Current Topics in Developmental Biology - Volume 7

CURRENT TOPICS IN

DEVELOPMENTAL BIOLOGY VOLUME 7

ADVISORY BOARD

JEAN BRACHET

ERASAlO MARRE

JAMES D. EBERT

JOHN .PAUL

E. PETER GEIDUSCHEK

HOWARD A . SCHNEIDERhL4N

EUGENE GOLDW ASSER

RICHARD L. SIDMAN

PAUL R. GROSS

HERBERT STERN

CONTRIBUTORS

ROBERT AUERBACH

H. HOLTZEIi

ROBERT L. DeHAAN

FOTIS C. KAFATOS

ERNST FREESE

R. AfAYNE

G. P. GEORGIEV

B. MOCHAN

JAMES E. H.4REIi

HOWARD G. SACHS

HARLYN

H. WEINTR.4UB

0

HALVORSON

C U R R E N T T O P I C S IN

DEVELOPMENTAL BIOLOGY EDITED BY

A. A. MOSCONA DEPARTMENT OF BIOUIGY T H E UNIVERSITY OF CHICAGO CHICAGO, ILLINOIS

ALBERT0 MONROY C.N.R. LABORATORY OF MOLECUWR EMBRYOLOGY

ARCO FELICE ( N A P L E S ) , ITALY

VOLUME 7

1972

@

ACADEMIC PRESS New York

London

COPYRIGHT 0 1972, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London N W l

L~BRARY OF

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CATALOG CARD NUMBER:66-28604

PRINTED IN THE UNITED STATES OF AMERICA

List of Contributors Preface

........................................................

.....................................................................

Contents of Previous Volumes CHAPTER

..............................................

ix xi

... Xlll

1. The Structure of Transcriptional Units in Eukaryotic Cells

G . P . GEORCIEV I. Introduction .......................................................... I1. The Patterns of Transcription in Eukarvotic Cells ...................... I11. Models of the Transcriptional Unit in Eukaryotic Cells ................ IV . General Aspects of Regulation of Transcription in Eukaryotes and the Role of Chromosomal Proteins ........................................ V. On the Possible Mechanism of Cell Transformation hy Oncogenic Viruses ................................................................ References ............................................................ CHAPTER

1

3 24

44 49 53

2. Regulation of Sporulation in Yeast

JAMESE. HABERA N D HARLYN 0. HALVORSON I. I1. I11. IV. V. VI .

Introduction ........................................................... From Vegetative Growth to Asrus ...................................... Morphological Changes during Sporulation ........................... Mutations Affecting Sporulntion ...................................... Sporulation-Specific Biochemical Events .............................. Cell Cycle Dependency of Sporulntion ................................ References ............................................................

CHAPTER

61 62 61 66 72 77 82

3 . Sporulation of Bacilli. a Model of Cellular Differentiation

ERNSTFREESE I. I1. I11. IV .

V. VI .

General Remarks about Differentiation ................................ Morphology and Genetics of Sporulation in Bacilli .................... Necessary Conditions for the Onset of Sporulation ....................... Suppression of Sporulation ............................................ Later Spore Development .............................................. Commitment to Sporulation ............................................ References ............................................................ V

85 88 91 114 116 117 120

vi

CONTENTS

CHAPTER

4. The Cocoonase Zymogen Cells of Silk Moths: A Model of Terminal Cell Differentiation for Specific Protein Synthesis

FOTISC. KAFATOS I . Introduction .......................................................... I1. Cocoonase Productrion: Morphological Studies ........................ I11. The Differentiation-Specific Product of the Galea: Biochemical and Enzymological Characterization ....................................... IV. Quantitation of Zymogen Synthesis and Accumulation during Development ................................................................. V. Transition Points in Zymogen Synthesis during Development .......... VI. Progressive Increase in Zymogen Synthesis during Phase I1 ............ VII . Concluding Remarks .................................................. References ........................................................... CHAPTER

125 129 142 145 159 161 185 187

5 . Cell Coupling in Developing Systems: The Heart-Cell Paradigm

ROBERTL . DEHAAN AND HOWARD G . SACHS I. I1. 111. IV. V.

Introduction .......................................................... Cell Coupling in Mature Cardiac Tissue .............................. Cont.acts and Junctions in the Early Embryo .......................... Contacts and Coupling in T i s u e Culture .............................. Conclusions and Speculations .......................................... References .............................................................

CHAPTER

193 194 205 209 222

225

6. The Cell Cycle. Cell Lineages. and Cell Differentiation

H . HOLTZER. H . WEINTRAUB. R . MAYNE. AND B . MOCHAN I. I1. 111. IV. V.

Introduction ........................................................... Aspects of Myogenesis ................................................ Asperts of Erythrogenesis ............................................. Aspects of Chondrogenesis ............................................ Disrussion ............................................................. References ............................................................

CHAPTER

229 232 239 246 251 254

7 . Studies on the Development of Immunity: The Response to Sheep Red Blood Cells

ROBERTAUERBACH I. Introduction ......................................................... I1. Ontogeny of Responsiveness .........................................

257 259

CONTENTS

I11. IV. V. VI . VII . VIII .

vii

Phylogenetic considerations .......................................... Cell Interactions during the Response to SRBC .................... Ontogeny of Cells Responding to Sheep Red Blood Cells (SRBC) ..... Immunological Tolerance to SRBC ................................... Ontogeny of Antibody Varinbility .................................... General Considerations .............................................. References ...........................................................

Author Index

..............................................................

281

Subject Index

..............................................................

296

This Page Intentionally Left Blank

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on whirh the authors' contributions begin.

ROBERT AUERBACH, Department of Zoology, University of Wisconsin, Madison, Wisconsin (257) ROBERT L. DEHAAN, Department of Einbryology, Carnegie Institution of Washingfon, Baltimore, Maryland ( 193) ERNSTFREESE, Laboratory of Molecular Biology, 'Yational Insfitufe of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland ( 8 5 ) G. P. GEORGIEV, Institute of Molecular Biology, Academy of Sciences of the USSR, Moscow, USSR (1) JAMES E. HABER,Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts (61) HARLYN 0. HALVORSON, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, W'althani, iMassachusetts (61) H. HOLTZER, Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (229) FOTIS C. KAFATOS, The Biological Laboratories, Harvard University, Cambridge, Massachusetts (125) R. MAYNE, Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (229) B. MOCHAN, Department of Anatomy, School of Medicine, {Jniversity of Pennsylvania, Philadelphia, Pennsylvania (229) HOWARD G. SACHS," Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland ( 193) H. WEINTRAUB,~ Department of Anatornu, School of Medicine, University of Pennsylvania, Philadelphia, Penns ylvania (229)

* Present address: Department of Anatomy, University of Illinois, Chicago, Illinois 60680. t Present address: MRC Laboratories, Molecular Biology, Cambridge, England. ix

This Page Intentionally Left Blank

The seventh volume of this series l~ringatogcther scven articles dealing with some of the most active areas in developmental biology, and spanning various cxpcrimentnl disciplines. Consistent with our editorial policy, the articles focus priniarily on the writers’ work and views and they aiiialganiate factual inaterial with discussions antl projections of provocative ideas. The Editors wish to thank thc contributors for tlicir cooperation i n meeting the aims antl st:tnd:trris of this publication ; thcy also wish to acknowledge with thanka Dr. Frecse’s initiative toward including i n this volume the articles by Ernst Freesc; H. Holtzer, H. \i’eintrnul), R. RIayne, and B. Rlochan; and James Habcr and Harlyn 0. Halvorson, which werc presented a t thc 1972 Annual AIectirig of thc American Society for hlicrobiology in hlinneapolis. \Ye are grateful to mcmbers of the Atlvihory Board for reviewing manuscripts and also to Dr. U. E. Locning for special help in review mnttcrs. Finally, we thank the staff of Academic Press for their cfforts to maintain the usefulncsa of this publication.

