst Robenek, we showed that one of our antibodies against hsp70, inhibited endocytosis after microinjection into tissue culture cells (Ref. [107] a finding hard to publish at that time, as the specific role of such a ubiquitous chaperone with pleiotropic functions in coated vesicle traffic was questioned). However, today it is well accepted that hsp70 is essential for the uncoating of clathrin-coated vesicles, and our anti-hsp70 is still in use in several laboratories in Europe, Australia, and the US. In the field of actin-binding proteins, we made progress in analyzing the molecular anatomy of vinculin, one of my pets. Again by using a set of monoclonal antibodies directed against different epitopes, we identified vinculin regions important for binding to actin and also to talin, another structural protein of cell attachment sites [108]. For non-muscle alpha-actinin, the other cell attachment protein which interested us, we made an unexpected discovery: this protein is widely expressed and highly conserved through evolution, as seen by data showing that alphaactinin from Dictyostelium can be incorporated into and used by the mammalian cytoskeleton. Furthermore, we found that this

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protein regulates its own synthesis. Blocking alpha-actinin mRNA by anti-sense probes resulted in an increase of endogenous synthesis, while elevating the cellular level by injecting the protein inhibited it [109]. This was the result of a collaboration with the groups of Angelika Noegel and Michael Schleicher from Munich and was very surprising at that time, as such a tight control of the cellular protein level had not been reported for actin-binding proteins, but was only recognized for tubulins. Today, it is well accepted that the intracellular level of many different actin-binding and other cytoskeletal proteins is controlled by such a mechanism. Since there were many bright students joining the group at that time, we could embark on two more topics. One concerned the role of actin filament severing proteins like gelsolin. This is a ubiquitous protein which, in mammals, comes in two flavors: it exists in an intracellular as well as in an extracellular variant, has a very high affinity for actin due to several binding sites and cuts actin filaments in a Caþþ-dependent manner. The biological role of gelsolin and the control of its activity remained a mystery for a long time. We reported that muscle phosphofructokinase and gelsolin colocalize at the thin filaments in sarcomeres of striated muscle [110], and characterized both proteins in a detailed immunological and biochemical study [111]. In several projects performed with Horst Hinssen and with Alan Weeds from the MRC in Cambridge, Elias Lazarides from Caltec and the Sangers [112–114], we analyzed the differential effect of gelsolin on actin filaments in various types of non-muscle and in differentiating muscle cells. These studies were elaborate and quite expensive with respect to the consumption of time, manpower, and money, but, in the end, we were not much the wiser, and the physiological role of gelsolin as an actin-severing protein is still not understood. The second topic I started to tackle at that time was the role of myosins in non-muscle cells and tissues. This was greatly stimulated by recent reports from several groups that the myosins known at that time had some unusual relatives. It turned out that the myosin family comprises, in addition to the ‘‘conventional’’ long, hexameric molecules capable to form filaments, numerous ‘‘unconventional’’ forms that are much shorter and do not polymerize. Furthermore, it was discovered that ‘‘conventional’’ myosins exist in an overwhelming number of isoforms, expressed in a species-, tissue-, and differentiation-dependent manner.


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It had been known for some time that non-muscle cells also express a conventional ‘‘non-muscle’’ myosin, but with the discovery of the mini-myosins being synthesized simultaneously in the same cell, it became necessary to understand the specific role of each myosin in the various types of cell motility and adhesion. In a collaboration with John Kendrick-Jones, MRC Cambridge, we found that microinjected rabbit antibodies, generated against a classical smooth muscle myosin from chicken gizzard, grossly interfered with actin filament bundles in epithelial and fibroblastic cells and thus with cell adhesion [115], while injection of antibodies against a mini-myosin from chicken brush border disturbed their cytoarchitecture and locomotory activity [116]. A PhD student, Barbara Zurek, generated a battery of monoclonal antibodies against different epitopes in pig brain myosin, and thus enabled an immunological comparison between conventional non-muscle myosins from pig brain and cultured cells from pig and other mammals. Barbara Barylko from Warsaw and, once more, the Sangers participated in two studies dealing with the role of different regions on the long myosin heavy chain in filament assembly [117] and for the dynamics of contractile actomyosin rings in cell division and epithelial sheet formation [117,118]. Interfering with a particular epitope on the myosin tail by injection of a corresponding antibody revealed that this motif is relevant for the contractile properties of submembraneous actomyosin networks [119]. Similarly, and using again our monoclonal, epitope-characterized anti-myosins, we found that specific motifs in the tail of the nonmuscle myosin heavy chain are important for unfolding the molecule [120]. This unfolding reaction is an essential prerequisite for filament formation of smooth muscle and non-muscle ‘‘conventional’’ myosins and for their subsequent interaction with actin filaments. The difficulties in obtaining high affinity antibodies against cytoskeletal antibodies prompted me to embark on a method that had been described in several immunological papers: production of antibodies in chicken. Indeed, we found that this was relatively easy: one had to immunize a chicken subcutaneously only twice, and antigen-reactive antibodies were secreted into the daily laid eggs. The titre was high, and the chicken deposited these antibodies in the egg yolk for about 1 year! The purification from yolk was easy, and we managed to obtain a particular anti-actin

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that recognized only a subpopulation of actin filaments, suggesting that its epitope was not accessible in other filament bundles [121]. So, we were quite happy with this approach, until we had to realize that avian antibodies are very difficult to handle in blots and immuno precipitation, and good antibodies against these chicken immuno globulins, as needed for indirect procedures, were not commercially available. And there was another rather dubious blessing: For several years, the students who were engaged in this project sent me dozens of fresh eggs and numerous bottles of egg liqueur, products of these ever industrious hens, for Christmas and my birthday! In one of the projects in the SFB, we had proposed to characterize physical properties of actin-binding proteins and actin-networks in solution, together with the physicochemistry group of Thomas Dorfmu ¨ ller in the Physicochemistry Department of the university. Using dynamic light scattering and photon correlation spectroscopy, we studied size and shape of G-actin, the subunit of actin filaments [122], and two isolated actin-binding proteins in solution, the small compactly folded profilin [123] and the asymmetrically shaped protein vinculin [123,124]. Furthermore, we found that the large actin filament cross linker filamin does not inhibit flexibility of the actin filaments, so that the actinfilamin network is highly dynamic [125]. The data we reported in these studies are still valid, but could not reach the resolution of more refined methods that are used today for analogous studies. Furthermore, I was a bit skeptical on how to relate these findings to what was really going on inside the living cell. In my last years in Bielefeld (1986–1993), several projects developed that I later on continued at Braunschweig: studies on the vasodilator stimulating protein, VASP, and on profilin. VASP is a phosphoprotein that had been studied for some years by the group of Ulrich Walter, University of Wu ¨ rzburg, as a protein involved in signaling during platelet activation. In a collaboration with the Walter group and using specific VASP antibodies, we identified VASP as a novel, prominent component of the actin cytoskeleton of non-muscle cells. It turned out to be another component of cell attachment sites but is also enriched in the leading lamella of locomoting cells, thus being an excellent candidate for transmitting extracellular signals to the dynamic submembraneous actin network [126]. VASP was to follow me on my way from Bielefeld to the last station of my scientific life.


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And there was another actin-binding protein that shared the same fate: the small, ubiquitously expressed and highly conserved actin-binding protein profilin. It was known that profilin bound with high affinity to G-actin and was considered to be responsible for keeping approximately half of the cellular G-actin bound in a complex, thus preventing it to polymerize to actin filaments, as these are favored by physiological conditions. With a polyclonal high-affinity antibody against profilin, Folma Buss, a PhD student in the Bielefeld laboratory, studied its expression in various species and tissues and compared the biochemical properties of isolated profilins [127]. She also localized profilin to the leading lamella of locomoting fibroblasts, thus supporting previous suggestions that it was involved in the dynamic regulation of actin filament networks in cells [128]. Together with Renata Dabrowska from Warsaw, we studied the dissociation of the profilin–actin (profilactin) complex by another Ca-regulated actin ligand, caldesmon [129]. We got further interested in profilin when Rudolf Valenta from the Medical University in Vienna informed us that he had identified profilin as a major allergen in tree and grass pollen, in vegetables and fruit. In collaboration with his group, we isolated plant and mammalian profilins, raised monoclonal antibodies highly specific for either species and subsequently compared their biochemical and immunological properties [130]. In addition to enjoying my rather well progressing research, I gradually became involved in an increasing number of ‘‘extramural activities’’ connected to science and science politics. I felt some pressure not to refuse when called to such offices, mainly for two reasons: First, I myself had greatly benefitted from grants awarded to me on the judgement of numerous referees, and thus I wanted to return their benevolence by serving now on similar bodies. Second, from the 1980s onward, Germany became sensitized to the fact that women were greatly underrepresented in higher academic positions, and wanted to remedy this situation by calling females in committees dealing with academic appointments or supporting specific programs for female scientists. To this day, it is not clear to me why females should prefer female scientists over males and not simply judge on qualifications other than sex, but in many federal states a law was established that required a certain proportion of females in every relevant committee. So, I served in numerous committees engaged in

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appointing university professors, and as a referee for the DFG, the Volkswagen Foundation, the Humboldt Foundation, the Studienstiftung des Deutschen Volkes, the Boehringer Ingelheim Foundation, and the Deutsche Krebshilfe (Mildred Scheel Stiftung). In 1991, I was appointed to the Minerva Fellowship Committee, a body of the Max Planck Society supporting an exchange program for young scientists between Israel and Germany. I served on his committee for 8 years and cherish many enjoyable encounters with Israeli scientists at the meetings that took place twice a year, alternating between Heidelberg and the Weizmann Institute in Rehovot.

HJ: Genes, Chromosomes, and Sick Neurons Ion channel and cytoskeletal diseases had kept us busy in the field of skeletal muscle research and enabled us to clarify a few issues in that field, but what about neurobiology, or neuromuscular interactions, my original field of interest? The mutation with which I had started my work in mouse neurobiology was motor endplate disease, med. After a tedious search that took several years, the gene was finally identified by the group of Miriam Meisler at the Department of Human Genetics of the University of Michigan Ann Arbor, with whom we later collaborated on the wobbler spinal muscular atrophy. I had received the wobbler mutant from Harvard University in 1979, while I was still at Heidelberg. Already at that time, the mutant had been widely used as a model for neurodegenerative diseases, especially spinal muscular atrophies (SMAs) and amyotrophic lateral sclerosis (ALS). In those years, the gene(s) responsible for hereditary SMAs had not yet been identified, and the same was true for the 20% of ALSs, the so-called ‘‘familial ALSs’’ (FALSs) that are hereditary. In the mouse, the wobbler gene (wr) had not yet been mapped. In the beginning of my work in mouse genetics, in 1975, I had used classical fur markers (like the caracul gene closely linked to med) and alloforms (usually electrophoretic variants) of enzymes. For the latter one had to set up agar or starch gel electrophoresis specifically for each marker enzyme and to buy corresponding sets of substrates. With the advent of DNA restriction fragment polymorphisms (RFLPs) as allelic markers, one would work with a limited set of restriction nucleases (then


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commercially available), 32P-labeled probes and spend lots of money on X-ray film. At least the technique was the same irrespective of the marker, but waiting for the exposures of the films in the deep freeze was time consuming. Nowadays all of this is done in a few hours, by using the polymerase chain reaction (PCR) on micro satellite or single nucleotide polymorphisms using customs made primers. I thought we might be as lucky with the wobbler (wr) gene as we were with the myotonia gene, that is that we could identify the gene by a combination of positional cloning and educated guesses with regard to functional candidates. This view turned out to be naı¨ve. As with muscle, prior to having identified the wr mutation, we analyzed its effects (in the homozygous condition) on the central nervous system and on spermiogenesis. In parallel, in 1987, Stephan Laage, then a PhD student, had carried out 2D gel electrophoresis on wobbler CNS and had found that in the regions affected by neurodegeneration, glial fibrillar protein (GFAP) was much more abundant than in control CNS. By immuno histochemistry he showed that this was due to both hypertrophy and hyperplasia of astrocytes [131], which had been overlooked in previous histological analyses of the wobbler CNS. Reactive astrogliosis is a well-known response of CNS tissue to any kind of insult. However, with the biochemical aspect of intercellular signaling, the histopathological processes in the wobbler CNS became a timely research project of our group 10 years later. Since its discovery in 1953, the wobbler mouse had been investigated, usually by scientists in medical institutions, with respect to the degeneration of motor neurons in brain stem and spinal cord. Its defect in sperm assembly was usually neglected, in many publications it was not even mentioned. For biologists, however, a defect in an extremely specialized pathway of cell differentiation, like spermatogenesis, is fascinating. In the doctoral thesis of Stephan Laage, there are 2D separations of radioactively labeled proteins and glycoproteins synthesized in testes of wobbler and control mice, but we did not find a reproducible difference that would have explained the pathological phenotype, round-headed spermatozoa. Normal mouse spermatozoa have an eagle beak shaped head in which the nucleus is capped by a large vesicle, the acrosome, which is formed by the fusion of small vesicles. Peter Heimann carried out a beautiful

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electron microscopic analysis on sperm maturation in wobbler testis and showed that the fusion of the precursor vesicles to yield the acrosome was blocked [132]. A prerequisite for positional cloning is conventional mapping to the chromosome. Klemens Kaupmann, in his PhD thesis set out to map the wr gene using the RFLP technique. In those days, I had close contacts with Jean-Louis Gueńet, a veterinarian by training and the mouse genetics expert of the Pasteur Institute. He would help us with some probes and advice. Gene mapping is a gamble: You might be lucky and find linkage with the correct chromosome in you first tests, or you might check one chromosome after the other (of the 20 mouse chromosomes) without finding linkage to the disease gene and only late hit the correct chromosome or chromosomal region. Klemens had to go through virtually all of the 19 autosomes before he discovered linkage with markers on Chr 11 [133]. After finishing his thesis Klemens joined a research laboratory at CIBA Geigy (now Novartis) in Basel, where he developed into a continuously productive scientist, publishing articles in the most renowned journals. As a reward for Klemens Kaupmann’s extensive mapping effort, we now had a panel of markers (in the form of DNA samples from individual F2 mice) spread over the mouse genome which made it easy to map other genes for which DNA probes were available. Due to known segments of homology (conserved synteny) between mouse and human chromosomes we could guess for a number of genes where the homologs should map in the human genome and this again could be the basis, on the principle of conserved synteny, of identifying genes responsible for human hereditary diseases. Usually we had to leave the identification of the human genes to others because there was no medical faculty at Bielefeld University to cooperate with. Earlier, using conserved synteny (on a somewhat shaky basis) I had been successful to predict the chromosomal localization of the human myotonia gene, but there seemed to be no human homolog to the wobbler disease of the mouse. Today, the mapping efforts of decades relating to single genes are only of historical interest, since the whole genome sequences of man and mouse are available, but at their time, especially in the 1990s they were very helpful in the analysis of hereditary diseases. The identification of the wr gene continued to be a tough issue. Thomas Schmitt-John, a young scientist who came from a


