GENE FAMILIES Studies of DNA, RNA, Enzymes and Proteins
f^
Editors ,iong Xue, Yongbiao Xue, Zhihong Xu, Roger Holmes, Graeme L Hammond & Hwa A. Lim
World Scientific
GENE FAMILIES Studies of DNA, RNA, Enzymes and Proteins
Published by World Scientific Publishing Co. Pte. Ltd. P O Box 128, Farrer Road, Singapore 912805 USA office: Suite IB, 1060 Main Street, River Edge, NJ 07661 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE
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GENE FAMILIES: STUDIES OF DNA, RNA, ENZYMES AND PROTEINS Copyright © 2001 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.
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GENE FAMILIES Studies of DNA, RNA, Enzymes and Proteins Proceedings of the October 5 - 1 0 , 1 9 9 9 Congress, Beijing, China The 10th International Congress on Isozymes
Editors
Guoxiong Xue Institute of Developmental Biology Chinese Academy of Sciences, Beijing, China
Yongbiao Xue, Ph.D. Institute of Developmental Biology Chinese Academy of Sciences, Beijing, China
Zhihong Xu, Ph.D. Peking University, Beijing, China
Roger Holmes, D.SC. University of Newcastle, New South Wales, Australia
Graeme L. Hammond, M.D. Yale University, School of Medicine, Connecticut, USA
Hwa A. Lim, Ph.D., MBA D'Trends Inc., California, USA
V f e World Scientific w l
Singapore • New Jersey'London • Hong Kong
Honorary President: Clement L. Markert Chair: Zhihong Xu Co-Chairs: Boqing Qiang Fangzhen Sun Guoxiong Xue (Executive) Laining Yu
International Executive Committee: Carla Frova (Italy) Erwin Goldberg (USA) Roger Holmes (Australia) Hans Jornvall (Sweden)
Masataki Mori (Japan) C. Schnarrenberger (Germany) John VandeBerg (USA) Guoxiong Xue (China)
National Advisory Committee: Songlin Chen Yongfu Chen Zhu Chen Miao Du Guofan Hong Yunde Hou Jifang Huang Kam-Len D. Lee Zhensheng Li Cheng Ma Bo Tian Guihai Wang Guanhua Xu Yongbiao Xue Longfei Yan Shaoyi Yan Qifa Zhang Rongquan Zhang Lihuang Zhu Zuoyan Zhu
International Advisory Committee Atonnio Blanco (Argentina) W. Richard Chegwidden (USA) Jacques Drouin (Canada) Clara Gozodezki (Mexico) Hwa A. Lim (USA) Jose Luis Millan (USA) Atsushi Nakazawa (Japan) Eviatar Nevo (Israel) P.R.K. Reddy (India) Francisco Salzano (Brazil) Maria de Fatima L. Santos (Portugal) John Scandalios (USA) Wolfgang Scheffrahn (Switzerland) Michael J. Siciliano (USA) Oleg Serov (Russia) Y.H. Tan (Singapore) Athanasios Tsaftaris (Greece) Jerry Wang (Canada) Diter von Wettstein (USA) Suren Zakian (Russia)
Sessions and Session Chairpersons: (In chronological order) Plenary Symposium:
John VandeBerg Gerald Stranzinger Erwin Goldberg Che-Kun Shen Roger Holmes Boqing Qiang Yongbiao Xue
Gene Families and Isozymes:
Desmond Cooper Henry Weiner Richard Chegwidden
Population Variation of Gene Families:
Robert Gracy
Gene Structure and Mapping:
Jiayang Li
Gene Families and Human Diseases:
Hans Jornvall
Gene Families and Evolution:
Eviata Nevo Shusen Liu
Genetic Mutations and Diseases:
Masami Muramatsu
Genomes and Bioinformatics:
Hwa A. Lim Cheng Jing
Gene Families and Plants:
Desmond Cooper Claus Schnarrenberger
Gene Families and Gene Expression:
Jacques Drouin John R. McCarrey Frederick Sweet
Mammalian Gene Families:
Yongfu Chen
Gene Families and Biotechnology:
Zuoyan Zhu P.R.K. Reddy
Young Scientists:
Fuchu He Fangzhen Sun Congress Liaison: Wang Ning
ACKNOWLEDGEMENTS Technical & Logistics Support for Proceedings Production DTrends, Inc., USA
Financial Support for Proceedings Production DTrends, Inc., USA
MS WORD Editor Hwa A. Lim
[email protected] Manuscript Review Committee Charles H. Blomquist (Health Partners Ramsey Clinic, Minnesota) Xiao Zhuo Chen (Ohio University, Ohio) Paolo Fortina (University of Pennsylvania School of Medicine, Pennsylvania) Erwin Goldberg (Northwestern University, Illinois) Robert Gracy (University of North Texas Health Science Center, Texas) Perry B. Hackett (University of Minnesota, Minnesota) Graeme Hammond (Yale University School of Medicine, Connecticut) Roger Holmes (University of Newcastle, Australia) Larry Kricka (University of Pennsylvania, Pennsylvania) Hwa A. Lim (DTrends, Inc., California) John R. McCarrey (Southwest Biomedical Foundation, Texas) Jose Luis Millan (The Burnham Institute, California) Peter Parsons (University of La Trobe, Australia) Tim Robbins (University of Nottingham, UK) Claus Schnarrenberger (Freie Universitat Berlin, Germany) Frederick Sweet (Washington University School of Medicine, Washington) Alan R. Templeton (Washington University, Missouri) John L. VandeBerg (Southwest Biomedical Foundation, Texas) Guoshun Wang (University of Iowa College of Medicine, Iowa) Henry Weiner (Purdue University, Indiana) Ditter von Wettstein (Washington State University, Washington) Edgar Wingender (Research Group Bioinformatics, GBF, Germany)
National Sponsoring Organizations Chinese Academy of Sciences National Natural Science Foundation of China Chinese Association for Science & Technology Chinese Committee for International Union of Biological Sciences Changjiang Fisheries Institute, Chinese Fisheries Academy of Sciences Molecular Developmental Biology Open Lab, Chinese Academy of Sciences Plant Molecular Developmental Biology Lab, Institute of Developmental Biology
Industry Organization Sponsoring Congress DTrends, Inc., USA http://www.d-trends.com
Industry Exhibitors Gene Company Ltd. Amersham Pharmacia Biotech Shanghai Sangon Co. Ltd. Bio-Rad LTI Co. Promega Co. EG&G Co. Perkin-Elmer Applied Biosy stems Eppendorf Olympus Co. Nikon Co. Novo Nordisk Beijing Tianxiangren Biotechnology Co. Ltd. Beijing Liuyi Instrument Factory Pall Co. Millipore Biotinge-Tech Co. Ltd. Beijing SBS Biotechnology Inc.
PREFACE As Chairman of the International Executive Committee of the 10 International Congress on Genes, Gene Families, and Isozymes, I am pleased to make the opening remarks for this special volume. The meeting, held in Beijing, coincided with the celebrations of the 50th anniversary of the founding of People's Republic of China. The Congress was organized by Dr. Guoxiong Xue and his Co-Chairs, Drs. Boqing Qiang, Fangzhen Su, and Laining Yu. The Chair of the Congress, Dr. Zhihong Xu, is Vice President of the Chinese Academy of Sciences, and since the Congress was appointed also as President of Peking University. As Presiding Chair of the Congress, and on behalf of all of the participants, I thank the organizers and the Chair for the effort they committed to making the meeting a huge success scientifically as well as socially and culturally. The 10th Congress continued the precedent, established at the 9th Congress, of emphasizing genes and gene families as the primary determinants of isozymes and of protein multiplicity. Toward that goal, the organizers structured the Congress around the following themes: Gene Families and Isozymes Gene Families and Enzymes Population Variation of Gene Families Gene Families and Human Disease Genetic Mutation and Disease Gene Families and Evolution Mammalian Gene Families Gene Families and Plants Gene Families and Gene Expression Gene Families and Biotechnology Genomes and Bio-information The 10th Congress also continued the tradition of exploring the interfaces among various biological disciplines, rather than focusing on individual disciplines as is most common at scientific meetings. Cross-fertilization was highly evident among cognate disciplines in the biological sciences in studies of disease, evolutionary biology, medical genetics and gene regulation. Moreover, research was presented involving a wide range of organisms including bacteria, protozoa, plants, and mammals. This volume contains selected papers from the Congress, all of which have gone through a rigorous peer review process. These papers are representative of the breadth of scientific topics discussed at the meeting and of the high scientific quality of the meeting. I thank the authors, the reviewers, and the editors for their IX
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commitment to the excellence of this volume as a lasting tribute to the Congress and to the field that was founded by Professor Clement Markert. The 10* Congress began with a tribute to Professor Markert, who passed away just four days before the opening of the meeting. Professor Markert had been named as the Honorary President of the Congress, in recognition of his role in discovering isozymes and pioneering the concepts of isozymes and gene families. Professor Graeme Hammond presented a moving tribute to Professor Markert and to the professional and personal contributions that he made to science and to society throughout his life. The 10* Congress was held 30 years after Dr. Markert and his colleagues first published the concept of isozymes. The Congress had a strong international flair, with presentations from scientists representing 29 countries and regions. They included Australia, Austria, Belorussia, Brazil, Canada, the Czech Republic, Denmark, Finland, Germany, Greece, Hong Kong Special Administrative Region of China, India, Ireland, Israel, Italy, Japan, Malaysia, The Netherlands, People's Republic of China, Portugal, Russia, Singapore, Spain, Sweden, Switzerland, Taipei, Thailand, United Kingdom, and USA. As has been traditional from this series of Congresses, the organizers arranged a variety of special social and cultural activities that complemented the scientific activities of the Congress. These included a superb welcoming reception, a visit to the Chinese Opera, and a day trip to the Great Wall and other local attractions. Since their inception in 1961, this series of congresses has provided an opportunity for scientists working in a wide variety of fields involving isozymes and gene families to interact and to learn from the isozyme concept as it is applied diversely to many biological disciplines. I look forward to the 11 International Congress on Genes, Gene Families, and Isozymes to be hosted by Dr. Hans Jornvall of the Karolinska Institute in Stockholm in 2001, the 30th anniversary of this series of congresses.
September 2000 JOHN L. VANDEBERG
Director, Southwest Regional Primate Research Center San Antonio, Texas, USA E-mail:
[email protected] OBITUARY Clement L. Markert (1917-1999) The concept of isozymes was developed by Clement Markert and Freddy Moller in 1959,' which paved the way for extensive studies on enzyme, protein and gene multiplicity across all living organisms. This important scientific discovery has had a profound influence on the biological sciences for more than 40 years, and provided the basis for regular international meetings to discuss the biological and biomedical implications of enzyme multiplicity. More recently, this concept has been extended to a wide range of gene families for DNA, RNA, proteins and enzymes. As the Honorary President of the 10th International Congress on Genes, Gene Families and Isozymes, recently held in Beijing China in October 1999, Dr Markert was planning to attend and participate in his 10th 'Isozyme' Conference. Unfortunately, it was announced at the Congress Opening by Clem's friend and collaborator from Yale University, Dr Graeme Hammond, that he had passed away four days earlier following a recent illness. As Clem would have wanted it, the Congress proceeded in his absence and was an outstanding success. All of us attending the meeting, however, were saddened by the absence of a wonderful scientist who established the field of gene families and isozymes as a fundamental concept of living organisms. We also missed his friendship, good humor and contributions of criticism, advice and commendation of the papers presented at the Congress. Clem Markert was born on April 11 1917 in Los Animas, Colorado USA, and passed away in Colorado Springs on October 1 1999. His 82 years were filled with hard work, adventure, outstanding science, international travel, love for his wife Margaret and their children Alan, Robert and Betsy, and in his younger years, controversy. Clem graduated from the University of Colorado in 1940, following activities in Spain fighting with the International Brigades against the fascist regime in that country in 1938. During the Second World War, he served in the U.S. merchant navy, following completion of his Masters degree at UCLA. After the war, he completed his Ph.D. in 1948 at John Hopkins University in Baltimore, followed by a two-year Postdoctoral at the California Institute of Technology. His first academic appointment as Assistant Professor was at the University of Michigan during 195056. It was during this period that he came under scrutiny by the Committee for Unamerican Activities, commonly referred to as the McCarthy Committee. The response from Clem and Margaret, and the outstanding support provided by his
Markert and F. Moller, Multiple forms of enzymes: Tissue, ontogenetic and species specific patterns. Proc. Natl. Acad. Sci. USA 45 (1959) pp. 753-763. XI
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scientific colleagues and friends, reflected their strength and resilience, and their desire to ensure that free speech was a protected right. His appointment at Johns Hopkins University during 1956-65 was enormously productive and rich in biological discovery and conceptual development. In 1957, the 'zymogram' technique was published jointly with Robert Hunter, a colleague from the University of Michigan. This method combined the resolution power of multiple forms of enzymes by starch gel electrophoresis with the specificity derived from histochemistry in the staining of enzymes. It was applied initially to esterases and subsequently to many other enzymes, including lactate dehydrogenase. It was the latter path-breaking work that led to the 'isozyme' concept, and many scientific discoveries around the world, with studies on micro-organisms, plants and animals revealing the extensive multiplicity of genes, gene families and isozymes in all biological species. In 1965, Dr. Markert moved to Yale University to become Chairman of the Department of Biology during 1965-1971, and continued on at Yale as Professor of Biology until 1986. During 1974-86, he served as the Director of the Center for Reproductive Biology, reflecting his pioneering role in the related field of transgenics. Together with his collaborator, Jon Gordon, Clem developed a powerful tool for developmental genetics, involving the microinjection or micromanipulation of nuclei of mammalian eggs. After 21 years at Yale University, Clem and Margaret moved to North Carolina State University, where he was appointed as a Distinguished University Professor during 1986-93. There, he joined a longstanding friend and colleague, John Scandalios, who had been appointed to a similar prestigious position supported by the State of North Carolina, in its enhancement program for scientific research. At the age of 76, Clem became an Emeritus Distinguished Professor of the University, and returned to live in Colorado Springs, which is near where the Markert family spent their summer break at their 'cabin' high in the Santa Cruz Mountains. During this distinguished career, Clem was recognized with many honors and awards, including election to the National Academy of Sciences (governing Council member during 1970-71,1977-1980); American Institute of Biological Sciences (President, 1965); American Genetic Association (President, 1980); American Society of Naturalists (Vice-President, 1967); American Society of Zoologists (President, 1967); and many other societies. He has served as a scientific editor on a number of journals and other publications, including positions as Managing Editor of the Journal of Experimental Zoology (1963-85); member of the Editorial Board of the Archives of Biochemistry and Biophysics; Differentiation; Cancer Research; Developmental Genetics; Transgenics; and Editor of the Proceedings of the 3 r , and
2
R.L. Hunter and C.L. Markert, Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels. Science 125 (1957) pp. 1294-1295.
Obituary
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5th ^th j S 0 Z y m e Congresses and of the Prentice-Hall Series in Developmental Biology. Ten international congresses have now been held on isozymes and gene families: the first (1961) and second (1966) in New York under the sponsorship of the New York Academy of Sciences, and the third (1974) at Yale University and Chaired by Clem, with outstanding support from Margaret. Subsequent Congresses have been regularly held at various locations, including Austin, Texas (4 , 1982); Island of Kos, Greece (5th, 1986); Toyama, Japan (6th, 1989); Novosibirsk, Russia (7th, 1992); Brisbane, Australia (8th, 1995); San Antonio, Texas (9th, 1997); and Beijing, China (10th, 1999). All of these Congresses, with the exception of the last Congress, were attended by Clem, who played major roles in them all by the delivery of Plenary Lectures as Congress President or Plenary/Symposium Chairman, and strong participation in the scientific and social activities of the Congresses. We have not only lost a great scientist and pioneer in field of isozymes and gene families, he has been a friend, mentor, student and postdoctoral supervisor, collaborator, editor, referee and supportive critic for many of us. Clem has been strongly supported throughout his distinguished career by his wife Margaret, who accompanied him to conferences, Isozyme Congresses and other visits with friends and colleagues around the world. They were always generous hosts, inviting us into their homes in Ann Arbor, Baltimore, New Haven, Raleigh and Colorado Springs, as well as to their mountain retreat in the Santa Cruz mountains. Clem was a strong person in every sense of the word, physically and mentally. He was an engaging conversationalist with a remarkably broad knowledge of the biological sciences, and with strong interests in science policy, which were expressed at the highest levels in government and scientific organizations. He also was an internationalist, with a healthy cohort of 'foreigners' undertaking graduate and postdoctoral study. His contribution to the development of science in many countries is well known, including China and Russia. Clem Marker! will be remembered by many scientists, colleagues and friends around the world and has given all of us a lasting legacy in the biological sciences with the isozyme concept now being applied in a broad range of gene families in molecular biology and biochemistry, cellular differentiation, developmental biology, biomedical science, gene regulation, structure and function of enzymes and isozymes, transgenics, and population biology. Farewell Clem. Our condolences go to Margaret and the Markert family with our love and best wishes. September 2000 ROGER S. HOLMES
Vice-Chancellor and President, The University of Newcastle Callaghan, New South Wales, AUSTRALIA Email: vc@newcastle,edu.au
TABLE OF CONTENTS Preface
ix
Obituary
xi
Clement Markert G. L. Hammond
1
Identification of Novel Gene Family Members Based on Efficient Full-Length cDNA Cloning J. Gu, X.-Y. Wu, M. Ye, Q.-H. Zhang, Z. G. Han, H.-D. Song, Y. -D. Peng and Z. Chen Strategies for Testis Specific Gene Expression E. Goldberg Oxidized Isoforms as Diagnostic Biomarkers of Alzheimer's Disease R. W. Gracy, J. M. Talent, C. Malakowsky, R. Dawson, P. Marshall and C. C. Conrad Transgenic Fish and Biosafety W. Hu and Z. Zhu
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21
29
39
Aldehyde Dehydrogenases of Human Corneal and Lens Epithelial Cells R. S. Holmes
49
X-Chromosome Inactivation During Spermatogenesis: The Original Dosage Compensation Mechanism in Mammals? J. R. McCarrey
59
Molecular Evolution and Environmental-Stress E. Nevo
73
Nitric Oxide Related Enzymes and Coronary Artery Disease X. L. Wang
89
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xvi Pathways, Compartmentation and Gene Evolution C. Schnarrenberger and C. F. Martin
103
Tomato CF Genes for Resistance to Cladosporium fulvum C. M. Thomas, M. S. Dixon and J. D. G. Jones
115
Gene Expression and Intermolecular Forces in Estrogen/Receptor Binding Q. Chen, S. Adler and F. Sweet
133
Probing for the Basic of the Low Activity of the Oriental Variant of Liver Mitochondral Aldehyde Dehydrogenase B. Wei and H. Weiner
141
S RNases and Self and Non-self Pollen Recognition in Flowering Plants Y. Xue, H. Cui, Z. Lai, W. Ma, L. Liang, H. Yang and Y. Zhang
149
The Roles of Carbonic Anhydrase Isozymes in Cancer W. R. Chegwidden, I. M. Spencer and C. T. Supuran
157
Biochip and Miniaturization J. Zhang, W. -L. Xing, Y. -X. Zhou and J. Cheng
171
Functional Genomics: A Platform for the Discovery of New Therapies D. Cohen
179
A Novel Mathematical Analysis of Human Leukocyte Antigen (HLA) Polymorphism B. Feng, D. Pan, S. Chen, Z. Ye and A. Xu
185
Characterization of a New Tissue-Specific Mutation of the Yellow Gene Which Supports Transvection J.-L. Chen, J. Liu, K. Huisinga, P. Geyer, J. Mossis and C.-T. Wu MHC Class II Suppression by Trophoblast cDNAs G. L Hammond, D. Mandapati, J. Davila, M. A. Coady and A. L. M. Bothwell
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Null Activity Mutation of Phenoloxidase in Drosophila melanogaster N. Asada, N. Kawamoto and T. Hatta Molecular Information Fusion for Metabolic Networks R. Hofestadt, M. Lange and U. Scholz Intron-Size and Exon Polymorphisms in the Mouse Tissue-Nonspecific Alkaline Phosphatase Gene N. Frohlander and J. L. Milldn Lipoxygenases and Cyclooxygenases of the Testis of Rat S. Neeraja, P. Reddanna and P. R. K. Reddy Effect of Heterogeneous Sperm and Hybridization of DNA Fragment in Allogynogenetic Silver Crucian Carp D. Xia, G. Xue and L. Zhang Gene Expression During Carrot Somatic Embryogenesis N. Wu, F. Diao, M. Qi, Y. Cheng, L. Zhang, M. Huang and F. Chen
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243
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Epigenetic Modifications in Maize Parental Inbreds and Hybrids and their Relationship to Hybrid Vigor and Stability A. S. Tsaftaris, A. N. Polidoros and E. Tani
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CIS-Elements and Transcription Factors Regulating Antioxidant Gene Expression in Response to Biotic and Abiotic Signals J. G. Scandalios and L. M. Guan
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Index
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Photo 1. A rest during a picnic on an island on the Ob Sea - a man-made sea in Siberia, Russia. (I to r): Mrs. and Erwin Goldberg, John Scandalios, Clement Markert, Mrs. and Athanasios Tsaftaris, (unidentified), Eviatar Nevo, Eobert Gracy, Michael Crawford. Prof. Ma&ert was an active participant in every single congress since the inception of the Conpess series in 1%!. (The 7 th International Congress on Isozymes, Novosibirsk, Russia, September 6-13,1992).