A. A. ~ I O S C O N A ALBERTOMONROY

xi

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CONTENTS OF PREVIOUS VOLUMES Volume 1

REMARKS Joshua Lederberg FORMS O F ME S S E NGE R RNA I N EARLY EMBRYOGENESIS AND IN OTHERDIFFERENTIATING SI-STEMS A . S. Spirin THETRANSCRIPTION OF GENETIC INFORMATION I N T H E SPIRALIAN EMBRYO J . R. Collier SOMEGENETICAND BIOCHEMICAL ASPECTSOF THE REGULATORY PROGRAM FOR SLIME MOLDDEVELOPMENT Maurice Suss m an THEh‘lOLECULAR BASISO F DIFFERENTIATION I N EARLY DEVELOPMENT OF AMPHIBIANEMBRTOS H . Tiedemann THECULTURE OF FREE PLANT CELLSAND ITSSIGNIFICANCE FOR EMBRYOLO N “hi A SK ED ”

R~ORPHOCENESIS F. C . Steztlarrl, A n n E. K e n t , and Marion 0 .Mnpes GENETICAND VARIEGATION MOSAICS IN THE EYEOF Drosophila

OGY A N D

Hans Joachim Becker

BIOCHEMICAL CONTROL OF ERYTHROID CELLDEVELOPMENT Eugene Golduwsser DEVELOPMENT O F RIAhlhIALIAN ERTTHROID CELLS Paul A . M a r k s and John S. Kounch GENETICASPECTS OF SKINA N D LIMBDEVELOPMENT P. F . Goetinck AUTHORINDEX-SUBJECT INDEX

Volume 2

THECONTROL OF PROTEIN SYNTHESIS IN EMBRYONIC D EV ELO PMEN T DIFFERENTIATION Paid R. Gross

...

Slll

AND

xiv

CONTENTS O F PREVIOUS VOLUMES

THE GENES FOR RIBOSOMAL RNA AND THEIRTRANSACTION DURING AMPHIBIANDEVELOPMENT Donald D. Brown RIBOSOME AND ENZYME CHANGES DURING ~ ~ A T U R A T I OANN D GERMINATION OF CASTOR BEANSEED Erasnzo MarrB CONTACT A N D SHORT-RANGE INTERACTION AFFECTINGGROWTHOF ANIMAL CELLSIN CULTURE Michael Stoker AN ANALYSISOF THE MECHANISM OF NEOPLASTIC CELLTRANSFORMATION BY POLYOMA VIRUS,HYDROCARBONS, A N D X-IRRADIATION Leo Sachs DIFFERENTIATION OF CONNECTIVE TISSUES Frank I(. Thorp and Albert Dorfman THEIGA ANTIBODYSYSTEM Mary A n n South, Max D. Cooper, Richard Hong, and Robert A . Good TERATOCARCINOMA: MODEL FOR A DEVELOPMENTAL CONCEPT OF CANCER G. Barry Pierce CELLIJLAR AND SUBCELLULAR EVENTS I N W O L F F I A N LENSREGENERATION Tuneo Yainada AUTHORIND E X ~ U JBECT INDEX

Volume 3

SYNTHESIS OF MACROMOLECULES AND MORPHOGENESIS IN Acetabularia J . Brachet BIOCHEMICAL STITDIES O F MALEGAMETOGENESIS I N IAILTACEOUS P L A N T S Herbert Stern and Yasiio Hotta DURING ORGANOGENESIS SPECIFIC INTERACTIONS BETWEEN TISSUES Etienne W01.f LOW-RESISTANCE JUNCTIONS BETWEEN CELLSI N EMBRYOS A N D TISSUE CULTURE Edwin J . Furshpan and David D . Potter COMPUTER ANALYSIS OF CELLULAR INTERACTIONS F. Hein m e ts C E L L AGGREGATION AND DIFFERENTIATION IN ~ i C t ~ O s f e l i U W l Giinther Gerisrh HORMONE-DEPENDENT DIFFERENT~AT~ON OF MAMMARY GLANDin Vitro Roger W . Tiirkington AUTHORINDEX-SUBJECT INDEX

CONTENTS O F PREVIOUS VOLUMES

xv

Volume 4

GENETICS AND GENESIS Clifford Grobstein THEOUTGROWING BACTERIAL ENDOSPORE Alex Keynan CELLULAR ASPECTSOF MUSCLE DIFFERENTIATION in Vitro David Yaffe hf ACROMOLECULAR BIOSYNTHESIS I N ANIMAL C E L L S INFECTED WITH CYTOLYTIC VIRUSES Bernard Roizinan and Patricia G. Spear THEROLEO F THYROID AND GROWTH HORhlONES I N NEUROGENESIS Max Ham burgh INTERRELATIONSHIPS OF NIJCLEAR A N D CYTOPLASMIC ESTROGEN RECEPTORS Jack Gorski, G . Shynmala, and D . Toft TOWARD A MOLECULAR EXPLANATION FOR SPECIFIC CELLADHESION Jack E . Lilien THEBIOLOGICAL SIGNIFICANCE O F TURNOVER O F T H E SURFACE h l E M B R A N E OF ANIMALCELLS Leonard Warren AUTHORINDEX-SUBJECT INDEX

Volume 5

DEVELOPMENTAL BIOLOGY A N D GENETICS : A PLEA FOR COOPERATION Albert0 Monroy

REGULATORY PROCESSES I N T H E AMPHIBIANEGGS L. D . Smith and R. E. Ecker

htATURATI0N AND

EARLY CLEAVAGE

OF

-

O N THE L O N G - T E R h l CONTROL O F NUCLEAR ACTIVITY DURING C E L L DIFFERENTIATION J . B. Gurdon and H . R . Woodland THEINTEGRITY OF THE REPRODUCTIVE CELLLINEIN THE AMPHIBIA Antonie W . Rlackler REGULATION OF POLLEN TUBE GROWTH H ansf erdin and Lins kens a n d Marianne Kro h PROBLEMS OF DIFFERENTIATION IN THE VERTEBRATE LENS Ruth M . Clayton RECONSTRUCTION OF h f u s c L E DEVELOPMENT AS A SEQUENCE O F hq ACROMOLECULAR SYNTHESES Heinz Herrmann, Stuart M . Heyuvod, and Ann C . Marchok

xvi

CONTENTS O F PREVIOUS VOLUMES

THIIS Y N T H E S I S AND ASSEMBLY O F MYOFIBRILS I N EMBRYONIC MUSCLE Donald A . Fischnaan THE T - L O C U S O F T H E h l O U S E : IMPLICATIONS FOR MECHANISMS OF DEVELOPMENT Salome Gluecksohn- Waelsch and Robert P. Erickson DNA ~’IASKING I N h/IAMMALIAN CHROMATIN : A MOLECULAR MECHANISM FOR DETERMINATION OF CELLTYPE J . Paul AUTHORINDEX-SUBJECT INDEX

Volume 6 THEINDUCTION AND EARLY EVENTS OF GERMINATION IN THE ZOOSPORE OF Blastocladiella einersonii Louis C . Truesdell and Edward C. Cantino STEPSOF REALIZATION OF GENETICINFORMATION IN EARLY DEVELOPMENT A . A . Neyfakh PROTEIN SYNTHESIS DURING AMPHIBIANI~ETAMORPHOSIS J . R. T a f a HORMONAL CONTROL OF A SECRETORY TISSVE H . Yomo and J . E . Varner GENEREGULATION NETWORKS: A THEORY FOR THEIR GLOBALSTRUCTURE AND BEHAX’IORS

Stuart Kauffiitan POSITIONAL INFORMATION A N D PATTERN FORMATION Lewis Wolpert AUTHORINDEX-SUBJ ECT INDEX

CHAPTER 1

THE STRUCTURE OF TRANSCRIPTIONAL UNITS IN EUKARYOTIC CELLS G. P. Georgiev INSTITUTE O F MOLECULAR BIOLOGY, ACADEMY O F SCIENCES OF THE USSR, MOSCOW, USSR

I. Introduction. . . . 11. The Patterns of Cells. . . . . . . . . . . . . A. Giant dRNA Is a Primary Product of Transcription.. . , , , . . . B. Cleavage of Giant dRNA and Translation of Monocistronic D. Processing of d R N A . . , , , . . . . . . . .