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non-university institution in Munich to our group, together with me, devoted much time and manpower of diploma and PhD students to the problem. All my clever suggestions for shortcuts failed, and it took the help of bioinformatics and the publication of the human and mouse genome sequences to finally identify the wr gene as Vps54. Like other genes involved in neurodegenerative diseases it is expressed in all tissues tested, albeit at a low level, and codes for a product that is involved in a general cellular process, namely retrograde vesicular transport of proteins. The protein is called vesicular protein sorting (factor) 54 (VPS54) and, together with VPS52 and VPS53, constitutes the so-called GARP (Golgi-associated retrograde protein) complex (Figure 12). The alteration of the VPS54 protein responsible for the wobbler phenotype, degeneration of motor-neurons and a spermiogenesis defect turned out to be a leucine to glutamine exchange, L967Q, introducing a hydrophilic in place of a hydrophobic residue; this change by itself would not have been exciting, but it turned out that the leucine residue was conserved in nine vertebrate species checked. The wobbler phenotype could be ‘‘cured’’ by introducing a wild type Vps54 transgene – we had performed this experiment together with Miriam Meisler’s group as additional proof that our identification of the wobbler gene was correct. Perhaps not unexpectedly, a knockout of the Vps54 gene caused embryonic lethality [134]. The nature of the gene product suggested at least a plausible mechanism for the pathology of the wobbler disease: Vesicular protein traffic is certainly important for neurons with long axons and for the formation of the acrosome of the sperm head, but nobody could have guessed this gene as a candidate by function. With the sequences of the Human Genome Project available, the localization of the human gene coding for VPS54 was confirmed, but up to now no human disease has been detected in which either VPS54 or another VPS (VPS52, VPS53) in the GARP complex (Figure 12A) is affected. There were several short popular articles, based on the press release of Bielefeld University, on the identification of the mutant gene in a mouse model for ALS, mentioning that we had used transgenic rescue to prove the identity of the genes wr and Vps54. Quite a number of patients and relatives of patients with ALS-like neurodegenerative diseases contacted us to ask whether now a gene therapy for ALS was available. Due to my experience in science journalism, I could at least clarify the issues muddled up

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in newspaper articles – of course with the consequence that I had to disappoint these desperate people. The most important catalyst for research is communication, both within and between research groups. After we had identified Vps54 as the gene affected in the wobbler mouse, we discovered that in 2002 a group of biochemical immunologists at the University of Go¨ttingen headed by Eberhard Gu ¨ nther (1941–2004) had published the structure and chromosomal localization of this gene in mouse and rat [135]. I suppose that the components of the GARP complex were only a sideline of the


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research interests of that group. Had they taken a quick look at a mouse gene catalog, they would have found Klemens Kaupmann’s mapping of the wr gene and could have suggested Vps54 as a positional candidate for wr. We could have started a BielefeldGo¨ttingen collaboration before 2002 and perhaps identified the wobbler gene nearly as quickly as we had identified the myotonia gene in collaboration with the Jentsch group in Hamburg. In any event, in 2005, after so many years of work with the wobbler mouse, I was relieved that the gene was finally identified before my regular work at the University of Bielefeld would end. Positional cloning depends to a great extent on good luck, but there is a correlation between gene function and the ease to guess a promising candidate gene. In knockout experiments (‘‘reverse genetics’’) the gene is known, but, despite the modest total number of mammalian genes, it often happens that the knockout of a given gene has no overt effect (in the present, rather ridiculous terminology: ‘‘The knockout mouse has no phenotype’’). At least, this disappointment cannot happen in a positional cloning project because it starts with a ‘‘phenotype,’’

Fig. 12. Neurodegeneration in the wobbler mouse. (A) Schematic representation of the vesicular traffic in an animal cell. In retrograde transport, the GARP complex, together with SNARES (hook-shaped molecules) mediates the tethering of vesicles to outer part of the Golgi apparatus. The GARP complex consists of three VPS (vesicular protein sorting) factors, one of which VPS54 (black), is the protein affected by the wobbler mutation in the mouse. Scheme by Carsten Drepper, Peter Heimann, and Harald Jockusch. (B) Pathways of proteolytic cleavage and signaling during neurodegeneration. Shown are parts of three cell types present in the CNS and the extracellular space containing ECM between them. Tumor necrosis factor alpha (TNF alpha) is released by ADAM17 (TNF alpha converting enzyme, TACE) from its membrane-bound precursor; it diffuses through the extracellular space and binds to its transmembrane receptor TNFR-I, thus activating intracellular JAK/STAT and NF-kappaB pathways; these in turn induce transcription of NF-kappaB responsive genes, one of which codes for ADAM8; thus TNF alpha stimulates the synthesis of membrane-bound ADAM8, which is activated by auto proteolysis and produces a soluble form which is able to degrade ECM. ADAM8 also cleaves membrane-bound TNFR-I to produce soluble TNFR-I, which diffuses into the extracellular space where it neutralizes soluble TNF alpha. This negative feedback reduces the cytotoxicity of TNF alpha during inflammatory processes in the brain and may thus rescue neurons from cell death. This model predicts that the wobbler neurodegeneration should be aggravated in ADAM8 knockout mice. Scheme by Jo¨rg W. Bartsch and Harald Jockusch.

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most often with a disease phenotype. The search for the responsible gene is still a challenge, despite the extensive sequence information on both mouse and man. Furthermore, once the gene is identified, one might find oneself in a field of cell biology and biochemistry in which one lacks expertise. Thus, none of the researchers engaged is the identification of the wobbler gene, be it at Bielefeld, Ann Arbor, or Braunschweig, had more than meagre textbook knowledge on retrograde vesicle transport. It is informative to compare the two cases of gene hunting for the ADR myotonia and the wobbler neurodegeneration: In the case of myotonia, the gene search was guided by a candidate by function: as there was a defect in excitability, an ion channel gene was a likely candidate, and of these a chloride channel gene was the favorite, and finally the winner. Neurodegeneration can have so many different causes that there was no convincing candidate by function, nobody had even thought of Vps54; thus the search had to progress via narrowing down the number of candidates by chromosomal position. Before the myotonia gene had been identified in the mouse, several alleles had been discovered. After 50 years of wobbler research, there was only one allele. What is the explanation for this difference? A functional knockout of the Clc1 gene, which is only expressed in mature skeletal muscle, is not lethal to the mouse but results in an easily recognizable disease phenotype, myotonia. In contrast, a knockout of the Vps54, the ubiquitously expressed gene affected in the wobbler mouse, causes embryonic lethality. It is possible that most mutations affecting the function of the protein VPS54 are lethal. The wr allele with a single amino acid exchange, although causing a severe disease, is apparently a comparatively mild, but very rare type of allele. This may explain why so far no additional allele in the mouse and no human homolog to the wobbler disease have been documented. Cases of embryonic lethality rarely become clinically manifest. Neuropediatricians do not check their male patients with spinal muscular atrophies for defects in spermiogenesis, the hallmark of the wobbler mutation. However, if one day a gene for spinal atrophy would map to Chr 2p13, then the VPS54 gene would be a hot candidate. In 1993, after Brigitte had left Bielefeld, I was elected speaker of the SFB 223. In 1996, after 12 years of successful work, this SFB came to an end. It took about 15 suggestions of titles and about


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the same number of sessions to outline the concept of a new SFB. My original aim as the coordinator was to replace about half of the project leaders with younger people. With the relatively small faculties for biology, biotechnology, and chemistry, though, it was difficult to come up with a thoroughly renewed crew. A big disadvantage of the academic scene at Bielefeld was (and still is) that there is no medical faculty at the university and no research institute outside the university nearby. Brigitte and I always had the policy to encourage young scientists to participate; but most of these had contracts for 6 years at the most and there were no tenure tracks in the German university system. Despite these drawbacks we managed to get another SFB granted by the DFG. This ‘‘SFB 549’’ started in 1998. I was elected speaker, probably because I had coordinated the planning and had the experience to moderate between biologists and chemists. In this SFB, the biochemist Harald Tschesche, though close to retirement, turned out one of the most active and productive project leaders, thus disproving my sociobiological hypothesis ‘‘younger is better.’’ The topic of this second SFB was ‘‘Processing of, and signalling by, extracellular macromolecules.’’ The idea was to investigate signaling processes dependent on the liberation of cell-surface bound signal sequences by lyases, proteases in case of polypeptides, and glycosidases in case of polysaccharides. In animals, the number of cases in which peptide signaling molecules are released by controlled proteolysis is ever increasing. In contrast, in plants, oligosaccharides play an important role, especially in the signaling between plant tissue and symbiotic bacteria. The groups of Alfred Pu ¨ hler and Karsten Niehaus (Genetics) were active in that field. Harald Tschesche and his group of the Biochemistry Department contributed with their expertise on extracellular metalloproteinases and their role in arthritic diseases and tissue alterations during pregnancy and birth. My own group did not have any experience with these enzymes, but we were fascinated by a biological problem which could perhaps be tackled by investigating extracellular proteolysis and signaling: neurodegeneration, astrogliosis, and tissue remodeling in the CNS of the wobbler mouse. But where to start? We faced a colorful zoo of members of the ‘‘metzincin’’ super family, zinccontaining extracellular proteases that come in different flavors, subfamilies according to the sequences around their active centers and their domain structure. To present some preparatory

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work to the referees, we quickly screened for extracellular matrix metalloproteinases (MMPs) and ADAMs (this acronym stands for ‘‘A disintegrin and matrix protease’’) that were expressed in the CNS and were significantly up or down regulated in the wobbler brain stem and spinal cord. Using the PCR technique to estimate the respective mRNA levels, we obtained interesting results within a few days: MT1-MMP and ADAM8 were expressed in the CNS and their mRNAs were drastically elevated in the CNS regions affected by neurodegeneration in the wobbler mouse. This was a good starting point, and our project was approved by the referees. In the following years, we discovered that ADAM8 expression is induced by tumor necrosis factor alpha (TNF alpha) [136]. This cytokine is liberated from its membrane-bound into a soluble form by ADAM17 ( ¼ TACE, TNF alpha converting enzyme), and the membrane bound receptor for TNF alpha, TNFR-I, is converted into a soluble form by ADAM8, which autoactivates by proteolysis [137]. Presumably, soluble TNFR-I neutralizes TNF alpha in the intercellular space, thus reducing the cytotoxic effect of high concentrations of TNF alpha (Figure 12). If this sequence of events, some of which have been reproduced under cell culture conditions, would happen in situ, a knockout of ADAM8, which itself does not lead to an overt disease phenotype, should aggravate neurodegeneration in the wobbler mouse. This was indeed found (Bartsch et al., unpublished). The negative feedback loop between TNF alpha signaling and the activity of MMPs and ADAMs continues to be a fascinating field of biochemical neuropathology, which was followed up by Jo¨rg Bartsch after he had moved to London. The topic of neurodegeneration provided an opportunity for one of the rare cooperations with Brigitte. I had invited Judith Melki, the French scientist who had identified the gene for human spinal muscular atrophy (SMA) on Chr 5, for a seminar. The product of this gene had been termed SMN (Survival Motor Neuron). It participates in the formation of small nuclear ribonucleoprotein (RNP) particles. When Judith Melki reported on the primary structure of SMN she mentioned a polyproline stretch. From Brigitte I had learned that her pet protein during those years, profilin, has a binding site for polyproline. Thus, I suggested that we should check whether SMN and profilin interact. Indeed, such a complex was found in vitro and colocalization of SMN and profilin was shown in mouse spinal cord sections. Not quite


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unexpectedly, immuno staining for profilin and SMN was found in nuclear inclusions, in the so-called gems [138].

BMJ: Greener Pastures in Lower Saxony In 1991, Arne left home to serve his time in the German military service, with the marines at the north shore of Germany. In 1992, when Wolf was approaching the end of his high school period, I decided to try my luck with applying for the position of a full professor. I concentrated on openings not too far away from Bielefeld, to be able to spend at least the weekends with Harald. Within a very short time, I had three options: I could either move to the private University of Witten-Herdecke, or stay in Bielefeld as a full professor, or accept the offer of the Technical University of Braunschweig, the ‘‘Carolo Wilhelmina,’’ to become the head of the Zoological Institute and leader of a Cell Biology group. The first opportunity turned out to be not attractive, for various reasons. The conditions offered to me were less favorable than what I already had in Bielefeld, and I figured that it would be difficult to attract a sufficient number of students and co-workers there. The second one was greatly favored and supported by Prof. Grotemeyer and the Ministery of Science of Nordrhein-Westphalia, but had the drawback that I would still have to remain in the Faculty of Biology. Many professors there were still the same colleagues who had given me quite a hard time when we arrived there, and they were rather reluctant to accept me now in a new position, with rights equal to their own. Furthermore, apart from my own position, my situation would not have changed: there were no offers as to positions for scientific or technical co-workers, lab space, equipment, or financial support, so in fact, I would still have been dependent on Harald’s institute. The third option turned out to be rather attractive: I was warmely welcomed and treated very well at the Technical University of Braunschweig. The position that had become vacant after the previous occupant had retired was for a full professor in Zoology, but the ‘‘Carolo Wilhelmina,’’ although rather conservative and very proud of its standing as the oldest Technical University in Germany, was inclined to change directions and appoint somebody working in cell biology. This was facilitated by the fact that the Zoological Institute comprised three more groups focusing on animal physiology,

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ecology and evolution, so that these topics were cared for in teaching. In addition, the TU was determined to increase its quota of female professors. And from their point of view, I had another advantage: I was indeed a trained zoologist, as demonstrated by my PhD thesis! Hence, I obtained a generous offer concerning positions for secretarial, technical, and scientific co-workers, all of them were vacant and I could choose to appoint the persons to my liking. Furthermore, I was offered laboratory and office space in the Biocenter, a rather new and well equipped building, where groups working in genetics, cellular, molecular and developmental biology were concentrated, who were interested to welcome a new colleague and share experience and equipment with her. The contrast to my previous reception in Bielefeld could not have been larger. So, I moved on to the Technical University of Braunschweig, with the full consent of my family – a decision I never had to regret. In Braunschweig, I lived in a small flat during the week and commuted to Bielefeld almost every weekend. Occasionally, we reversed this custom and Harald would come for a weekend to Braunschweig. We enjoyed this rather attractive city that, although much of it had been destroyed in the World War II, had still conserved many traits of its aristocratic past (for centuries, it had been the capital of the independent Duchy of Braunschweig). Located rather close to the East German border, it had been kind of a sleeping beauty until the collapse of the East German government in 1989, and not too many people knew of its interesting historical past and carefully restored architectural treasures. When I moved there in 1993, things had already changed, and the Braunschweig inhabitants seemed determined to make up for the long eclipse – I found a lively city with a thriving cultural life, offering numerous excellent theatre performances, art exhibitions, and concerts.

HJ: The Resurrection of TMV In recent years, due to threat by BSE and Alzheimer’s disease, the concept of ‘‘protein misfolding diseases’’ has become a hot topic of neuropathology. Since the early days of TMV research, the ¨mme’’) had fasciso-called ‘‘yellow’’ mutants of TMV (‘‘Gelbsta nated phytopathologists. Of course, it is not the virus that is


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yellow, but the systemically (i.e. in the absence of a hypersensitive reaction) infected leaf tissue turns yellow due to the destruction of the chloroplasts by these strains. Not all TMV mutants with destabilized coat proteins are ‘‘yellow strains,’’ but all yellow strains have thermo labile or defective coat proteins [11]. Due to problems with the isolation of revertants this observation provided a correlation rather than a proof. Seven years later, this proof has been provided by in situ mutagenesis of the coat protein which excluded mutations in other genes as the cause of chloroplast destruction [139]. With an old shoebox full of TMV preparations (they last for decades, even at þ 41C) and new technical possibilities I revived the program ‘‘cytopathic effects of mutant TMV coat proteins.’’ New mutants were generated by in vitro mutagenesis and we extended the notion of position specific destabilizing effects of proline to leucine replacements. We showed that denatured TMV coat protein (accumulating when ts mutants are grown at non-permissive temperatures) induces elevated levels of heat shock proteins [140] and dramatic ubiquitinylation reactions in leaf tissue of tobacco [141]. More recently, Christiane Wiegand transfected mammalian cells, especially cell lines with a neuron-like phenotype, with constructs coding for TMV coat proteins with different stabilities, in order to see whether there are pathogenic effects similar to those observed in plant tissue. The idea is to use a protein as a model substance for a misfolding disease which in its native form has absolutely no function in the host cell so that there is only a ‘‘dominant garbage effect.’’ There is presently a rapidly growing literature on misfolding diseases dealing with tangles of filaments with a highly ordered but nonfunctional multimeric structure. In contrast, the destabilized TMV coat proteins, when synthesized in the cell or re-assembled in vitro at high, non-permissive temperature, form predominantly disordered aggregates (Figure 4). In the living cell, they are probably related to ‘‘aggresomes,’’ remnants of the unsuccessful attempts, by ubiquitinylation and subsequent proteolysis in the proteasome, to get rid of denatured proteins.