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Photo 2. A business dinner at Oleg Serov's residence. (1 to r): Leonid Korochkin (Congress Co-Chair), Roger Homes, Oleg Serov (Congress Co-Chair), John Scandalios and Clement Markert. (The 7th International Congress on Isozymes, Novosibirsk, Russia, September 6-13,1992).
Photo 3. Clement Markert opening the 8th International Congress on Isozymes. (The 8th International Congress on Isozymes, Brisbane, Australia, June 25-July 1, 1995).
Photo 4. Margaret and Clement Markert, between sessions at the Congress Auditorium. (The 8!h International Congress on Isozymes, Brisbane, Australia, June 25-July 1,1995).
Phot© S. Sidney Atanan and Clement Markert Sid, 1989 Chemistry Mobe! laureate for the disccwery of catalytic properties of RNA, is Clement's longtime colleague and Mend. Sid was one of the six Nobel keynote speakers at the Congress to commemorate Clement's 80th birthday. (The 9 th International Congress on Isozymes, San Antonio, USA, April 14-19,1997).
Photo 6. Clement Markert speaking at a dinner to celebrate his 80 birthday. (The 9th International Congress on Isozymes, San Antonio, USA, April 14-19,1997).
Photo 7. Guoxiong Xue and Clement Markert. Guoxiong is Executive Co-Chair of the 10* International Congress on Isozymes, Genes, and Gene Families. Clement was Honorary President of the Congress. This photo was taken in 1995, Brisbane, Australia.
Photo 8. Congress banquet at a Yunan restaurant in Beijing. The Congress Chairman, Professor Zhihong Xu (President, Peking University), is taking part in one of the entertainment programs. Conspicuously absent is Prof. Clement Markert, who passed away four days before the 10* Congress commenced. (The 10* International Congress on Isozymes, Beijing, China, October 5-10,1999).
CLEMENT MARKERT 1917- 1999 GRAEME L. HAMMOND Yale University School of Medicine, Department of Surgery, 333 Cedar Street -121 FMB, New Haven, CT 06510 USA E-MAIL:
[email protected] Thank you Dr. VandeBerg, members and guests of the Chinese Academy of Sciences and participants in the Congress. In April 1997, Clem Markert was diagnosed with carcinoma of the colon and underwent right hemi-colectomy. The pathology report showed 19 positive lymph nodes in a tumor that had invaded through the bowel serosa. He underwent three months of chemotherapy and was then discovered to have widespread pleural pulmonary metastases. For the next two years, he led a very active life with trips to Alaska and Africa, and boating on the Columbia and Snake Rivers. His course for the past three months, however, was one of steady deterioration - to the point that he knew he would be unable to attend the 10th International Congress. He died on the evening of October 1st, 1999 in Colorado Springs. As a surgeon and member of the Department of Surgery at Yale, I collaborated with Clem for many years during his tenure as Chairman and member of the Department of Biology at Yale. This collaboration began in an unusual way. I was investigating how the ischemic myocardium worked - an issue of great importance to medicine and to patients with coronary artery disease. During these studies, I recognized that there must be fundamental changes in the way the heart uses energy - in short, that it must be able to function anaerobically and, as the terminal step in glycolysis, be able to convert pyruvic acid to lactic acid. However, the heart never normally makes this conversion. I searched the literature for an explanation and came across papers by Clement Markert from Johns Hopkins describing the theory of isozymes and showing that the LDH isozyme pattern differed from organ to organ depending upon its energy requirements. I tried to contact Dr. Markert at Johns Hopkins and was told finally that he was, in fact, at my own institution. When I broached the idea to him that the LDH isozyme pattern in ischemic myocardium must be changing to favor lactic acid formation, he responded that this would be tantamount to Lamarckian biology. However, we later showed that the LDH pattern did change and he quickly said, "We both learned a lot from this." His openness, honesty, and high principles attracted many followers from around the world and was responsible, along with such work as his discovery of isozymes, for his election to The National Academy of Sciences.
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G. L. Hammond
Because of the unfortunate terminal nature of his disease in late September, his wife, Margaret, asked if I would give his Presidential Address which follows and which he entitled: Isozymes: A Brief Historical Perspective "Since the initial recognition of multiple molecular forms of enzymes (isozymes) by Markert and Moller (1959), isozymes have been extensively studied or used as markers in a wide range of studies, with virtually every known organism, and at all levels of biological organization. On numerous occasions since the establishment of the isozyme concept, researchers from around the world have gathered in international congresses to discuss their work and to be brought up to date on the technologies employed and the novel applications of isozymes. Each Congress has resulted in significant publications that have proved helpful to scientists from a wide range of disciplines. The study of isozymes has provided insights into the structure and function of the genome, the regulation of gene function during cell differentiation and development, and the structure, function, and evolution of isozymes and their encoding genes per se. With the advent of new technologies and developments in molecular biology came a rapid expansion in the dissection of genes encoding isozymes. Prior knowledge of isozyme structure and function served as a critical foundation of information on which research at the DNA and RNA levels could be based. The significance of gene families encoding functionally related isozymes is becoming increasingly apparent. As the genomes of various organisms from microbes to higher eukaryotes are being resolved, the question of the product of the various genes will greatly be impacted by the early and current studies with isozymes and gene families. Since the first Isozyme Congress was held in New York thirty-eight years ago (1961), there have been several revolutions in biology. Each development has had an impact on our science, and the work presented at each of the subsequent Congresses has in turn impacted biology, medicine, and agriculture in significant ways. The International Congress on Genes, Gene Families, and Isozymes has provided and will continue to provide a unique forum for international communication among biologists. As in the past, I am certain that the 10th Congress being held here in Beijing will prove to be another significant milestone in the dissemination of important information that will further enhance the use of isozymes as basic to all aspects of the biological sciences. It is obvious that isozymes will be a continuing part of biological research and will play a central role in
3
Clement Markert enlarging and deepening our understanding of biological organization. A rich, rewarding, expanding, and exciting future is clearly in store for the field of isozymes as we analyze the structural and functional organization of the genome that creates the metabolic patterns which collectively make all organisms what they are." Clement L. Markert, 1999
In closing, I would like to add two personal notes. The first is from Dr. Erwin Goldberg, Professor of Biochemistry, Molecular Biology, and Cell Biology at Northwestern University, who has known Clem for many years. Dr Goldberg writes: "While discoveries in biology move the field forward, it is rare that an individual's accomplishments can have such an impact on an entire discipline. That is Clem Markert's legacy for present and future biologists." Finally, from myself, I would like to add that Clem Markert significantly affected the lives and careers of many people in this audience, including my own, and that his imprint on clinical medicine and surgery was just as great as it was on biology. His identification of isozymes is used every day in every major hospital in the world for diagnosing pulmonary emboli, myocardial infarction, and ischemia in virtually every organ. His ability to conceptualize led to our present understanding of cell and organ stress, the impending or actual presence of cell death and how to reverse these effects before death of the patient. This is Clem Markert's legacy for present and future clinicians.
IDENTIFICATION OF NOVEL GENE FAMILY MEMBERS BASED ON EFFICIENT FULL-LENGTH CDNA CLONING JIAN G U ' ' 2 , X I N - Y A N W U \ M I N Y E 2 , Q I N G - H U A Z H A N G 2 , Z E - G U A N G H A N 1 , H U A I - D O N G S O N G 3 , Y O N G - D E P E N G 1 , 3 , AND Z H U C H E N 1 ' 2
'Chinese National Human Genome Center at Shanghai, 351 Guo Shoujing Road, Pudong, Shanghai, 201203, China Shanghai Institute of Hematology, Rui-Jin Hospital, Shanghai Second Medical University, 197 Rui-Jin Road II, Shanghai, 2000250, China 3 Shanghai Institute of Endocrinology, Rui-Jin Hospital, Shanghai Second Medical University, 197 Rui-Jin Road II, Shanghai, 200025, China Email:
[email protected] A combination of EST analysis, application of bioinformatics, primer walking, reversetranscription PCR and RACE has been widely used in obtaining novel full-length cDNAs. By applying this method we have cloned 600 novel full-length cDNAs sequences mainly from endocrine and hematopoietic systems. Some of these genes can be categorized into several gene families, which included some transcription factors and those involved in vesicle trafficking and signal transduction. There are also many novel genes showing homology to genes discovered in relatively lower creatures. The bioinformatic analysis combined with experimental methods were used for identifying new members of known gene superfamilies.
1
Introduction
Human genome project now is at a historic turning point, from structural genomics to functional genomics. According to announcement from both public sector and private company sequencing efforts, a working draft of the human genome sequence will be obtained soon, though the finishing will take some longer time [1,2]. The gene discovery and understanding of genetic information will require annotation of the sequence data using bioinformatic tools [3]. On the other hand, cloning of fulllength cDNA has been listed as one of the major tasks of the next phase of genomic science [1]. The integration of cDNA sequences into the genomic ones will greatly facilitate the identification of transcriptional units, the gene isoforms, and the mRNA level and specificity in cell/tissues as a result of genome expression. On the other hand, cDNA project links directly to the protein structural biology and exert significant impact to the medical genetics and biotechnology/pharmaceutical industries. Several approaches have been used to identify full-length cDNA, but the most efficient and popular way is EST sequencing. The conception of EST project is first proposed in the early 1980s, when some scientists recognized that short stretches of cDNA sequences could be used as marker for genes [4]. Until ten years later did this conception turn into reality with large flow of EST data output as the sequencing technique became more automatic and efficient [5]. The dbEST database bulked up 5
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J. Gu et al.
in the last decade when EST projects from different tissue and diverse collection of organisms have been conducted. Along with the finishing of genomic sequencing and gene identification of some model organisms, rapid cloning of important human genes by leaping over taxonomic boundaries, from genes identified in model organisms to those embedded in the more complex genomes of human and mouse is now a possibility. Moreover, ESTs based STSs have been widely used in the construction of gene-based physical map. It also offers valuable experimental evidence of transcription when compared with other computational programs used to predict exons. So that it is a powerful tool for the prediction of exon-intron organization of genes, identification of alternative splicing events and unusual genome organization cases. Gene expression profiles in specific tissue or cell type reflect the functional features of them, hereby identification of novel genes preferentially expressed in this tissue or cell type would be important to clarify the molecular basis of certain physiological process. Functional assays of those obtained novel genes could be of great value to both research and commercial domains. Over the last 3 years, we have been undertaking projects of cataloging the expressed sequence tags (ESTs) from cDNA libraries relatively enriched in full-length cDNA of CD34+ hematopoietic stem/progenitor cells (HSPCs) populations and Hypothalamus-Pituitary-Adrenal (HPA) endocrine system. This approach turned out to be very successful in terms of both gene expression profiling and the discovery of novel genes in an efficient way [6]. Based on bioinformatic analysis, important clues could be obtained with regard to the structural and functional characteristics in the context of the cell compartments of each open reading frame (ORF) in large amount of putative fulllength cDNA sequences. Gene families and groups were identified through homology search across wide range of species through evolution. Application of bioinformatic information from public database allowed to assign the chromosomal localizations for the majority of the novel genes and to obtain the genome organizations in part of the genes, and to all at last in future. Moreover, the gene expression patterns were further approached using both "electronic Northern" and cDNA array so that genes with cell/tissue specific expression could be picked up for further functional studies. 2
2.1
Materials and Methods
EST sequencing and data analysis
CD34+ cells were harvested from cord blood and bone marrow, with gradient separation and anti-CD34 McAb-conjugated MACS (Miltenyi Biotec, Germany) separation twice. RNA extraction, lambda ZAPII cDNA libraries construction,
Identification of Novel Gene Family Members
7
Bluescript phagemid templates preparation, sequencing strategy and data management were manipulated as previous reports of our group [6]. Libraries of HPA endocrine system were constructed using classical strategy according to manufacturer's protocol (Stratagene, USA). The sequencing primers were universe primers including Ml3 Reverse and/or Forward, T3 and/orT7 primers, sequencing mix was BigDye Terminator (Perkin Elmer). 5' or 3' end ESTs generated were categorized into known-gene, known-EST and novel EST groups by BLAST searching against GenBank database with Blast and Fasta programs integrated in GCG package (Madison Wisconsin) (release 108 and later due to working time). 2.2
Full-length cDNA open reading frames cloning
The unknown-gene clones were candidates for novel full-length cDNA ORF cloning. The HSPC clone inserts sequences were obtained with combination of primer extension, partial deletion and subcloning sequencing. AutoAssembler (Perkin Elmer) was applied to assemble the sequences to get the contigs, DNA Strider (Version 1.0) was employed to analyze the reading frames of the contigs. To those partial reading frames clones, 'in silico' EST assembly and rapid amplification of cDNA ends (RACE) was efficiently applied. Proper Marathon-ready cDNA libraries (Clontech, Palo Alto, CA) were selected as RACE template, and the gene specific primers (GSP) were generated from the sequences from HSPC clone. The whole open reading frames were thus obtained and confirmed by RT-PCR. 2.3
Chromosomal mapping
Electronic mapping - For novel genes, dbEST were searched to find the hit EST, then UniGene database (http://www.ncbi.nlm.nih.gov/UniGene) was applied to determine the tissue expression pattern and chromosomal mapping of these novel genes. Those cDNA matched genomic DNA sequence data can also provide mapping information. Radiation hybrid - In addition to the electronic mapping results, Stanford G3 and GeneBridge 4 Radiation Hybrid panels (Research Genetics Inc, Huntsville, AL) was applied as a complementary method to map the novel genes [7]. The results were obtained by submitting the PCR results to the Radiation Hybrid Mapping Server at Stanford Human Genome Center (http://www-shgc.stanford.edu) and Whitehead Institute / MIT center for Genome Research (http://www-genome.wi.mit.edu/cgibin/contig/rhmapper.pl). SHGC or MIT framework markers linked to the subjected genes with a LOD score >6.0 were returned from the auto-servers.
8 2.4
J. Gu et al. Preliminarily structure and function analysis with bioinformatics
Sequence Similarity Comparison - GCG package contains the release versions of EMBL and GenBank databases where the known genes and predicted ORFs were deposited. All amino acid sequences encoded by our novel genes were searched against the nucleic acid sequence sub-databases of some important model organisms such as E.coli, S.cerevisiae, C.elegans, Drosophila, Arabidopsis, and mammals (excluding primates) with the tfasta program in GCG package, respectively. There were two reasons to choose this strategy for homology search: first, there were much more nucleic acid sequences than amino acid ones in the databases; second, through evolution, the amino acid sequences are more conservative than those of nucleic acid ones. In this study, two amino acid sequences were considered as homologues when they shared more than 25% similarity over a region of 50-100 amino acids or the Z-score value higher than 200. Fundamental Structural and Functional Elements Searching - Programs including motifs, profile scan in GCG package and prosite at the Expacy website (http://www.expacy.ch/tools/scnpsite.html) were employed to scan for the motifs on primary structure of the peptides. Programs like peptide structure, plotstructure, pepplot, coilscan and hthscan in the GCG package were applied to analyze the secondary structure of the proteins, and spscan (GCG package), signalP (http://www.cbs.dtu.dk/services/SignalP/) as well as TMHMM(http://www.cbs.dtu.dk/services/TMHMM-l-0/) were used to predict the signal peptide and the a-helix transmembrane domains in those novel ORFs so as to characterize the secreted or membrane anchored proteins. In order to acquire more information about some genes, the psort (http://www.psort.nibb.ac.jp.8800) and NNPSL (http://www.predict.sanger.ac.uk/nnpsl_mult.cgi) were chosen to predict their subcellular localization. Gene Expression Pattern - Unigene and dbEST databases were used to search for the gene expression patterns, namely as Electronic Northern. Gene expression patterns of part of these novel genes were also performed by applying Northern blotting and semi-quantitative RT-PCR. Functional assays of zinc finger genes Functional Analysis of Putative Transregulatory Domain of Construction Expression Plasmid - In order to define the transregulatory properties of zinc finger genes, we select three of them, namely ZNF191, ZNF253 and ZNF255, for further functional assay. Non-zinc finger regions of these genes were inserted into yeast plasmid pGBT9 and mammalian cell plasmid pM (Clontech). Both pGBT9 and pM contain DNA-binding domain (GAL4-BD)(l-147aa) of GAL4, which was driven by ADH1 and SV40 promoters respectively. pGBT9 or pM vector inserted with target sequences were constructed to generate fusion genes encoding GAL4-ZNF191,
Identification of Novel Gene Family Members
9
GAL4-ZNF253 and GAL4-ZNF255 chimeras, respectively. The amplified regions and the junctions in these constructs were verified by DNA sequencing. Yeast One-Hybrid System - Yeast one-hybrid system was used to detect DNAprotein interaction. Yeast reporter strain Y187 (CLONTECH), which contains an integrated lacZ reporter construct protein, was transformed with hybrid expression plasmids containing different GAL4 fusion protein, the negative control pGBT9, weak positive control pGBT9-HA (hemagglutinin) and strong positive control pCLl encoding the full-length wild-type GAL4 according to the protocol of TransActTM Assay Kit (Clontech). Qualitative and quantitative analyses of p-galactosidase were performed with the colony-lift filter assay and liquid culture assay using onitrophenyl (3-D-galactopyranoside (Sigma) as substrate, respectively. Mammalian Cell Transfection-In the mammalian assay system, the recombinant pM with different GAL4 fusions, the negative control pM and the positive control pM3-VP16 encoding herpes virus protein were cotransfected by lipofectAMIN (Gibco/BRL) into NIH3T3 or CHO cells with reporter plasmid pGAL45tkLUC containing five consensus GAL4 binding sites and thymidine kinase (TK) minimal promoter upstream of the luciferase. Different ratios of plasmids to be tested and reporter plasmid were compared in transfection assays. Analyses of luciferase were performed according to the protocol of Luciferase Assay System (Promega) and relative light unit (RLU) was measured on luminometer (Lumat LB9507). 3
Results
Totally, 50000 ESTs were generated from both CD34+ cells and endocrine system, from which 750 novel open reading frames (ORF) were obtained, which included 600 full ORFs and 150 partial ORFs. (Available on website http://www.chgc.sh.cn) Only full ORFs were submitted for further functional analysis. After homology and motifs searching, the 600 ORFs were divided into 7 functional categories according to the functions of their homologue genes or possible functional domains they contain as shown in table 1. Regarding those genes with unknown functions, we compared them to genes discovered in relatively lower model organisms from virus to plants and 151 of them showed homology as shown in table 2. While considering the average length of either full-length cDNA or their deduced peptides, we found that most of them were around 500-1500 in nucleotides and 100-300 amino acids respectively, suggesting a more efficient full-length cloning strategy should be developed to obtain longer genes.