Eukaryotic Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. The Selective Inhibition of Transcription from Repetitive Base Sequences in the Cell-Free System, , , . . . . . . . . . . . . . . . . . . . . . 111. Models of the Transcriptional Unit in Eukaryotic Cells.. . . . . . . . A. Cascade Regulation Hypothesis. . . , , , . . . . . . . . . . . . . . . . . . . . . B. The Activator RNA Model.. , . . . . . . . C. The Author's Model of Operon Structure in Eukaryotes. . . . . I).Tandem Repetitions in the Genome and Their Possible Role. E. Genetic Data on the Structure of Operons in Eukaryotes.. . . , IV. General Aspects of Regulation of Transcription in Eukaryotes and the Role of Chromosomal Proteins.. . . . . . . . . . . . . . . . . . . . . . . .......... A. Histones as Inhibitors of Transcription. B. Nonhistone Proteins as Possible Repressors. . . . . . . . . . . . . . . . . C. The Possible Regul I). Three-Dimensional .............. (Crick Model). . . , . V. On the Possible Mech

..........................

References. . . . . . . . . . .

1 3 3 8 12 14 17 18 21 23 24 24 26 27 39 42 44 45 47 48 48 49 53

1. Introduction During the last decade great progress was made in understanding the structure of transcriptional units in prokaryotes: phages and bacteria. According to the classical studies by Jacob and Monod (1961, 1963), and as developed by others (Ippen et al., 1968; see review by Martin, 1969), the general scheme of the transcriptional unit may be 1

2

G. P. GEORGIEV

drawn as follows (Fig. 1). It consists of a promoter, which is a short base sequence recognized by RNA polymerase; an operator, also a short base sequence recognized by repressor ; and some structural genes, the products of which are usually related functionally. By combining with operator, the repressor prevents the movement of RNA polymerase along the DNA strand and thus provides a negative control of RNA synthesis. There is also a positive control of RNA synthesis which depends on special protein factors combining with RNA polymerase and influencing its affinity to different kinds of promoters (Khesin et al., 1962; Burgess e t al., 1969; Zillig e t al., 1970; Losick et al., 1970). LAC OPERON-

7;

-I

\

Pronwter Operator PO’

c

2

,

i

Y

7

0

DNA

3----+ i

~7

RNA pOlymemS5

Represux

mRNA

Ribosomes

fl -Golactosidose

Thiogolactoside transacetylose Permeose

FIQ. 1. The scheme of the lac operon structure in Escherichia coli (Jacob and Monod, 1961; Ippen et al., 1968).

It is now well known that the interaction of the repressor with the operator depends on the presence of some metabolites-effectors (inductors or corepressors). The concentration of these metabolites in the medium determines the rate of transcription of the operon. In the operons studied up to now, the greater part of their length consists of structural genes that carry the information for protein synthesis. The “service” sequences-promoter and operator-comprise not more than &lo% of the total length of the operon. Immediately after the start of RNA synthesis, the ribosomes begin to interact with the mRNA after the RNA polymerase. As a result a complex structure is formed containing DNA, RNA polymerase, nascent mRNA, ribosomes combined with the latter, and growing polypeptide chains (see review by Martin, 1969; 0. L. Miller, 1970). Very soon after the completion of RNA synthesis and its dissociation from DNA, its degradation from the 5’ end begins (Morikawa and Imamoto,

1.

TRANSCRIPTIONAL UNITS I N EUKARPOTIC CELLS

3

1969; Morse et al., 1969; Kuwano et al., 1970). Thus in prokaryotic cells transcription and translation are coupled, and no intermediate steps take place. The eukaryotes differ from the prokaryotes in many respects. First, they have nuclei separated by a membrane from the cytoplasm. This membrane effectively separates transcription (which takes place in the nucleus) from translation (which proceeds mainly in cytoplasm). Thus transcription and translation in eukaryotes are uncoupled, and for this reason an additional step in gene expression appears-namely, mRNA transport. Second, in eukaryotes the process of the regulation of gene expression is complicated. Various cells differ greatly in their properties and these differences are stable and do not depend on the conditions of medium; i.e., the cells are differentiated. Third, the chromosomes in eukaryotes contain many proteins. I n particular the strongly basic proteins, histones, are complexed with DNA. This may be connected with the appearance of differentiation and the general complication of regulatory processes. From this simple description, one can see that the regulation of transcription in eukaryotes should be more complex than in prokaryotes. Two main questions arise: (1) How is the elementary transcriptional unit, or operon, organized in eukaryotes? (2) What is the role of different proteins of chromatin-histones and nonhistone proteins-in the regulation of transcription? In this paper I shall discuss in detail the data concerning the first question and only briefly describe the main concepts in the second field. The rapid progress in understanding of the operon structure in bacterial cells depended on combined genetic and biochemical analysis. Unfortunately, in the case of eukaryotes the most convenient systems from the genetic point of view, such as Drosophila, are very difficult for biochemical studies. For this reason the main results are obtained from biochemical studies on DNA and especially on newly formed RNA. The latter may be taken to represent copies of transcriptional units. It should be pointed out that the term “operon” is used in this paper in the sense of “transcriptional unit” or the DNA sequence transcribed as an uninterrupted RNA chain. No other functional meaning is included. I n this sense the term was used by Jacob and Monod (1963). II. The Patterns of Transcription in Eukaryotic Cells

PRIMARY PRODUCT OF TRANSCRIPTION D - R N A or R N A with DNA-like base composition in animal cells was a t first discovered in 1961 by means of a phenol fractionation tech-

A. GIANTdRNA Is

A

4

Q. P. GEORGIEV

nique (Georgiev, 1961; Georgiev and Mantieva, 1962a). I n the first experiments the nuclei isolated by cold phenol treatment of tissue homogenates were subsequently extracted by phenol-sodium-p-aminosalicylate and by phenol-0.14 M NaCl a t 60OC. The first treatment led to liberation of RNA of AU-type (G C/A U 0.9) that resembled cellular DNA in base composition. Then the technique was improved, namely a hot phenol fractionation procedure was elaborated. The latter consisted of sequential extractions of the interphase layer (layer formed between water and phenol phases after shaking and centrifugation with the mixture of phenol, pH 6, and 0.14 M NaCl) at stepwise increased temperatures. At 40°C nuclear rRNA was liberated, a t 55°C a mixture of dRNA and rRNA, and at 65O and 85°C dRNA, was extracted into the water phase (Georgiev and Mantieva, 196213; Arion et al., 1967). The method allows one to obtain dRNA of about 80-90% purity. The existence of nuclear dRNA was confirmed by a number of authors using hot phenol fractionation or some other methods (Sibatani et al., 1962; Scherrer et al., 1963; Brawerman, 1963; Ellem and Sheridan, 1964; Samis et al., 1964; Tyndall et al., 1965). I n 1966 it was redescribed by Darnell’s group as heterogeneous nuclear R N A (Warner et al., 1966; see also Attardi et al., 1966). Another name recently suggested for nuclear dRNA is “messengerlike RNA” (mlRNA) (Scherrer and Marcaud, 1968). All these terms define the same nuclear RNA fraction. Probably the best designation for this RNA is pre-mRNA, precursor of mRNA (see below the data confirming this nature of nuclear dRNA). Nuclear dRNA is rapidly labeled. Its specific activity after short pulses is much higher than that of cytoplasmic RNA (Georgiev and Mantieva, 1962a,b; Sibatani et al., 1962). The investigation of the physical-chemical properties of this RNA showed it to have a very high molecular weight. Hiatt (1962) and Scherrer and Darnel1 (1962) found that most of the rapidly labeled nuclear RNA had a very high molecular weight and sedimented faster than the main components of cellular RNA-28 S and 18 S ribosomal RNA’s. However, the base composition of this RNA was high in GC, and thus related to rRNA (Hiatt, 1962; Perry, 1962). Only indirect evidence that some of heavy RNA was newly formed dRNA was obtained (Scherrer et al., 1963; Georgiev et al., 1963). A more careful investigation of sedimentation properties of nuclear dRNA isolated by the hot phenol method showed that although the peak of UVabsorbing material is localized in the 18 S region, the rapidly labeled dRNA is in the heavier. The peak of radioactivity was about 30 S, a significant part of label sedimenting even faster (40-70 S) . On the basis of the empirical equation M = 1550 S2.’(Spirin, 1963), the molecular weight of the newly formed dRNA was found to be about 2 X lo6 with

+

+

-

1.