BMJ: Research at the ‘‘Carolo Wilhelmina’’ Five persons moved with me from Bielefeld to the ‘‘Carolo Wilhelmina’’: Martin Rothkegel, who had just finished his PhD

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with me in Bielefeld and whom I could offer a permanent position as a senior co-worker in the group, and the PhD students Klaudia Giehl, Kathrin Schlu ¨ ter, Martina Kroemker, and Annette Menkel. Martin proved invaluable as my ‘‘state secretary of financing,’’ setting up the institute for our needs in cell and molecular biological research in very short time. He also organized teaching by designing lab courses for students at various levels in biology and biotechnology, and soon became my deputy in running the institute during the periods of my absence. These grew over time with the increasing number of my appointments in various committees. In addition, Martin never lost his footing in research, and with him and the PhD students we managed to follow up very quickly the research projects we had started in Bielefeld. Furthermore, I was still heading the service project on antibody production in the SFB 223 in Bielefeld, and thus the production of useful monoclonal antibodies never ceded. We were now well known in the international community for our research on the role of the actin cytoskeleton in cell attachment and thus invited to write a review on this topic – which is widely cited today, 12 years after its appearance in print [142]. In Braunschweig, as in Bielefeld, there is no medical faculty, which I, as well as Harald, always considered a disadvantage for our research. However, there is the German Research Center for Biotechnology (now: Helmholtz Center for Infection Research, HZI) with several groups working on topics related to my own interests. There were especially close contacts between my group and the group of Ju ¨ rgen Wehland, who is now heading the Division of Cell and Immune Biology at the HZI. Ju ¨ rgen and I had known each other for a long time, and we both were pleased to find ourselves now in neigboring institutes. His presence helped a lot to speed up our start at the Technical University. I could always rely on his help and support in various crises – regardless of whether they were of political, scientific, or financial nature. Shortly after my appointment at the TU, I was elected member of the Advisory Board of the HZI, which was meant to demonstrate the close connection between these two institutions, but Ju ¨ rgen and myself never had to shape these connections into an official document – we just became scientific partners and collaborated for the years to come. One topic we tackled together was the intracellular mobility of a human pathogen, Listeria monocytogenes. This bacterium


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penetrates the intestinal epithelium of a person who has ingested contaminated food, for example raw milk cheese. Once inside the epithelial cell layer, Listeria multiplies and hijacks the cytoplasmic actin system of the host cells to move through the cytoplasm, thus laterally spreading the infection while escaping discovery by the immune system of the host. The actin filaments are shaped into a ‘‘comet tail’’ protruding from one pole of the bacterium. Movement is achieved by the rapid turnover of these dynamic filaments which grow by addition of new actin subunits at the proximal site and disassembly at the distal site. We found that soon after their formation these comet tails already contain crosslinking proteins like alpha-actinin [143] and are linked to the bacterial wall by a complex formation between the bacterial protein ActA and VASP [144]. A few years later, Marie France Carlier from Paris reported in a collaborative study with Ju ¨ rgen’s and my group that the essential role for VASP in Listeria movement can also be demonstrated in cell free assays [145]. Another actin ligand well known to us is also involved in controlling this movement: profilin [146]. In 1994, I organized the 45th Mosbacher Kolloquium on ‘‘The Cytoskeleton,’’ together with Klaus Weber from Go¨ttingen and Eckhard Mandelkow, Hamburg. In accordance with our own research interests and expertise, Klaus was responsible for the program on intermediate filaments, Eckhard for that on microtubules and I arranged the presentations in the actin field. We were pleased that all the internationally acknowledged ‘‘big shots’’ in these fields responded positively to our invitations and assembled there, and the conference was considered a great success. I also recall one of the less serious but quite amusing events: The hotel managers of Mosbach, a small town in the hilly region of the ‘‘Odenwald,’’ were not used to accomodating guests from abroad. Most of them did not speak English, and, much worse, the rooms did not meet international standards. Prompted by the reports of our American speakers, Eva Mandelkow and myself dashed to all the relevant shops in town to buy soap bricks for the bathrooms of these colleagues! In 1995, I organized a Minerva ‘‘Gentner Symposium’’ in Israel, together with Benjamin Geiger from the Weizmann Institute, on ‘‘Mechanisms of Cellular Morphogenesis.’’ Benny selected the Kibbuz in Ein Gedi as the conference venue, and we enjoyed an excellent program supported by many international speakers, several well

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organized and guided tours to the most impressive sites in Israel, like Massada and the Dead Sea, and a visit to a Beduin tent. The role of profilin and its partners in cytoplasmic motility and cell attachment kept us busy for the years to come in Braunschweig. Klaudia Giehl spent some time in Vienna with Rudolf Valenta, comparing immunological properties of birch with mammalian profilin [147]. In Braunschweig, Oleg Mayboroda from St Petersburg and Manfred Ru ¨ diger had joined our group and contributed to investigating the role of plant and animal profilins in stabilizing the animal microfilament system [148,149]. Structural and functional features of profilins and the properties of their distinct and well conserved binding sites for actin, acidic lipids, and poly-proline stretches were unraveled in biochemical analyses using recombinant profilins and mutants thereof, and these studies were primarily the work of Martin Rothkegel, Kathrin Schlu ¨ ter, and Klaudia Giehl, in collaborations with the groups of Dieter Schomburg, HZI [150], Michael Schleicher, Munich [151], and Hans Georg Mannherz, Bochum [152]. A widely recognized result of our collaboration with the Walter group in Wu ¨ rzburg was that the polyproline binding motif of profilin forms complexes with a proline-rich stretch in VASP [153]. This was the first biological ligand identified for this motif exposed in all profilins (Figure 13), and there were many more such ligands to be discovered in the future. Slowly the profilin community came to the conclusion that this small, widely expressed protein, so easy to express in and purify from bacteria, is apparently involved in multiple functions: in the regulation of the submembranous actin filament system (Figure 13), but also in modulating acidic phospholipid turnover and the activity of polyproline-stretch proteins of quite diverse families. Thus, it serves as an important adaptor between the cytoskeleton and several signaling pathways ([154]; Figure 13). More recently, the concept emerged that the different profilin isoforms that are either the products of different genes or of differential splicing, may specialize in cell-specific tasks and thus grossly inflate the number of profilin functions in a cellular context. It is clear now that even within the three conserved binding sites on profilin, for actin, acidic phospholipids, and polyproline-stretch proteins, the structural fold is conserved, but the sequences are only moderately homologous. For profilins from different kingdoms, this had been known earlier, and on this basis, we had generated

Fig. 13. Profilin, a regulator of the actin cytoskeleton. (A) Schematic view on the main three ligand binding sites and their topographic location on the folded polypeptide. The mass of profilins ranges between 14 kD and 17 kD, depending on species and isoform. The binding motif for poly-L-proline stretches (PLP) covers the N- and C-terminal folds (left-hand hatching), while the G-actin-binding site is located on the opposite side of the compactly folded molecule (right-hand hatching). The binding site for acidic phospholipids like phospho-inositol 4,5 bisphosphate (PIP2) forms an elongated band (shaded) overlapping with both, the PLP- and actin-binding sites. Consequently, G-actin and PLPcontaining proteins can bind simultaneously, while PIP2 binding competes for the binding of G-actin or PLP-containing proteins (modified after Ref. [154,156]). (B) Fluorescence image of actin filament bundles (left panel) and the profilin distribution (right panel) in a HeLa cell settling and spreading on a culture dish. Profilin, as seen in an indirect fluorescence staining with a monoclonal antibody, is concentrated primarily at the ends of actin filament bundles at the cell periphery, and also in the center of the cell, in the nuclear compartment (modified after K. Giehl and B.M. Jockusch). Bar: 10 mm.

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a monoclonal antibody highly specific for a birch profilin epitope, which, when fused to the cDNA for any candidate protein, serves as a tag faithfully and specifically detected by the epitope antibody [155]. Nowadays, more subtle differences in the affinity of profilin isoforms, expressed in the same species, for their ligands have been described and seem relevant for cell-type specific differences in profilin function [156]. Profilin was also recognized as a tumor suppressor protein. In a collaboration with the group of Siegfried Scherneck, Max Delbru ¨ ck Center Berlin, and funded by the German Cancer Foundation, we found that several human breast cancer cell lines contain a significantly lower level of profilin than their normal, non-tumorigenic epithelial counterparts. Overexpressing profilin in the breast cancer cell line CAL51 resulted in a normalization of cell growth, actin filament organization and cell attachment, and suppressed tumor growth in subcutaneous transplants in nude mice [157]. In a subsequent study, using CAL51 cells transfected with various profilin mutants defective in ligand binding we could demonstrate that only profilin with a functional actin-binding site is capable to rescue the epithelial phenotype and prevent tumor growth in nude mice [158]. Already in 1995, I had interested several of my colleagues in the Biocenter of the TU and the HZI in setting up an application to the DFG for support of a joint research project in the frame work of the DFG funding program ‘‘Research Units’’ (Forschergruppen). This DFG program is less demanding with respect to coherence and number of participating groups than the SFBs and thus was more appropriate for the situation in Braunschweig. In 1996, we were granted such a unit, with the title ‘‘Regulation, Modification and Organisation of Structural Proteins,’’ first for a 3-year period, and, after a successful evaluation, for another 3 years. I was elected as speaker, until in 2002 it terminated after 6 years, which is the allotted life span according to the DFG rules. For a period of 8 years (1996–2004), I was elected as referee in the DFG panel evaluating grant applications in cell and molecular biology, genetics, and microbiology, and served for the same time period as an elected member in the ‘‘Wissenschaftsrat,’’ the scientific advisory board to the German Federal and the State governments. In 1996, I was also elected member of the ‘‘Braunschweigische Wissenschaftliche Gesellschaft’’ – an assembly of elderly gentlemen who treated me with charmingly good


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manners and stressed that my addition to their congregation lowered the average age and increased the number of female members considerably. In 2000, I became an elected member of the German Academy of Sciences Leopoldina, on a proposal by Rainer Jaenicke and the zoologist Bernd Ho¨lldobler from the University of Wu ¨ rzburg. In 2002, Ju ¨ rgen Wehland and myself managed to establish an International Graduate College, funded by the State of Lower Saxony, which was a joint research unit of the TU and the HZI of Braunschweig, and the Weizmann Institute in Rehovot. The project title of this college, which is still ongoing, is ‘‘Molecular Complexes of Biomedical Relevance.’’ We were granted a number of PhD fellowships, a position for a postdoctoral fellow and a generous amount of money for research, and I, once more, was to serve as speaker. In 2002, I was elected member of the Advisory Board of the VW Foundation, a body deciding on the distribution of money of the VW Foundation for support of science, in Lower Saxony, but also nation and world wide. In 2003, I was called to serve on the Advisory Board of the Biocenter of the University of Wu ¨ rzburg. These multiple duties required much of my time outside the laboratory, and I was very lucky in that my co-workers at the institute tolerated this with grace and managed to keep up the high reputation of our research. This was mainly achieved by the more experienced scientists: Martin Rothkegel, Kathrin Schlu ¨ ter, Manfred Ru ¨ diger, Jo¨rg Winkler and, later on, Susanne Illenberger, and Wolfgang Ziegler, but also involved a number of highly motivated students. Our studies on the cell attachment protein vinculin continued to prosper in Braunschweig. We characterized it further as a multi-ligand protein (Figure 14), defined its actin filament binding motif in the tail sequence [159,160], and reported on the gross conformational changes that govern its capacity to bind to different ligands [161–164]. The intramolecular head-to-tail interactions which we postulated on the basis of biochemical data and which allow the molecule to adopt the shape of a curled up or extended ‘‘tadpole,’’ thus covering or exposing ligand binding sites [162], could be visualized in a study using sophisticated electron microscopy ([165]; Figure 14), and was elegantly confirmed much later by other groups using X-ray analysis. Furthermore, we compared actin binding of vinculin with alpha-catenin, which is a relative of vinculin restricted to

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Fig. 14. The cell attachment protein vinculin. (A) Molecular anatomy of vinculin and its ligand binding sites. The polypeptide comprises 1,066 amino acids. The large N-terminal head is depicted as an ellipsoid (grey), followed by a proline-rich neck region (grey) and an elongated tail comprising five alpha helical folds (black) and terminating in a hydrophobic finger (grey). A selection of ligands is shown, positioned close to the binding regions identified. MVI: the position of the insert for metavinculin, a larger splice form of vinculin. (B) Electron micrographs of isolated vinculin, as obtained by electron spectroscopic imaging and negative stain. Different conformations can be seen, demonstrating the five alpha helical globules in the tail and head-to-tail interactions in the folded molecule (modified after Ref. [165]). Bar: 10 nm.


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cell–cell attachment sites in epithelia [166]. In biochemical and immunological studies, we studied the interaction of metavinculin, a larger vinculin splice form (Figure 14), expressed specifically in muscle with actin [167,168] and showed in a collaborative study with Tim Olson from the Mayo Clinics that a hereditary form of dilated cardiomyopathy displays point mutations in the actin-binding site of metavinculin. Regulation of vinculin conformation and thus of ligand-binding was studied by analyzing vinculin-protein kinase C interactions [169], and the significance of phospholipid binding to vinculin for the dynamics of cell attachment sites was shown somewhat later [170]. And there was one finding which finally linked the studies on profilin and vinculin to actin filaments in cell attachment sites: VASP, a ligand for profilins [153], forms also complexes with vinculin [171] and both proteins interact with actin [172]. Based on these results, Stefan Hu ¨ ttelmaier, a highly motivated and very bright PhD student investigated the dynamics of cell attachment sites in spreading cells. He could show that VASP–vinculin complex formation depends on the activation of vinculin, which is achieved by the disruption of the head-to-tail interaction by acidic phospholipids [173]. Detailed biochemical analyses of the VASP actin-binding ability by Stefan Hu ¨ ttelmaier and Birgit Harbeck demonstrated that VASP contains discrete binding sites for both, filamentous actin and for their monomeric subunits, G-actin [172,174]. It stimulates actin polymerization, and bundles the newly generated filaments. These processes are regulated by VASP phosphorylation [175]. Considering the close spatial relationship between the G-actin and the profilin-binding motif on the elongated VASP molecule (Figure 15), one can envision the following sequence taking place at the nascent attachment site: Profilin–G-actin complexes are collected to the profilin-binding site of VASP where they dissociate, delivering G-actin to the neighboring G-actin binding site, thus generating a cloud of highly concentrated of polymerization-competent actin. Filament polymerization can then set in, and the growing, tightly bundled filament ends are stabilized by complexes of VASP with activated vinculin (Figure 15). The resulting attachment structures are dynamic and can be regulated by various signals, modulating VASP as well as vinculin. This concept is still hypothetical, but it is at least consistent with the available data.