J. Gu et al. Table 1. Functional category of genes. categories Gene number Cell division 26 Cell signaling 50 Cell structure/mobility 13 Cell/Organism defense 9 Gene/Protein expression 99 Metabolism 69 unclassified 334 Table 2. Genes with Homology to those from Lower Creatures. Creature Gene number cowpox virus 3 Bacillus subtilis 3 Haemophilus somnus 1 Saccharomyces cerevisiae 41 Caenorhabditis elegans 79 Drosophila melanogaster 13 Arabidopsis thaliana 11
Further analysis of those genes with homology to known genes reveals that part of them belong to several gene families. The biggest gene family is zinc finger and leucine zipper family, with 17 members respectively. Vesicle transporting related gene families are also abundant in our catalogues, which included 6 ras-related protein, 3 VAMP proteins, 2 sec22 protein and so on. We also identified some gene families involved in signal transduction, for example, the PKA and PTP family with 6 and 1 members respectively. Zinc finger gene family belongs to one of the largest human gene families and plays an important role in the regulation of transcription [8]. This large family may be divided into many subfamilies such as Cys2/His2 type, glucocorticoid receptor, ring finger, GATA-1 type, GAL4 type and LIM family [9-10,13]. In Cys2/His2 type zinc finger genes, there are highly conserved consensus sequence TGEKPYX (X representing any amino acid) between both zinc finger motifs. The zinc finger proteins containing this specific structure are named after kriippel-like zinc finger proteins because the structure was firstly found in the Drosophila kriippel-protein [11]. In our study, we found 14 typical C2H2 zinc finger genes and 3 ring finger ones. Bioinformatics analyses revealed that ZNF191, ZNF253, ZNF254, ZNF255, ZNF256, and ZNF257 were novel genes belonging to Kriippel-like zinc finger gene family. The deduced amino acid sequences of these genes contain 3-18 tandemly repeated zinc finger motifs related to Drosophila Kriippel gene family at the Cterminal and possible transcriptional regulatory elements such as KRAB and SCAN box at their N-terminal. The amino acid "knuckle" between zinc finger motifs, typified by the amino acid sequence TGE(R/K) P (F/Y) X, was also highly conserved in all six deduced amino acid sequences. From these features it was
11
Identification of Novel Gene Family Members
reasonable to predict that all six genes could encode DNA-binding proteins with transcriptional regulatory properties. A Novel Trans-regulatory Domain KRNB Analysis of Non ZF Regions Deduced 368 amino acid sequence of ZNF191 had 4 continuous typical krilppel-like zinc finger motifs in C-terminal and contains rich acidic amino acids in non-zinc finger region. An 81 amino acid stretch at the N-terminal of these genes was highly conserved and has been designated as the SCAN box [12]. In addition to 3, 14, 13 and 4 tandemly arranged typical Cys2Mis2 zinc finger motifs respectively, ZNF253, ZNF254, 3^F256f and 7NF257 genes contained Krtippel-associated box (KRAB) in their non-zinc finger regions. These domains consisting of approximately 75 amino acids are all located at the N-terminal moiety of the genes and enriched in hydrophobic and negatively charged residues with the L (X6)L at its core. This core isflankedby certain residues "(e.g. E, L, V, and C) that arefrequentlyfound in ahelices. Although ZNF2S5 has 18 continuously tandem zinc finger motifs homologous with KrOppel-like zinc finger, its deduced amino acid sequence contains a previously undefined domain, which consists of approximately 81 amino acids, at the N-terminal of the protein. This region was homologous with a few zinc finger genes such as FDZF2 (GenBank accession number U95044) and Q14588, which are enriched in hydrophobic amino acids (e.g. G, I, A, L, F) and negatively charged acidic amino acids (e.g. D) (Fig. 1). This new domain was thus nominated as Krappel-related novel box (KRNB). *
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figure 1. Amino acid alignment of non-zincfingerregionfromZNF255 and related proteins. Conserved amino acid residues are in same color.
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/. Gu et al.
Expression Pattern of ZNF191, ZNF253, ZNF254, ZNF255, ZNF256, and ZNF257- Northern blots and semi-quantative RT-PCR were carried out to examine the tissue or cell expression pattern of these zinc finger genes (data not shown). ZNF191 gene was expressed in almost all tissues and cell lines except for heart. The other five genes were selectively expressed in different tissue. Within the hematopoietic system, ZNF191 and ZNF255 were expressed in all lineages, whereas ZNF253 expression was restricted to monocyte (U937) and immature erythroid (K562). ZNF256 and ZNF257 tended to be expressed in myelomonocytic lineages (HL-60 and U937), although a low expression level could be detected in Tlymphocytes (MOLT-4) and early erythroid cells (K562). ZNF253 expression was observed in all lineages except for K562 cells. Functional Analysis of KRNB in Comparison with SCAN and KRAB Transcriptional Regulatory Domain To further address the function of the six genes isolated in the present work, ZNF191, ZNF253 and ZNF255 were chosen to study their transcriptional regulatory activities, since these genes contain SCAN, KRAB and KRNB, respectively. The recombinant pGBT9-ZNF191 containing GAL4ZNF191 chimera and control plasmids pGBT9, pGBT9-HA, and pCLl were then used to transform Yeast strain Y187. The qualitative and quantitative analyses of Pgalactosidase indicated that ZNF-191 might be a transactivator in Y187, since a substantial activity of P-galactosidase from the GAL4-ZNF-191 chimera was observed, as compared to the controls (Fig. 2A). However, when a recombinant pM containing GAL4-ZNF191 chimera was cotransfected with a luciferase reporter plasmid into mammalian cells CHO and NIH3T3, it failed to stimulate the expression of the reporter gene. The luciferase activity was even lower than that of pM with basal activity (Fig. 2B and C). Analysis using yeast one-hybrid system and mammalian cell transfection for defining the functions of KRAB domain from ZNF253 generated, nevertheless, coherent results. After Y187 was transformed with pGBT9-ZNF253 containing GAL4 BD-ZNF253 (l-174aa) chimera, both qualitative and quantitative assays of P-galactosidase displayed a suppressive effect of ZNF253 non-zinc finger region on the transcription of reporter gene lacZ, making the galactosidase activity lower than that from pGBT9 with minimal basal stimulation. A similar transcriptional repressor effect was also observed in mammalian cells in that recombinant pM containing GAL4-ZNF253 fusion gene inhibited significantly the expression of reporter plasmid pGAL45tkLUC in CHO and NIH3T3 cell lines (Fig. 2A, B and C).
Identification of Novel Gene
Figure 2. Functional analysis of putative transregulatory domain of ZNF191, ZNF253, ZNF255 by yeast one-hybrid system and mammalian cell transfection. Each value represents the mean of three replicate assays. The error bars indicate standard deviation from the mean. Where the error bars are not visible, the standard deviation was smaller. A, quantitative analysis of |3-glactosidase in yeast reporter strain Y187 transformed with hybrid expression plasmids containing different GALA fusion protein such as GAL4 BD-ZNF191 (1-25laa), GAL4 BD-ZNF253 (l-174aa) and GAL4 BD-ZNF255 (l-81aa). Y187 were also transformed with the negative control pGBT9, weak positive control pGBT9-HA (hemagglutinin) and strong positive control pCLl encoding the full-length wild-type GAM simultaneously. B and C, analysis of luciferase in CHO and NH3T3 cells cotransfected by constructive plasmids derived from pM containing GAL4 BD with reporter plasmid pGAL45tkLUC, respectively. The recombinant pM including GAL4 BD-ZNF191 (l-251aa), GAL4 BD-ZNF253 (l-174aa) and GAL4 BD-ZNF255 (l-81aa), the negative control pM and the positive control pM3-VP16 encoding herpes virus protein were cotransfected into CHO and NM3T3 cells with different molar ratios of plasmids to be tested and reporter plasmid. Open columns and filled columns represent ratio of 1:1 and ratio of 1:3, respectively.
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To approach the property in transcriptional regulation of ZNF255 containing KRNB domain, the same experimental procedures were performed. Non-zinc finger region (l-81aa) including the KRNB domain was subcloned into the pGBT9 and pM to form in frame fusions, which were used to transform Y187 and transfect mammalian cell lines, respectively. It is interesting to note that the fusion protein GAL4-ZNF255 can stimulate the expression of reporter gene lacZ in yeast. However, slight transcriptional suppression was observed in both CHO and NIH3T3 cell lines (Fig.2A, B and C). Chromosome Localization of ZNF191, ZNF253, ZNF254, ZNF255, ZNF256, and ZNF257. Using FISH, ZNF191 was mapped on chromosome 18ql2.1. Interestingly, ZNF253, ZNF254, ZNF255, ZNF256, ZNF257, ADCAHAOl and CBCBHDIO were all mapped on chromosome 19, ZNF253, ZNF254, ZNF257 being located at 19pl3 and ZNF255, ZNF256 ,ADCAHA01 and CBCBHDIO at 19ql3 by RH technique (Fig 3).
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Figure 3. 7 novel zinc finger genes were localized on chromosome 19 by using RH and STS searching.
Identification of Novel Gene Family Members
15
Ring Finger is another subfamily of zinc finger family which contains two subtypes of C3HC4 and C3H2C3 ring finger [13]. This subfamily contains several functional important tumor related genes such as PML and BRCA1 [14,15]. However, we have identified three members in our library. Further functional analysis of these genes are now undertaking. Leucine zipper is a kind of transcription factor with a characteristic L-X(6)L pattern [16]. 17 novel full-length cDNA were found to have this pattern while 6 of them have this pattern localized at a-helix or coil region. When NLS searching was performed, only 3 of them showed this signal. 4
Discussion
Since tissue- or development stage-related differential expression exists for many genes, cloning of full-length cDNA based on EST analysis in different tissues represents a useful approach for gene identification, especially for those subject to temporal-spatial regulation. In strict sense, a full-length cDNA should cover both the ORF and the complete 5' and 3' UTR. Though a number of methods have been used to surmount the technical obstacles for getting the 5' end of cDNA [17], it is still difficult to reach the transcription start site in many cases. However, as the most important functional information of the mRNA is contained in the ORF, cDNAs containing entire ORFs are often considered as being full-length. By combining several technologies including construction of full-length cDNA enriched libraries, in silico cloning and RACE, a relatively efficient working system has been established to obtain full-length cDNAs, or more precisely cDNAs including entire ORFs, in a cost-effective way. This system has enabled the first resource of cDNAs with entire ORFs to be generated for novel genes whose expression is found in human CD34+ HSPCs and neuro-endocrine system. One strong challenge to the genomic science nowadays is to elucidate the function of the newly discovered huge amount of genes. In this work, we tried to apply the currently available bioinformatic tools to the analysis of the structural and functional characteristics of each ORF. Some experimental assays were also performed to explore the functions of some important genes. Using BLAST search, totally 266 out of 600 ORF were found to share homology to genes with known functions, offering thus important clues for the choice of appropriate functional assays in further study. Hereby we divided them into several gene families involved in transcriptional regulation, vesicle transporting, signal transduction and so on. Cys2/His2 type zinc finger gene family is one of the largest gene families and each member has repeated zinc finger motifs containing finger-like structure by 2 cysteine and 2 histidine covalently binding to one zinc ion[18]. It is estimated that in this huge family, about one third of the members are Kriippel-like genes as characterized
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by the presence of highly conserved connecting sequences "TGEKPYX" between adjacent zinc finger motifs. Substantial evidence indicates that Kriippel proteins are important players in many physiological processes as transcriptional regulators. Kriippel-like zinc finger family has many subfamilies based on non-zinc finger regions and these subfamilies play distinct roles in terms of transcriptional regulation of target genes. So far several domains are found in non-zinc finger regions, such as KRAB, FAX (finger-associated box), POZ (poxvirus and zinc finger), SCAN, FAR (finger-associated repeats) and PR domains [12,19-22]. These domains may affect transcription directly or indirectly. There is evidence to show that KRAB domain, present in one-third of the Kriippel-like zinc finger genes, functions as transcriptional repressor [23]. Of note, 4 genes cloned in the present work contain KRAB domain and the experiments on ZNF253 containing KRAB domain did show transcriptional repressive activities. In view of its wide existence, it is reasonable to suggest that the KRAB domain play an important role in transcription regulation. However, results on the transregulatory properties of other domains from different authors could be controversial. In this study, the SCAN domain from ZNF191 showed distinct activities in different experimental systems, slight transactivator in yeast cells but transrepressor in CHO and NIH3T3 cells. It is possible that the properties of SCAN are determined by gene and/or cell context with distinct transcriptional machineries. A previously undefined domain, nominated here as KRNB, was discovered in ZNF255. This domain, when fused with GAM BD, upregulated the transcriptional expression of luciferase reporter gene in yeast, although no obvious effect was observed in both CHO and NIH3T3 cells. It is thus possible that the KRNB functions as a conditional transactivator. Previous work showed that in human being, more than 40 zinc finger genes aggregated on chromosome 19pl3 and more than 10 genes on chromosome 19ql3 [24-25]. Chromosomal localization also supports this conclusion because 7 of them are aggregated on chromosome 19pl3 or 19ql3 regions except for ZNF191 that has been mapped to chromosome 18. The precise functions of these genes should be further elucidated. However, exploring the function of these novel genes with homology to genes of known functions may provide an insight into their novel functions as well as confirming their known functions. One imporant clue to the possible functions of these novel genes was their expression pattern (available on website: www.chgc.sh.cn) . It is interesting to note that that ZNF253, ZNF254, ZNF256, and ZNF257 are selectively expressed in certain leukemia cell lines representing different lineages, and thus could be related to the differentiation and maturation of hematopoiesis. In contrast, ZNF191 and ZNF255 show ubiquitously expression in leukemia cell lines Regarding these novel genes without ascertained functions, bioinformatic tools are used to search the functional motifs and domains as well as their possible subcellular localization, thus speculate the possible pathways it may involve in.
Identification of Novel Gene Family Members
17
The difficulty was how to deal with the majority of the ORFs without obvious functional information. We therefore attempted to evaluate the conservatism of the sequences through evolution. As a result, 151 ORF show over 25% similarity at amino acid level to those identified in organisms including E.coli, S.cerevisiae, C.elegans, Drosophila, Arabidopsis and mammals. Though a large proportion of these evolutionarily conserved genes are of unknown function, this analysis can provide at least the following information: on one hand, they are most likely to exert important biological function; and on the other hand, the low organisms containing homologous sequences can be used as models in the functional study with gene knock-out or other methods. Moreover, efforts have been made to approach the gene function by search of distinct motifs, including those related to the subcellular localizations. Regarding those orphan genes with no homologous genes available, de novo functional analysis should be taken while keeping comparison to genetic information of any newly sequenced genomes of model organisms. New approach such as more efficient functional analysis assays and 3D modeling software needs to be developed, in order to speed up the shift from structural genomics research to functional genomics research. 5
Acknowledgement
This work was supported in part by the Chinese High Tech Program (863), the Chinese National Key Program for Basic Research (973), the National Natural Science Foundation of China, Shanghai Commission for Science and Technology, and the Clyde Wu Foundation of SIH. The authors thank all members of SIH, SIE and CHGC for their constructive discussion and encouragement. Reference 1.
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STRATEGIES FOR TESTIS SPECIFIC GENE EXPRESSION ERWINN GOLDBERG Department of Biochemistry,
Molecular Biology and Cell Biology, Northwestern Evanston, 1L 60208 U.S.A. E-mail:
University,
[email protected] The mammalian testis is a unique organ programmed for both endocrine and germ cell production, functions that are clearly interdependent. The germ cell component displays distinctive developmental properties illustrated by programmed molecular events that occur with the onset and during spermatogenesis and include activation and inactivation of numerous genes yielding protein products with distinct or modified properties. Genes expressed during spermatogenesis can be classified as "housekeeping" or structural. Both categories include testis specific isozymes and isoforms. One such example is LDHGt, a member of the lactate dehydrogenase gene family that is transcribed only during prophase of the first meiotic division. We have cloned and sequenced the promoter of this gene and demonstrated functionality. Even though this gene and protein are well-studied, there remains the question of why LDH-C4 supplants the other lactate dehydrogenases in testis and sperm metabolism. A second example of an unique protein is provided by calpastatin. This protein is the endogenous inhibitor of calpain, a cytoplasmic cysteine protease. The calpastatin gene, unlike ldhc, is the product of alternative promoter usage by which a truncated testis specific isoform of the somatic calpastatin is produced. Testis calpastatin (tCAST) is transcribed and translated in round spermatids. The promoter region and coding exon is located within an intron of the somatic gene. We have co-localized the testis calpastatin and calpain to the region of the sperm between the plasma membrane and outer acrosomal membrane where presumably it may be a player in the events associated with the acrosome reaction and/or with sperm-egg fusion. A third example is UDP-N-acetylhexosamine pyrophosphorylase, described originally as AgX, the product of an alternatively spliced mRNA. A 16 amino acid deletion in the protein product results in a change in substrate specificity. The large number of testis specific and testis abundant isozyme and protein isoforms suggests that they are not a biological curiosity, but rather are required for both full and complete spermatogenesis and for sperm function. Mechanisms regulating testis-specific gene expression, and structure/function aspects of testis gene expression will be addressed in this report.
1
Specific Gene Expression During Spermatogenesis
Development of an undifferentiated stem cell to the highly specialized spermatozoan is a complex process. Spermatogenesis occurs in three stages. First a stem cell, the spermatogonium undergoes a series of mitotic divisions resulting in renewal of the stem cell population, apoptosis, or commitment to enter the meiotic pathway. This in turn leads to the spermatocyte which in the meiotic phase undergoes two divisions that yield the haploid spermatid. The spermatid then undergoes extensive
21
22
E. Goldberg
remodeling involving cytoplasmic reduction and nuclear chromatin condensation for delivery to the egg during fertilization. In addition to well-studied morphological changes spermatogenesis is characterized by activation and repression of many genes including those encoding isozymes and protein isoforms unique to the testis [1,2]. This paper describes three examples of regulatory strategies resulting in expression of testis specific proteins. 2
Alternative Splice Variants
There are a number of examples of alternative splice variants in germ cells and other tissues 1. AgX subsequently named SPAG2 for Sperm Antigen 2, was discovered in a screen of a human testis cDNA expression library with a pool of sera containing antibodies that agglutinated spermatozoa [3]. AgX cDNAs isolated from testis and placenta cDNA libraries (AgX-1 and AgX-2, respectively) differed by a 48-bp deletion in the open-reading frame (ORF). The AgX-1 and AgX-2 ORF's encoded putative peptide chains of 505 and 521 amino acids (-55.5 and -67.3 kDa), respectively. Both AgX isoforms occur in the testis, but AgX-1 appears to be the only species present in spermatozoa. Immunofluorescence analysis of human spermatozoa detected AgX in the principal piece of the tail. Subsequently, we showed by immunoelectronmicroscopy, localization to the outer dense fibers, structural filaments associated with the mammalian sperm axoneme [4]. Southern analysis of human genomic DNA with a probe common to both AgX isoforms indicated a single AgX gene, therefore alternative splicing is the likely mechanism for production of these variant mRNAs. The AgX isoforms differed by a 16 amino acid deletion suggesting that the AgX-1 mRNA resulted from splicing out of a "miniexon", as has been suggested for mRNAs that differ by a small insertion, e.g. the CI and C2 heterogeneous ribonuclear proteins [5]. Furthermore, alternative splicing of short exons has been proposed as an "on/off switch" for the testis isoforms of other proteins such as the cyclic adenosine 3',5'-monophosphate response element binding protein (CREB) [6]. The tight control of gene expression by alternative splicing occurs frequently in differentiating tissues such as the testis [7]. When we originally described AgX3, the cDNA nucleotide and derived amino acid sequences were not similar to any sequences in the Genbank, EMBL SwissProtein, or PIR data libraries. Recently Mio et al. [8] reported that the cDNA for human UDP-N-acetylglucosamine pyrophosphorylase (UAP1) was identical to AgX. The substrate for this enzyme, N-acetylglucosamine-1-phosphate (GlcNAc-1-P), is a ubiquitous and essential metabolite and plays important roles in several metabolic processes. Subsequently, Wang-Gillam et al. [9] found that substrate specificity of these isoforms differ. The amino acid deletion in AgX-1 changes UDP-N-
Testis Specific Gene Expression
23
acetylhexosamine pyrophosphorylase specificity from UDP-GlcNAc to UDPGalNAc. The significance of this shift in substrate specificity is not immediately apparent but identification of AgX-1 as UDP-GalNAcPP should help unravel its function in spermatozoa. 3
Alternative Promoter Usage: Testis Specific Calpastatin (tCAST)
A testis specific isoform of calpastatin was identified in a screen of a human testis cDNA expression library with serum from an infertile woman [10]. Three transcripts were detected in human testes by Northern blots; the smallest of which (1.9 kb) was specific to testis [11]. The testis specific transcript contained 186 bp of unique sequence at the 5' end. The remainder of the molecule was virtually identical to somatic calpastatin. Calpastatin is the endogenous inhibitor of calpain, a widely distributed cysteine protease. The calpain/calpastatin system is Ca+2 activated and plays important roles in membrane fusion events and vesicle formation. Calpain has been detected in porcine and human sperm [12,13], suggesting critical involvement in sperm function and fertilization. The importance of Ca+2 to calpain activation and the absolute requirement for Ca+2 influx in initiating the acrosome reaction [14,15] support this contention. Isoforms of calpastatin have been described in several tissues. Differences arise from alternate splicing and exon skipping. The testis isoform of calpastatin, however, is the product of unique promoter usage and a single exon residing within intron 14 of the somatic calpastatin gene 11. The overall structure of tCAST is similar to that of the testis specific isozyme, angiotensin-converting enzyme (ACE). A testis specific mRNA encodes t-ACE [16,17] which arises from a unique promoter and single exon in intron 12 of the somatic ACE gene [18,19]. A similar transcriptional strategy generates calspermin from the gene encoding a Ca+2 /calmodulin-dependent protein kinase IV. The calspermin transcript is produced by utilization of a testis-specific promoter located within an intron of the calmodulin kinase IV gene [20]. Functional data for the testis isoforms including tCAST have yet to establish a specific role for each during spermatogenesis. In the case of tCAST, we have learned that this isoform localizes to the space between the plasma membrane and outer acrosomal membrane of the sperm [21] and appears to be associated with the acrosomal vesicle during spermiogenesis (Li & Goldberg, in preparation). As noted above, a functional role in the acrosome reaction seems plausible and is amenable to testing. The more compelling question concerns the selection of the intronic promoter that initiates t-CAST transcription. Possibly, a testis specific trans-activation factor(s) is involved in this regulatory strategy.