TRANSCRIPTIONAL UNITS I N EUKARYOTIC CELLS

5

a distribution between 1 x lo6 and 6 x lo6. This is much higher than the expected size for average monocistronic mRNA (0.2to 0.6 X lo6). The conclusion was drawn that the dRNA is synthesized in the cell nucleus in the form of very large, probably polycistronic, entities (Georgiev and Lerman, 1964; Samarina, 1964; Samarina et al., 1965b). Similar results were also obtained by Yoshikawa et al. (1964, 1965), who isolated total cellular RNA and then fractionated it on a methylated albumin-kieselguhr column. I n 1965, Scherrer and Marcaud, using a milder method for RNA isolation, found that newly formed dRNA from erythroblasts had even Sedimentation coefficient, S 28 48

10 Froction No

20

FIG.2. Sedimentation properties of total nuclear dRNA isolated a t 5585°C from rat liver after pulse labeling. 0-0, Optical density (in the top fractions it depends on the presence of degraded DNA). 0-0, 'Radioactivity ("P). Data of Georgiev et al. (1972a).

higher sedimentation coefficients, i.e., about 5&70 S. This corresponds to a molecular weight of about 4 to 10 x lo6. Similar results were obtained later by a number of groups (Attardi et al., 1966; Warner et al., 1966; Gazaryan et al., 1966). In all these studies, the total RNA was isolated and then immediately ultracentrifuged in sucrose gradients. It was shown that the high molecular weight of newly formed RNA does not result from aggregation. Granboulan and Scherrer (1969) measured the length of dRNA chains by electron microscopy and found good agreement between the sedimentation coefficients and the average chain length. Finally, some modifications of the hot phenol fractionation procedure allowed the isolation of very heavy, purified dRNA (see Figs. 2-4) (Ryskov and Georgiev, 1970; Mantieva et al., 1971; Georgiev et al.,

6

G. P. GEORGIEV

1972a). I n a preparation of total nuclear dRNA (obtained in the temperature interval 55-85OC), one can find a peak of UV absorption in the 18 S zone trailing into the heavy zone. The peak of radioactive RNA after short pulse labeling is localized a t 3 0 4 0 S, but much radioactivity may be found in the 40-70 S, and even heavier, zone. Furthermore,

5 I

Fraction No. 10

(5

1

A

Fraction

No.

FIG.3. Sedimentation properties of the fractions of nuclear dRNA isolated from Ehrlich ascites carcinoma cells after pulse labeling. (A) 55-65°C fraction. (B) 6585°C fraction. ( C ) Resedimentation of heavy fraction from experiment B. 0-0, Optical density. 0-0, Radioactivity. Data of Gcorgiev et al. (1972a).

the hot phenol fractionation allows the separation of the most rapidly labeled RNA from a less active RNA (Arion et al., 1967). The less active fraction extracted a t 65OC consists mainly of 18 S material (UV absorption or radioactivity after long-term labeling). On the other hand, the most rapidly labeled fraction extracted a t 85OC (after removal of the more stable dRNA a t 65OC) contains RNA molecules of higher molecular weight. After short labeling the radioactive material in the

1.

TRANSCRIPTIONAL UNITS I N EURARYOTIC CELLS

7

6 5 O fraction is in a heavier zone than the bulk of UV peak. I n the 8 5 O fraction the radioactivity follows the optical density closely. Thus

the latter fraction is metabolically homogeneous. One can conclude that dRNA is synthesized in the form of giant, presumably polycistronic, chains. However, it remained unclear whether the giant dRNA is the only primary product of dRNA synthesis or whether short chains are also synthesized. One can always, even after

1

I ,

I

10

20

Fraction No.

FIG.4. Sedimentation properties of the nuclear dRNA fractions isolated after long labeling from rat liver ( A , B) or Ehrlich ascites carcinoma cells (C, D). (A, C) 55-75°C fractions. (B, D) 75-85"C fractions. 0-0, Optical density. 0-0, Radioactivity: (A, B). "P, (C, D), "C. The marked zones have been taken for 'H end labeling (see below) : H-heavy (>35 S), I-intermediate (20-30 S),L-light (10-18 S). Data of Georgiev et al. (1972a).

a very short pulse, find a fraction of labeled RNA of rather low molecular weight (18 S ) , and it is not possible to exclude its independent synthesis. To resolve this question, the nature of 5' ends in different fractions of isolated nuclear dRNA was studied. It is known from the experiments with cell-free systems that in the first nucleotide of the growing RNA chain the 5'-triphosphate group is conserved. Later 7- and &phosphates are removed by special enzymes (Maitra and Hurwitz, 1965; Maitra and Dubnoff, 1967). 5'-Triphosphate ends have been also found in vivo in a number of viral RNA's and in a newly formed

8

0. P. GEORGIEV

5 S RNA. (Takanami, 1966; Hatlen et QZ., 1969). Thus triphosphate 5' ends, if present, may be considered as markers of newly synthesized molecules. Ryskov and Georgiev (1970) analyzed the nature of 5' ends in newly formed nuclear dRNA (Table I). After alkaline hydrolysis, triphosphorylated ends give nucleoside tetraphosphates (pppxp) ; monophosphorylated ends, nucleoside diphosphates (pXp). Both kinds of ends were found in dRNA, but triphosphate ends (pppxp) were observed only in the heavy fraction of dRNA ( 3 3 5 S). Monophosphorylated ends are present in all dRNA fractions, and their concentration is much TABLE I THENATURE OF 5' ENDSI N NUCLEAR dRNA FRACTIONS FROM RAT LIVERS Nuclear dRNA fractions Expt. No. 1

x 10-6

>30 10-30 35 20-30 10-18

>2 0.2-2 22.5 0.7-2.0 0.2-0.7

>

2

mw

520

(S)

Percent of total 32Pb In In PPPXP PPXP PXP

+

0.041 0.004 0.022 . .. %

8060

*it! .

.

* .

J 100 -

Ga

..

'I *****

7D 0

..*

140-

40

%

3 180-

2

*.

.*

Z' I 160 -

10'

I

..

.

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These two types of experiments serve to demonstrate that the interaction or cardiac myocytes, which 'have the inherent capacity to beat (about 50% of the cells isolated from an embryonic chick heart do so spontaneously), is not predictable from a simple pacemaker hypothesis.

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CELL COUPLING I N DEVELOPING SYSTEMS

The second controversy noted was that of the presence of electrotonic coupling between myocytes, and the morphological correlates of any coupling found. Even from early studies it was clear that myocardial cells were mononucleate, and were separable into single viable cells. I0'

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Thus cytoplasmic continuity did not seem to be an appealing explanation for synchronization. When Barr et al. (1965) described the physiology and morphology of the nexal junction in adult myocardium, the question arose whether myocytes in vitro are coupled. Lehmkuhl and Sperelakis

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ROBERT L. DEHAAN AND HOWARD G. SACHS