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In the following years, we learned from our own results but also from the studies of other groups that VASP is the prototype of many large, elongated multidomain proteins with a linear sequence of binding motifs for profilin, monomeric actin, and actin filaments. Their conformation, and thus their ligand binding abilities can be regulated by phospholipids, phosphorylation, or small GTPases, and they may contain additional binding sites for cell-type specific structural proteins in the submembranous region. Hence, they serve as adaptors between the peripheral, dynamic actin cytoskeleton, and various signaling


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cascades. Two candidate proteins of this type were studied in our own laboratory. One of these is p140mDia, a member of the large family of formin-related proteins which also are elongated, multidomain proteins located in the actin-rich periphery of mammalian cells. In a collaboration with us, Shu Narumiya, Kyoto University, had reported that p140mDia is a target for Rho small GTPases and, mediated via its extensive poly-proline stretch, binds to profilin. These results suggested that Rho regulates actin filament dynamics by targeting profilin via p140mDia to the plasma membrane [176]. In a follow-up study, we analyzed the molecular architecture of p140mDia in detail and could support this conclusion [177]. The second interesting protein concerned the VASP relative Mena, expressed primarily in neuronal cells. In a collaboration with the groups of Ralf Mendel, Braunschweig and Joachim Kirsch, Heidelberg, we showed that Mena, forming trimeric complexes with actin and with profilin, binds also to gephyrin, a structural component of the postsynaptic scaffold in inhibitory neurons which is also a profilin ligand. Actin, profilin, Mena, and gephyrin participate in the formation of larger complexes and may thus contribute to the microfilament-dependent receptor packing density and dynamics

Fig. 15. The cell attachment and signaling protein vasodilator stimulating phosphoprotein VASP. (A) Molecular anatomy of VASP and its ligand binding sites. The VASP polypeptide, comprising 380 amino acids, is organized in three discrete domains (like its larger relatives Ena, Mena, and Evl): an N-terminal EVH1 domain and a C-terminal EVH2 domain (grey) flank a central proline-rich domain (light grey). The central domain contains a threefold repetitive GP5 motif. Of the many ligands of VASP, only a selection is shown of those identified at the cell membrane during focal adhesion formation, and positioned close to their binding region. Profilin and profilactin (a complex containing profilin and ATP-charged actin) can bind to the GP5 motif, and the adjacent G-actin binding site may bind to and concentrate the ATP-actin subunits released from this complex. Subsequently, actin filament formation is favored in a dense cloud of polymerization-competent G-actin, and the nascent filaments bind to and are stabilized by VASP and its ligands vinculin and alpha-actinin. ActA: a protein expressed by Listeria monocytogenes which anchors VASP to the bacterial wall during comet tail formation (see text). (B) Double fluorescence image of a spreading myoblast expressing a fusion protein of VASP and EGFP, green fluorescent protein (upper panel), and stained for vinculin with a specific antibody (lower panel). The arrow-head shaped structures at the cell periphery are focal complexes, where actin filament bundles terminate and VASP and vinculin colocalize (modified after Ref. [175]). Bar: 10 mm.

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at inhibitory synapses [178,179]. Hence, the number of proteins participating in organizing, regulating, and modulating the actin filament networks leading to cell motility or adhesion, either by serving as building blocks or as members of signaling cascades, is steadily increasing (Figure 16). In Braunschweig, I also came back to my old hobby: cytoskeletal proteins in the nucleus. In several cell types, either with cells expressing fluorescent proteins or by using specific profilin antibodies for localization, we had occasionally observed profilin not only concentrated in the dynamic submembraneous actin networks of the cytoplasm (Figure 13), but also in the nuclear compartment [148]. Subsequently, we identified two nuclear proteins that colocalize with profilin and form complexes with it. One is SMN, a protein which participates in RNA processing in

Fig. 16. Hypothetical scheme depicting the formation of actin filament suprastructures at the cell periphery. On the left side, a nascent focal complex is depicted, with the main features of actin filament formation, bundling and stabilization of the resulting suprastructure and anchoring it to the transmembrane proteins of the integrin family. On the right side, the growth of a dynamic, branched actin network is seen, as required in the front part of cells for locomotion. Symbols of many players in this these processes are depicted in the box, and the putative role of profilin, profilactin, vinculin, VASP, and mDia140 is described in the text (modified after Ref. [156]).


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spliceosomes and whose mutations cause spinal muscle atrophy in man. Harald, interested in neurodegenerative diseases, brought SMN to my attention, pointing out its conspicuous poly-proline stretches. Our collaboration confirmed the initial suspicion: SMN is another ligand of the polyproline binding motif in profilin. Furthermore, SMN colocalizes with profilin in ‘‘nuclear gems,’’ specific RNA storage compartments within the nuclei of many different cells [138]. However, we never found out whether this interaction has any consequence for SMN function. The second nuclear profilin ligand we identified was quite a surprise to us: it is a transcription factor that belongs to the Myb-family but had not been described previously. We coined it p42POP, for ‘‘partner of profilin,’’ with an apparent molecular mass of 42,000. We could show that p42POP acts as a repressor of transcription and that this activity is substantially reduced by profilin binding, indicating that profilin is involved in gene regulation [180]. Another interesting, hitherto unknown nuclear protein was discovered by Stefan Hu ¨ ttelmaier. Searching for ligands that might specifically interact with metavinculin, the mysterious splice form of vinculin, he identified a protein that bound to metavinculin, but also to vinculin and alpha-actinin. Surprisingly, this new protein contains specific RNA-recognition motifs (RRMs) that identified it as a member of the hnRNP family that is primarily involved in RNA splicing. Stefan, a fan of the German ‘‘Love Parade,’’ an annual event taking place in Berlin a few years back, where the hottest techno music was promoted and stimulated people to dancing (‘‘raving’’) in the streets, baptized his new protein ‘‘raver1.’’ As we found out later by using monoclonal antibodies against raver1, this was really a well chosen name: raver1, widely expressed in different species and tissues, contains nuclear import and nuclear export signals and shuttles between the nucleus and the cytoplasm of cultured cells. In the perinucleolar compartment, it forms complexes with another hnRNP (PTB). During skeletal muscle differentiation, it leaves the nucleus and migrates to the alpha-actinin– and vinculin/metavinculin-rich positions in the periphery of myofibrils [181], and persists as an integral component of the contractile machinery of skeletal, heart, and smooth muscle [182]. Due to the unique motif combination of this dual compartment protein, we speculated that raver1 may be involved in mRNA transport, targeting mRNA to peripheral sites and

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subsequent localized protein translation [183], but until today, we could not prove this hypothesis. Raver1 developed into the founder of an hnRNP subfamily: We identified raver2 as a relative of raver1, but this protein lacks the binding motifs for cytoskeletal proteins and shows a more restricted tissue expression [184]. A raver1 null mouse mutant, generated by and analyzed in collaboration with Hans Henning Arnold and Martin Korte in Braunschweig, is viable and quite healthy, with only a slight alteration in neuronal plasticity [185]. It seems that raver1, although widely expressed and highly conserved through evolution, is not essential for life subsidy, but may serve a more subtle function. And what about nuclear actin, my special pet from the old days? Well, I never abandoned it completely, and right now, it is experiencing a prominent revival. In a collaboration with Horst Hinssen from Bielefeld, we had generated a specific monoclonal actin antibody by injecting mice with a profilin–actin complex. This anti-actin displayed a rather unusual reactivity against actin: it recognized a non-sequential epitope consisting of three stretches adjacent to each other within in the region of the nucleotide-binding cleft of the folded actin polypeptide. This antibody does not bind to actin filaments, consistent with the fact that its epitope is not surface-exposed but buried in the filament. However, quite surprisingly, it selectively recognizes non-polymerized actin in the cytoplasm and prominent patches of actin in the nucleus of myogenic cells [186]. This form of actin is also a component of nucleoplasmic filaments in Xenopus and Pleurodeles oocyte nuclei, as seen in immuno gold electronmicrographs. When microinjected, this antibody inhibited the export of several nuclear components to the cytoplasm, indicating that actin at these nucleoplasmic filaments is involved in transport through the pores [187].

HJ: Morphogenesis and a Bit of Mathematics I have for many years been interested in the morphogenesis of tissues: Which effects are tissue intrinsic, which are determined by the environment? Which role does the coherent expansion of clones play in comparison to migration and intermingling? In order to demonstrate tissue autonomy of pathogenic phenotypes,


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I transplanted genetically diseased muscle onto genetically normal hosts and vice versa. Thus we had shown (see above) that myotonia is a muscle intrinsic disease, a myopathy [56]. Transplantation experiments have always been important in immunology and developmental biology. They often lead to unexpected and interesting results. I had transplanted ventricular myocard tissue from neonatal rats into the muscle bed as a control to skeletal muscle, because cardiomyocytes in contrast to satellite cells do not fuse with host muscle cells during regeneration. Small pieces of ventricular muscle tissue unexpectedly formed, at the ectopic site of a leg muscle bed, autonomously beating heart-wall like structures [188]. When the same experiment was done with minced atrial tissue, blood filled beating atria formed, indicating that the morphology at least of the atria is intrinsically determined by their muscle cells [189]. After this publication, I got a phone call at 2 o’clock in the morning: Alexander Mauro, the discoverer of the muscle satellite cell, invited me to participate in a workshop on cardiac regeneration, at the Rockefeller University. This workshop was to commemorate Pavel Rumyantsev, a Russian specialist on cardiomyocyte dynamics, who had died prematurely of a heart attack. I spontaneously accepted the invitation, which I considered an honor, especially since I had met Rumyantsev at a muscle meeting in Hungary and was impressed by this friendly and most openminded scientist. I reported on the cell biology and physiology of self-organized atrial tissue, as described in a book on this highly stimulating meeting [190]. The book not only commemorated Pavel Rumyantsev but also Alexander Mauro who died shortly after the workshop, The result of grafting tissue from one individual onto another is a chimera. The analysis of a chimera requires the distinction of the cells according to their origin, and this was done for many years using electrophoretic differences between allo-enzymes (e.g. glucose phosphate isomerase (GPI) [56]. A biochemical assay, however, requires the homogenization of the tissue, and even if it is so sensitive that it can be performed on minute samples from frozen sections, as in the case of GPI, the procedure is tedious and information is lost. A label that can be used on histological sections would be preferable. Nowadays, usually transgenes are used for that purpose, and jellyfish green fluorescent protein (GFP) and its variants as well as beta-galactosidase are now the

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most popular markers of origin. Using these labels, we analyzed both transplantation and embryo aggregation chimeras and obtained information on myoblast migration during muscle regeneration [98] as well as on the behavior of cell clones during the histo- and morphogenesis of skeletal muscle [191], heart [192], and pancreas (Eberhard and Jockusch, unpublished) in embryo aggregation chimeras. In addition to providing new insights, these analyses produced some of the most aesthetic pictures I encountered in my scientific career. The analysis of a chimera comprising tissues of different origin presents a mapping problem that can be treated mathematically. Andreas Dress with other members of the Faculty of Mathematics of the University of Bielefeld had founded a graduate school (financed by the DFG), which dealt with the ‘‘formation of structures,’’ a topic very relevant for biology. My PhD student Daniel Eberhard (now postdoc in Bath, UK), who had studied biology and mathematics, was financed by this program and I, as teaching member of this school enjoyed close contacts with colleagues from mathematics and informatics . As a student I got hold of a new edition of D’Arcy Thompson’s famous book On Growth and Form (original edition 1917) and my dream was to contribute new ideas to that topic. In 2001, I got an invitation to talk at the MPI for the Physics of Complex Systems in Dresden, about a self-chosen subject relating biology to mathematics. I had not published anything in the field and saw this invitation as an enormous challenge. While in Philadelphia for a short visit I bought Ian Stuart’s marvellous book Concepts of Modern Mathematics. When I read it, it occurred to me that mathematical topology should provide new insights into morphology and morphogenesis. In my talk ‘‘From sphere to torus,’’ in which I explained orientable (the sphere, the torus) and non-orientable ¨bius band, the Klein bottle) surfaces, ‘‘permitted’’ and (the Mo ‘‘forbidden’’ membrane morphologies of cells and body plans of metazoa. In cells which contain complicated membrane systems and in the metazoan body plan, the morphology of which is determined by cell sheets (epithelia), only closed, orientable surfaces are permitted. I had great fun with these ideas and obviously entertained the audience. When the formal discussion was over, a small elderly gentleman approached me and ¨bius.’’ Mo ¨bius? – a descendant of introduced himself as ‘‘Mo ¨bius? No, he was not – just a coincidence. I was famous Mo


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disappointed and cannot remember what his question was. Together with the mathematician Andreas Dress of Bielefeld University, I published these ideas [193], however, not much later it turned out that very similar ideas had been published a few years earlier by a Russian group. Such surprises can happen when one steps into foreign fields like a naı¨ve ‘‘Percival.’’

BMJ and HJ: Returning to Southern Germany When the end of our university duties was in sight, we decided that shuttling between Bielefeld and Braunschweig should not continue endlessly. We discussed several possibilities of setting up a common household again: Bielefeld, Braunschweig, or somewhere else? Finally, we seriously considered the third possibility in more detail. We had always been very fond of the ‘‘Regio,’’ the region between Freiburg, Basel, and Colmar where Germany, Switzerland, and France border each other. We had already fallen in love with this region during our Basel period. The landscape is very attractive, the Rhine valley bordered by the Black Forest in the East and the Vosges in the West, the Swiss Jura and the Alps close by. Both of us enjoy the many aspects of the rich history of this region, reflected in the beautiful old towns and cathedrals as well as the fantastic art collections in Basel, Colmar, and Strasbourg. So, when Harald found a small, rather rundown house in a winegrower’s village, suburbanized by Freiburg, we bought it. Our new home is directly below the vineyards of the ‘‘Tuniberg,’’ a hill range nicknamed ‘‘the vineyard of Freiburg’’ with a view to the Black Forest. Freiburg city, with its beautiful old town and a famous cathedral, but also an excellent university with impressive activities in the life sciences, is close. We renovated the house step by step while using it during our holidays. In fall of 2004, we moved from Bielefeld to Freiburg: It was the first time that we had to transfer our belongings from a large into a smaller home, a more than educational experience! Harald rented a simple studio for painting, and Brigitte devotes more of her time to music again. However, this house is a base again from which we communicate by electronic equipment and travel quite frequently to our scientific collaborators and colleagues. Harald has revived his interest in popularizing science while Brigitte is finishing up some joint projects in Braunschweig

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and Basel. Last but not least, we enjoy the frequent visits of friends and family, and never regretted the move.