24 4
E. Goldberg Unique Gene Expression
The testis specific isozyme of lactate dehydrogenase (LDH-C4) has been studied extensively and has served as an important model for testis specific gene expression. Lactate dehydrogenases became the foundation for the isozyme concept which Clement Markert formulated in 1959. Since that time the LDH literature has increased logarithmically and studies on LDH have been applied to evolution, protein structure, function and diversity, clinical manifestations and gene expression. The evolution of the LDH gene family, tissue distribution of LDH isozymes and physiological implications, have been described on numerous occasions and in exquisite detail (see, for example [22]). Molecular cloning technology applied to the ldhc gene in my laboratory has confirmed the origin in mammals of ldhc as a duplication of ldha [23]. Additionally we have cloned and sequenced the promoter region of ldhc and demonstrated its function by in vitro transcription assays [24] and in vivo as a transgene construct [25]. Surprisingly, our transgene studies revealed that the promoter was active only during the pre-meiotic stages of spermatogenesis even though the protein accumulates throughout germ cell development and differentiation. Whether this is due to stable mRNA or low turnover of the protein remains to be established. Lack of a reliable germ cell culture system makes difficult analyses of testis gene expression in general and ldhc gene expression in particular. Additionally, the question of LDH-C4 function during spermatogenesis or as a sperm enzyme remains open. Our approach to this question, therefore is to target disruption of the gene by homologous recombination. Difficulties in preparing a suitable targeting construct have been resolved by sequencing the entire gene. The gene is large (14 kb) and contains an abundance of intronic repetitive elements (Olssen, unpublished observations) which tend to confound the analyses of targeting construct. Nevertheless, we (Goldberg & Millan, unpublished observations) are completing this project to obtain the ldhc-/-mutant for phenotypic analysis. 5
Summary
Specific gene expression during spermatogenesis seems to have become the norm rather than the exception. The variety of strategies reflect the complexity of the process. Alternative splice sites, alternate promoter usage and cell specific gene expression occur in many cells during development and differentiation. The uniqueness of these gene regulatory paradigms in the testis lies in timing, distribution and specialization of the final product, the spermatozoan.
Testis Specific Gene Expression 6
25
Acknowledgements
The many students who contributed to studies on LDH-C4 are acknowledged as coauthors of the publications from my laboratory. As a personal reflection, I recall that it was my first meeting with Clem Markert at an AIBS meeting in Bloomington, Indiana in 1961 that turned me on to look for multiple forms of LDH in spermatozoa. Blanco and Zinkham and I reported in Science papers simultaneously the discovery of LDH-X (its operational designation) in 1963. Subsequently, I visited with Clem at Johns Hopkins University and clarified the existence of the C subunit of lactate dehydrogenase. Clem's perception, interest and collaboration were instrumental in supporting my long association with this isozyme. This work was supported by NIH HD05863, NIH Sub-5-U54-HD29099, and P 30 HD28048. References 1. 2. 3. 4.
5.
6.
7. 8.
9.
Goldberg E., Minireview: Transcriptional regulatory strategies in male germ cells. J. Androl. 17 (1996) pp. 628-632. Hecht N.B., Molecular mechanisms of male germ cell differentiation. BioEssays 20 (1998), pp. 555-561. Diekman A.B. and Goldberg E., Characterization of a human antigen with sera from infertile patients. Biol Reprod. 50 (1994) pp. 1087-1093. Diekman A.B., Olson G., and Goldberg E., Expression of the human antigen SPAG2 in the testis and localization to the outer dense fibers in spermatozoa. Molec. Reprod. Develop. 50 (1998) pp. 284-293. Nakagawa T.Y., Swanson M.S., Wold B.J., and Dreyfuss G., Molecular cloning of cDNA for the nuclear ribonuclear particle C proteins: A conserved gene family. Proc. Natl. Acad. Sci. USA 83 (1986) pp. 2007-2011. Waeber G., Meyer T.E., LeSieur M., Hermann H.L., Gerard N., and Habener J.F., Developmental stage-specific expression of cyclic adenosine 3',5'monophosphate response element-binding protein CREB during spermatogenesis involves alternatiave exon splicing. Mol. Endocrinol. 5 (1991) pp. 1418-1430. Smith C.W.J., Patton G., and Nadal-Ginard B., Alternative splicing in the control of gene expression. Annu. Rev. Genet. 23 (1989) pp. 527-577. Mio T., Yabe T., Arisawa M., and Yamada-Okabe H., The eukaryotic UDP-NAcetylglucosamine pyrophosphorylases. /. Biol. Chem. 273 (1998) pp. 1439214397. Wang-Gillam A., Pastuszak I., and Elbein A.D., A 17-amino acid insert changes UDP-N-Acetylhexosamine pyrophosphorylase specificity from UDP-GalNAc to UDP-GlcNAc. J. Biol. Chem. 273 (1998) pp. 27055-27057.
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10. Liang Z.G., O'Hern P.A., Yavetz B., Yavetz H., and Goldberg E., Human testis cDNAs identified by sera from infertile patients: a molecular biological approach to immunocontraceptive development. Reprod. Fertil. Develop. 6 (1994) pp. 297-305. 11. Li S., Liang Z.-G., Wang G.-Y., Yavetz B., Kim E.D., Ngai K.-L., and Goldberg E., Characterization of a membrane associated mouse testis calpastatin. Biol. Reprod. (in Press, 2000). 12. Rojas F.J., Brush M., and Moretti-Rojas I., Calpain-calpastatin: a novel, complete calcium-dependent protease system in human spermatozoa. Molec. Human Reprod. 5 (1999) pp. 520-526. 13. Schollmeyer J.E., Identification of calpain II in porcine sperm. Biol. Reprod. 34 (1986) pp. 721-731. 14. Green D.P., The induction of the acrosome reaction in guinea-pig sperm by the divalent metal cation ionophore A23187. J. Cell. Sci. 32 (1978) pp. 137-151. 15. Talbot P., Summers R.G. Hylander B.L., Keough E.M., and Franklin L.E., The role of calcium in the acrosome reaction: an analysis using ionophore. J. Exp. Zool. 198 (1976) pp. 383-392. 16. Ehlers M.R.W., Fox E.A., Strydom D.J., and Diordan J.F.,. Molecular cloning of human testicular angiotensin-converting enzyme: the testis isozyme is identical to the carboxyl-terminal half of endothelial angiotensin-converting enzyme. Proc. Natl. Acad. Sci. USA 86 (1989) pp. 7741-7745. 17. Bernstein K.E., Martin B.M., Bernstein E.A., Linton J., Striker L. and Striker G., The isolation of angiotensin-converting enzyme cDNA. J. Biol. Chem. 263 (1988) pp. 11021-11024. 18. Howard T.E., Shai S.-Y., Langford K.G., Martin B.M., and Bernstein K.E., Transcription of testicular angiotensin-converting enzyme (ACE) is initiated within the 12th intron of the somatic ACE gene. Mol. Cell. Biol. 10 (1990) pp. 4294-4302. 19. Langford K.G., Shai S.-Y., Howard T.E., Kovac M.J., Overbeek P.A., and Bernstein K.E., Transgenic mice demonstrate a testis-specific promoter for angiotensin-converting enzyme. J. Biol. Chem. 266 (1991) pp. 15559-15562. 20. Means A.R., Cruzalegui F., LeMangueresse B., Needleman D.S., Slaughter G.R., and Ono T., A Novel Ca2+/calmodulin-dependent protein kinase and a male germ cell-specific calmodulin-binding protein are derived from the same gene. Molec. Cell. Biol. 11 (1991) pp. 3960-3971. 21. Yudin A.I., Goldberg E., Robertson K.R., and Overstreet J.W., Calpain and calpastatin are located between the plasma membrane and outer acrosomal membrane of cynomolgus macaque spermatozoa. /. Androl. (in Press, 2000) 22. Markert C.L, Isozymes: model systems for analyzing the origin, evolution, regulation, and function of gene families. In: Gene Families: Structure, Function, Genetics and Evoluton. Holmes, R.S. and Lim, H.A. (Eds.) (World Scientific Publishing Co, New Jersey, 1995) pp. 3-7.
Testis Specific Gene Expression
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23. Millan J.L., Driscoll E.E., LeVan K.M., and Goldberg E., Epitopes of human testis-specific lactate dehydrogenase deduced from a cDNA sequence. Proc. Natl. Acad. Sci. USA 84 (1987) pp. 5311-5315. 24. Zhou W., Xu J., and Goldberg E., A 60-bp core promoter sequence of murine lactate dehydrogenase C is sufficient to direct testis-specific transcription in vitro. Biol. Reprod. 51 (1994) pp. 425-432. 25. Li S., Zhou W., Doglio L., and Goldberg E, Transgenic mice demonstrate a testis-specific promoter for lactate dehydrogenase (LDH). J. Biol. Chem. 273 (1998) pp. 31191-31194.
OXIDIZED ISOFORMS AS DIAGNOSTIC BIOMARKERS OF ALZHEIMER'S DISEASE ROBERT W. G R A C Y , JOHN M. TALENT, CHRISTINA MALAKOWSKY, RACHEL D A W S O N , P A M MARSHALL, AND C R A I G C. CONRAD
Molecular Aging Unit, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas, 76107, USA Email:
[email protected] Senile plaques consist of beta-amyloid (AfJ), and is the major pathology found with Alzheimer's Disease (AD). Ap\ is particularly sensitive to oxidation, but can also produce reactive oxygen species (ROS) during Afl-fibril formation. Cells from AD subjects are more sensitive to oxidation than non-AD age-matched controls, and it appears that a number of proteins are preferentially oxidized in plasma samples from AD compared to non-AD. We are using immunoprobes specific for oxidized proteins to elucidate the mechanism of oxidative damage and apoptosis in the neuron and to evaluate the potential of oxidized isoforms as biomarkers for early detection of AD.
1
Introduction
1.1 Oxygen and ROS Oxidative metabolism is more efficient than anaerobic metabolism, however, incomplete oxygen metabolism leads to cytotoxic reactive oxygen species (ROS). There are many different types of ROS, including oxygen radicals (e.g., superoxide anion, hydroxyl radical), non-radical oxygen species (e.g., hydrogen peroxide, ozone), reactive lipids and carbohydrate derivatives (e.g., hydroxynonenal, malondialdehyde, ketoamines, or ketoaldehydes), as well as others. These ROS can spontaneously react with virtually all cellular macromolecules (e.g., proteins, lipids, and nucleic acids), causing undesirable damage and cell death. For recent reviews see: [1,2,4,5,8]. As seen in Figure 1, damaging ROS also occur from environmental exposure. For example, cigarette smoke, air/water pollutants, ozone, some food additives, and medications all contain powerful oxidizing compounds that directly cause ROS or indirectly generate ROS during breakdown and catabolism. Furthermore, low-level cosmic irradiation, x-rays and other types of electromagnetic irradiation can generate ROS. Even ultraviolet light produced by sunlight can induce photooxidations.
29
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R. W. Gracy et al.
In vivo ROS activated astrocytes. or glial cells, etc.
EXTERNAL ROS Pollutants, UV radiation, etc.
Cell Death Figure 1. The production of Reactive Oxygen Species (ROS). Damaging oxygen/nitrogen species can be generated in vivo or from the environment. Severe damage can result in cell dealth via Necrotic, or Apoptotic pathways.
ROS are also produced by the cellular immune system to combat infections. Macrophages kill invading microorganisms by generating toxic ROS. Because ROS can damage cells indiscriminately, some of the host's cells also succumb to the macrophage attack on the invading microorganism. In the case of chronic inflammations, such as autoimmune responses, much of the tissue damage is due to ROS generated by the immune system. 1.2 ROS Damage and Aging Susceptibility to oxidative stress is more pronounced with age. Organisms accumulate oxidative damage with age, and ROS are implicated in the fundamental process of aging. This has been substantiated both in vitro and in vivo. Cells and tissues exposed to low-level ROS accumulate oxidized proteins similar to those observed in aged cells and tissues. Furthermore, when laboratory animals are fed a
Oxidized Isoforms as Diagnostic Biomarkers of Alzheimer's Disease
31
caloric-restricted diet (to increase life span), the amount of oxidative damage to cellular components is reduced [2]. This evidence supports a correlation between age and the accumulation of oxidatively damaged proteins. ROS react with DNA and cause mutations that can lead to cancer. Beckman and Ames [1] have estimated that a steady state level of DNA damage is approximately 150,000 oxidative adducts per cell, and these oxidative modifications may contribute to half of all human cancers. Furthermore, oxidized proteins may represent 30-50% of the total cellular protein of an old individual [2]. These modifications can result in peptide fragmentation, cross-linking, and amino acid modifications. Essentially every amino acid in a protein is potentially susceptible to chemical modification by oxidation. Such modifications can result in changes in the protein secondary and tertiary structures, and these conformational changes may expose previously shielded regions to further oxidations, or other types of spontaneous modifications such as deamidation [6]. The turnover of modified or damaged proteins also decreases with increasing age. Modified or damaged molecules are more readily degraded in young cells and tissues compared to similar proteins in old cells and tissues, which may interfere with the cell's ability to maintain homeostasis. The accumulation of such oxidized proteins with age was originally thought to result from random oxidation events. However, different ROS are not equally damaging to all amino acids, and different proteins exhibit different susceptibilities to such damage. Schoneich and Yang [13] have pointed out the importance of peptide sequence and neighboring groups in the oxidation potential of methioninecontaining peptides. In addition, protein oxidation can result in free radical propagation. The amyloid beta peptide (A(3) in the brains of patients with Alzheimer's Disease is an example. The A(J peptide contains 40 amino acids, of which only one methionine residue (Met35) can be "easily" oxidized [15]. In contrast, peptides that contain the same amino acids, but in the reverse sequence or scrambled sequences, do not become oxidized. This emphasizes the importance of specific amino acid sequences for susceptibility to oxidation. Because A(3 can also generate free radicals, it is believed to contribute to oxidative damage and neurotoxicicity that occur in the brains of Alzheimer's patients [7,9,10]. Substitution of cysteine for Met35 eliminates the toxic effects of Ap"s toward cells in culture [16].
32 2
R. W. Gracy et al. Results
2.1 Oxidative Damage and Alzheimer's Disease We now recognize that many of the age-related neurodegenerative diseases such as Parkinson's, Alzheimer's and other dementias are either caused, or exacerbated by oxidative damage. This can be explained because the brain is particularly susceptible to ROS damage. First, the brain relies on very large amounts of oxygen (e.g., approximately 20% of the total body oxygen consumption is for brain metabolism). Secondly, brain tissue contains a high concentration of unsaturated fatty acids that are highly susceptible to oxidation. Thirdly, the brain contains high levels of iron but has a relatively low capacity for iron binding. Iron catalyzes the spontaneous generation of ROS. Finally, the brain has relatively low levels of antioxidants. Thus, ROS may play an important role in the etiology of many types of chronic neuropathies. Figure 2 shows the pathological cascade believed to take place during the development of AD. Mutations in several different genes give rise to the abnormal production of the AP peptide, which can lead to increased ROS as discussed above [14]. For example, mutations in genes for the Amyloid Precursor Protein (APP), or in genes encoding the enzymes that cleave this protein, result in the accumulation of the Ap\ AP is believed to mediate the oxidative damage, but it is not clear whether it does this directly or indirectly (or both). Some data suggest that as the peptide undergoes aggregation to oligomers, it generates ROS as a consequence of packing of the nontoxic AP monomers into a toxic oligomer. Other studies suggest that AP causes the stimulation of glial cells (AP is not toxic to glial cells), and that the resulting hyperactivity of the glial cell generates ROS. Mutation m APR l'S-1 FS-2
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Oxidized Isoforms as Diagnostic Biomarkers of Alzheimer's Disease
33
In young cells, these processes do not result in large amounts of accumulated oxidized proteins, but in old cells, the oxidation is greater and leads to neurodegeneration. This could be due to an age-related lack of neuroprotective agent(s), the loss of antioxidants with age, or the failure of old cells to recognize and destroy oxidized proteins. Both genetic pre-disposition and environmental factors play key roles in the age of onset of AD. The addition of AB cells in culture can cause cell death (Figure 3). The cells from AD patients are more susceptible to oxidative damage than non-AD controls. 125
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Figure 3. Survival of AD and control fibroblasts following exposure to the AP peptide (residue 25-35), Hyperbaric Oxygen (HBO), or both. Control and AD cells were grown to a density of 125,000 cells, then incubated as follows: No Treatment (NT); 50 um of A|3; 3ATM of hyperbaric oxygen (HBO); and 50 um of AP + HBO (Ap/HBO). Each bar represents the average and SEM of three separate 30 mm culture dishes.
At the subcellular level, damage caused by exposure to ROS and AB may lead to increased membrane permeability, which results in calcium leaking into the cell. The elevated intracellular calcium may activate calmodulin and stimulate the inducible isoform of nitric oxide synthetase. Nitric oxide in the presence of superoxide spontaneously forms peroxynitrite, which can modify tyrosine to nitrotyrosine. Such nitrotyrosine modifications could effect phosphorylation cascades, such as the hyper-phosphorylation of tau in neurofibrilary tangle formation. Ultimately, the consequences of ROS damage lead to apoptotosis of the neuron causing dementia.
34 2.2 Oxidized Isoforms as Biomarkers of Alzheimer's
R. W. Gracy et al.
Two forms of AD have been characterized; early onset (familial) and late onset (sporadic, greater than 97% of all cases). Because the pathology for both forms of AD are similar, the mechanism(s) that lead to the neuronal death due to excessive Ap* deposition are thought to be similar. The initial stages of AD begin long before clinical symptoms are apparent. The ability to detect pre-clinical AD would offer opportunities to develop and test preventative measures (e.g. antioxidants). Unfortunately, postmortem observation of brain tissue is the only reliable method to date for the 100% confirmation AD. Postmortem confirmation of AD relies on the presence of senile amyloid plaques and neurofibrillary tangles of the aggregated and phosphorylated tau protein. Clearly, predictive diagnostic biomarkers for AD are needed. Genetic biomarkers can be used to predict familial AD, but this is only a small (less that 3%) subset of patients likely to develop the disease. Furthermore, genetic biomarkers are of little use for monitoring the development, progression or prevention of AD. For such purposes, oxidized protein isoforms would offer the best potential diagnostic test. Also, the oxidative damage of AD may not be restricted to proteins in the brain since the ROS may damage cells that make up the blood brain barrier. Moreover, the antioxidant defense systems are compromised in AD brains. Thus, it is likely that specific oxidized proteins may be found in the blood or cerebral spinal fluid (CSF) of persons susceptible to or suffering from AD. The identification of such blood or CSF oxidized protein biomarkers may be the key to diagnosing AD. Furthermore, the degree of oxidation of such isoforms might be reflective of the level of progression of the disease similar to the glycosylation of hemoglobin (HbAlc) in the diabetic. We are using Western blots coupled with immunological staining to identify specifically oxidized AD protein biomarkers. We have found several potential biomarkers in the blood serum. Figure 4 shows a western blot that has been immunostained and quantified for oxidized proteins. Although the protein fingerprints are similar when stained for total protein (not shown), immunostaining reveals specific proteins were more oxidized from the AD samples compared to the age-, gender-, and race- matched controls. Figure 4A (band 1) represents one possible biomarker. The data in figure 4B show that the level of oxidation of band 1 is increased nearly 3-fold in the serum from AD patients compared to Non-AD controls. This increase in oxidation appears to be specific for the protein(s) in band 1. This specific oxidation of band 1 is apparent when band 2 is quantified. Band 2 is not specifically oxidized because there is no apparent oxidation changes when AD and non-AD controls are compared.