(1965) used a two-electrode approach with cultured chick cells. They concluded that electrotonic coupling did exist between some, but not all, cells in the culture. However, they postulated that the intercalated disc was the membrane specialization required for coupling. The presence of nexuses between myocytes was later demonstrated convincingly by Hyde and his colleagues (1969), and such junctions were implicated as the sites for electrical coupling. However, coupling with a lower efficiency (that is, with a higher junctional resistance) was also seen between fibroblasts and myocytes; nexuses were not seen in these cases. It should be remembered, however, that in the embryonic chick, nexal junctions were not demonstrated for many years because of inadequate fixation or staining. Goshima suggested that coupling may take place in absence of nexuses, an idea reminiscent of the suggestions about the early chick embryo by Sheridan and Hay. Goshima (1970) reported that two myocardial cells could be coupled across a nonexcitable cell (HeLa, FL) and that nexuses were present. However, his pictures do not show convincing junctions, probably because they were not blockstained to heighten membrane contrast. More recent micrographs by Pinto de Silva and Gilula (1972) demonstrate convincingly that nexal junctions do indeed exist between chick fibroblasts in culture. I n our own studies on cell pairs, we have assumed that when cells synchronize they have become electrically coupled. The logic of this assumption is based on the evidence reviewed in Section 11. However, in the case of the aggregates, the evidence that the majority, if not all, of the cells are electrically coupled stems from microelectrode (intracellular) recordings. Impalement of a number of cells in an aggregate yields action potentials that are all virtually identical (Sachs and DeHaan, 1973). In addition, the widespread presence of focal close junctions in newly formed aggregates (24 hours) and a similar widespread distribution of well defined nexuses some days later (Fig. 11) is also consonant with the idea of coupling. The rate setting process in assemblages of embryonic myocardial cells is still not understood. The often cited dominance of the fastest cell over all others has not been found in our studies. We would conclude that the coupling of cells, and the reciprocal interactions it permits in pairs and in aggregates, is important to the rate determination. It has been shown (Woodbury and Crill, 1961) that not all cells in the adult myocardium are coupled to the same extent. We do not know whether the coupling (Hyde et al., 1969) and the consequences of that coupling (DeHaan and Hirakow, 1972; Goshima and Tonomura, 1969) seen in cultured heart cells reflect accurately the situation in the early embryonic heart. We have already cited the evidence that the plasma membranes of

5.

CELL COUPLING IN DEVELOPING SYSTEMS

22 1

heart cells are relatively impermeable to ion flow. Estimates of specific resistance range from 500 to 9000 ohm cm2. The resistance of the nexuses found between coupled cells, on the other hand, is calculated to be less than 5 ohm emz. In our experiments with synchronizing cell pairs, i t seems safe to assume that each isolated member of the pair before contact had a high-resistance membrane. An isolated cell with a functional

FIG.11. The contact between two adjacent myocytes in an aggregate of 7-day embryonic cells, 4 days in culture ; glutaraldehyde-osmium fixation, uranyl acetate en bloc stain; calibration scale in B equals 0.4 p ( A ) and 0.1 p (B). (Courtesy of John E. Rash.) (A) A nexal-like junction is indicated by the arrow; (B) High magnification of the same junction; the intercellular gap is about 20 A.

“heminexus,” i.e., a spot 0.1-0.2 p in diameter with a resistance on the order of 5 ohm cm2, would be so leaky that it could not continue to beat or even survive for long. If indeed synchronization requires electrical coupling, and coupling in turn is dependent on the presence of nexal junctions, then the punctate close junctions observed in the newly synchronized cells (DeHaan and Hirakow, 1972) must have been nexuses or nexal precursors. The nexus has a different molecular architecture than nonjunctional surface membrane, as revealed in freeze-etch prepara-

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tions (McNutt and Weinstein, 1970; Lorber and Raynes, 1972), and has a different chemical composition, as indicated by its solubility properties (Goodenough and Revel, 1970; Goodenough and Stoeckenius, 1971). Since, in the cell-pair experiments, synchrony was achieved in from 4 minutes to an hour after initial contact, it also follows that the transition from the molecular state of a high-resistance outer membrane to that of a nexal plaque can take place in that brief time. We would predict, therefore, that the formation of a nexal junction does not require de no210 synthesis but represents the insertion of prebuilt material into the membrane, or some kind of a phase transition process. V. Conclusions and Speculations

It should be apparent from the previous discussion that our ignorance about cell coupling is vastly greater than our knowledge. We know essentially nothing about the molecular structure or function of the nexus: its specificities, mechanisms of formation, or of action. Nonetheless, there are a few conclusions that can be derived from the evidence presented.

A. CONCLUSIONS 1. Electrical coupling among cells is a widespread phenomenon in the animal kingdom.* It is found in all excitable tissues, and among many nonexcitable cells, in both embryos and adults. Since coupled cells can pass electrical signals bidirectionally and without appreciable delay the major role of coupling in electrically active tissues is in mediating the propagation of impulses from cell to cell and the subtler interactions t,hat underlie synchronized activity of cells. 2. The nexus or a related type of close membrane apposition is the anatomic pathway for electrical coupling and the passage of ions in all excitable tissues (heart, smooth muscle, and electrotonic neural junctions), and very probably between most nonexcitable cells in the adult and embryo. Rigorous experimental proof for this conclusion is still lacking, but the body of supportive evidence seems compelling. 3. When embryonic cells come into contact, small regions of their high-resistance surface membranes can become modified to form points of low electrical resistance within the junctional area, and the cells become electrically coupled. This alteration, which can be completed in minutes, is accompanied by the appearance of points of close membrane apposition, visible in the electron microscope. Whether these punctate

* Electrical coupling between plant cells has also been reported recently (Spanswick and Costerton, 1967; Spitzer, 1970) ; however, the low resistance intercellular path is probably different than between animal cells.

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gap junctions are a primitive form, or a precursor of nexuses, has not been established. The initial coupling may be labile or transitory. 4. Some degree of electrical communication can exist among a group of cells that are not joined by gap junctions, provided that the electrical resistance of the outer membrane which establishes the boundary of the group is high relative to that of the cell membranes within the group. Cells within such a group would be coupled via the intercellular fluid. A measurable potential difference would exist between the inside and outside of the group. 5 . There are hints and suggestions that the anatomic route for the transfer of larger molecules between cells is the same as for electrical communication, but there is as yet no compelling evidence in support of this contention.

B. SPECULATIONS Electrical coupling among embryonic cells has been recognized for less than a decade, yet the phenomenon seems to have excited the interest of many developmental biologists as the rapidly growing number of publications on the subject would attest. Nonetheless, no satisfactory theoretical framework regarding the possible relevance that such communication might have to the processes of development yet exists. Previous workers have suggested that coupling may provide a means for tissue homeostasis and functional control, and even be a t the “root of cellular differentiation during embryogenesis” (Loewenstein, 1966, p. 467) . Low resistance junctions would allow for rapid distribution of inorganic ions, nutrients, and ‘[substances that control movement, rate of division and differentiation” (Furshpan and Potter, 1968, p. 115). They could also mediate interactions necessary for pattern formation in embryos (Wolpert, 1971). Although these are attractive ideas, as Wolpert points out there has been no experimental verification that cell coupling is in any way involved with developmentally significant intercellular communication. The question of concern here is, of what advantage is it to an embryo to transmit ions or molecules via an intracellular route, rather than permitting them to cross the cell membrane and diffuse through the intercellular fluid to surrounding cells. Whether these factors serve as transcellular nutrients, inducers, metabolic or mitotic regulators, or for other functions need not concern us. It is of interest to consider several possibilities on a purely speculative basis. One advantage derives from the dilution problem. Molecules inside cells can be maintained at high concentrations and can be transferred to neighboring cells without substantial loss via a nexal route. Molecules

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passed across a nonjunctional surface membrane, however, are subject to drastic dilution by diffusion into the extracellular volume. Crick (1970) has attempted to quantitate this factor by estimating the maximum distance over which a steady concentration gradient of a hypothesized “morphogen” could be established in some reasonable time within an embryo. On the assumption of diffusion from cytoplasm to cytoplasm along a chain of coupled cells, of a compound with molecular weight of 200-1000 daltons, such a gradient would be established over a distance of about 70 cells in 3 hours. Wolpert (1971) has estimated that most embryonic “fields” involve distances of less than 100 cells. We may also postulate, for example, that a t some point in the life of a cell-related either to its mitotic cycle or its developmental pathway-it becomes transiently leaky. A cell insulated from all neighbors would tend to lose intracellular ions and other components, conceivably to levels dangerous to its economy. A t the end of the leaky period, the cell would have to expend substantial energy manning its metabolic pumps to restore appropriate transmembrane concentration levels. I n contrast, a cell coupled to a number of neighbors by ion-permeable junctions would suffer less of a reduction in intracellular concentrations during the postulated leaky period. Ions lost would come, in effect, from the ion pool of the total coupled population. Moreover, a t the end of the leaky period any given cell would need to expend less energy pumping itself back up again since this task would be shared by all of the coupled group. The capacity to pass compounds from cell to cell across low-resistance junctions may also provide fewer restrictions on the kinds of molecules cells can use to influence each other. Nonelectrolyte molecules that can cross plasma membranes, from cytoplasm to extracellular fluid and back, must be rather small and hydrophobic in character (Lieb and Stein, 1969). Moreover, they must remain unaltered by passage through the extracellular milieu. Cytoplasm is characterized by a high concentration of potassium, magnesium, and organic anions, and little or no free calcium. Interstitial fluid, in contrast, is low in potassium, magnesium, and organic anions, and has substantial free calcium. It is not difficult to imagine that many kinds of molecules would be altered by the transition from one compartment to the other. Furthermore, proteases and nucleases which, inside the cell, appear to be bound or packaged by membranes may be free in solution in the extracellular fluid. Any molecule that would find exposure to the extracellular environment deleterious could pass from cell interior to cell interior via a nexal route under constant intracellular conditions. I n this review we have tried t o pose questions that may be answered by current techniques. The finding that coupling is widespread in the