HJ: Shop Closed, Business Continues In spring 2004 I became 65, and thus reached the official age of retirement (presently being gradually changed to 67). In December 2004 I arranged a multidisciplinary workshop with friends from other faculties as speakers. Fortunately, we could use the beautiful auditorium of the Center for Interdisciplinary Research (ZiF), an institute affiliated with, but separate from, the main University building, on the slope of the mountain range of the ‘‘Teutoburger Wald.’’ The title of this mini-symposium was ‘‘Leonardo is Alive. Images and Associations in Sciences and Linguistics,’’ and presentations were given by my friends Andreas Dress (Mathematics), Eberhard Neumann (Physicochemistry), Horst Mu ¨ ller and Gert Rickheit (Linguistics), Andre´ Stoll (Literature), and by my former PhD student Ernst-Martin Fu ¨ chtbauer, now professor at the University of Aarhus, Denmark (Molecular Embryology). Chair persons were Rainer Jaenicke (Schwalbach) and Anna Starzinski-Powitz (Frankfurt/Main), and there was a large audience including my former co-workers J.W. Bartsch and Thomas Schmidt-John, and Karl Peter Grotemeyer, the former rector of the University, and many colleagues from different faculties. I gave an introduction with an ‘‘exploded view’’ of Leonardo’s famous sanguine-drawn selfportrait. The ‘‘exploded view’’ was to symbolize today’s so popular belief, not to say superstition, that everything has to be executed in detail by specialists and experts. A ‘‘universal genius’’ like Leonardo is not conceivable and would not be acknowledged today. The presentations from different research fields of the University of Bielefeld were both sound and entertaining. I closed the workshop with a ‘‘Son et Lumie`re’’ (PowerPoint) performance, combining my own graphics and paintings with music by Mahler, ´k, and others. I have good reasons to believe that Stravinsky, Barto this event is memorized by the participants as a good mixture of high level academic entertainment, and has successfully conveyed the message ‘‘Science is fun and one should not take the borders between disciplines too seriously.’’ ‘‘LEONARDO LEBT’’ (Leonardo is alive) was the text of my introductory transparency,


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and most people in the audience knew that it was on purpose, and not due to butterflies in the stomach, that it appeared in mirror scripture. What were my feelings and thoughts when the time of retirement approached? Was I forced to be tired? Since many young people are looking for permanent positions in academia, I felt obliged not to oppose to retire, although shortly after the date of my retirement, 2004, the possibility of a prolongation by 2 more years came up in the State of North Rhine-Westphalia. However, for research purposes, a span of 2 years is too short, as at least 3 years would be required to employ collaborators for a postdoctoral period or a PhD thesis. It was a bit disappointing how long it took until our successors finally took over, due to the intolerably long procedures for appointing professors at German Universities. Furthermore, the groups, chairs, and departments of retired professors often disperse completely, because there are very few positions with tenure tracks for young scientists between 35 and 40 years in the German academia. Thus, many of the younger German scientists migrate to Switzerland, England, or Scandinavia where tenure track positions are offered, or they do not return from their postdoc positions in the United States. There is much discussion about Germany suffering from ‘‘brain drain,’’ but not much change in university politics, and we experience an exodus of welltrained and experienced young scientists. The experience of the elder is likewise frequently wasted. While still in office, professors are burdened with a heavy load of teaching, administration, and the supervision of thesis work. Consequently, they have very little time for informal chats with beginners, and there is no tutoring system for small numbers of students in Germany. It is obvious that the potential of retired professors, from age 65 onwards, with many of them in an excellent state of health, could be exploited for this purpose, on a voluntary basis. Il moto è causa d’ogni vita – Motion is the cause of all life. On earth, there is no motion without frictions. I believe that I have moved too many times, both geographically and with respect to my research topics. I could have abbreviated my scientific odyssey by changing from virus to the nematode Caenorhabditis elegans early on – I had read the papers by Sidney Brenner on this new ‘‘model organism’’ as soon as they had appeared. For a microbiologist and an expert in temperature-sensitive mutants it

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would have been a technically easy, but conceptually radical step. There would be no need to kill vertebrates – and nematode genetics is by orders of magnitude less expensive than mouse genetics. Of course, without detailed genomics it was not obvious for a long time how useful C. elegans would be for biomedical research. This is not to say that I am not happy with my work on mouse models, yet it cannot be continued without an elaborate infrastructure, that is an expensive breeding colony with animal caretakers. For me it is fortunate that I have developed sufficient interest in theoretical considerations and mathematics as well as contacts to people competent in mathematical modeling, which should enable me to keep busy without a budget. And, last but not least, my enthusiasm for morphogenesis will find its application in the creation of complex forms, ‘‘Gestalten,’’ that grow in my mind, without being subject to the limitations by the laws of thermodynamics.

BMJ: Closing the Circle In September 2004, I turned 65, and on November 5, the group of my co-workers, with Susanne Illenberger and Martin Rothkegel as the senior members, organized a mini-symposium in my honor, with the title ‘‘The Cytoskeleton – more than a Backbone.’’ It was a charming event, where Alan Weeds, Cambridge, Mary Osborn from Go¨ttingen, Werner Franke from Heidelberg, and Ueli Aebi reported on their various encounters with me, and an overwhelming number of the former students and friends from Basel, Heidelberg, Bielefeld, and Braunschweig attended – including Harald and our son Wolf, who had just finished his PhD in neurobiology at Cambridge, UK. I was quite touched, but presumed already then that this would not mark the end of my scientific life. In 2006, Ju ¨ rgen Wehland organized the Annual Meeting of the German Society for Cell Biology in Braunschweig, and I, as a senior person with previous experience in this task (cf. Figure 9), was happy to help. Right now, in 2007, two DFGfunded projects in Braunschweig are still ongoing, including the project on antibody production, and I still serve as consultant for the International Graduate College. Furthermore, I hold a


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mandate as a consultant for the president of the TU, Ju ¨ rgen Hesselbach, which was promoted by the Secretary of the State for Science and Culture of Lower Saxony, Lutz Stratmann. On his recommendation, I received the Cross of Merit on Ribbon of Lower Saxony in 2005. In 2007, my successor, Martin Korte, a specialist in the cell biology of neuronal cells, was inaugurated, and his chair is now termed ‘‘cellular neurobiology.’’ I feel still warmly welcome in my former laboratory and office, due to his generosity. Fortunately, we share a number of scientific interests, like the role of profilin isoforms in neuronal cells. Nuclear actin is still haunting me, and I have taken up a collaboration with the group of Ueli Aebi in Basel to tackle this project further. So, I travel occasionally from our home in Freiburg to Basel – the distance being only 60 km – to work again in the Biozentrum, like 35 years ago. Together with Ueli and his co-worker, Cora-Ann Schoenenberger, we investigate the question whether nuclear actin might display a conformation slightly different from the conventional, filament-forming G-actin. This hypothesis is greatly stimulated by early findings from the Aebi laboratory, showing that actin is a polymorphic protein capable of adapting different shapes and suprastructures. In this assumption, G-actin, the subunit of the ‘‘conventional,’’ double-helical actin filament marks only one of several possibilities, and nuclear actin, which is involved in functions different from the cytoplasmic tasks, may thus interact with ligands like RNA polymerases or nuclear pore components [194]. So far, the data we obtained with specific actin antibodies support this assumption: in the ongoing cooperation between Braunschweig and Basel, we have generated and characterized another actin antibody which shares some properties with the one described previously [186]: it is also directed against an epitope buried in the conventional actin filament, but exposed in non-filamentous actin. The conformation recognized by this antibody is also present in the nucleus, but not identical with the first one [195]. Our findings support once more the notion that actin exists in the nucleus and suggest that there is more than one conformation involved in the numerous tasks already described for actin and actin ligands in the nuclear compartment. I am curious to see whether I, together with the Basel group, will finally be able to prove this concept.

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HJ and BMJ: Special Places, Special People Both of us participated in many visits and conferences, and there were quite a few that we attended together. From these, we will describe some which are deeply engraved in our memories, for scientific but also for other reasons. In 1973, when our first son was about 20 months old, we left him with Harald’s mother near Frankfurt to follow an invitation from the northernmost university of the world, Tromsø University 300 km north of the polar circle. This university had been founded to counteract the depopulation of Northern Norway and one of our scientific friends from the Madison days, Finn Haugli, was appointed a professor of cell biology in Tromsø and organizing a laboratory course supported by the European Cell Biology Organization. The course, possibly influenced by its setting, had attracted an international crowd of participants from California, Italy, Israel, the Lebanon, and also from Finland, which is located ‘‘just over the hills’’ from Tromsø. The program comprised an ambitious catalog of experimental laboratory work, but there were additional activities offered to us, ranging from piano concerts, hiking trips on the ‘‘fjells’’ where we were eaten by mosquitoes, and fishing trips in the midnight sun. The 24 hours of sunshine, the heat and the continuous crying of the sea gulls were hard to tolerate. After a week or so, most people suffered from deprivation of sleep and thus were prone to make mistakes in their experiments, like exposing the emulsions for 3 H-thymidine autoradiography to the bright sunlight. However, a few of us were greatly rewarded by a boat trip to the outer islands, where we experienced the breathtaking beauty of this place: eider duck nests made from their silky feathers, a couple of sea eagles feeding their young, and myriads of wonderful flowers softly swinging their petals in the incredibly clear light with. In the end, new acquaintances and friendships evolved from the ‘‘sciencenorth-of-the-polar-circle’’ experience. Twice, in 1986 and 1989, we, including our sons, had the opportunity to spend several weeks in Woods Hole, invited by the Sangers, to work and give lectures at this famous Marine Station. Jean Sanger is a native from this area, and the Sangers own a lovely house there. For years, they also rented a laboratory during the summer. For our visits, they managed to rent one of the spacious wooden guest houses and made our stay as pleasant as


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possible. In the mornings, Brigitte, who had taken some of her antibodies from Germany, would spend some time in the Sanger lab, doing antibody microinjection and immuno fluorescence on tissue culture cells, while Harald was writing manuscripts in the library. Everybody seemed at leisure at Woods Hole, and our afternoons were devoted to pointing out the beauties and amenities of American life to the boys, and to chatting with scientists at the wide Atlantic beach. Such conversations often continued at parties in the evenings. We loved to absorb the specific atmosphere at Woods Hole, a concoction of reports on the newest scientific views and downright gossip. At our second stay in Woods Hole, Harald’s mother visited us and all of us went to Provincetown for a whale watch trip. We looked right into the eye of one of the huge but playful humpback whales, an unforgettable experience! We met many people whom before we knew as authors from the literature; their fame was only surpassed by the deceased celebrities on Woodshole churchyard, where geneticists may contemplate at the tombstone of Thomas Hunt Morgan. Due to our related, but discrete research fields, Brigitte was more attached to the ‘‘Cyoskeletal Club,’’ whereas Harald was more at home in the ‘‘Muscle Club.’’ But in earlier years, every once in a while there were joint meetings of both ‘‘clubs.’’ One of these was held under the rather baroque title ‘‘European Club for Muscle and Motility and the European Cytoskeletal Club on Cellular Dynamics,’’ in Tiberias, Israel, 1987. The main organizers were Marcus C. Schaub (University of Zu ¨ rich) and Benjamin Geiger (Weizmann Institute). The program boasted an impressive list of international speakers, and we found it most rewarding to compare muscle and non-muscle systems. The fringe program included excursions to the historical sites at Lake Tiberias and many opportunities to discuss with Israelis their personal fates. We were taken to the Golan heights and were pointed out the Syrian tanks, burnt out and abandoned in the Israeli–Syrian wars in 1967 and 1973. In 1988, we followed an invitation of Jacek Kuznicki to visit his home institute, the Nencki Institute of Experimental Biology in Warsaw. At that time, Jacek had already turned from a shy, introvert postdoctoral fellow who had visited Brigitte at EMBL, into a self-confident well informed young scientist. Supported by our SFB 223, he had been several times to Bielefeld, participating in the various conferences we organized, and comparing the

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biochemical properties of the Caþþ-binding protein calcyclin with parvalbumin and a Caþþ-binding isoform of alpha-actinin. We cherish many memories on his energetic attempts to adapt to the ‘‘western’’ style of doing science, and on his indefatigable thrive to learn about German history, culture, and language. When we visited him, we adopted his style of traveling and took the train Paris-Moskow, which stops at night in Bielefeld and arrives in Warsaw the next evening. The train was poorly lit, and comprised poorly aired compartments. Jacek picked us up at the station and showed us the city which had terribly suffered during World War II. We were treated as VIPs and were offered a special guided tour through the reconstructed city castle, which is one of the marvels of historical restoration and made the Polish stucco workers highly esteemed all over Europe. In the Nencki Institute Jacek introduced us to many people, and with some of them, for example Renata Dabrowska and Barbara Barylko, ties were established that led to later visits at Bielefeld University. We were impressed by the motivation of these people and their determination to continue with their scientific projects under grossly adverse conditions. In the future, we were able to alleviate a few of these by sending small parts of equipment and chemicals to the Nencki Institute, usually by messenger. When we were about to return, we wanted to buy some food and small presents to take along, but had to learn that in the huge department stores of Warsaw there were only a few non-food items available, but these by the ton. We purchased a combined tin opener and corkscrew that was on offer – for tinned and bottled goods that did not exist! When we were to depart from the main station, one of the lady scientists from the institute came to bid us farewell and presented us with a sunflower studded with dry seeds. This, together with some tea we could buy on the train, was our food supply on the way back. We had not been aware of the poor living conditions in Warsaw at that time. The opening device in its brown plastic case is still kept as a souvenir. After several years in the US, Jacek was promoted as a director of the rather new Institute for International Molecular and Cell Biology, set up by the Polish government in Warsaw. In 1993, we flew to St Petersburg, in a mission supported by the DFG. The DFG, which can finance joint projects between German and non-German groups, wanted some information on whether it was still advisable to support such projects involving groups in the


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former Soviet Union, in particular in the Institute of Cytology of the Russian National Academy (RAS) in St Petersburg, headed by Nikolay N. Nikolsky (the library at this RAS institute was then, and still is, supported by Germany). The DFG proposed that Harald should evaluate conditions for research on muscle, while Brigitte was supposed to examine the cell biology groups. We were taken care of by Sergej Krolenko, whose research topic was the sarcolemmal membrane system of skeletal muscle, that is a field related to Harald’s research. Already at that time Sergej had a close collaboration with British colleagues. Both of us gave seminars, Harald on the excitability of muscle and myotonia, Brigitte on the dynamics of actin networks in non-muscle cells. In his introduction, Harald thanked the Russian colleagues for their warm welcome in a city that had lost more than 1 million civilians by starvation and exposure during World War II when it was besieged for almost 900 days by the German Army. During our 5-day stay, the hospitality of the members of the Institute was overwhelming. Sergej took us to Puschkin with its colorful architectural marvels with gilded roofs and fed us sandwiches that his wife had prepared. We also spent a charming evening in the Krolenko’s home with Russian food and, when we left, were given an old German edition on ‘‘Schiller’s Poems,’’ kept and cherished in the family of our host for generations, for a farewell present. We were taken to the ‘‘Winter Palace’’ with its immense art collections and to a performance of the famous ballet. Although all of this must have taken a lot of preparation, organization and money from our hosts, we enjoyed it. Younger members of the Institute, Olga Antropova and Oleg Mayboroda, guided us through the city and explained the historical sites. We also visited the Pavlov Institute for Physiology where we met the electrophysiologist Grigorij Nasledov and his co-workers. All these people visited us later in Bielefeld, some of them several times, and got involved in collaborations. In the setting of a western scientific laboratory, with few restrictions on financial means for research, they displayed a motivation and vigor in working in the laboratory that grossly impressed the technical and student members of the group. A number of Bielefeld-St Petersburg joint publications appeared afterwards. Only Sergej Krolenko did not make his way to Bielefeld, on the basis of ongoing collaborations he preferred to move to England. So, regrettably, we could not return his hospitality.