Oxidized Isofarms as Diagnostic Biomarkers of Alzheimer's Disease
35
2.3 Antioxidants Many antioxidants exist in vivo. These include metabolites (e.g., glutathione, NAD(P) H, cysteine), enzymes (superoxide dismutase, catalase), and vitamins (e.g., vitamin A, C, E). It has also been proposed that some proteins contain specific regions of antioxidant amino acids that serve as the last line of protection against EOS damage [11]. Since the antioxidant defenses may become compromised with age, and especially in potential AD subjects, 'antioxidants may prove to be useful in preventative therapy. For example, Vitamin E has been shown to slow the progression of the AD [12]. Estrogen replacement therapy has also been used for prevention and treatment of AD. It is now recognized that this is 'due to the antioxidant properties of estrogen. In animal models, powerful antioxidants have been reported to- reverse the damage cause by ROS. Furthermore, these compounds when administered to senile animals decreased levels of oxidized proteins in their brains, and restored short-term memory [3]. However, more research is needed before the optimal antioxidants can be prescribed. For example, it is not known which antioxidants may work best, or the optimal dosage or delivery routes.
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Figure 3. Gene expression promoted by the steroid 16,17-isoxazole derivative V in human HeLa cells transfected with ERa. Compound V (•) was compared as an estrogen with estradiol (A) and I (O). Similarly, 16-(hydroxy-methylene) estrone (X), the synthetic precursor of both I and V [1], was tested as an estrogen. Results are not shown from testing its antiestrogen activity under conditions similar to those for I (see Figure 2).
4
Discussion
When the heterocyclic pyrazole group is fused with the D-ring in estrone, it imparts strong hydrogen bonding properties to that part of the steroid molecule. This causes it to bind with increased affinity for certain steroid-binding proteins, as for example in compound I (Table I). The sterically similar isoxazole group is chemically different from pyrazole; it does not hydrogen bond. The isoxazole group in V increases the hydrophobic characteristics of the D-ring relative to estradiol. These differences can explain why compound I is a much more powerful competitive inhibitor of human placental 17 $-hydroxysteroid dehydrogenase than V, shown in earlier experiments [1]. When I was compared to V with respect to estrogen receptor binding in the present gene expression studies, the hydrophobic isoxazole ring in V appeared to cause a reversal relative to I in the binding affinities for the enzyme and estrogen receptor. Alternatively we must conclude, when D-ring analogs of estradiol interact with estrogen receptors, the effect of hydrogen bonding near the steroid D-ring does not very significantly affect receptor binding.
Gene Expression and Intermolecular Forces in Estrogen/Receptor Binding 5
139
Acknowledgments
We thank Mary Ann Mallon for technical assistance. This work was supported by grants from the National Institutes of Health in the United States R03 CA70515 (SA) and 5R01 DK15708 (FS), and from the Chinese Academy of Sciences (QC). This project was also supported in part by the State Basic Research Development Program (973), G200016107. References 1.
Sweet F., Boyd J, Medina O., Konderski L. and Murdock G.L., Hydrogen bonding in Steroidogenesis: Studies on new heterocyclic analogs of estrone that inhibit human estradiol 17|3-dehydrogenase. Biochem & Biophys Res Commun 180 (1991) pp. 1057-1063. 2. Murdock G.L., Warren J.C. and Sweet F., Human placental estradiol 17(Jdehydro-genase: Evidence for inverted substrate orientation ("wrong-way" binding) at the active site. Biochemistry 27 (1988) pp. 4452-4458. 3. Sweet F. and Murdock G.M., Affinity labeling of hormone-specific proteins. Endocrine Reviews 8 (1987) pp. 154-174. 4. Anstead G.M., Carlson K.E. and Katzenellenbogen J.A. The estradiol pharmaco-phore: ligand structure-estrogen receptor binding affinity relationships and a model for the receptor binding site. Steroids 62 (1997) pp. 268-303. 5. Meyer C.Y., Lutfi H.G. and Adler S., Transcriptional regulation of estrogenresponsive genes by non-steroidal estrogens: Doisynolic and allenolic acid. J Steroid Biochem 62 (1997) pp. 477-489. 6. Adler S., Waterman M.L., He X. and Rosenfeld M.G., Steroid receptormediated inhibition of rat prolactin gene expression does not require the receptor DNA-binding domain. Cell 52 (1988) pp. 685-695. 7. Ramkumar T. and Adler S., Differential positive and negative transcriptional regulation by tamoxifen. Endocrinology 136 (1995) pp. 536-542.
PROBING FOR THE BASIC OF THE LOW ACTIVITY OF THE ORIENTAL VARIANT OF LIVER MITOCHONDRIAL ALDEHYDE DEHYDROGENASE BAOXIAN W E I , AND HENRY W E I N E R
Department of Biochemistry,
1153 Biochemistry Building, Purdue University, West Lafayette, Indiana 47907-1153, USA E-mail:
[email protected] Many Asia people possess a variant form of liver mitochondrial aldehyde dehydrogenase where a lysine replaces a glutamate at position 487 in the 500 amino acid homotetrameric enzyme. From the three-dimensional structure of the enzyme, it appeared that residue 487 interacts with two arginine residues, 264 in the same subunit and 475 in a different one. We used site directed mutagenesis to probe for why the Oriental variant had a high Km for NAD and a low specific activity. The results show that these interactions are not the sole reason for the altered properties of the Oriental variant.
1
Introduction
Abusive consumption of alcohol is a problem that exists in all populations. Though investigators have tried to explain why some individuals consume intoxicating amounts of alcoholic beverages, no definitive explanation has been presented. In contrast, it has been found that there are populations who for non-religious reasons do not consume alcohol, or if they do, it is at very low levels compared to other members of the community. It turns out that these people can not metabolize well ethanol to acetate [1,2]. The normal metabolic pathway for alcohol involves ingested ethanol being converted in the liver by the action of cytosolic alcohol dehydrogenase [3] to acetaldehyde. The acetaldehyde in turn is converted into acetate, catalyzed by liver mitochondrial aldehyde dehydrogenase (ALDH) [4]. Acetate is then utilized by liver or other tissue. Both dehydrogenases use NAD as the electron acceptor producing one mole of NADH per mole of compound oxidized. The cell must convert these NADH molecules back to NAD [5]. A person might not convert ethanol to acetate for a variety of reasons. These would include decreased activity of either dehydrogenase or impaired ability to regenerate NAD from NADH. All three of these systems have been studied and it appears that the major reason for some populations to avoid drinking alcohol containing beverages is that they are deficient in an active form of liver mitochondrial ALDH [6]. Though there are many isozymes of the enzyme in liver, it appears that the mitochondrial form is primarily responsible for acetaldehyde
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oxidation [7]. Apparently, it is the aversion to acetaldehyde that causes people to choose not to drink alcohol containing beverages. Many people from the Orient, particularly of Chinese, Japanese and Korean ancestry, "flush" after drinking ethanol. That is, their faces tend to become reddish [1, 2]. Enzyme analysis was performed on many of these individuals and they were found to be lacking the active form of liver mitochondrial ALDH. Later it was shown that these people possessed a variant of the active enzyme that differed in just one of the 500 amino acids from the active form of the enzyme. At residue 487, a lysine replaced the glutamate that is found in the active form of the 500 amino acid containing homotetrameric enzyme [6]. It was assumed that the Oriental variant of the enzyme was inactive since investigators could not detect any catalytic activity when performing gel assays normally used by investigators studying ALDH. When the tools of molecular biology became available, our laboratory cloned and expressed the active form of the human ALDH [8]. We then mutated the cDNA so it would code for the Oriental variant and expressed it in E. coli. Unexpectedly, the mutant enzyme after purification was found to possess catalytic activity. The specific activity of it was 10% of the non-Oriental form of the enzyme while the Km for NAD increased to nearly 7700 uM compare to 30 uM for the highly active enzyme [9]. Thus, under either physiological conditions or even standard assay conditions, the Oriental enzyme would essentially exhibit so little activity that it would appear to be inactive. When some Asian people who were deficient in ALDH were genotyped, it was found that they possessed genes coding for both the 487 glutamate active variant as well as the 487 lysine low-active form [10]. We later showed that these people express both forms of the enzyme indicating that the Oriental subunit was dominant over the glutamate-form [11]. Crabb's laboratory demonstrated using HeLa cells that the Oriental variant caused a decrease in activity of the glutamate containing enzyme [12]. This was the first proof that the Oriental variant was dominant in a heterotetramer. Before our knowing the structure of the enzyme, we postulated that the reason for the high Km of the Oriental enzyme could be that the positive nicotinamide ring of NAD was located near the glutamate at position 487. The binding of NAD would become difficult when the glutamate residue became a lysine. Consistent with this proposal was our finding that if we placed a neutral glutamine (Q) at this position (residue 487) a low Km, high activity mutant was produced [9]. This shows that it was not the absence of the glutamate residue (E), but the presence of the positively charged lysine (K) that caused the Oriental variant to become inactive.
Oriental Variant of Liver Mitochondrial Aldehyde Dehydrogenase
R475 in B E487 in A R264 in A
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A-siibunit
B-subunit
Figure 1. Human liver mitochondrial aldehyde dehydrogenase. Panel A shows one subunit of the homotetrameric enzyme. Residue 487 in a glutamate (E) in the active form of the enzyme'and is a lysine in the Oriental variant. Also shown are two arginine (R) residues. Panel B shows a dimer of aldehyde dehydrogenase. One of the arginines that interacts with residue 487 is found in the same subunit (R264) while the other (R475) is found in a different subunit. For purpose of illustration, one subunit is shown as a ribbon. Panel C shows the tetrameric arrangement. Two pairs of dimers make of the tetrameric enzyme.
In 1997 the three dimensional structure of the corresponding beef liver enzyme was solved by Thomas Hurley's laboratory [13], It was found, much to our surprise, that residue 487 was not located near the nicotinamide ring as we postulated. Instead, this residue was located on the surface of the subunit and formed salt bonds with two different arginine residues (R). One was located at position 264 in the same subunit and the other was at position 475 in a different subunit. This structural arrangement is presented in Figure 1 along with an illustration of just one subunit of the enzyme. The enzyme actually is a dimer of dimers. That is, two subunits interact as shown in panel B to form the tetrameric enzyme, shown in panel C. The important interactions with respect to the Oriental variant take place between subunits that form one of the dimer pairs. Since the altered properties of the Oriental variant of ALDH were not a result of the lysine at position 487 directly interfering with the binding of NAD, we undertook an investigation of the importance of the interaction between it and the
Oriental Variant of Liver Mitochondrial Aldehyde Dehydrogenase two arginine residues. Site directed mutagenesis was employed. results are presented in this chapter.
2
145 Some of the
Methods
Single mutations were created by use of oligonucleotides and polymerase chain reaction techniques. The mutant colonies were selected by double stranded DNA sequencing with a thermocycler sequencing kit. Double and triple mutants were constructed by exchanging the cDNA fragments containing the single mutants with the corresponding fragments of the native or E487K cDNA from pT7-7 plasmid. All mutants forms of the enzyme were purified as described previously[14]. The purity of the enzymes was checked by SDS-polyacrylamide gel electrophoresis using the Coomassie Blue staining procedure. The final protein concentration was determined with a Bio-Rad protein assay kit with bovine serum albumin as a standard. Dehydrogenase activity assays were performed by measuring the rate of increase in the fluorescence of NADH formation in 100 mM sodium phosphate(pH7.4) at 25 °C with an Aminco filter fluoro meter. Concentrations of NAD were l-10mM for native and different mutant enzymes. The propionaldehyde concentration was 14 uM. All kinetic measurements were performed at least three times, and the mean values were used for calculations or plots. Kinetic parameters were obtained from the MicroMath scientist program. All detailed description of the methods and materials can be found in our recent publication [15]. 3
Results and Discussion
We previously demonstrated that the recombinantly expressed version of the Oriental variant of human mitochondrial liver aldehyde dehydrogenase was active but had a very high Km for NAD. It bound NAD poorly but bound NADH well [9]. Thus it appears that the enzyme has difficulty in interacting with the positive charge of the nicotinamide ring of NAD. The properties of the enzyme as well as that of the E487Q mutant are presented in Table 1. The latter construct, mentioned above, was studied so we could determine if it was the loss of the glutamate residue or the presence of the lysine that caused the enzyme to possess altered properties. From the data, it is apparent that the presence of a positive lysine hurt the enzyme and not the loss of the negative glutamate.
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From the structure shown in Figure 1 it was apparent, so we thought, that the disruption of the interactions between the glutamate at position 487 and the arginines at positions 264 and 475 were the cause of the altered properties of the Oriental variant. To test for this, a number of mutations to the enzyme were made. These included changing the arginines so that there would not be positive charge repulsions between the groups as well as trying to restore the salt bonds by introducing a negative charge in place of the positive arginines. First, simple controls were made which included changing the arginine in the native enzyme. All the data is summarized in Table 1. The arginine at position 264 does not seem to be important for the activity of the enzyme. This is not the case with the arginine at position 475. The mere removal of this arginine caused the enzyme to have altered properties. Thus, it was not possible for us to determine how important were the interactions between residue 487 and 475. It is of possible interest to note that the glutamate at 487 and the arginine at position 475 are not conserved in all the members of the ALDH family. Every form of the enzyme that has a glutamate at 487 has an arginine at position 475. This interaction, then, is critical for the enzyme. It was our hope to be able to explain why the Oriental variant of the ALDH had a low activity and a high Km for NAD. Based on structural information it appeared that the interaction between the lysine at position 487 in the Oriental variant and the arginine 264 in the same subunit or arginine 475 in the subunit making up the dimer-pair was critical. The mutational approach did not allow us to unravel this interesting question for any change made to the arginine at position 475 caused the enzyme to have altered properties. Thus in spite of knowing the three dimensional structure of the enzyme, we still cannot explain how the one amino acid replacement caused the Oriental variant to have such different properties. Table 1 Kinetic properties of various mutant of human liver mitochondrial ALDH.
Mutant E487a (Native enzyme) E487Q E487,R475Q E487.R264Q E487,R475Q, R264Q E487,R475E E487,R264E E487,R475E, R264E E487K E487K, R475Q E487K, R264Q
KM NAD (pM) 37 90 850 60 1300 1300 740 16000 7400 1500 1400
*C<JA
(min1) 200 120 120 125 nd 18 12 4 16 27 27
Hill coefficientb (n) 1.0 1.0 1.8 1.0 1.6 2.0 1.0 ndc 1.0 1.5 1.0
Oriental Variant of Liver Mitochondrial Aldehyde Dehydrogenase
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E487K, R264Q, R475Q 1500 nd 1.4 E487K,R475E 3200 40 1.5 E487K.R264E 700 31 1.0 E487K, R264E, R475E 7700 7 L4_ a E487 is the Native ALDH possessing a glutamate at position 487. E487K is the Oriental variant ALDH in which glutamate 487 was replaced by a lysine. The other mutants, except for E487Q, represent double or triple mutants that were designed to test for the restoration of activity by reestablishing salt bonds between residues at position 4887 with those at 264 and 475. b Hill coefficient is a measure of subunit interaction. Mutation of R475 caused the enzyme to be one that exhibits cooperativity in coenzyme binding as noted by the value being greater than 1.0. c the activity was too low to determine accurately a Hill coefficient. 4
Acknowledgements
This work was supported in part by a grant from the National Institute of Alcohol Abuse and Alcoholism (AA05812). References 1. Wolff, P.H. Ethnic differences in alcohol sensitivity. Science 175 (1972) pp. 449-50. 2. Harada, S., Agarwal, D.P. and Goedde, H.W., Aldehyde dehydrogenase deficiency as cause of facial flushing reaction to alcohol in Japanese [letter]. Lancet 2 (1981) pp. 982. 3. Crabb, D.W., Bosron, W.F. and Li, T.K., Ethanol metabolism. Pharmacol Ther 34 (1987) pp. 59-73. 4. Svanas, G.W. and Weiner, H., Use of Cyanamide to Determine Localization of Acetaldehyde Metabolism in Rat Liver. Alcohol 2 (1985) pp. 111-115. 5. Lehninger, A.L., Phosphorylation coupled to oxidation of dihydrodiphosphopyridine nucleotide J. Biol. Chem. 190 (1951) pp. 345-359. 6. Ikawa, M., Impraim, C.C., Wang, G. and Yoshida, A., Isolation and Characterization of Aldehyde Dehydrogenase Isozymes from Usual and Atypical Human Livers. J. Biol. Chem. 258 (1983) pp. 6282-6287. 7. Svanas, G.W. and Weiner, H., Aldehyde Dehydrogenase Activity as the Ratelimiting Factors for Acetaldehyde Metabolism in Rat Liver. Arch. Biochem. Biophys. 236 (1985) pp. 36-46. 8. Zheng, C.-F., Wang, T.T. and Weiner, H., Cloning and Expression of the Fulllength cDNAs Encoding Human Liver Class 1 and Class 2 Aldehyde Dehydrogenase. Alcohol. Clin. Exp. Res. 17 (1993) pp. 828-831.
148 9.
10.
11.
12.
13.
14.
15.
B. Wei & H. Weiner Farres, J., Wang, X., Takahashi, K., Cunningham, S.J., Wang, T.T. and Weiner, H., Effects of Changing Glutamate 487 to Lysine in Rat and Human Liver Mitochondrial Aldehyde Dehydrogenase. A Model to Study Human (Oriental Type) Class 2 Aldehyde Dehydrogenase. 7. Biol. Chem. 269 (1994) pp. 13854-13860. Crabb, D.W., Edenberg, H.J., Bosron, W.F. and Li, T.-K., Genotypes for Aldehyde Dehydrogenase Deficiency and Alcohol Sensitivity. The Inactive ALDH2(2) Allele is Dominant. J. Clin. Invest. 83 (1989) pp. 314-316. Wang, X., Sheikh, S., Saigal, D., Robinson, L. and Weiner, H., Heterotetramers of Human Liver Mitochondrial (class 2) Aldehyde Dehydrogenase Expressed in E. coli. A Model to Study the Heterotetramers Expected to be Found in Oriental People. J. Biol. Chem. 271 (1996) pp. 3117231178. Xiao, Q., Weiner, H. and Crabb, D.W., The mutation in the mitochondrial aldehyde dehydrogenase (ALDH2) gene responsible for alcohol-induced flushing increases turnover of the enzyme tetramers in a dominant fashion. J Clin Invest 98 (1996) pp. 2027-32. Steinmetz, C.G., Xie, P.G., Weiner, H. and Hurley, T.D., Structure of Mitochondrial Aldehyde Dehydrogenase: the Genetic Component of Ethanol Aversion. Structure 5 (1997) pp. 701-711. Jeng, J.J. and Weiner, H., Purification and Characterization of Catalytically Active Precursor of Rat Liver Mitochondrial Aldehyde Dehydrogenase Expressed in Escherichia coli. Arch. Biochem. Biophys. 289 (1991) pp. 214222. Wei, B., Ni, L., Hurley, T.D. and Weiner, H., Cooperativity in nicotinamide adenine dinucleotide binding induced by mutations of arginine 475 located at the subunit interface in the human liver mitochondrial class 2 aldehyde dehydrogenase. Biochemistry 39 (2000) pp. 5295-302.
S
RNASES
AND SELF AND NON-SELF POLLEN RECOGNITION IN FLOWERING PLANTS
YONGBIAO X U E 1 , HAIYANG C U I , Z H A O L A I , WENSHI M A , LIZHI LIANG, HUUUN Y A N G , AND YANSHENG ZHANG
Laboratory of Plant Genetics and Developmental Biology, Institute of Developmental Biology, The Chinese Academy of Sciences, Beijing 100080, China ('Authorfor
correspondence)
Email: ybxue @public3. bta. net. en Self-incompatibility (SI) is an important intraspecific reproductive barrier to prevent selffertilization in flowering plants. In many cases, SI is controlled by a single multi-allelic locus, the 5 locus. Molecular analysis of self-incompatible species of the Solanaceae, Scrophulariaceae and Rosaceae have shown that a class of ribonucleases encoded by the S locus, known as S RNases, determine the stylar expression of SI but not its pollen expression. A different gene is thought to control pollen expression of SI (pollen S gene). Here, we present some progress made towards molecular cloning of the pollen S in Antirrhinum using two approaches, 5-locus directed transposon tagging and map-based cloning. Possible pathways of how S RNases interact with pollen S gene product to achieve self and non-self pollen recognition are discussed.