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embryo during early embryogenesis but decreases during organogenesis suggests that a t some point cells no longer need to be coupled. Is uncoupling the cause, or the result, of differentiation, or is it indeed related a t all? Does interruption of coupling cause improper development? Clearly these questions should be answerable now that cell biologists are beginning to apply the techniques of ultrastructural and electrophysiological analysis with which the role of coupling can be readily investigated. Given the present techniques for examining coupling and junctions from a morphological and physiological point of view, we should be able to progress from the current largely descriptive approach to one based on incisive developmental questions. REFERENCES Aidley, D. J. (1971). “The Physiology of Excitable Cells.” Cambridge Univ. Press, London and New York. Asada, Y., and Bennett, M. V. L. (1971). J . Cell Biol. 49, 159. Ashman, R. F., Kanno, Y., and Loewenstein, W. R. (1964). Science 145, 604. Azarnia, R., and Loewenstein, W. R. (1971). J . Membrane Biol. 6, 368. Baldwin, K. M. (1970). J . Cell Biol. 46, 455. Barr, L. (1963). J . Theor. Biol. 4, 73. Barr, L., and Berger, W. (1964). Pfluegers Arch. Gesamte Physiol. Menschen Tiere 279, 192. Barr, L., Dewey, M. M., and Berger, W. (1965). J . Gen. Physiol. 48, 797. Bennett, M. V. L., and Dunham, P. B. (1970). Biophys. J . 10, 117a. Bennett, M. V. L., and Spira, M E. (1971). Biol. B d l . 141, 378. Bennett, M. V. L., and Trinkaus, J. P. (1970). J . Cell Biol. 44, 592. Borek, C., Higashino, S., and Loewenstein, W. R. (1969). J . Membrane Biol. 1, 274. Brightman, M. W., and Reese, T. S. (1969). J . Cell Biol. 40, 648. Burrows, M. T. (1911). J . Exp. 2001.10, 63. Cavanaugh, M. W. (1955). J . Ezp. Zool. 128, 573. Crick, F. (1970). Nature (London) 225, 420. DeHaan, R. L. (1967). Develop. Biol. 16, 216. DeHaan, R. L. (1968). Deuel. Biol. Sup& 2, 208. DeHaan, R. L., and Hirakow, R. (1972). Exp. Cell Res. 70, 214. Deleze, J. (1970). J . Physiol. (London) 208, 547. Dewey, M. M., and Barr, L. (1962). Science 137, 670. Dewey, M. M., and Barr, L. (1964). J . Cell Biol. 23, 553. Dixon, J S. (1971). J . Physiol. (London) 218, 97P. Draper, L r ~ . H., and Mya-Tu, M. (1959). Q u a ~ t .J . Exp. Physiol. Cog. M e d . Sci. 44, 91. Dreifuss, J. J., Girardier, L., and Forssmann, W. G. (1966). PfEziegers Arch. Gesamte Physiol. Menschen Tiere 292, 13. Eberth, C. J. (1866). Arch. Pathol. Anat. Physiol. Klin. Med. 37, 100. Fambrough, D., and Rash, J. E. (1971). Develop. Biol. 26, 55. Farquhar, M. G., and Palade, G. E. (1963). J . Cell Biol. 17, 375. Fawcett, D. W. (1966). “The Cell: Its Organelles and Inclusions,” p. 374. Saunders, Philadelphia, Pennsylvania. Fawcett, D. W., and McNutt, N. S. (1969). J . Cell B i d . 42, 1.

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CHAPTER 6

THE CELL CYCLE, CELL LINEAGES, AND CELL DIFFERENTIATION* H . Holtzer, H . Weintraub,t R. Mayne, and B. Mochan DEPARTMENT OF ANATOMY, SCHOOL OF MEDICINE, UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA,

PENNSYLVANIA

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Aspects of Myogenesis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Aspects of Erythrogenesis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11’. Aspects of Chondrogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

229 232 239 246 2.51 254

I. Introduction “Cell differentiation” means different things to biologists of different persuasions. For many biologists the term denotes a change in the behavior or structure of a cell which correlates with a sharp rise in the synthesis of certain macromolecules. This definition is of dubious value, for it accommodates such mechanistically unrelated phenomena as the induction of (1) “axonation” in neuroblastoma cells by withdrawal of serum components, X-irradiation, or addition of bromodeoxyuridine (BrdUrd) or cyclic AMP (Schubert and Jacob, 1970; Prasad, 1971; Furmanski et al., 1971 ; (2) tyrosine aminotransferase in hepatoma cells or of glutamine synthetase in embryonic retinal cells by glucocorticoids (Tomkins et al., 1969; Sarkar and Moscona, 1971) ; (3) hyperplasia and hypertrophy in sympathetic ganglion cells by NGF (Levi-Montalcini, 1963) ; and (4) somitic chondrogenesis by embryonic spinal cord or notochord (Holtzer and Matheson, 1970). The core problem of cell differentiation is not the identification of the inducing molecules, nor even the charting of the de novo biochemical pathways they elicit in responding cells. These are problems for studies in cell nutrition or cell physiology, for they deal with modulations of a cell’s synthetic activity within the constraints of a single developmental *This work was supported by Research Grants from the American Cancer Society, National Science Foundation and from the National Institutes of Health (HD-00189). t Present address : MRC Laboratories, Molecular Biology, Cambridge, England. 229

230

H. HOLTZER, H. WEINTRAUB, R. MAYNE, B. MOCHAN

program. The core problems of cell differentiation relate to mechanisms whereby cells acquire from their progenitor cells the machinery to respond to a variety of unspecific inducers so as to produce an axon; or to synthesize tyrosine aminotransferase rather than glutamine synthetase when exposed to glucocorticoids; or to produce more and bigger sympathetic ganglion cells when exposed to NGF rather than to deposit chondroitin 4-sulfate. The central problem of differentiation relates to those endogenous mechanisms that make available in daughter cells genetic information that was not readily available in the mother cell. Differentiation, in contrast to cell physiology, involves the emergence of daughter cells that synthesize molecules their mother cell did not and could not synthesize. Elsewhere (Holtzer, 1963, 1968, 1970a) the theme has been developed that the cell cycle subserves two distinct functions: (1) It can yield two daughter cells with the same synthetic pathways as those of the mother cell or (2) it can yield one or two daughter cells with synthetic pathways very different from those active in the mother cell. Cell cycles leading to duplication of the mother cell’s phenotype have been termed “proliferative” cell cycles, whereas cell cycles leading to cells with new pathways have been termed “quantal” cell cycles. Proliferative cell cycl 1s are responsible for increases in numbers of similar cells; quantal cell cycles are postulated as the means whereby genetic diversity is introduced into replicating systems. Only quantal cell cycles are believed to lead to rearrangements in chromosomal structure required to reprogram daughter cells. The events measured to mark terminal differentiation in the descendents of a zygote occiir over relatively large periods of time and involve many generations of cells of intermediate stages of differentiation. This was appreciated by earlier investigators and they stressed that a cell synthesizing myosin, hemoglobin or chrondroitin 4-sulfate was the terminal cell of a lineage that emerged very early in development. To state that a cell in the blastula stage was “determined” to become a muscle cell is not, however, an accurate statement. It is more probable that the first event establishing a “myogenic” lineage is followed by a second and third event in subsequent generations until a generation emerges that has accumulated the requisite machinery to produce all those molecules characteristic of fully differentiated muscle. By focusing on the transition between penultimate and ultimate generations in the skeletal myogenic lineage, the concept was developed that DNA synthesis, followed by nuclear division was an obligatory condition, not only for the terminal events in normal myogenesis, but also for the terminal synthetic events for all cell types (Holtzer, 1970a; Holtzer

6.