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What was our impression of the research activities and qualification of scientists in St Petersburg, to be reported to the DFG? We had mixed feelings. The people we met were well informed and tried to keep pace with the western standard of their scientific field, but it was obvious that most of the younger people had already left, and some were preparing their imminent departure, to the US or South America. The instrumental equipment and the means for consumables were both rather depressing. Furthermore, the most basic needs for the institute were not secured. Four elderly ladies shared the single position of a secretary, and during our stay, we overheard a repetitive whispering ‘‘Did it arrive? Did it finally arrive?’’ This related to the cheque expected from Moscow on a weekly basis – essential for paying salaries and electricity for the building! Our final conclusion was that it would need a strong effort from an external funding organization to repopulate the institute with young people and re-establish research at a level which would enable cell biologists, molecular biologists, and electro physiologists to cope with the international scientific community. In the years that followed our visit, the situation of this RAS Institute deteriorated further, and there were periods when it could not be heated in winter. Hence, almost all young people left, and went abroad to the US, the UK, or continental Europe. Alternatively, they left science and changed professions. One of these persons, while in Bielefeld, bluntly declared that the communist Soviet Union had a political system much superior than present day Russia or the Western democracies. Rather surprisingly for us, a total collapse of the Petersburg Institute was somehow avoided, and there are new projects sprouting there now. During the time of the former German Democratic Republic (GDR), the ‘‘Deutsche Akademie der Naturforscher, Leopoldina’’ (German Academy of Sciences Leopoldina), situated in Halle, was one of the few organizations allowed to keep close communication with the international and even with the West German academic community. One of its activities included the performance of annual conferences at the Institute of Plant Genetics and Crop Plant Research in Gatersleben, a tiny settlement east of the Harz mountains, organized by the director, Ulrich Wobus, and his wife ¨che’’ proAnna. For many years, these ‘‘Gaterslebener Gespra vided a forum for meetings between eastern and western scientists, as well as philosophers, theologists, and artists. The


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setting of this institute, famous for its collection of seeds and its beautiful garden, is very attractive. Ulrich Wobus is a plant geneticist, Anna Wobus is a renowned specialist on animal stem ¨che’’ continued cells. For a few years, the ‘‘Gaterslebener Gespra after the reunification of Germany, and we participated twice. In 2003 Harald was invited as an artist, not as a scientist (Anna Wobus knew about his alter ego from the Muscle Conference in Bielefeld). He considered this as a challenge and for this occasion prepared a ‘‘Son-et-Lumie`re’’ power point presentation of his paintings, underlined with music, and framed by cartoons. We arrived at Gatersleben a few hours ahead of the performance, with a number of paintings in the boot of our car. The paintings had to be hanged and the media installed for the performance. All of this was done voluntarily by highly competent retired members of the institute. During the exhibition, participants could get a ¨del sie zerbarsten’’ free copy of the booklet ‘‘Hal Jos: Ei und Scha (egg and skull they burst), a dadaistic and philosophical variant of developmental biology. Harald had never again such an enthusiastic audience and everybody asked ‘‘the artist’’ for an autograph in his/her copy of his booklet. Both of us were impressed with the atmosphere of these meetings and the hospitality of Anna and Ulrich Wobus and their crew, and we still are in contact with some of the participants. In April 2003, when our son Wolf was a PhD student at the Molecular Research Center in Cambridge, UK, we experienced another special meeting: the 50th anniversary of the discovery of the DNA double helix. As expected, it was an excellent scientific conference, with the added benefit to see many of the famous founders of molecular geneics. Whereas James Watson advertised ‘‘big science,’’ Francis Crick was unable to atttend but had sent a surprisingly soft-gloved video message, calling on the audience to better acknowledge the bodies financially supporting science. From 2004 onwards, we participated in the still ongoing annual ‘‘Manfred Eigen Winter Seminars on Biophysical Chemistry.’’ We were invited by one of Eigen’s pupils and long-term organizer of this series, Eberhard Neumann from the University of Bielefeld. The conference has reached its 41th sequel in series, and is a 2 week affair taking place in Klosters, Switzerland, always in January, so that the participants can enjoy skiing there in the mornings. Eigen himself, who turned 80 in 2007, was an active

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skier until 2 years ago. Originally strictly devoted to biophysics, the scientific program now incorporates also topics in biochemistry, and molecular and cell biology, with Brigitte having contributed to the latter. The evening events have a wider scope, including archaeology, medicine, concerts – even Harald’s biotopology and ‘‘Son et Lumie`re’’ were tolerated. Harald is only moderately interested in skiing and Brigitte does not find it attractive, but we both enjoy the walks and especially the conversations with Manfred Eigen, who is always sharp, well informed, widely interested, and a charming host.

HJ and BMJ: A Privileged Life Both of us consider ourselves privileged in many respects. As children, we survived and grew up in a country from which the worst atrocities in the 20th century originated, as witnessed by several articles in this series, for example by the contribution by ´ra ńy (Vol. 44 of this series). Among German Michael and Kate Ba families, many had lost their fathers, and whole families and many children lost their lives in the last years of the war and during the first months after it had ended. Although it took a number of years, both of us succeeded to become professors at a university. Both of us had the chance to build up research groups and to choose our own field of research. In a peaceful period of a democratic country, we enjoyed a traditional family life and raised two sons without major problems. They both went to university and finished their professional training successfully. The elder turned to economics and specialized in financial risk management, the younger, after some detours, ended up in molecular neurobiology [196], not very far from the fields of his parents. They presented us with extremely nice daughters-in-law and, so far, two lovely grandchildren. The privilege of being a University professor is the daily work with young people, in the lecture hall, in the seminar room and in the laboratory. And, in contrast to one’s own children, this population does not age: it is constantly rejuvenated. These young people create an environment of a permanent challenge, which forces one to stay flexible and tolerant, in this way: to stay young.


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ACKNOWLEDGMENTS

We are very grateful to the various funding organizations mentioned in the text, in particular to the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie, and to all the people who contributed to our work over the past decades. We are indebted to our son, Wolf J. Jockusch, for invaluable help with assembling the figures, and to Giorgio Semenza for carefully editing this chapter.

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[175]

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[178]

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[182]

[183] [184] [185]

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[187]

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Appendix Abbreviations and Symbols Chr DFG MPI SFB

chromosome Deutsche Forschungsgemeinschaft (German Research Council) Max-Planck Institute Sonderforschungsbereich (Special Collaborative Program)

Symbols in genetics: italics, genes, alleles, for example wr, wobbler allele of the gene Vps54 of the mouse; CAPs, gene

564


products, phenotypes, diseases: VPS54, vesicular protein sorting factor (a protein); WR wobbler phenotype (neurodegenerative disease) of homozygous wr/wr mice. Technical Information Relating to HJ Home pages: www.uni-bielefeld.de/biologie/Entwicklungsbiologie, www.hal-jos.org The mutants described in the contribution by HJ can be obtained as follows: - Mutants of tobacco mosaic virus by e-mail contact to HJ - Mouse mutants (Clc1 and Vps54 derived, respectively) from European Mouse Mutant Archive (EMMA), Monterotondo, I-00015 Italy (www.emma.rm.cnr.it). Information on booklets by Hal Jos: Deutsche Nationalbibliothek (www.d-nb.de).

565

Index Abelev, G. 6 Achstetter, T. 378, 387 Adams, J. 268 Adenauer, K. 426 Aebi, M. 378 Aebi, U. 470, 542–543 Agerberth, B. 397–416, 401, 408 Akhmatova, A.A. 22 Alder, K. 284 Alexandrov, A.P. 37 Alkalaeva, E.Z. 83, 90 Altman, S. 292 Anderson, R.W. 375 Andre´, B. 375 Anfinsen, C.B. 143, 188, 241 Ansorge, W. 312 Antonov, A.S. 44 Antropova, O. 547 Armstrong, L. 156 Armstrong, N. 455 Arnold, H.H. 536 Arnold, V.I. 95 Arnon, D.I. 341 Artzimovich, L.A. 37 Attenhuber, F. 252 Aumailley, M. 259, 260 Autrum, H. 429, 431, 449 Autrum, H.-J. 337 Avery, O. 42 Avner, P. 214

Babczinski, P. 377–378, 387 Bach, A.N. 47

Bach, J.S. 422, 424 ¨chinger, H.P. 242, 260 Ba ¨chinger, H.R. 241–242 Ba Bahrens, N. 344 Baker, B. 439 Baker, R. 322 Baldwin, E. 38 Baldwin, R.L. 147, 152, 165, 174, 193 Baldwin, T.O. 190 Ball, A. 456 Baltimore, D. 73 Banting, F. 411 ´ra ńy, K. 418, 550 Ba Barbaric, S. 315 Baron, B. 213–214 Barrell, B. 291, 304 ´k, B. 540 Barto Bartsch, J.W. 517, 520, 540 Barylko, B. 509, 546 Barz, W. 359 Batalov, A.V. 34 Bauer, C. 349 Bauer, H. 450 Bause, E. 378 Bayev, A.A. 40, 43, 57, 75, 76, 95 Bayliss, W. 402, 403 Becker, P.E. 498 Becker, U. 235 Beckwith, J. 163 Beevers, H. 338–340, 354–355, 358, 362 Beevers, J. 355 Behrens, N. 343

566

Beissel, H. 283 Be´jard, M. 129 Belehev, B. 70 Belenitskaya, V.V. 49, 51 Belitzer, V.A. 41 Belozersky, A.N. 28, 95 Berg, A.I, 29, 296 Berg, P. 148, 296 Berger, A. 240 Berger, R. 213 Bergman, I. 282 Bergstro¨m, S. 400 Beritashvili, D.R. 50 Bernardi, G. 310, 313 Bernheim, A. 213 Berridge, M.J. 373 Best, C. 411 Birkofer, L. 284 Birstein, J.A. 28 Bishop, C. 214 BleifuX, C. 294 Blobel, G. 353 ¨ck, B. 400 Blomba ¨ck, M. 400 Blomba Blond, S. 171, 173–174, 181 Blondel, A. 191, 221 Bo¨ck, A. 344 Bockstahler, L. 451 Bodenstein, M. 419 Boeck, W. 350 Boehringer, C.F. 289 Bohm, A. 277 Boman, H. 407 Bonner, J. 45, 46 Borisy, G. 453 Bork, P. 246 Boshyan, G.M. 22, 94 Bowles, D. 378 Boyer, H. 350 Branton, D. 244 Braun, R. 470

INDEX

Brauner, L. 337 Braunstein, A.E. 40, 43, 48, 49, 50, 81 Brecht, B. 444 Breidenbach, R.W. 339 Bresch, C. 285 Bresinsky, A. 370 Brezhnev, L.I. 48 Brinkmeier, H. 483 Brown, D.A. 375 Bruckner, P. 241–242, 252–253 Bru ¨ ning, H. 277 Brunner, H.G. 383 Buc, H. 167, 210 Bu ¨ cher, T. 308 Buchner, J. 203 Buckel, W. 349 Bulfield, G. 498 Bu ¨ nning, E. 432 Burger, M. 465, 470 Burger, M.M. 466–470 Burkardt, D. 448–449 Bush, D.R. 372 Buss, F. 511 Bussey, H. 304 Butler, J. 445–457

Calvin, M. 362 Cambell, N.A. 357 Campbell, I.D. 246–255 Canvin, D. 339 Carlier, M.F. 525 Carlquist, M. 401 Caskey, T. 81, 82, 316 Caspari, T. 363, 369 Caspersson, T. 401 Cavaille`s, J. 120 Cavaille`s, R. 120 Celada, F. 168 Celis, J. 315

INDEX

Chabbert, Y. 219. Chaffotte, A.F. 176, 186, 189, 221 Chambon, P. 67 Changcau, J.-P. 79 Changeux, J.P. 138, 207 Chapeville, F. 45, 52, 67, 69, 71, 72, 77 Charles, R. 156 Charpak, G. 182, 216 Chen, C.C. 373 Chen, Z.-W.407 Chousterman, M. 71 Chousterman, S. 71 Chu, M.-L. 250, 263 Chung, A.E. 249 Clark, B. 310, 313, 317 Cohen, G.N. 359 Cohen, J. 262 Cohen, R. 133, 350 Cohen, S. 350 Columbus, C. 407 Conte Camerino, D. 490 Cori, C.F. 156 Cori, G.T. 156 Courcel, N. 130 Coursaget, J. 133–135 Cox, G.A. 501, 502 Cramer, F. 310 Crane, R. 360 Crawford, I.P. 150, 170 Creighton, T.E. 150, 152, 174 Crick, F.H.C. 42, 44, 51, 52, 125, 142, 286, 549 Crowfoot, J. 99 Cruces, J. 382 Czihak, G. 450

D’Ari, R. 78 da Vinci, L. 418

567

Dabrowska, R. 511, 546 Dahlstro¨m, A. 404 Dainty, J. 347, 361 Dam, J. 191 Darrieurx, D. 130 Darwin, C. 356 Darwin, F. 356 Datta, S.P. 314 Dautry, A. 209 David, C. 290 Dc Duve, Ch. 43 Dc Gaulle, Ch. 63 De Beauvoir, S. 130 Debussy, C. 422 Decker, K. 314 Decker, M. 363 Dedonder, R. 158–159, 217, 222 Deering, N. 322 Degenne, P.G. 166 Delbru ¨ ck, M. 285, 287, 290, 353, 435, 443, 462, 463 Delepierre, M. 177, 221 Dementyev, G.P. 28 Denosˇl, A. 71 Derzavin, M.M. 8 Desson, A.G. 21 Deutzmann, R. 385 Dirheimer, G. 71, 292, 294–295, 314 Djavadi-Ohaniance, L. 179, 189, 190 Dobson, C.M. 175 Doerfler, W. 199 Donadieu 119 Dorfmu ¨ ller, T. 510 Doty, P. 44, 45, 52, 53 Douzou, P. 132 Dove, B. 458 Dowhan, W. 374 Drepper, C. 517

568

Dress, A. 538–540 Dubinin, N.P. 30 Dubowitz, V. 498 Dujon, B. 303 Dutrochet, H. 360 Du ¨ tting, D. 288 Dutzler, R. 485 Dvoretskayu, T.M. 20 Dvoryankin, F.A. 28 Dyatchkov, N.N. 47 Dziadeh, M. 250

Ebel, J.-P. 56, 71, 72, 73 Eberhard, D. 538 Edelman, G. 236 Edelstein, S.J. 162 Edgar, D. 246, 248 Edman, P. 400 Edsall, J.T. 239 Eggerer, H. 348 Ehrenberg, M. 87, 89 Eibl-Eibelsfeld, I. 431 Eidelman, N.Ya. 48 Eigel, A. 294, 322 Eigen, M. 240 Eign, M. 549, 550 Ekblom, P. 244, 246 Ellington, D. 156 Elson, E.L. 149 Endo, T. 384 Engel, J. 231–273 Engelhardt, W.A. 1, 34, 35, 37–41, 43–51, 58, 59, 61, 62, 64, 66–68, 70, 72–76, 93, 95 Entian, K.-D. 305 Epifanova, O.I. 50 Erenburg, J.G. 33 Erspamer, V. 407 Ertl, G. 350 Esser, K. 340

INDEX

Eto, K. 156 Evangelopoulos, T. 315

Falck, B. 404 Falter, H. 322 Farkas, U. 385 Fasiolo, F. 73 ¨ssler, R. 263 Fa Favre, A. 89 Fedorov, A.N. 181 Feiginson, N.I. 28 Feizi, T. 384 Feldmann, B. 290 Feldmann, H. 275–333 Feldmann, M. 290 Feldmann, W. 277 Fessler, J. 249 Fessler, L, 249 Fischer, E.H. 156 Fischer, E.O. 337 Fischer, E.P. 353 Fisher, H.F. 166 Fittler, F. 318 Fleischmajer, R. 250 Folkman, J. 261 Forssmann, W.G. 497 Fox, C.F. 373 Fradin, A. 322 Franke, W.W. 475, 493, 542 Franz, G. 349 Fresco, J. 44, 52, 53, 60–62 Frey, J. 507 Friedrich, B. 350 Friedrich-Freksa, H. 435 Friguet, B. 179, 181, 182, 189, 190 Frisch, K.v. 337 Frolova, L.Yu. 52, 74, 75–79, 81, 99 Fromageot, P. 71