1
Introduction
Fertilization in flowering plants involves several cell-cell recognition events. The pollen first adhere on the stigma surface and then germinate and grow intercellularly within transmitting tissues of the style and finally deliver sperm cells into a structure, called ovule, to fuse with the egg cell and central cells to form the embryo and endosperm, respectively. However, in many species of angiosperms, selffertilization is prevented by an intraspecific reproductive barrier, known as selfincompatibility (SI) [4]. In many cases, SI is controlled by a single multi-allelic locus, termed the S locus, which is also referred to as the monolocus SI. There are also di- or multi-locus SI. The number of S alleles within an S locus is quite large, reaching hundreds in some species [4], and the S locus is the biggest multi-allelic one in plants described so far, similar to animal MHC (major histocompatibility) and fungal mating type loci. Based on floral morphs (style lengths and anther levels) of self-incompatible species, SI is divided into homomorphic or heteromorphic [4]. In the former, floral morphs are the same between individuals with different S genotypes, whereas individuals with different S genotypes among a heteromorphic SI species display different floral morphs. Homomorphic SI is a predominant form and can be further classified into gametophytic and sporophytic based on modes of genetical control of pollen SI phenotype [4]. 149
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Gametophytic Self-Incompatibility and S RNases
In gametophytic self-incompatibility (GSI), the haploid S genotype of pollen determines its phenotype. When an S allele carried by pollen matches either of the stylar ones, pollen tube growth is arrested within the style after germination. Species from the Solanaceae, Rosaceae and Scrophulariaceae are often of GSI. Biochemical studies on GSI began in 1950s when Lewis identified pollen antigens related to S alleles in evening primroses {Oneothera organesis) [14]. But until 1986, Anderson et al. [2]were able to clone the first gene product encoded by the S locus, known as S locus glycoproteins (SLGs), from Nicotiana alata. So far, over 100 related SLG genes have been isolated from several species of the Solanaceae, Rosaceae and Scrophulariaceae [1, 11, 20, 23, 24]. Based on DNA sequence analysis, the SLGs showed high homology to a class of ribonucleases in fungi (Rh and T2) [10] and contained similar active sites. Because of that, they were refereed to as self-incompatibility (S) ribonucleases (RNases) [17]. S RNases are usually 250 amino acids in length and contain 5 conservative (C1-C5) and 2 hypervariable (Hva and HVb) regions [9] (Figure 1). CI, C4 and C5 are hydrophobic domains and may be related to the formation of the spatial structure of S RNases; C2 and C3 are hydrophilic enzymatic sites. However, recent results showed that C4 domain is not well conserved in the S RNases from the Scrophulariaceae and Rosaceae [20, 24]. The expression of S RNases are developmentally regulated and mainly located in extracellular matrix of styles with a distribution below top 1/3 of style length, where self-pollen tube growth arrest occurs [3, 24]. Using in vivo 32P labeled pollen RNA, McClure et al. [18] demonstrated that labeled RNA remained intact after outcrossing and were degraded after selfing, suggesting that S RNases function selectively in vivo and only degrade RNAs after selfing. This result also indicated that the S RNases possibly inhibit self-pollen tube growth through a cytotoxic effect. In fact, removal of ribonuclease activity of S RNases in Petunia inflata leaded to the loss of their ability to reject self-pollen [8]. Further, transgenic results from P. inflata using both loss-of-function and gain-of-function approaches provided direct evidence that S RNases are responsible for the stylar expression of SI [12].
NH2-COOH
CI
C2
HVa
HVb
C3
C4
C5
Figure 1. Schematic illustration of S RNase organization. CI: 5 Conservative domains. Hva and HVb, Hypervariable domains.
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It is still not clear how S RNases accomplish pollen recognition. The transgenic results showed that the pollen product of the S locus (pollen S) is not S RNases because down-regulation of S RNase only affected the style SI phenotype but not pollen [12]. Recent results have demonstrated that the HV regions determine S RNase recognition specificity [15], though other regions could not be excluded from their involvement[26]. However, Matton et al. [16] showed that a single amino acid change within the HV region between two S RNases converted their respective pollen recognition specificity. The current models of S RNase action postulate that the pollen component of the 5 locus encodes receptor or inhibitor for S RNases [5, 19]. In the receptor model, the pollen S produces a membrane-bound receptor which specifically internalizes self-RNase and cause self-pollen destruction. In the inhibitor model, the expression of pollen S leads to production of an S ribonuclease inhibitor which inhibits all other S RNase activity except self-S-RNase which then arrest the growth of self-pollen tubes. Currently, both models are lacking molecular or biochemical evidence. However, the inhibitor model could explain the generation of pollen part mutations obtained previously [13] and the result that GSI diploid plants normally become self-compatible after being made tetraploids [1]. In Antirrhinum, a pollen part mutant obtained through transposon mutagenesis experiments was likely produced by an S allele duplication [25]. Recently, Golz et al. [7] have studied pollen part mutants in Nicotiana alata generated through irradiation and clearly shown that some of them resulted from the S allele duplication. In these cases, two different S alleles are present in the pollen, two different S RNase inhibitors would be generated and, therefore, S RNase activities encoded by either 5 alleles would be inhibited. Consequently, pollen carrying two different 5 alleles would overcome the action of S RNases produced in the self-style and allow normal pollen tube growth to occur. However, it is possible that the inactivation of pollen 5 encoded inhibitor could be lethal to pollen tube growth because of the inability to inhibit any RNases from the style. If this were the case, it would be difficult to clone the pollen S gene through conventional transposon mutagenesis approaches. Thus, strategies that select specifically for gametophytic lethal mutations or more physical approaches might be useful for cloning the pollen component of the S locus. 3
Approaches to Clone Pollen S Gene in Antirrhinum
Considering the possibility that pollen S inactivation might lead to gametophytic lethality, indirect approaches should be adopted to clone it. In Antirrhinum, the following two approaches are being actively pursed in order to clone the pollen S.
152 3.1
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DNA transposons are known to transpose to linked loci more frequently. Therefore, linked transposons are more useful to mutate target genes. Further, if a transposon is close enough to a known target gene, polymerase chain reaction (PCR) can be applied to screen a large pool of individuals using gene- and tranposon-specific primers for transposon insertions into the target gene. This approach is known as site-directed transposon tagging which is widely used in plant functional genomics. In order to implement this approach, Antirrhinum plants carrying functional S alleles and an S locus-linked transposon were obtained (Xue et al., unpublished data). Molecular genetic analysis showed that the transposon was highly mobile with a germinal excision rate of about 30%. The plants also carried a non-functional S allele, Sc, which allows a large number of progeny to be generated by selfing. It is possible to screen directly for the S RNase gene insertion because inactivation of S RNase does not lead to lethality but self-compatibility [12]. Currently, we are screening for transposon insertions into an S RNase gene. Once the transposon jumps within or close to the S RNase gene, it will be further mobilized to mutagenise the S locus and, in particular, to screen for gametophytic lethal mutations. The mutations will be further analyzed to determine if they affect pollen expression of SI. 3.2
Map-based approach
Genetic and molecular evidence indicate that the S locus is highly polymorphic [6]. Recent developments in DNA marker and cloning technology, e.g., bulk segregant analysis, and AFLP (amplified fragment length polymorphism) and bacterial artificial chromosomes (BAC), made a map-based cloning approach to the pollen S possible. In this approach, BAC contigs covering the S locus are constructed. Fine mapping of the S locus can be done using a large number of AFLP markers linked to the S locus and an S allele segregating population, allowing the determination of its physical limits. Genes with the pollen S gene signatures, e.g., polymorpic and pollen-specific expression, can be analyzed in details to determine if they encode the pollen S gene product. Up to now, we have made a BAC library of a selfincompatible Antirrhinum line with 52S4 and a BAC clone (ca. 64 Kb) containing a complete S2 RNase gene was obtained. Molecular analysis of this clone has identified over 10 genes tightly linked to the S2 RNase gene (Lai et al, unpublished data).
S RNases and Self and Non-Self Pollen Recognition in Flowering Plants 4
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Perspectives
Genes encoding S RNases from several plant families have been isolated and their roles in determining stylar expression of SI demonstrated. However, a complete picture of GSI is still missing because the identity of the pollen product of the S locus is still elusive. Major efforts are needed to clone this gene. It appears that several approaches including those described for Antirrhinum are applicable for reaching this goal. Recent identification of a pollen determinant of sporophytic SI in Brassica is very revealing [21, 22]. Pollen S gene from a GSI system will certainly lead to more insights into how flowering plants accomplish self and non-self pollen recognition. 5
Acknowledgements
Financial support by the National Natural Science Foundation of China (grant nos.39670387 and 39825103), the National Climbing Programme of the Ministry of Science and Technology of China and the Chinese Academy of Sciences is gratefully acknowledged. We are also grateful for Drs E. S. Coen and R. Carpenter of John Innes Center, UK for their generous helps. References 1.
2.
3.
4.
5.
Ai, Y., Singh, A., Coleman, C.E., Ioerger, T.R., Kheyr-Pour, A., and Kao, T.H., Self-incompatibility in Petunia inflata: Isolation and characterization of cDNAs encoding three 5-allele-associated proteins, Sex.Plant Reprod. 3 (1990) pp.130-138. Anderson, M. A., Cornish, E. C , Mau, S. -L., Williams, E. G., Hogart, R., Akinson, A., Bonig, I., Grego, B., Simpson, R., Roche, P. J., Haley, J. D., Penshow, J. D., Niall, H. D., Tregear, G. W., Coghlan, J. P., Crawford, R. J. and Clarke, A. E., Cloning of cDNA for a stylar glycoprotein associated with expression of self-incompatibility in Nicotiana alata, Nature 321 (1986) pp. 3844. Cornish, E. C , Pettitt, J.M., Bonig, I., and Clarke A.E., Developmentally controlled expression of a gene associated with self-incompatibility in Nicotiana alata, Nature 329 (1987) pp. 99-102. de Nettancourt, D., Incompatibility in Angiosperms: Monographs on Theoretical and Applied Genetics, Vol. 3. (1977) (Heidelberg: SpringerVerlag). Dodds, P.N., Clarke A. E., Newbigin, E., A molecular perspective on pollination in flowering plants, Cell 85 (1996) pp.141-144.
154 6.
7.
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Y. Xue et al. Ebert, P. R., Anderson, M A., Bernatzky, R., Altshuler, M., Clarke, A. E., Genetic polymorphism of self-incompatibility in flowering plants, Cell 56 (1989) pp.255-262. Golz, J. F., Su, V., Clarke, A. E., Newbigin, E., A molecular description of mutations affecting the pollen component of the Nicotiana alata S locus, Genetics 195 (1999) pp.1123-1135. Huang, S., Clark, A.G., Kao, T.-h., Ribonuclease activity of Petunia inflata S proteins is essential for rejection of self-pollen, Plant Cell 6 (1994) pp. 10211028. loerger, T. R., Clark, A.G., Kao, T.-h., Polymorphism at the self-compatibility locus in Solanaceae predates speciation, Proc. Natl. Acad. Sci. USA 87 (1990) pp.9732-9735. Kawata, Y., Sakiyama, F. and Tamaaoki, H., Amino-acid sequence of ribonuclease T2 from Aspergillus oryzae, Eur. J. Biochem, 176 (1988) pp. 683697. Kaufmann, H., Salamini, F., and Thompson, R.D., Sequence variability and gene structure at the self-incompatibility locus of Solanum tuberosum, Mol. Gen. Genet. 226 (1991) pp. 457-466. Lee, H.-S., Huang, S., Kao, T.-h., S-proteins control rejection of incompatible pollen in Petunia inflata, Nature 367 (1994) pp.560-563. Lewis, D., Structure of the incompatibility gene. Ill, types of spontaneous and induced mutations, Heredity 5 (1951) pp.399-414. Lewis, D., Serological reactions of pollen incompatibility substances, Proc.Roy.Soc. (Lond.) B 140 (1952) pp. 127-135. Matton, D. P., Maes, O., Laublin, G., Xike, Q., Bertrand, C , Morse, D., Cappadocia, D., Hypervariable domains of self-incompatibility RNases mediate allele-specific pollen recognition, Plant Cell 9 (1997) pp. 1757-1766. Matton, D. P., Luu, D. T„ Xike, Q., Laublin, G., O'Brien, M., Maes, O., Morse, D., Cappadocia, M., Production of an S RNase with dual specificity suggests a novel hypothesis for the generation of new S alleles, Plant Cell 11 (1999) pp.2087-2097. McClure, B. A., Haring, V., Ebert, P.R., Anderson M.A., Simpson, R.J., Sakiyama, F., Clarke, A.E., Style self-incompatibility gene products of Nicotiana alata are ribonucleases, Nature 342 (1989) pp. 955-957. McClure, B. A., GrayJ.E., Anderson, M.A., Clarke, A.E., Self-incompatibility in Nicotiana alata involves degradation of pollen rRNA, Nature 347 (1990) pp.757-760. Ren, D., Zhang, Y., Xue, Y., Molecular controls of self-incompatibility, Adv. Plant Sci. 1 ( 1998) pp.95-106 (In Chinese). Sassa, H., Nishio, T., Kowyama, Y., Hinano, H., Koba, T., Ikehashi, H., Selfincompatibility^ alleles of the Rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily, Mol.Gen.Gene. 250 (1996) pp.547-557.
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21. Schopfer, C. R., Nasrallah, M. E., Nasrallah, J. B., The male determinant of self-incompatibility in Brassica, Science 286 (1999) pp.1697-1700. 22. Takayama, S„ Shiba, H., Iwano, M., Shimosato, H., Che, F. S., Kai, N., Watanabe, M., Suzuki, G., Hinata, K., Isogai, A., The pollen determinant of self-incompatibility in Brassica campestris, Proc. Natl. Acad. Sci. USA 97 (2000) pp. 1920-1925. 23. Xu, B., Mu, J., Nevins, D.L., Grun, P., Kao, T.-h., Cloning and sequencing of cDNAs encoding two self-incompatibility associated proteins in Solanum chacoense, Mol. Gen. Genet. 224 (1990) pp.341-346. 24. Xue, Y. Carpenter, R., Dickinson, H.G., Coen, E.S., Origin of allelic diversity in Antirrhinum 5 locus RNase, Plant Cell 8 (1996) pp.805-814. 25. Xue,Y. Carpenter, R., Dickinson, H.G., Coen, E.S., Mutational analysis of the self-incompatibility locus in Antirrhinum, submitted to Heredity (2000). 26. Zurek, D. , Mou, B., Beecher, B., McClure B., Exchanging sequence domains between S-RNases from Nicotiana alata disrupts pollen recognition, Plant. J. 1 (1997) pp.797-808.
THE ROLES OF CARBONIC ANHYDRASE ISOZYMES IN CANCER W. RICHARD CHEGWIDDEN 1 , I A N M . SPENCER 2 , A N D C L A U D I U T . SUPURAN 3
'Lake Erie College of Osteopathic Medicine, 1858 West Grandview Boulevard, Erie, PA 16509, U.S.A. Email:
[email protected] 2
Division of Biomedical Sciences, Sheffield Hallam University, Sheffield, SI 1WB, UK.
3
Universita degli Studi, Laboratorio di Chimica Inorganica e Bioinorganica, Capponi 7,1-501221, Florence, Italy.
Via Gino
Specific sulphonamide inhibitors of carbonic anhydrase also inhibit the growth, and suppress the invasion, of certain types of cancer cells in culture, suggesting potential for cancer therapy. Inhibition of cell growth may be mediated through a reduction in bicarbonate provision by the cytosolic CA II and mitochondrial CA V isozymes, for the synthesis of nucleotides and other cell components. It is hypothesized that suppression of invasive properties may be mediated through inhibition of the cancer-associated, cell surface isozymes, CA IX and CA XII, resulting in a less acidic extracellular pH. CA DC may be a useful marker for renal clear cell and cervical carcinomas and a valuable adjunct to PAP screening. CA XII may be a useful marker for colorectal tumours.
1
Introduction
The zinc metalloenzyme carbonic anhydrase (CA: EC 4.2.1.1) catalyses the reversible hydration of carbon dioxide to bicarbonate (C02 + H20 methazolamide > acetazolamide against all oc-CAs examined. All the a-CA isozymes examined have been shown to possess additional general esterase activity, to which no physiological significance has been ascribed hitherto [37]. In recent years a range of evidence has accumulated suggesting that the active isozymes, CA II, V, IX and XII may be involved in oncogenesis and tumour growth or invasion. The activity of intracellular CA isozymes may be required to support the enhanced rate of biosynthetic processes characteristic of cancer cells, whilst activity of extracellular isozymes may be involved in the creation of an extracellular milieu that is more conducive to cell invasion. Finally, circumstantial evidence has also prompted speculation that CA-RP(RPTPy) may be a tumour suppressor gene. It is also a tenable supposition that both CA-RP(RPTPp) and CA-RP(RPTPy) may be involved in the control of oncogenesis or cell growth by a mechanism mediated through the dephosphorylation of tyrosine.
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Roles of Carbonic Anhydrase Isozymes in Cancer Table 1. Expression and distribution of mammalian carbonic anhydrase isozymes. ISOZYME
SUB-CELLULAR LOCALIZATION
MAJOR KNOWN SITES OF TISSUE EXPRESSION
CAI CAII CAIII CAIV
cytoplasmic cytoplasmic cytoplasmic membrane-bound (extra-cellular)
CAVA
mitochondrial
CAVB CAVI CAVII
mitochondrial secreted cytoplasmic
CA-RP VIII
unknown
CAIX
transmembrane (extra-cellular domain) unknown
CA-RP X CA-RP XI CAXII CA XIII1 CAXIV CA-RP (RPTPp) CA-RP (RPTPy)
secreted transmembrane (extra-cellular domain) unknown transmembrane (extra-cellular domain) transmembrane (extra-cellular domain) transmembrane (extra-cellular domain)
red blood cell, intestine ubiquitous red muscle, adipose tissue kidney, lung, gut, brain, eye, widespread in capillary endothelium liver (also skeletal muscle, kidney) widespread (except liver) saliva brain, salivary gland, lung, probably widespread at low levels brain (esp. Purkinje cells) (widespread at lower level) various tumours, gastric mucosa brain (also pineal gland, placenta) brain widespread, especially colon, kidney, prostate unknown widespread, especially kidney, heart central & peripheral nervous system brain, lung2 (widespread in mouse)
To date CA XIII has been identified only from ESTs (expressed sequence tags) from a mouse cDNA library. 2 Human tissue distribution has not been fully investigated.
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Table 2
Inhibition of some mammalian CA isozymes by sulphonamides
K, (nM) CA ISOZYME Acetazolamide Human CA I Human CA II Human CA III Human CA IV Murine CA V
Methazolamide
200 7 3 x 105 66 60
10 7 1 x 105 33
Ethoxzolamide 1 0.5 5 x 104 13 5
Data are from [24] for the human isozymes and from [11] for the murine isozyme. 2
CA Isozymes and Cell Growth
Bicarbonate, not C02, is the true substrate for early carboxylation steps in biosynthetic pathways which involve biotin-dependent carboxylases or carbamoyl phosphate synthetases. Use of specific sulphonamide inhibitors has furnished evidence that carbonic anhydrase activity is required for provision of bicarbonate for gluconeogenesis [9,29], lipogenesis [6,39,40] and ureogenesis [29,10]. It may be that a low flux through pathways may be accommodated by the uncatalyzed rate of bicarbonate provision, whilst the catalytic activity of carbonic anhydrase is necessary to sustain a higher level of metabolic flux [6]. Such a high level of flux occurs in cancer cells, where the enhanced rate of cell proliferation necessitates a higher level of synthesis of nucleotides and other cell components, such as membrane lipids, than that required in normal cells. Inhibition of the growth of human cancer cells in culture by specific carbonic anhydrase inhibitors was first reported by Chegwidden and Spencer [5], who drew attention to the possible potential of CA inhibitors in cancer therapy. Table 3 shows the effectiveness of three different sulphonamide inhibitors in inhibiting the growth in culture of two different cell lines: U927, a line established from a diffuse, histiocytic, human lymphoma, and Raji, a line of lymphoblast-like cells established from a Burkitt lymphoma. The relative effectiveness of the three inhibitors in inhibiting cell growth correlates well with their effectiveness as inhibitors of CA activity (Table 2). This supports the premise that the effects observed do, indeed, result specifically from inhibition of carbonic anhydrase. Although the GI50 values obtained are much higher than the Ki values for purified CA isozymes, the inhibitor concentrations present in the cell, and especially in the mitochondrion, are likely to be much lower than that of the medium.