CELL CYCLE, CELL LINEAGES, AND CELL DIFFERENTIATION

231

and Sanger, 1972; Holtzer et al., 1973). The abrupt transition from the penultimate generation to the terminal generation can always be coupled to one particular round of DNA synthesis. By extrapolating back we would argue that a small number of quanta1 cell cycles is responsible for establishing the successive states of determination in the covertly differentiated cells in any lineage. The first step in effecting this succession has been postulated to occur in the DNA synthetic period of the mother cell. The actual expression of that decision may occur immediately following mitosis in the daughter cell’s G, cytoplasm. Alternatively, the altered state may be covertly transmitted through several proliferative cycles, only to be expressed many generations later by way of cues from accumulated endogenous or exogenous inducers or the dilution of inhibitors. Virtually nothing is known about the mechanisms by which the emergence of new synthetic programs is coupled to the prior or concurrent synthesis of DNA. However, we would speculate that the proposed coupling between DNA synthesis and differentiation in eukaryotes performs the function of “slowing down” the unfolding of the developmental programs. This would allow a step-by-step readout of different mRNA’s which correlate with different generations of covertly differentiated cells, each generation functioning as a precursor to the next. The correlation with generations rather than with time per se would also preclude the development of cells engaging in mutually exclusive synthetic activities, i.e., schizoid cells attempting to synthesize hemoglobin and myosin and albumin concurrently. The fertilized egg contains essentially all the information required to generate an organism. If we define an organism as some function of say 20 basic cell types, then the system is confronted with the problem of differentially retarding the flux of information a t least until some 20 cells are generated. This is further compounded by the possibility that the flux in eukaryotes is probably not greatly slower than that observed in prokaryotes. From readout to the production of considerable numbers of p-galactosidase molecules may take seconds in Escherichia coli. But even in myogenic or erythrogenic cells, less than 5 hours elapses from the mitosis which yields a myoblast or erythroblast containing no myosin or hemoglobin to a cell rich in one or the other of these molecules. The differential retardation clearly required for orderly development would result from the coupling of an emergent event to DNA synthesis. Implicit in this model of an emerging lineage are the following: (1) On a molecular level there are no biochemically “undifferentiated,” genetically unprogrammed cells (Holtzer, 1968). All eukaryotic cells are specialized for specific functions and possess cell-unique mRNA’s.

232

H. HOLTZER, H. WEINTRAUB, R. MAYNE, B. MOCHAN

Zygote, morula, or blastula cells have evolved synthetic programs that are as unique to their physiological niche as are the synthetic programs of mature cells. Accordingly, two of the grand generalizations relating cell differentiation to cell division are meaningless ; namely, “undiff erentiated cells replicate” whereas “differentiated cells do not replicate.” (2) There must be stringent controls operating in all cells which preclude the synthesis of inappropriate luxury molecules.* Early embryonic cells do not and cannot translate for molecules such as myosin, hemoglobin, or insulin. Consequently, it is misleading to state that early cells may lose the capacity to synthesize myosin, hemoglobin, or insulin, since they never had the machinery for assembling such molecules. (3) The chromosomal changes that are postulated to occur in quantal cell cycles are not likely to involve individual structural genes, but probably involve rearrangements a t a higher order genetic unit, a unit for “terminal” myogenesis, or “terminal” erythrogenesis, etc. At the chromosomal level, quantal cell cycles are likely to lead to “derepression” rather than “repression.” (4) The term “equipotential” may better apply to the nucleus than to the cell cortex, cytoplasm, or cell per se (Gurdon, 1970; Schneiderman and Bryant, 1971), for the metabolic options open to any one cell a t any one time are very limited. I n what follows, we describe three systems, the myogenic, the erythrogenic, and the chondrogenic. Each has its own peculiarities and each contributes its own specific solution to an aspect of differentiation. They all emphasize, however, the cumulative racial differences between cells with different cell cycle histories. And in all three instances the concept of progressive determination is emphasized and linked to our idea of quantal cell cycles by stressing the stepwise decisions daughter cells must make as a lineage evolves. II. Aspects of Myogenesis

The following observations have been confirmed in many laboratories: (1) Presumptive myoblasts synthesizing DNA do not translate for contractile proteins, whereas mononucleated or multinucleated skele-

* “Essential” molecules have been defined aa those ubiquitous molecules synthesized by most cells and are molecules that are required for the viability of the cells that synthesize them. “Luxury” molecules are those cell-unique molecules responsible for the state of differentiation of the cell that either synthesizes them or has inherited them. Luxury molecules are not generally required for the viability of the cell. Obvious luxury molecules are myosin or hemoglobin; others are informational RNA’s in the oocyte that are transmitted to early embryonic cells, pole plasm in germinal cell lineages, etc.

6.

CELL CYCLE, CELL LINEAGES, AND CELL DIFFERENTIATION

233

tal myogenic cells translating for contractile proteins do not synthesize DNA. (2) Multinucleated myotubes result from fusion, but there is no obligatory relationship between fusion and the translation for contractile proteins. (3) Within 5 hours after the terminal quantal mitosis, daughter myoblasts have synthesized sufficient quantities of myosin, actin, and tropomyosin to be detected with fluorescein-labeled antibodies or with the electron microscope. (4)If the coordinated translation of these proteins is not obligatory, their syntheses are a t least coupled. (5) Neither nerves, known hormones, nor exogenous molecules such as collagen are required for the programming of these basic myogenic events (Okazaki and Holtzer, 1965, 1966; Bischoff and Holtzer, 1968, 1969; Ishikawa et al., 1968, 1969; Holtzer, 1970b; Fischman, 1970; Holtzer et al., 1972). The underlying premise of our experiments is that when a given postmitotic myoblast translates for the first molecule of myosin, actin, and tropomyosin, the major decisions regulating myogenesis have long been made. Accordingly, the experiments focus on the mother of the myoblast, the presumptive myoblast, and the precursors to the presumptive myoblast, the postulated myogenic beta cell (Holtzer, 1970a,b). The following experiments stress the obligatory role of DNA synthesis in programming precursor myogenic cells to produce terminal cells in the myogenic lineage. Approximately 30% of the mononucleated cells from 10-day chick breast muscle are capable of fusing in vitro without further rounds of DNA synthesis. The remaining 70% normally divide, yielding one to several generations of myogenic cells. It is these cells that divide that make up the majority of the cells fusing to form myotubes in vitro. The following experiments suggest an obligatory requirement for DNA synthesis if the bulk of the myogenic cells in the original inoculum are to fuse: Cells were plated out in high and low densities and cultured for 24 or 48 hours. During this period the great majority of cells in the low density cultures divided, whereas only a modest fraction divided in the high density cultures. The 2 types of cultures were trypsinized and subcultured a t either high or low densities. I n both series, subcultures of the progeny from low density cultures formed many more myotubes than did subcultures prepared from the high density cultures (Holtzer et aZ., 1973, Bischoff, 1970; Dienstman and Holtzer, unpublished data). Apparently many myogenic cells in 10-day breast muscle do not fuse unless they undergo one or more rounds of DNA synthesis. The following experiments were designed to learn when in normal development the first quantal cell cycles occur resulting in the ability of myogenic cells to fuse: Mononucleated cells from 5-, 6, 7-, and 8-day breast muscle were cultured in dThd-YHfor 24 hours. These labeled cells

234

H. HOLTZER, H. WEINTRAUB, R. MAYNE, B. MOCHAN

were then challenged to fuse with 10-day unlabeled myogenic cells as described in Bischoff and Holtzer (1968). Within 24 hours large numbers of %day labeled myogenic cells fused with 10-day cells; few fused from the 6-day embryos and virtually none from 5-day embryos. These findings are consistent with the proposition that 5-day myogenic cells from breast consist primarily of presumptive myoblasts and myogenic beta cells,

t Fdyd

0

(

2

3

4

Days in culture

FIQ.1. Creatine phosphokinase activity of chick breast muscle cultures grown in the presence and absence of bromodeoxyuridine (BrdUrd). Creatine phosphokinase was assayed according to the method of Coleman and Coleman (1968). Assays were performed on the supernatant of cultured breast muscle derived from l l d a y old embryos (cf. Bischoff and Holtzer, 1968). BrdUrd (10 pglml culture medium) was added at day zero, and cultures were fed daily. Fluorodeoxyuridine (FdUrd) M ) was added after 48 hours in culture. Velocities ( v = AAJ4ammsec-I) at each point were determined in triplicate, corrected for a small background rate in the control (-BrdUrd) : (A--A) 10 absence of creatine, and averaged. (0-0) pglml BrdUrd.