INDEX

Fruton, J. 402 Fru ¨ hwald, W. 350 Fuchs, I. 375 Fu ¨ chtbauer, A. 493 Fu ¨ chtbauer, E.-M. 540 Fujiwara, S. 248 Furthmayr, H. 235–236, 240–241, 244, 253 Fuxe, K. 404

Gabbiani, G. 470, 475 Gaill, F. 260 Gall, L. 350 Gancedo, C. 304, 314 Garcia-Bellido, A. 382 ˚rdh, T. 401 Ga Garfunkel, A. 453 Garner, E. 156 Garoff, H. 473 Garrone, R. 239 Gauze, G.G. 44 Gebhardt, S. 338 Geiger, B. 222–223, 525, 545 Gentzsch, M. 382, 387 Georgescu, R.E. 190 Georgiev, G.P. 41 Gerisch, G. 470, 492 Gerl, M. 250, 255 Geronimo, J. 340 Giehl, K. 524, 526–527 Gierer, A. 434–435 Giraudeau 125, 133, 159 Goebbels, J. 276 Goertz, G. 294 Goethe, J.W. 425 Goffeau, A. 302, 304 Gohen, G.N. 220 Goldberg, J.J. 132 Goldberg, M.E. 115–230 Goldwater, B. 148

569

Goodman, H.M. 292 Gorbachev, M.S. 3 Gottikh, B.P. 40, 58, 67–71 Gouaux, E. 372 Graber, R. 347 Granit, R. 400 Grassl, R. 363 ´ssmann, W. 240 Gra Grechko, V.V. 50 Gregory, B.P. 131, 132, 133 Grell, K. 432, 433 Griffith, G. 474 Griffiths, F. 4 GroXkopf, R. 294 Gros, D. 70 Gros, F. 67, 70, 79, 209, 219, 223, 313 Grossmann, G. 375 Grotemeyer, K.P. 494, 495, 506, 521, 540 Gruhl, H. 291, 325 Grunberg-Manago, M. 44, 65, 67, 69, 70, 94, 313, 315 Grzeschik, K.H. 488 Gueńet, J.-L. 514 Gudmundsson, J. 290 Guenet, J.L. 212, 213, 214 Guez, V. 188 Guijarro, J.I. 177 Gu ¨ nther, E. 516 Guschlbauer, M.-P. 71 Guschlbauer, W. 71 Gvozdev, V.A. 54

Haase-Pettingel, C. 190 Hade, E.P. 161 Haemmerli, G. 492 Haenni, A.X. 71–73, 77, 82 Hagen, I. 385 Hall, B.D. 292

570

Hamdorf, K. 449 Hamilton, A. 360 Hancock, J.-F. 376 ¨nggi, U. 318 Ha Hannig, K. 240 Happe, M. 462 Harbeck, B. 531 Hardman, J. 150 Hartmann, G. 338 Hartwig, J. 507 Haselbeck, A. 379 Haselbeck, T. 380 Hasilik, A. 378, 387 Hatano, S. 464, 475 Hauber, J. 302 Haugli, F. 544 Hauska, G. 345, 348–349, 371, 387 Hausmann, R. 285 Heidelberger, C. 458 Heimann, P. 434, 500, 513, 517 Heinegard, D. 248 Heinisch, J.J. 385 Heinz, E. 366 Heizmann, C.W. 482, 483 Helenius, A. 473 Helmreich, H. 200 Hemming, F.W. 378 Henderson, P.J. 371 Henrich, D. 346–347 Hershey, J. 316 Hertel, R. 339 Hesselbach, J. 543 Hikmet, N. 33 ˚ . 404 Hillarp, N.-A Hindemith, P. 425 Hindenburg, P.V. 276–277 Hindennach, I. 462 Hinssen, H. 504, 508, 536 Hitler, A. 8, 50, 276–278 Hoagland, M. 52

INDEX

Hodge, A. 238 Hoffmann, L. 233 Hoffmann-Berling, H. 427 Hogan, B. 251 Ho¨gberg, A. 168, 173 Hogness, D. 148 Hohenester, E. 255–256, 261 Ho¨kfelt, T. 404, 413 Hollan, S. 360 Ho¨lldobler, B. 529 Holley, R.W. 289 Hoppe, W. 240 Ho¨rz, W. 318, 320 Huber, R. 255 Huisgen, R. 337, 428 Hunt, T. 458 Hunter, T. 445 Hurt, E. 387 Hu ¨ ttelmaier, S. 531, 535 Hutter, O.F. 499 Hynes, R.O. 239, 259

Illenberger, S. 529, 542 Isenberg, G. 474, 477 Ivanov, V.I. 50 Ivanova, E.V. 83

Jackson, M. 177 Jacob, F. 67, 69, 134–135, 147–148, 162, 197, 212, 435 Jacobs, W. 448 Jacquelin, M. 118, 121 Jacquez, J.A. 365 Jaeckle, H. 320 Jaeken, J. 384 Jaenicke, R. 199, 200–201, 203–204, 347, 349, 418, 427, 445, 452, 466, 529, 540 Janeway, C. 408

INDEX

Jarrett, N.M. 189 Jelkmann, W. 349 Jennekens, F. 474, 492 Jentsch, T. 485–488 Jockusch, B.M. 417–564 Jockusch, H. 417–564 Jockusch, W.J. 551 Johnson, L. 148 Johnston, M. 304 Joly, M. 442 Jo¨rnvall, H. 397–416, 398, 400, 408 Jorpes, E. 399, 401, 402 Jos, H. 549 Jung, P. 377

Kaback, H.R. 359, 361–362, 370–372 Kaesberg, P. 451, 453, 456, 462 Kaestner, A. 337 Kaiser, D. 148 Kalatozov, M.K. 34 Kandler, O. 337, 340–342, 344, 359, 379 Kaplan, N.O. 66 Karau, W. 288 Karlson, P. 285 Karpeisky, M.Ya. 40 Karplus, M. 191 Karpov, V. 321–322 Karzinkin, V.M. 44 Katchalski, E. 240 Kaupmann, K. 514, 517 Kauss, H. 344 Kauzmann, W. 166, 442 Kaverin, N.V. 68 Kaverin, V.A. 4, 26, 27 Keldysh, M.V. 62 Kellenberger, E. 470 Kelley, K. 470

571

Kelly, F. 213 Kendrew, J.C. 237, 473 Kendrick-Jones, J. 509 Kennedy, E.P. 66, 373 Khesin, R.B. 35–38, 95 Khomutov, R.M. 40 Khorana, H.G. 453 Kiefhaber, T. 203 King, J. 190 Kingsman, A.J. 298 Kirsch, J. 533 Kisselev, A.P. 10 Kisselev, L.L. 1–113 Kisselev, P.E. 8 Kisseleva, V.P. 3, 9 Kisseljov, F.L. 6, 11, 12, 13, 17, 18 Klebl, F. 385, 538 Klein, G. 6 Kleinkauf, H. 314 Kleinmann, H.K. 248 Kleinow, W. 293 Klis, F. 378 Klobeck, G. 318 Klug, A. 53, 443 Knoche, K.-F. 350 Knorre, D.G. 56, 73, 95 Knunyantz, J.L. 37 Koch, M. 488 Kochetkov, N.K. 67, 343 Koffler, H. 339 Kohler, G. 179 Kolmogorov, A.N. 29 Komor, E. 359–364, 387 Kondler, O. 345 Koning, W. 372 Kononenko, A.V. 86, 88 Kornberg, A. 148, 153, 155, 226 Kornberg, H. 339 Korte, M. 536, 543 Kostka, G. 263

572

Kotyk, A. 361, 363 Kourilsky, P. 208 üXlich, H. 319 Kra Kramer, P. 354 Kratky, O. 236 Krebs, H. 353–354 Kretsinger, R.H. 251 Kreutzberg, G.W. 476 Kriedemann, P. 362 Krieg, R. 300 Kritsky, M.S. 44 Kroemker, M. 524 Krolenko, S. 547 Krushehev, N.S. 32, 40, 43, 95 Ku ¨ hn, K. 240–242, 251, 262 Kuhn, R. 322 Kunau, W. 306 Kunitz, M. 442 Kunkel, L.M. 496 Kuperman, F.M. 28 Kurchatov, I.V. 37 Kusumi, A. 376 Kuwajima, K. 173 Kuznicki, J. 545

Laage, S. 513 Labeit, S. 500 Labouesse, B. 81, 158 Labouesse, J. 81 Lafon, J. 118, 119 Lamb, C. 369 Lamport, D. 362 Lange, O. 357–358 Langer, H. 235 Langer, T. 306–307 Lapeyre, Dr. 119 Laplante 156 Larson, L. 340 Lauffer, M. 441, 452 Laurent, T. 401

INDEX

Lazar, D. 79 Lazarides, E. 508 Lehle, L. 345, 349, 379, 387 Lehman, R. 148 Leibniz, G.W. 424 Leloir, L. 343–344 Lengeler, J. 387 Lennox, E. 138, 140, 142–143 Leopold, C. 338 Lepeshinskaya, O.B. 22, 94 Leprince-Ringuet, L. 132 Levin, D.E. 382, 385 Levina, E.S. 6 Lexell, L. 401 Libbert, E. 370 Liberman, E.A. 364 Lilly, E. 458 Lils, H. 383 Lindauer, M. 431 Linden, J. 378, 387 Lindenmann, J. 481 Linderstrøm-Lang, K. 419 Link, T. 149 Lipmann, F. 52, 63–66, 81 Lis, H. 384 Lissitsky, S. 71 Littauer, U. 293 Lochmu ¨ ller, H. 300 Lockau, W. 349, 387 Lodisch, H.F. 369 Lo¨ffler, G. 349 Lohmann, K. 479 Loibl, M. 375 London, J. 194–195 Loos, E. 341, 349, 363 Lorenz, K. 337, 431 Lowey, S. 469 Luft, R. 401 Luitpold, H. 252 Lwoff, A. 67, 134–135, 221–222 Lyapunov, A.A. 29, 31, 32

INDEX

Lyapunova, E.A. 29, 31, 95 Lyapunova, N.A. 29 Lynen, F. 236, 337–338, 344, 437, 449, 487 Lyon, M. 467 Lysenko, T.D. 2, 22, 28, 29, 37–40, 43, 44, 47, 50, 64, 94, 96 Lyubimov, Yu.P. 48

MacKinnon, R. 372, 485 MacLennan, D. 340 MacLennan, L. 340 MacLeod, C.M. 42 Macmillan, H. 51 Madsen, N.B. 168 Maelicke, A. 506 Mahler, G. 422, 540 Makarov, A.A. 87 Maldener, I. 345 Malinska, K. 375 Malinsky, J. 375 Malygin, E.G. 73 Mandelkow, E.M. 493, 525 Mandelkow, K.M. 493 Mannhaupt, G. 300, 302–304 Mannheim, B. 309 Mannherz, H.G. 526 Mansen, A. 277 Mansen, H. 277 Mantsch, H.H. 177 Marriott, K. 378 Marriott, M. 378 Marshall, E. 262 Martin, G. 243, 257 Martin-Blanco, E. 382 Matthaei, H. 448, 456 Matthews, B.W. 165 Matveev, B.S. 28 Maurer, P. 251, 255 Mauro, A. 537

573

Mayboroda, O. 526, 547 Mayer, U. 250, 255 McCarthy, M. 42 Mechnikov, I.M. 81 Medzhitov, R. 408 Meerwein, H. 419 Me´gnin, J.P. 211 Mehrke, G. 483 Me´jean, A. 192 Meisel, M.N. 40 Meisler, M. 512, 515 Meister, J. 116 Meixner, E. 322 Melchers, F. 288, 290 Melchers, G. 434–438, 441–444, 446, 451, 459–461, 463–465, 469 Melki, J. 520 Mendel, G. 99, 360 Mendel, R. 533 Menke, A. 499 Menkel, A. 524 Mereouroff, O. 80 Mereouroff, V. 80 Merkulova, T.I. 79 Merkuryev, V.V. 34 Merxmu ¨ ller, H. 337 Meselson, M. 44, 45, 63 Meshkova, N.P. 42 Messelsohn, M. 133 Me´te´zeau, P. 210 Mewes, W. 303 Meyer zum Bu ¨ schenfelde, K.-H. 350 Michelson, A.M. 70, 285 Michurin, L.V. 28, 29, 47 Miles, E.W. 154, 176, 214 Milgram, V.D. 92, 93 Milstein, C. 179 Minchenkov, L.E. 50 Mirzabekov, A.D. 50, 76, 93

574

Mitchell, P. 360, 362–363, 365–366 Mitirev, G.A. 19 Mitkevich, V.A. 88 Mlynek, J. 350 ¨bius, A.F. 538 Mo Monier, R. 67, 71 Monod, J. 67, 69, 72, 77, 82, 115–230, 359, 435 Monod, O. 135 Morgan, T.H. 545 Morozov, Yu.V. 50 Moscona, A.A. 466 Moser, H. 235 Mosher, D.F. 239, 244 Mothes, K. 359 Mowbray, J. 314–315 Mrosek, V. 369 Mrsa, V. 385, 387 Muhlbock, O. 8 Mu ¨ ller, H. 540 Mu ¨ ller, K. 339 Mu ¨ ller-Hill, B. 204, 369 Mundry, K.-W. 436 Murry-Brelier, A. 172–173, 181 Mutt, M. 399, 413 Mutt, V. 397–416

¨geli, C.W. 360 Na Nadukavukaren, M. 339 Nakamura, Y. 83, 85, 90, 91 Narumiya, S. 533 Nasledov, G. 547 ¨ck, P. 302, 305 Nelbo ¨ck-Hochstetter, P. 302 Nelbo Nester, E. 358 Neumann, E. 540, 549 Neumann, P. 485 Neupert, W. 306 Neurath, H. 156

INDEX

Neuve´glise, C. 299 Newton, I. 424 Niehaus, K. 519 Nikitin, S.Ya. 48 Nikitina, T.Kh. 48 Nikolsky, N.N. 547 Nirenberg, M. 44, 448 Nischt, R. 250 Noegel, A. 508 Nomura, M. 453 ˚ . 398 Norberg, A Norris, A. 153 Northcote, D.H. 378 Notz, S. 288 Novakova, L. 375 Nowak, H. 235 Nurse, P. 458 Nussbaum, G. 73 Nu ¨ sslein-Vollhard, C. 473

Oaks, A. 340 Obermeier, U. 300 Ochoa, S. 456 Oertel, W. 349 Okazaki, T. 443 Okorokov, L. 387 Olah, J. 296 Olivecrona, H. 401 Oliver, S. 302 Olson, B. 261 Olson, M.V. 292 Olson, T. 531 Oosawa, F. 475 Oparin, A.I. 47, 59, 60, 67 Oparina, N.P. 59 Opekarova, M. 363–375 O’Reilly, M.S. 261 Orekhovich, V.L. 238 Orekhovich, V.N. 44 Orff, C. 422

INDEX

Orgel, L.E. 142 Orlean, P. 378, 387 Orsini, G. 196, 198 Osborn, M. 542 Ott, U. 245 Otto, J. 308 Ovchinnikov, Yu. A. 95