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Roles of Carbonic Anhydrase Isozymes in Cancer
Acetazolamide, especially, does not readily penetrate the cell membrane [25] and, although ethoxzolamide is the most lipophilic of the three inhibitors employed, a serum protein has been identified to which it binds strongly [8]. Consequently its effectiveness may be much reduced by the fetal calf serum present in the culture medium. Table 3 Inhibition of cell growth by sulphonamides GI 50 (uM) Cell Line U937 Raji
Acetazolamide 230 230
Methazolamide 20 18
Ethoxzolamide 0.5 0.4
Cells were cultured, in the presence of a range of sulphonamide concentration, in RPMI 1640 medium containing fetal calf serum (10%), glutamine (4 mmol/1) and penicillin and streptomycin (lOOug/ml). They were incubated at 37°C under an atmosphere of 5% C0 2 for a period of two days. Cell viability was confirmed by trypan blue exclusion. GI50 is the concentration of sulphonamide that inhibited cell growth by 50% as measured over the two day period. Chegwidden and Spencer [5] hypothesized that the inhibition of cell growth may be attributed to inhibition of nucleotide synthesis. This may be a consequence of sulphonamide inhibition of either the cytosolic isozyme, CA II, or the mitochondrial isozyme, CA V, or indeed, of both of these isozymes. Inhibition of cytosolic CA would limit bicarbonate availability for the cytosolic isozyme of carbamoyl phosphate synthetase (CPS II) that catalyses the first step of de novo pyrimidine synthesis. CPS II glutamine + HC03" + 2ATP ^. carbamoyl phosphate + glutamate + 2ADP + Pj This enzyme forms part of a multi-enzyme complex which appears to specifically direct cytoplasmically produced carbamoyl phosphate into pyrimidine synthesis. However, there is evidence that carbamoyl phosphate produced in the mitochondrion by the mitochondrial isozyme CPS I, may also be used in pyrimidine synthesis [31]. CPS I NH 4 + +HC0 3 " + 2ATP — ^
carbamoyl phosphate + 2ADP + Pj
Inhibition of the mitochondrial CA isozyme, CA V, would also reduce availability of bicarbonate for CPS I.
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The hypothesis that cell growth is limited by inhibition of nucleotide synthesis is supported by our observation that supplementation of the culture medium with nucleotide precursors (hypoxanthine and thymidine) resulted in no significant inhibition of growth of either U935 or Raji cells by either acetazolamide (up to 5 mM) or methazolamide (up to 0.2 mM) and only slight inhibition by ethoxzolamide (up to 5 uM) [7]. An intra-mitochondrial source of bicarbonate is also required for pyruvate carboxylase, which catalyses the carboxylation of pyruvate to oxaloacetate.
pyruvate + HC03" + ATP
pyruvate carboxylase biotin ^. oxaloacetate + ADP + Pj
This is an early step in the production of aspartate, glutamine and glycine, all of which are precursors of purines and pyrimidines. However, this is unlikely to be relevant in cell culture, since these amino acids are supplied in the medium. Nonetheless, reduction of mitochondrial oxaloacetate levels may well contribute to cell growth inhibition through a different mechanism. Oxaloacetate is required for the transport of acetyl groups out of the mitochondrion (as citrate) into the cytoplasm, where they serve as substrate for the synthesis of lipids, required as membrane components. There is a wealth of evidence that sulphonamide inhibitors of CA also inhibit lipogenesis [23,39,40]. Metabolic labelling experiments have demonstrated that the mitochondrial isozyme, CA V, plays a role in lipogenesis by providing bicarbonate ions for the production of oxaloacetate [23]. However, the possibility that cytosolic CA plays an additional role in lipogenesis, by providing bicarbonate for acetyl CoA carboxylase, cannot be excluded. 3
CA Isozymes and Tumour Invasion
The recent discovery of two a-CA isozymes (CA IX and CA XII) that are associated with, although not exclusive to, certain types of tumours, suggests that these isozymes may be involved in oncogenesis, cell growth or tumour invasion [35,45,49]. Since each of these two isozymes exists as an extracellular domain of a larger transmembrane protein, it is a tenable hypothesis that, by hydrating carbon dioxide, they produce a more acidic extracellular environment, which may facilitate the invasive and migratory properties of cancer cells. There is evidence that the invasive properties of some types of cancer cells are enhanced at more acidic pH [26]. It is now well established that the extracellular pH of solid tumours (pHe) is often lower than in normal tissues, whilst their intracellular pH (pHi) is generally higher. A lower pHe appears to promote invasiveness, whilst a higher pHi is likely to give a competitive advantage over normal cells for growth [42]. By non-invasive techniques, pHe values in solid
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tumours of 0.2 to 0.5 pH units lower than those in normal tissues have been measured [41,48]. Since each of these isozymes constitute part of a transmembrane protein, the possibility cannot be excluded that their role in cancer may, alternatively or additionally, be mediated through ligand binding and signal transduction. In addition to their association with cancer cells, there are several additional pieces of evidence that imply the involvement of CA IX and CA XII in cancer. Firstly, transfection of cultured NIH33T3 fibroblasts, which do not normally express CA IX, with a vector containing the CA IX gene, changes both their morphology and growth characteristics. They replicate faster, in a less controlled manner, and are less dependent on growth factors [33]. Secondly, in certain renal carcinoma cells in which both CA IX and CA XII are over-expressed, these isozymes are both down-regulated by the product of the von Hippel-Lindau (VHL) tumour suppressor gene [13]. Most renal carcinomas of the clear cell type are caused by inactivation of this gene [18]. Moreover, the human CA IX and CA XII genes have been mapped to regions of the chromosome (bands 17q21.2 and 15q22 respectively) that appear to be amplified in a number of human cancers [30]. Suppression of the invasion of four renal carcinoma cell lines by the specific CA inhibitor acetazolamide ( 1 - 1 0 |0.M) has been demonstrated recently in vitro [32]. However, the pattern of different CA isozymes detected in each cell line did not clearly indicate the identity of the specific isozymes involved. The only cell line in which CA IX was expressed showed the least suppression, and only CA II was expressed strongly in all four cell lines [32]. The authors concluded that the effect of acetazolamide was most likely attributable to the inhibition of the cytoplasmic CA II and/or CA XII. 4
Inactive CA Isoforms and Oncogenesis
Two inactive a-carbonic anhydrases CA-RP(RPTP|3) and CA-RP(RPTPy) form part of the extracellular domain of two receptor-type protein tyrosine phosphatases (RPTPP and RPTPy). These protein tyrosine phosphatases (PTPs) are transmembrane proteins with an extracellular ligand-binding region connected to a cytoplasmic tyrosine phosphatase domain [16,19]. It is considered that the CA domains are involved in signal transduction [1,36] and mutations in this domain of RPTPymay result in loss of response to external ligands [43,47]. The importance of tyrosine phosphorylation by protein tyrosine kinase in controlling such processes as cell growth and differentiation is well established and the transient nature of signaling by phosphorylation requires the additional action of PTPs for its regulation. Since hyperphosphorylation of protein tyrosine residues can cause malignant transformation, inactivation of PTPs may also be oncogenic. The gene encoding RPTPy has been mapped to a chromosomal region that is frequently
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deleted in certain renal cell and lung carcinomas, and as a consequence of this it is considered to be a candidate tumour suppressor gene for these cancers [14,17]. The expression of RPTPP is much narrower and seems to be confined to the CNS. Its CA domain binds to a number of ligands where it is intimately involved in intracellular signaling and cell growth. 5
CA Isozymes as Tumour Markers
No firm data was produced to support the use of any CA isozyme as a tumour marker until the discovery of CA IX and CA XII. CA IX was originally identified as a tumour-associated cell surface antigen in HeLa cells, a line established from a human carcinoma of the cervix. At that time it was not known that the antigenic protein possessed an active carbonic anhydrase domain and it was named MN protein. Initial studies demonstrated the expression of MN protein/CA IX in human carcinomas of ovary, endometrium and cervix, but not in the corresponding normal tissues [49]. No expression was observed in normal human heart, lung, kidney, prostate, peripheral blood, brain, placenta and muscle, but message was detected in liver and pancreas [27]. Since it appears to be strongly expressed in most dysplastic and neoplastic cervical tissues, CA IX may well prove to be an important new biomarker [2,20]. There is also strong preliminary evidence that CA IX expression may prove to be a valuable adjunct to cytological diagnosis in improving the discrimination of significant lesions in Papanicolaou (Pap) screening [22]. CA IX is widely expressed in renal cell carcinomas (RCCs), especially those of the clear cell type, but not in normal kidney nor in benign kidney lesions such as cysts, adenomas and oncocytomas. Consequently its potential has been suggested as a biomarker for certain renal cell carcinomas [21,27,28]. Recent evidence suggests that CA IX may also serve as a useful marker of cell proliferation in colorectal neoplasms [38]. The picture is less clear, however, in some other tissues. Whilst CA IX expression is abundant in normal gastric mucosa, it is reduced or absent in gastric tumours [34] Loss of CA IX expression may also be correlated with progression from dysplasia to adenocarcinoma in Barrett's oesophagus [46] and one report also correlates it with several adverse prognostic features in cervical carcinoma [2]. In contrast to the situation with CA IX, the CA XII transcript has been detected in a wide range of normal human tissues, with high levels in kidney, colon, pancreas and prostate [13,45]. Although increased expression of CA XII has been observed in some renal cell carcinomas (RCC) of the clear cell type [13] and in certain cell lines derived from human lung carcinoma [44,45] currently there is no additional data to support the use of CA XII as a tumour biomarker in these tissues. However, there is evidence that this isozyme may be of value in the histopathological diagnosis of colorectal tumours. Furthermore, CA XII expression appears to be
Roles of Carbonic Anhydrase Isozymes in Cancer
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directly correlated to grade of dysplasia, suggesting the possibility of prognostic application [15]. 6
Concluding Comments
Specific sulphonamide inhibitors of carbonic anhydrase clearly inhibit growth in culture and suppress invasiveness of certain cancer cell lines. The mechanisms of inhibition may be different for each process and have yet to be firmly established. Identification of the specific CA isozymes involved will facilitate the synthesis of more potent isozyme-selective inhibitors which may prove effective in cancer therapy. Work is currently in progress in our laboratories on the synthesis of new CA inhibitors with potential for cancer therapy, and on the effects of CA inhibitors on the growth of both cancer cells in culture and of human tumours implanted into immunodeficient mice. References 1.
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32. Parkkila S., Rajaniemi H., Parkkila A-K., Kivela J., Waheed A., Pastorekova S., Pastorek J. and Sly W.S., Carbonic anhydrase inhibitor suppresses invasion of renal cancer cells in vitro. Proc. Nat. Acad. Sci. USA 97 (2000) pp. 22202224. 33. Pastorek J., Pastorekova S., Callebaut I., Mornon J.P., Zelnik V., Opavsky R., Zat'ovicova M., Liao S., Portetelle D., Stanbridge E.J., Zavada J., Burny A. and Kettmann R., Cloning and characterization of MN, a tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loophelix DNA binding segment. Oncogene 9 (1994) pp. 2877-2888. 34. Pastorekova S., Parkkila S., Parkkila A.K., Opavsky R., Zelnik V., Saarnio J. and Pastorek J., Carbonic anhydrase IX, MN/CA IX: analysis of stomach complementary DNA sequence and expression in human and rat alimentary tracts. Gastroenterology 112 (1997) pp. 398-408. 35. Pastorekova S., Zavadova Z., Kostal M., Babusikova O. and Zavada J., A novel quasi-viral agent, MaTu, is a two-component system. Virology 187 (1992) pp. 620-626. 36. Peles E., Nativ M., Campbell P.L., Sakurai T., Martinez R., Lev S., Clary D.O., Schilling J., Barnea G., Plowman G.D., Grumet M. and Schlessinger J., The carbonic anhydrase domain of receptor tyrosine phosphatase P is a functional ligand for the axonal cell recognition molecule contactin. Cell 82 (1995) pp. 251-260. 37. Pocker Y. and Sarkanen S., Carbonic anhydrase: Structure, catalytic versatility and inhibition. Adv. Enzymol. 47 (1978) pp. 149-274. 38. Saarnio J., Parkkila S., Parkkila A.K., Haukipuro K., Pastorekova S, Pastorek J., Kairaluoma M.I. and Karttunen T.J., Immunohistochemical study of colorectal tumors for expression of a novel transmembrane carbonic anhydrase, MN/CA IX, with potential value as a marker of cell proliferation. Am. J Pathol. 153 (1998) pp. 279-285. 39. Spencer I.M., Hargreaves I. and Chegwidden W.R., Effect of the carbonic anhydrase inhibitor acetazolamide on lipid synthesis in the locust. Biochem. Soc. Trans. 16 (1988) pp. 973-974. 40. Spencer I.M., Dawson M. and Chegwidden W.R., The role of carbonic anhydrase in biosynthetic processes. Isozyme Bulletin 27 (1994) p. 42. 41. Stubbs M., McSheehy P.M. and Griffiths J.R., Causes and consequences of acidic pH in tumours: a magnetic resonance study. Adv. Enzyme Regul. 39 (2000) pp. 13-30. 42. Stubbs M., Sheehy P.M., Griffiths J.R. and.Bashford C.L., Causes and consequences of tumour acidity and implications for treatment. Mol. Med. Today 6 (2000) pp. 15-19. 43. Sun H. and Tonks K., The coordinated action of protein tyrosine phosphatases and kinases in cell signaling. Trends Biochem. Sci. 19 (1994) pp. 480-485. 44. Torczynski R.M. and Bolton A.P. U.S. Patent 5,589,579 (1996).
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45. Tiireci O., Sahin U., Vollmar E., Siemar S., Gottert E., Seitz G., Parkkila A-K., Shah G.N., Grubb J.H., Pfreundschuh M. and Sly W.S., Human carbonic anhydrase XII: cDNA cloning, expression and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cancers. Proc. Natl. Acad. Sci. USA 95 (1998) pp. 7608-7613. 46. Turner J.R., Odze R.D., Crum C.P. and Resnick M.B., MN antigen expression in normal, preneoplastic and neoplastic esophagus: a clinicopathological study of a new cancer-associated biomarker. Hum. Pathol. 28 (1997) pp. 740-744. 47. Wary K.K., Lou Z., Buchberg A.M., Siracusa L.D., Druck T., LaForgia S. and Huebner K., A homozygous deletion within the carbonic anhydrase-like domain of the Ptprg gene in murine L-cells. Cancer Res. 53 (1993) pp. 14981502. 48. Webb S.D., Sherratt J.A. and Fish R.G., Alterations in proteolytic activity at low pH and its association with invasion: a theoretical model. Clin. Exp. Metastasis 17 (1999) pp. 397-407. 49. Zavada J., Zavadova Z., Pastorekova S., Ciampor F., Pastorek J. and Zelnick V., Expression of MaTu-MN protein in human tumor cultures and in clinical specimens. Int. J. Cancer 54 (1993) pp. 268-274.
BIOCHIP AND MINIATURIZATION JIAN Z H A N G , WAN-LI X I N G , YU-XIANG Z H O U , AND JING C H E N G
Biochip R&D Center, Biology Department, Tsinghua University, Beijing, 100084, China E-mail: jcheng @ tsinghua. edu. en A typical bioanalytical system usually consists of three classical steps, i.e., sample preparation, chemical reaction and detection. The total integration of these three steps has been the dream for many years for both academic researchers and entrepreneur. The marriage between molecular biology and the semiconductor industry for the first time brings hope to the scientific community. This presentation will describe the efforts towards the construction of microchip-based total analytical system or laboratory-on-a-chip. Progress made on microscale separation and isolation of cells, DNA amplification (PCR or strand displacement amplification) in microchips, and detection of specific sequence information on chips (via either chip-based capillary electrophoresis or electronic hybridization) will be presented.
1.
Introduction
No more than one decade has passed since the first DNA microarray was fabricated, however, great progress has been made and a lot of attention attracted. For microarrays on a 1 cm2 surface, thousands of or tens of thousands of spots can be manufactured. Molecules on these spots can be many different types, such as DNA, peptide, protein, cell or even tissue. DNA microarray so far is the most commonly used one. Through hybridization with target DNA, a large quantity of bioinformation can be obtained. Besides the explosive data it can generate, biochip has many other merits as well which include small size, little consumption of sample, no contamination, fast speed in analysis, etc. The rapid development in biochip study benefits mainly from two aspects, the great demanding of efficient tools from human genome project (HGP) and pharmaceutic companies' desire in new drug discovery. A good example of the development is the chip-base capillary electrophoresis. The use of capillary electrophoresis chips saves a lot of time in DNA sequencing comparing to the traditional electrophoretic method. Other relevant technologies, especially micro electro mechanical system (MEMS) technology, have been pushing research in biochips going forward quickly, facilitated researchers to construct biochip-based micro total analytical system (uTAS), or so-called lab-on-a-chip system. Usually bioanalytical procedure consists of three conventional steps: sample preparation, biochemical reaction and detection. To integrate all of them in one small system is a dream of many researchers and entrepreneur. In a completed uTAS system, heaters, detectors,
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microfluidic devices including micropumps and mierovalves are all integrated with biochips. Traditional instruments cannot finish all three steps described above in a systematic manner. Moreover these instruments are large in size and expensive as well. All these hindered their wide use. Portable instruments made with biochips may efficiently overcome these difficulties faced by the traditional industry. Before integration of all three steps on one chip, feasibility study of each step on chip can be done first.
Figure 1. Silicon micropost-type filter designs and filter chips. (A) Offset array of simple microposts (13 x 20 pm spaced lym apart) set across a 500~nm-wide x 20-jim-deep silicon channel. (B) Array of complex microposts (73 pm wide) separated by 7-nm-wide tortuous channels spaced 30 p n apart and set across a 500-|im-wide x 5.7-(im-deep silicon channel fabricated using conventional wet etching procedures. (C) Filter chip with three test channels containing different designs of flow deflector and serial filters. (D) Isolation of 5.78-um-diameter latex microspheres by a post-type filter (5 um channels between 73-um-wide posts set across a 500-pm-wide, 5.7-um-deep channel). (E) Comb-type filter formed from an array of 120 posts (175um long x 18 um wide) separated by 6-um channels set across a 3-mm-wide x 13-um-deep silicon channel. (F) New methylene blue-stained WBCs isolated by comb-type filter (cells released from the front surface (upper) of the filter by reversing the flow through the filter). From Wilding et al. [1]. With permission.
2,
Sample Preparation
Sample preparation has proven to be the most difficult processes among the total three, which includes isolating target cells from a mixture* obtaining DNAS RNA or protein from the target cells. To separate different types of cells, two methods have been attempted, i. e., microfiltration and dielectrophoresis.
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The filters process different shapes and sizes. To isolate white blood cells (WBC) from red blood cells (RBC), the size of filter is critical. The filter type shown in Figure 1A was fabricated on silicon substrate with 7 fim gaps between each two posts. To reduce the flow of material through the filter bed and aid capturing of large cells, filters having more complex shapes were designed also (Figure IB). The filter spacing was designed to be larger than the average diameter of RBC and less than that of WBC. However, when used with blood, no WBCs were captured, and both WBCs and RBCs passed through the filters. The main reason for the unsuccessful attempt is cells are deformable and the concept of average diameter is not really a true value for spheres. New comb-type filter was designed as shown in Figures IE and F. It consists of a series of closely positioned narrow pillars aligned in one row. Spacing between two neighboring pillars is 3.2 |im or 5 pm. Experiments proved 3.2-nm filter was the most efficient one. The shortcoming of comb-type filter is that it is easy to reach the saturation in recovery of cells, resulting low recovery yield. A weir-type filter was then fabricated and proved to be more effective (figure 2). A narrow gap of 3 fim was formed between the dam on chip and the top covering glass. With the application of certain pressure RBCs could pass through the gap, leaving the WBCs retained on the surface of the dam [1,2].
Figure 2. Weir-type silicon rciicrofilter.
Dielectrophoresis has been widely used in the isolation of cells [3, 4, 8]. The experiment of separating E. coli from blood cells using this technology was performed. Figure 3 shows an array of 5 by 5 electrodes which can be addressed individually. Figure 3A indicates that the electrodes are addressed in a checkerboard mode, which means one electrode's polarity is opposite to adjacent one's. Figure 3B shows the simulation result of electric field distribution by computer. Impelled by dielectrophoretic force, blood cells and bacteria cells move towards different field zones. Figure 4 shows separation result accorded with the calculation. Blood cells accumulated in field minima (red color) and bacteria cells in field maxima (white
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color). Another square-wall mode was also tested. Electtodes can be addressed in another square-form mode, which means polarity of electrodes that in one square form is different from that of electrodes in the neighboring square forms. The studies indicate that checkerboard pattern can get more distinct separation. After isolation, the cells were lysed by applying high voltage pulses. Then the lysate was digested by proteinase K to degrade contaminating proteins. Electrophoresis result shown in Figure 5 indicates that no obvious damage to nucleic acids through the lysis process.
Figure 3. (A) Checkerboard addressing of the five-by-five electrodes. (B) Corresponding computer models of the alternating current electric field distribution. From Cheng et al. [3], With permission.
Figure 4. (A) Separation result of checkerboard format (field maxima are at white-shaded areas and field minima are at the red-shaded areas). (B) Checkerboard format at the completion of the washing process. From Cheng et al. [3]. With permission.
The two methods of microfiltration and dielectrophoresis have their own advantages and disadvantages. It is obvious that microfiltration is only adaptive for separating cells with different size, and while cells are alternated, the filter's size should be changed. Only cells with large size can be isolated by now due to the difficulty of fabricating filters having smaller size. Dielectrophoresis, dependent on dielectrophoretic characteristic of cells* is discriminative for cells with various sizes. But the appropriate frequency, at which one kind of cells are subjected to positive dielectrophoretic force and the other subjected to negative dielectrophoretic force, is
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empirically obtained. The two methods are expected to integrate for separation various kinds of cells.
* V%yjm ••$ j«^ sis.-& #2. \ i
Figure 5. Agarose gel analysis of nucleic acids released from E. coli by electronic lysis. Lane 1: ADNA Hind III digest marker, lane 6: $X 174 Hae III digest marker. Lane2: supercoiled plasmid pCR2.1, lane 3: the corresponding linear plasmid DMA. Lane 4: the electronic lysate with and, lane 5: without RNase treatment. From Cheng et al. [3]. With permission.
3.
Biochemical Reaction
Biochemical reaction includes various Mads of reactions, such as chemical labeling, receptor-ligand binding, reverse transcription, nucleic acid amplification, etc. As an example PCR carried out on chip will be discussed as representative of biochemical reaction.
Figure 6. Microfabricated silicon-glass chip used for PCR (reaction volume 12 |il, surface area is 210 mm2). From Cheng et al.[5]. With permission.
Silicon-glass chips for nucleic acid amplification were fabricated using wet etching procedures and anodic bonding (Figure 6). First a sink was etched, which had a depth of 115 pm, followed by thermal growing a silicon dioxide layer with a thickness of 2000 A. Then a top glass was bonded with the chip, forming a reaction cell.
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To amplify DNA on chip, Taq DNA polymerase was mixed with C. jejuni bacterial DNA. 10 silicon-glass chips were filled with 12jxl of PCR reaction mixture containing 200 urn each dNTP, 0.6 urn each primer and 1.2 ng C. jejuni DNA. The reaction mixture was initially heated to 94°C for 1 min and cycled for 28 cycles: 15 s at 94 °C, 1 min at 55 °C and 1 min at 72 °C. A final extension was performed at 72 °C for 10 min. The amplifications on chip were paralleled with reactions in tubes, using the same mixture and under the same thermal cycling condition. Products generated from both chips and tubes were examined by gel electrophoresis. The result indicates that yield of chip PCR was equivalent to that of PCR in tubes [5]. Apart from simple PCR reactions, other amplifications have been accomplished on chips too. These include multiplex PCR, single-step reverse transcriptase (RT)PCR, ligase chain reaction (LCR), and strand displacement amplification (SDA) [6]. 4.
Detection
The detection schemes used in biochip-based nucleic acid analysis are of two types. One is based on the detection of the separated nucleic acid molecules, and the other, so called microarray, is based on the examination of the hybridization characteristics between immobilized oligonucleotide probes and the target DNA molecules. Capillary electrophoresis (CE) chips are used to sequence DNA or examine DNA polymorphisms. Comparing with traditional electrophoretic method, CE chips' analysis is about 10 to 100 times faster. On the other hand, its reproducibility is relatively equivalent with traditional methods [7]. Various types of CE chips have been developed using different materials [9, 10]. Microarray can be used for gene expression study, mutation research and resequencing. Probe DNA molecules are pre-immobilized on chip, and then target DNA are introduced and hybridized with probes. Most of the chips that have been developed so far are passive type, however, active chips (using all types of forces) are starting to catch up. As an example of bioelectronic active chips, arrays of electrodes are fabricated on silicon substrate, which can be addressed individually by applying electric signals. After probes have been immobilized onto chips and targets are introduced, the electrodes are positively biased. As DNA molecules carry negative charges, with the application of a positive electric field, the target molecules can have access to the probes more easily and quickly. Hence, the hybridization speed is accelerated greatly. When the hybridization reaction is completed, the chip is made negatively biased, thereby the mismatched target DNA molecules are pushed away and washed off. By controlling the intensity of the electric field, different stringencies can be obtained [3, 6].
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Integration
After each of the three steps proven to be functional, a complete system Integrating all of the three was constructed. As an example, a chip having 100 electrodes is used for sample preparation. Applying alternating electric signals, target cells can be isolated from the mixture by dielectrophoretic force. When the separation is accomplished, high frequency pulses are applied to the electrodes and the cells lysed. The chip's backside-has a ceramic heater attached. By switching to three different resistors, the chips temperature can be set at 60!, 90!, 95!, when powered by a 11 volt D.C. supply. This satisfies temperature condition of- strand displacement amplification (SDA). Connected in tandem is another chip having 25 electrodes for electric controlled hybridization. The fluidic controlling unit consists of a pump and a series of valves which operate under programmed commands. A battery driven laser emitter (2 mW)5 with wavelength set at 635 nm Is utilized for Induction of fluorescence. A CCD camera Is employed for fluorescent Imaging (Figure 7).
Figure 7. The prototype of the sample to answer portable lab-on-a-chip system.
Using the system, micrococcus lysodeikticus was separated from whole blood* then lysed by applying high frequency pulses and deproteinated using Proteinase K. Employing SDA method, 81 base pairs of the Salmonella entericaspaQ gene In the digested product were amplified. Then the amplicant was denatured. Introduced Into the second chip and hybridized with pre-immobilized probes. Hybridization result was detected by the CCD camera. The entire process took approximately one hour.
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References: 1. Wilding, P., Cheng, J., et al., Integrated Cell Isolation and Polymerase Chain Reaction Analysis Using Silicon Microfilter Chambers. Analytical Biochemistry 257 (1998) pp. 95-100. 2. Cheng, J., et al., Sample preparation in microstructured devices. Topics in Current Chemistry 194 (1998) pp. 215-231. 3. Cheng, J., et al., Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips. Nature Biotech. 16 (1998) pp. 541546. 4. Cheng, J., et al., Isolation of cultured cervical carcinoma cells mixed with peripheral blood cells on a bioelectronic chip. Anal. Chem. 70 (1998) pp. 23212326. 5. Cheng, J., et al., Chip PCR. II. Investigation of different PCR amplification systems in microfabricated silicon-glass chips. Nucleic Acids Res. 22 (1996) pp. 380-385. 6. Cheng, J., et al., Fluorescent imaging of cells and nucleic acids in biochips. SPIE, 3600 (1999) pp. 23-28. 7. Cheng, J., et al., Degenerate oligonucleotide primed-polymerase chain reaction and capillary electrophoretic analysis of human DNA on microchip-based devices. Anal. Biochem. 257 (1998) pp. 101-105. 8. Wang, X.B., Huang Y., Becker F.F. and Gascoyne P.R.C. Gascoyne. A unified theory of dielectrophoresis and travelling-wave dielectrophoresis. Appl. Phys. 27(1994)pp.l571-1574. 9. Becker, F.F., Wang, X.B., et al., Separation of human breast cancer cells from blood by differential dielectric affinity. Proc. Nat. Academ. Sci. (USA) 29 (1995) pp.860-864. 10. McCormick, R.M., et al., MicroChannel Electrophoretic Separations of DNA in Injection-Molded Plastic Substrates, Anal. Chem., 69 (1997) pp.2626-2630.
FUNCTIONAL GENOMICS: A PLATFORM FOR THE DISCOVERY OF NEW THERAPIES
DALIA C O H E N
Novartis Pharmaceuticals Corporation, 556 Morris Avenue, Summit, New Jersey, 07901, USA E-mail: dalia. cohen @pharma. novartis. com Functional genomics can be described as scientific and technological approaches which are being applied to bridge genomic research with the discovery and development of diseaserelevant therapeutic targets. These scientific approaches can offer significant opportunities in the search for causal and disease modifying therapies to better treat society's most outstanding medical needs. The use of functional genomic approaches is demonstrated in our recent efforts to elucidate cellular events leading to tumor cell cycle arrest in response to the inhibition of histone deacetylase, an important regulator of gene transcription.
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Comprehensive Functional Genomics Platforms
Large scale DNA sequencing in the public and private sectors will enable the elucidation of a detailed genetic and physical map of the human genome. Furthermore, completion of the human genome sequence containing an estimated 100,000 genes [22, 3, 6] should identify molecular targets for disease-modifying therapies that are novel and designed to satisfy unmet medical needs. In the 1970's, relatively few characterized pharmacological proteins such as enzymes and receptors were available to researchers. However, advances in molecular biology in the 1980s resulted in an exponential growth in the number of genes and gene products as potential drug disease targets. Currently, marketed drugs interact with about 400 genes or gene products and an estimated number of important genes for disease predisposition, onset and progression range from 3,000 - 10,000. Therefore, many novel genes and proteins can still be identified as potential targets for pharmaceutical research and development. Among the classical drug targets are enzymes, receptors, channels, DNA, growth factors and hormones. Currently marketed drugs for the treatment of a number of diseases can be found in the list assembled by [5]. For example, for the treatment of nervous system disorders, eight channels, twelve enzymes and one hundred and fifteen receptors are the targets for drugs on the market. For the treatment of neoplastic diseases, drug targets include five DNA sequences, twenty enzymes, six factors and seven receptors. For the treatment of inflammation, one-channel, nineteen enzymes and twenty-six receptors are common drug targets.
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To identify novel disease relevant targets that can be exploited for therapeutic discovery, new scientific avenues and technologies are being explored. Functional genomics approaches are being applied to link genomic research with the process of drug discovery and development. These approaches encompass technological platforms ranging from in silico biology (computational biology) to the study of whole organisms. Computational biology encompassing data analysis and interpretation [9] is of major importance in the discovery of potential drug targets. New gene family members, such as secreted factors, orphan receptors, GPCR, kinases, phophatases, and proteases that are associated with diseases can be identified using informatic tools. In addition, linking genes to chromosomal positions associated with specific disorders can be performed. Furthermore, using bioinformatics tools to identify gene homologues that are evolutionarily conserved is likely to give insight into gene function. In the molecular biology arena, key technologies for the elucidation of potential drug targets have been developed. High throughput of particular mRNAs within the pool of cellular messages can be achieved using several approaches based on differential display, reverse transcriptase PCR and DNA array [12, 13, 18]. These technologies allow for the measurement of differential gene expression in healthy versus diseased tissues, or in drug treated versus control cells. Proteomics approaches enable the analysis of differential protein expression, post translation modification and protein regulation [15]. Studying the proteome of a cell is an important companion to gene expression studies since there is often an insufficient correlation between the level of expression of different genes and the relative abundance of the corresponding proteins. Furthermore, the same gene does not directly encode for a protein and its post translational modifications; therefore the complete structure (s) of an individual protein cannot be determined by reference to its gene sequence alone. Moreover, these proteins form cellular networks comprised of numerous signalling pathways in a living cell, which may be altered in disease. Proteomics technologies are likely to identify the components of these altered pathways. In the cell biology arena, gene function is assessed using high-throughput cellbased assays. For example, cDNAs, antisense oligonucleotides, and peptide libraries expressed in cells followed by selection of biologically relevant phenotypes allows identification of genes mediating the expected phenotypes. In addition, in situ and immuno-hybridization methodologies and the use of specific antisense sequences are widely used to determine and validate gene function. Finally, yeast, C. elegans, Drosophila and mouse represent some of the most important experimental systems for understanding gene function and are being used for in vivo gene profiling experiments. [14]. In addition, comparative genetics studies and the opportunity to genetically manipulate homologous genes in these
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organisms can identify important components of gene functions and give valuable clues as to their potential role as a disease mediator in humans. These technologies and approaches need to be used in an interactive manner in order to successfully assign gene function, place individual genes into biological pathways, predict initiating the disease process and screen and optimise therapeutic leads. Moreover, applying functional genomics approaches with the extensive knowledge and availability of in vitro and in vivo model systems to study disease pathophysiology as well as integration of functional genomics technologies with more established scientific disciplines (e.g. protein chemistry, biochemistry, pharmacology, physiology) is a major strength of the pharmaceutical industry. To complement the in-house efforts in functional genomics, Novartis has ongoing external collaborations with Celera (genome information and data-bases), Incyte (gene-chip technology and the LifeSeq database), Affymetrix (gene-chip technology), Protana (Proteomics), and Rigel (high throughput target discovery and validation). In addition, Novartis is a member of the Wellcome Trust / Industrial Consortium to generate a Single Nucleotide Polymorphism (SNPs) map for the public domain. 2
Studying Transcription Regulation and Cell Cycle Control Employing Functional Genomics Approaches
The application of functional genomic is demonstrated in our recent efforts to elucidate cellular events leading to tumor cell cycle arrest in response to the inhibition of histone deacetylase, an important regulator of gene transcription. Histone acetylation is a key regulatory mechanism thought to modulate gene expression by altering the accessibility of transcription factors to DNA. Histone deacetylases (HDACs) repress gene transcription and their enzymatic activity is inhibited by trapoxin, a microbial derived cyclotetrapeptide. We [17] have demonstrated that treatment of human tumor cells with trapoxin, causes induction of mRNA and protein levels of the p21 gene, the inhibitor of cyclin-dependent protein kinases. Furthermore, changes in the transcription of a small subset of genes that regulate the cell cycle were also observed. To monitor additional genes with altered transcription in response to trapoxin in human tumor lung cells, DNA microarrays were used. Selective modulation, greater than 3 fold, was observed for 32 out of -7000 genes and the results were confirmed by Real Time PCR. These included the p21 watl and gelsolin genes previously shown to be trapoxin inducible [7]. These genes are currently being evaluated for their role in cell cycle and proliferation of tumor cells. To study further the function and regulation of HDAC1, we searched for novel cellular factors that interact with HDAC1 using a yeast two-hybrid screen [2]. A large N-terminal region of HDAC1 from amino acids 53 to 285 out of a total of 482
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residues was used as the bait to search for interacting cellular factors in a HeLa cDNA library. A human gene was identified that demonstrated a specific interaction with HDACl. The gene, husl+p, encodes a polypeptide that is about 30% identical to S. pombe protein (for hydroxyurea sensitive) and it was therefore named human Husl. Husl homologues have also been identified in mouse, C. elegans and Drosophila [4]. S. pombe husl+p was reported to be a checkpoint rad protein that together with five other known rad proteins, relays a signal from DNA-damage or replication block to downstream effectors [10, 16]. This resulted in a G2/M growth arrest in cells suffering DNA damage or replication block. The interaction between HDACl and hHusl was characterized in vitro and in vivo. The HDACl putative region that interacts with hHusl encompasses amino acids 53 to 240. In transfected cells, immunoprecipitation of tagged HDACl precipitated co-expressed tagged hHusl. Furthermore, tagged HDACl was found to co-immunoprecipitate with rad 9, which is one of the checkpoint rad proteins. The finding that hHusl interacted with radl and rad9 [11, 19, 21], suggests the existence of a functional complex between HDACl, hHusl, radl and rad 9. This HDACl-rad 9 interaction might be stabilized by hHusl, which could act as a bridge between HDACl and rad 9. Our findings that HDACl interacts with G2/M checkpoint rad proteins suggests an involvement of HDACl in cell cycle regulation. Interestingly, bioinformatics analysis indicated that both hHusl [1] and radl [20] might contain the so-called PCNA motif responsible for the trimerization and binding to DNA of the proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerase [8]. This analysis suggests that checkpoint rad proteins employ a mechanism similar to that of PCNA binding to DNA. The interaction of Husl with HDACl could lead to chromatin structure modifications that facilitate DNA repair. 3
Conclusion
In conclusion, the genomics revolution is now entering a new phase whereby the pioneering efforts to map and sequence the human genome, and the enormous wealth of data they have generated, are being converted into information on gene and protein function in normal and disease states. The progress of functional genomics will focus pharmaceutical research towards disease relevant targets and provide a starting point for discovery of causal and disease modifying therapies to address society's most outstanding medical needs.
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A NOVEL MATHEMATICAL ANALYSIS OF HUMAN LEUKOCYTE ANTIGEN (HLA) POLYMORPHISM BINGJIAN F E N G , DEJING P A N , SHANGWU C H E N , Z H E N Y E , AND ANLONG X U
Department of Biochemistry,
Zhongshan Email:
University, Guangzhou,
510275,
CHINA
[email protected] In 1970, Wu and Kabat proposed an algorithm to calculate the variability of a specific site, defined by the number of different amino acids of a given position divided by the frequency of the most common amino acid of the site. This algorithm is then widely applied to MHC and TCR systems to understand their polymorphism and their relationship to diseases. Consider that the Wu-Kabat and it's modified index are not sensitive enough to evaluate polymorphism contributed by scarcely appeared members in a set of entities, and are excessively sensitive to one or two of the most common members, we propose a new algorithm to evaluate the variability of a given site with greatly improved accuracy. This new index is applied to HLA-DRB1 sequences to make further understanding of this gene.,predicting that residues 9 to 13 and residue 31,33 may correlate with antibody binding of HLA-DRB molecule which is well documented.
1
Introduction
Class II molecules of the Human Leukocyte Antigen (HLA) are cell surface a and p" heterodimeric proteins that presents peptides derived from extracellular antigens. These molecules play a central role in tissue compatibility and autoimmune diseases as well as immune response against cancer and infectious diseases [1]. Among the HLA class II locus (DR, DP and DQ), the most polymorphic one is the HLA-DRB which exhibit both allelic and haplotypic polymorphism. The former is manifested by the great number of alleles discovered nowadays, according to the resent data from the IHWS's database (1999, Jan), the numbers of alleles in HLA class II genes up to 260 DRB, 2 DRA, 38 DQB1, 20 DQA1, 82 DPB1, 13 DPA1. The latter is manifested by the existence of variable numbers of expressed as well as nonexpressed DRB genes [2]. In 1970, Wu and Kabat [3] proposed an algorithm to calculate the variability of a specific site, defined by the number of different amino acids of a given position divided by the frequency of the most common amino acid of this site. This algorithm is then widely applied to MHC and TCR systems to understand their polymorphism and its relationship to diseases. Noted that, the Wu-Kabat's algorithm pays too much attentions to the most common amino acid, Rita Jores and etc. [4] has modified it to improve its sensitivity. This new diversity index is defined as the number of distinguishable amino acid pairs occurring at a given position divided by the frequency of the most common amino acid pair at that position. Although it is really 185
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better than the Wu-Kabat one, but because of accounting for only two of the most common amino acid, it is not sensitive enough to evaluate the polymorphism contribute by the minor ones. At the same time its numerator is too much sensitive to the scarcely appeared amino acid at a specific site. This will cause underestimate of variability in some cases and overestimate in other cases. Here we propose a new algorithm to calculate the variability index of a given site with greatly improved accuracy. The new algorithm is applied to HLA-DRB1 sequences to make further understanding of this gene. 2
2.1
Material and Methods
Sequence data source
All of the HLA class II gene sequences including 213 HLA-DRB1 alleles, 19 DRB3, 7 DRB4, 13 DRB5, 3 DRB6, 2 DRB7, 1 DRB2, 1 DRB8 and 1 DRB9 are downloaded from the IHWS's (International Histocompatibility Workshop) database (www.anthonynolan.com/HIG/). This sequence file contains all the exons but no introns of the alleles. The exon2 encoding residue from 6 to 94 of the first domain of DRB molecule is the most polymorphic region on DRB locus, and is closely related to peptide binding and antibody recognition. With this consideration, we design a program to extract the second exon sequence segment from the sequence file offered by IHWS. These sequence segments are input into another program "Polysis" to analysis their polymorphism with the new algorithm as well as the WuKabat's. All programs were written in C++ and run on a Pentium PC. 2.2
Variability index algorithm
Here we describe the deduction of the algorithm for nucleic acid sequence data. As for the amino acid sequence data, there should be some modifications (not shown here). Suppose that the numbers of nucleotides at a certain position are: A:X, T:X 2
C: X3 G: X,
n=4
Then ~ _ X] + X2 + X3 + X4 4
/(X, -X)2 + (X2 -X)2+(X, Standard Deviation = , V 4
-X)2 +(X 4 -X)2
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4
|X2+X2+X2+X42-4X
1 1 + X2 + X3 + X 4 ) 2 <X 1 2 +X|+X 3 2 + X 4 2