Pain, R.H. 175, 185 Palamarczik, G. 378, 387 Parienti, R. 222 Pasteur, L. 81, 116, 118, 120–122 Patzelt, G. 288 Paulin, D. 502 Pauling, L. 38, 39 Paulsson, M. 248 Pavlov, L.P. 94 Peaud-Lenoel, C. 343 Perrin, D. 142–143, 161, 163 Perrin, F. 143 Perrin, J. 143 Perutz, M. 237 Pervushina, A.K. 10, 12, 14, 19, 21 Pervushina, S.A. 19 Pestova, T.V. 89 Peterson, A.C. 466 Petrovsky, L.G. 2, 32 Pette, D. 488, 490 Peyerimhoff, S. 350 Pfeffer, W. 360 Philippe, M. 91 Philippsen, P. 304, 318 Piccoli, M. 130 Piepersberg, W. 302 Pilz, I. 181 Pirro, G. 322 Plu ¨ ckthun, A. 203 Poljak, R.J. 202, 221

575

Pollok, K.-H. 344 Porter, R. 236 Po¨schl, E. 255 Preobrashinskaia, O. 322 Prezent, L.I. 28 Prockop, D.J. 231, 242 Prokofiev, M.A. 95 Prokofiev, S.S. 65 Prokofieva-Belgovskaya, A.A. 41 Prosvirnina, A. 21 Prusiner, S. 207 Ptitsyn, O.B. 166, 172, 175–176, 178, 185, 194 Pu ¨ hler, A. 519

Quigley, F. 378

Rachel, R. 349 Racker, E. 371 RajBhandary, T. 291 Rambach, A. 197–198 Rashi 117 Ratzinger, J. 347 Rebinder, E.P. 52 Refai, E. 398 Reichard, P. 401 Reichardt, W. 435 Reiter, M. 252 Rentschlcr, L. 438, 440, 442–443 Resek, H. 232, 234 Resek, M. 232, 234 Resnais, A. 130 Rich, A. 45–46, 52, 53, 60, 63, 238 Rickauer, M. 387 Rickheit, G. 540 Riethmu ¨ ller, G. 236

576

Riman, J. 74 Robbins, P. 343 Robenek, H. 507 Robl, I. 363, 372 Roder, H. 189 Rohde, H. 242 Roitsch, T. 345, 358, 359, 387 Roseman, S. 359 Roskin, G.I. 28 Rossellini, R. 282 Rossmann, M.G. 164 Rotenburg, M. 117, 523, 526, 529, 542 Rottenberg, E. 117 Rotter, M. 362 Rouviere-Yaniv, J. 70 Rubin, A.B. 44 Ru ¨ diger, M. 526, 529 Rudolph, R. 203 Rudzinski, K. 435 Rumyantsev, P. 537 Ruoppolo, M. 189 Ruoslahti, E. 259 Rusch, H.P. 453, 457, 458 Ryhage, R. 400

Sabinin, D.A. 28 Sage, H. 251 Sakharov, V.V. 28, 30 Samoilova, T.E. 34 Samuelsson, B. 400 Sandmeyer, S. 299 Sanger, F. 81, 291, 295–296 Sanger, J.M. 474, 479, 492, 504, 508–509, 544–545 Sartre, J.P. 130 Sasaki, M. 245 Sasaki, T. 246–247, 259–261 Sauer, N. 349, 363, 369, 387

INDEX

Schachman, H.K. 162, 166 Schachner, M. 471, 476 ¨fer, W. 435 Scha Schaller, C. 473 Schaub, M.C. 503, 545 Scheer, U. 493 Schekman, R. 379 Schenk, B. 423, 433, 437 Scheraga, H.A. 189 Scherneck, S. 528 Schiller, F. 547 Schleicher, M. 508, 526 Schlotman, B. 338 Schlu ¨ ter, K. 524, 526, 529 Schmid, F.X. 349 Schmid, V. 239 Schmidt, G. 124 Schmidt-John, T. 540 Schmitt, F.O. 238 Schmitt, R. 349 Schmitt-John, T. 514 Schneider, R. 129 ¨gl, G. 369 Schnelbo Schoenenberger, C.-A. 543 Schomburg, D. 526 Schpiller, N.D. 47 ¨dinger, E. 426 Schro Schramm, G. 435 Schwab, G.M. 240 Schwartz, L. 130–131, 137, 217 Schwartz, M. 133–134, 138, 148, 206 Schwarz, G. 240 Schwarz, R. 378 Schwarzlose, C. 300 Searle, T. 467 Seckler, R. 203 Seelig, J. 373 Seifert, G. 419 Sela, M. 196, 223, 242

INDEX

Semenza, G. 94, 99, 314, 336, 362, 418, 551, Semisotnov, G.V. 173 Senser, M. 342 Sentandreu, R. 378 Serebrovsky, A.S. 28 Serrano, R. 364 Sestak, S. 385 Severin, E.S. 40 Severin, S.E. 34, 35, 37, 38, 42, 60, 81 Shankar, R. 156 Sharma, B.C. 387 Sharma, C.B. 378 Sharon, N. 383–384 Shemyakin, M.F. 36 Shemyakin, M.M. 95 Shevlyagina, M.L. 4 Shirvindt, A.A. 48 Shostakovich, D.D. 22, 65 Sidman, R. 471, 483, 485 Siegel, A. 457 Simmonds, S. 402 Simon, P. 453 Simonov, K.S. 33 Simons, K. 375, 473 Sixma, J. 507 Sjo¨vall, J. 400, 413 Skoog, F. 358 Skou, J.C. 366 Skulachev, V.P. 35, 37, 38, 41, 42, 94, 99, 364 Skulacheva, N.A. 35 Smith, P. 322 Snell, E.E. 153 Sobolev, S.L. 29 Sonnenberg, A. 260 Souciet, J.-L. 321 Spirin, A.S. 52, 53, 95, 181 Spiro, M. 215–216 Splittstoesser, W. 340

577

Stahl, F.W. 133 Stahlschmidt, W. 375 Stalin, L.V. 21, 23, 32, 40, 64, 65, 70, 95 Stark, G. 148–149, 153 Starling, E. 402, 403 Starlinger, P. 287 Starzinski-Powitz, A. 506, 540 Steensma, Y. 302 Steffen, C. 236, 240 Steiner, D. 412 Steinmeyer, K. 487 Stenhagen, E. 400 Stent, G.S. 157 Stetefeld, J. 255–256 Stetter, K.O. 349 Stoll, A. 540 Stolz, J. 363, 375 üli, P. 492 Stra Strahl, M. 382 Strahl, S. 380–381, 387 Stratmann, L. 543 Stravinsky, I. 129, 156, 422, 427, 453, 540 Streeck, R. 318 Strominger, J. 343 Stryer, L. 148, 150, 153, 172 Stuart, I. 538 Stucka, R. 299–300, 302, 303, 305 Stuhlfauth, I. 482 Sumper, M. 348–349 Svoboda, J. 7 Szybalasky, W. 458

Tamm, I.E. 37, 48 Tanford, C. 161 Tang, M. 70 Tanner, B. 338 Tanner, G. 338

578

Tanner, R. 338 Tanner, W. 335–396 Tasayco, M.L. 190 Tate, W.P. 82, 91 Tatemoto, K. 401, 406 Taturskaya, R.L. 41 Taubes, G. 206 Tauer, R. 306 Tavitian, A. 79 Temin, H. 6, 7, 73, 458 Termenius, K. 408 Theorell, H. 400, 411 Thiebe, R. 286, 288 Thierry, J. 132, 134 Thomsen, A.J. 485–486 Timofeyev-Resovsky, N.V. 30 Timofeyeva, M.Ya. 50 Timpl, A. 232 Timpl, E. 232 Timpl, R. 231–275 Tiselius, A. 448 Todd, L. 285 Tokatlidis, K. 182, 183 Tomkins, 296 Tooze, J. 185 Tovarnitsky, V.I. 40 Trautner, T. 290 Travnic˘ek, M. 74 Trebst, A. 371 Trollope, A. 49 Tschaikowski, P. 422 Tschesche, H. 494, 507, 519 Tsvetkov, V.N. 52 Tudermann, L. 252 Tuite, M. 83 Tulkes, S.G. 50, 51 Tumerman, L.A. 40, 43, 52 Tuppy, H. 236 Turk, V. 314–315 Turner, W. 422 Tvardovskaya, S.E. 99

INDEX

Ullmann, A. 140, 159, 161, 163, 193, 195, 197, 206, 220 Urbanek, D. 369 Urry, D.W. 238 Urusevsky, S.P. 34 Utkin, I.A. 40

Vaheri, A. 239, 244 Valenta, R. 511, 526 van Delden, V. 252 van’t Hoff, J.H. 322 ´ , D. 435, 462 Varju Varshavsky, A. 307 Varshavsky, Ya.M. 40 Vavilov, N.I. 22 Vecchiali, P. 130 Veil, S. 222 Venkstern, T. 320 Venkstern, T.V. 40 Verkhovtseva, M.I. 44 Vermel, E.M. 29 Vespucci, A. 407 Vetter, I. 303 Vielmetter, W. 435 Vogel, H.-J. 338 Volkentstein, M.V. 40 von Bertalanffy, L. 426 von der Mark, K. 235, 252 von Euler, U. 400 von Frisch, K.R. 430–431 von Randow, T. 435 Voronin, L.G. 29 Vorontsov, N.N. 31 Vysotsky, V.V. 48

Wajda, A. 33 Walker, I. 464 Walter, P. 377 Walter, U. 510, 526

INDEX

Walther, T.C. 377 Warburg, O.H. 41, 42, 341 Ward, L. 340 Ward, O.G. 340 Warren, G. 473, 475 Watson, J. 42, 44, 51, 63, 70, 75 Watson, J.D. 125, 549 Watts, D. 480, 481, 488 Watts, R. 481, 488 Weber, K. 475, 500–501, 525 Weeds, A. 508, 542 Wehland, J. 524, 525, 542 Weidel, W. 337, 442–443, 461 Weig-Meckl, I. 375 Weill, K. 444 Weissenbach, J. 292 Welker, E. 189 Werner, B. 399, 409 West, I. 360 Wetlaufer, D.B 164, 165 Wheals, A. 464 Whitworth, A. 262 Wiberg, E. 428 Wick, G. 236, 240–241 Wiedemann, H. 248, 252 Wiegand, C. 523 Wieland, F. 349 Will, A. 363, 370 Willer, T. 382 Wilson, T.H. 365–366, 373 Winkler, H.H. 365–366 Winkler, J. 529 Winnacker, E.-L. 350 Wintermeyer, W. 293, 318 Wirtz, K. 313 Witt, C. 501 Witting, S. 350 Wittmann, H.-G. 436–438, 441–443, 445–456

579

Wittmann-Liebold, B. 437, 441 Wobus, A. 504, 548–549 Wobus, U. 548, 549 Woese, C. 344–345 Wold, I. 236 Wolf Frommer, W. 376 Wolf, E. 382 Wolfrum, R. 350 Wollman, E. 134, 136 Worton, R.G. 497 Wrobel, K.-H. 347 Wu ¨ nsch, E. 240 Wurtz, T. 463

Yamada, Y. 245 Yamashita, A. 372 Yamazaki, H. 456 Yaniv, M. 70 Yankelevich, L. 9 Yanosfsky, C. 149, 152 York, S. 154 Yurchenco, P. 248–249

Zabarovsky, E.R. 77 Zachau, H. 45, 64, 286–294, 291, 316, 320 Zachau, H.G. 285 Zaitlin, M. 457 Zamecnik, P. 52 Zasloff, M. 397–416 Zavilgelsky, G.B. 50 Zech, H. 355 Zenk, M. 338–339, 381 Zenkevich, I.A. 28 Zetina, C.R. 170–171, 173, 179 Zhebrak, A.P. 30 Zheltovsky, I.V. 40

580

Ziegler, H. 358 Ziegler, M.M. 190 Ziegler, W. 529 Zilber, L.A. 2–7, 9, 19, 38, 47, 100

INDEX

Zimelka, D. 281 Zimmermann, U. 361 Zipser, D. 163 Zoschenko, M.M. 22 Zurek, B. 509

Stories of Success, Volume 46: Personal Recollections XI (Comprehensive Biochemistry)

Stories of Success, Volume 45: Personal Recollections. X (Comprehensive Biochemistry)

Selected Topics in the History of Biochemistry VII: Personal Recollections (Comprehensive Biochemistry, Vol 42)

The Biochemistry of Archaea (New Comprehensive Biochemistry)

Glycoproteins I, Volume 29 (New Comprehensive Biochemistry)

Glycoproteins II, Volume 29 (New Comprehensive Biochemistry)

Bioenergetics (New Comprehensive Biochemistry, Volume 9)

New Comprehensive Biochemistry, Volume 2: Membrane Transport

Photosynthesis (New Comprehensive Biochemistry)

Volume XI

Bioenergetics New Comprehensive Biochemistry

Science Fiction Stories 46

SF-Stories 46 v3

How They Achieved: Stories of Personal Achievement and Business Success

Advances in Carbohydrate Chemistry and Biochemistry, Volume 46

Phospholipids (New Comprehensive Biochemistry v4)

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Separation Methods (New Comprehensive Biochemistry)

RECollections

Grantville Gazette Volume XI

Biochemistry of Lipids, Lipoproteins and Membranes, Volume 31, Third Edition (New Comprehensive Biochemistry)

New Comprehensive Biochemistry, Volume 12: Sterols and Bile Acids

Modern Physical Methods in Biochemistry, Part B (New Comprehensive Biochemistry)

Modern Physical Methods in Biochemistry, Part A (New Comprehensive Biochemistry)

Advances in Agronomy, Volume 46

Soil biochemistry, Volume 10

Recollections Of Anton Chekhov

Advances in Immunology Volume 46

Advances in Chromatography, Volume 46

Midnight Recollections

Progress in Optics, Volume 46

Stories of Success, Volume 46: Personal Recollections XI (Comprehensive Biochemistry)

Stories of Success, Volume 45: Personal Recollections. X (Comprehensive Biochemistry)

Selected Topics in the History of Biochemistry VII: Personal Recollections (Comprehensive Biochemistry, Vol 42)

The Biochemistry of Archaea (New Comprehensive Biochemistry)

Glycoproteins I, Volume 29 (New Comprehensive Biochemistry)

Glycoproteins II, Volume 29 (New Comprehensive Biochemistry)

Bioenergetics (New Comprehensive Biochemistry, Volume 9)

New Comprehensive Biochemistry, Volume 2: Membrane Transport

Photosynthesis (New Comprehensive Biochemistry)

Volume XI

Bioenergetics New Comprehensive Biochemistry

Science Fiction Stories 46

SF-Stories 46 v3

How They Achieved: Stories of Personal Achievement and Business Success

Advances in Carbohydrate Chemistry and Biochemistry, Volume 46

Phospholipids (New Comprehensive Biochemistry v4)

Molecular Genetics of Immunoglobulin (New Comprehensive Biochemistry)

Separation Methods (New Comprehensive Biochemistry)

RECollections

Grantville Gazette Volume XI

Biochemistry of Lipids, Lipoproteins and Membranes, Volume 31, Third Edition (New Comprehensive Biochemistry)

New Comprehensive Biochemistry, Volume 12: Sterols and Bile Acids

Modern Physical Methods in Biochemistry, Part B (New Comprehensive Biochemistry)

Modern Physical Methods in Biochemistry, Part A (New Comprehensive Biochemistry)

Advances in Agronomy, Volume 46

Soil biochemistry, Volume 10

Recollections Of Anton Chekhov

Advances in Immunology Volume 46

Advances in Chromatography, Volume 46

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