+

and that cells of both these generations must pass through, respectively, one and two different quanta1 cell cycles to yield cells programmed for fusion. The striking effects of BrdUrd on myogenesis and the fact that they occur only in replicating precursor cells have been described by Stockdale et al. (1964), Okazaki and Holtzer (1965), Coleman et al. (19701, and Bischoff and Holtzer (1970). The claim by Schubert and Jacob (1970) that the paradoxical effects of BrdUrd on cell differentiation

6.

C E L L CYCLE, CELL LINEAGES, A N D CELL D I F F E R E N T I A T I O N

235

does not require substitution in the DNA has not been confirmed in studies of myogenic, chondrogenic, or amnion cells (Bischoff, 1971; Mayne et al., 1971) or with normal nerve or pigment cells (Biehl and Holtzer, unpublished observations). BrdUrd has no readily detectable effect on postmitotic myoblasts synthesizing myosin or its associated low molecular weight polypeptides (Sreter et al., 1972), actin or tropomyosin, or on the behavior of the developing sarcolemma. At low concentrations (ca. 1 pg/ml) the analog suppresses fusion but does not block the synthesis of the above molecules,

Brd Urd

(pg/ml)

FIG.2. Creatine phosphokinase activity of chick breast muscle grown in the presence of varying concentrations of bromodeoxyuridine. Assays for creatine phosphokinase activity were performed as described in Fig. 1 on 4-day-old cultured breast muscle. BrdUrd (pglml culture medium) was added at zero day, and muscle cultures were fed daily for 4 days. Percent activity represents the specific activity (v/mg protein) of the BrdUrd-treated cultures compared to the control culture. (a)and (H)represents two separate experiments.

or their arrangement into myofilaments, nor does it interfere with cell replication. At higher concentrations (ca. 5-15 pglml) the synthesis of all terminal luxury molecules is greatly depressed, including the 3 low molecular weight polypeptides, C1, C2, and C3, though cell division is only moderately depressed. At still higher concentrations BrdUrd interferes with the frequency of cells entering S (Pujara and Whitmore, 1970; Bischoff and Holtzer, 1970) and has many other deleterious effects, particularly on the cell surface (Abbott and Holtzer, 1968; Chacko et al., 1969a). The differential suppressive effect of BrdUrd on the synthesis of creatine phosphokinase (CPK) was first demonstrated by Coleman and Coleman (1968). Figures 1-3 summarize additional data on this

236

H.

H. HOLTZER,

WEINTRAUB, R. MAYNE, B. MOCHAN

subject. At low concentrations of BrdUrd contractile protein synthesis is not significantly blocked, but fusion is dramatically suppressed. At these low concentrations (Fig. 2), CPK activity is not significantly blocked. At the concentrations that block the synthesis of all myofibrillar proteins and myoglobin, CPK is maximally blocked. The sharp cutoff between virtually no effect of the analog and maximum effect makes it less likely 900

-

000

-

700

-

600

-

300

-

19.5 hr I

200 -

'OOk I

0

t ,i,,

, , , , , 2 4 6 8 1 Days after subculturing

, 0

FIQ.3. Pattern of creatine phosphokinase activity of muscle cultures after treatment with bromodeoxyuridine (BrdUrd) for 19.5 and 68 hours. BrdUrd (10 pg/ml culture medium) was added a t zero day. The cultures were then subcultured by trypsinization after 19.5 hours (@--@) and 68 hours (A-A) and plated out a t 0.5 x 10' celldm1 in normal media. Creatine phosphokinase activity was measured as in Fig. 1 over several days. The abscissa represents time after subculturing.

that BrdUrd acts as a result of random substitution of bromouracil for thymidine. It will be of interest to learn whether the background level of CPK activity is found ,only in postmitotic cells and whether it is the isoenzyme of the neural or muscle type. Figure 3 illustrates the appearance of CPK activity in the descendants of BrdUrd-suppressed cells that have been removed from the analog and grown in normal medium. Although only one or two rounds of DNA synthesis are required to suppress myo-

6.

CELL CYCLE, CELL LINEAGES, AND CELL DIFFERENTIATION

237

genic cells (Bischoff and Holtzer, 1970) anywhere from 3 to 5 replications are required to yield nortnal functioning progeny. Total alkaline phosphatase, glucose-6-phospliatase, LDH, and cytochrome oxidase are similar in controls and BrdUrd-suppressed myogenic cells. Figure 4 is a comparison of the absorption spectra of a series of cytochromes in controls and BrdUrd-suppressed cells. The paradoxical effect of BrdUrd on myogenic cells is that it (1I blocks the synthesis of contractile proteins for tnyofibrils but not those for the cell surface (Ishikawa et at., 1969) or the mitotic apparatus; (2) blocks niyoglobin Muscle Cells

490

520

550

580

610

Wavelength ( m p )

FIG.4. A comparison of the absorption spectra based OR equivalent amounts of DNA from control cultures and cultures treated with bromodeoxyuridine (BrdUrd) (Estabrook and Holtzer, unpublished observations).

synthesis but not the synthesis of hemeproteins for the cytochromes; and (3) blocks CPK synthesis but not the synthesis of several other cytoplasmic enzymes. There are approximately 1 X lo3 mononucleated, postmitotic, crossstriated myoblasts in the anterior myotomes of the 3-day chick embryo (Holtzer et al., 1957; Allen and Pepe, 1966; Pryzbalski and Blumberg, 1966; Holtzer and Sanger, 1972).Assuming an inordinately brief cell cycle of 5 hours, it follows that when a myotome is first formed on day 2 it is populated with between 16 and 128 presumptive myoblasts, and probably many hundreds of their precursors, the myogenic beta cells.

238

H. HOLTZER, H. WEINTRAUB, R. MAYNE, B. MOCHAN

When these figures are considered along with earlier reports of when cells become "determined" in the chick embryo (e.g., Rudnick, 1948), it is likely that (1) when the zygote has yielded 11-13 generations of cells, the lineages of the major families of cell types (nerve, blood, gut, skin, etc.) are irreversibly established, and (2) clusters of precursor skeletal muscle cells are widely distributed in the 24-hour embryo. An obligatory requirement for DNA synthesis in the transition from presumptive myoblast to myoblast is shown by the following experiments: The thoracic segments of 3-day chick embryos were transected in the midline, and either the left or right halves incubated for 10 hours in normal medium, whereas the contralateral halves were incubated in norTABLE I

NUMBER OF STRIATED MYOBLASTS PER POSTERIOR %DAYMYOTOME'

A B

c

D

Control

FdUrd-treated

960 1220 810 1350

520 830 410 750

Three-day trunks were transected into right and left halves and reared as organ cultures for 10 hours in normal medium plus 10-6 M fluorodeoxyuridine (FdUrd) plus 10-4 uridine for 10 hours. After treatment with fluorescein-labeled antimyosin, the myotomes were squashed and the individual striated mononucleated myoblasts counted.

'

ma1 medium plus fluorodeoxyuridine (FdUrd) (lo-" M ; 95% inhibition of DNA synthesis). During this period there was no detectable fall in l e ~ c i n e - ~ incorporation H into total protein or autoradiographic evidence of a drop in the incorporation of ~ r i d i n e - ~ HAfter . 10 hours the somites were glycerinated, treated with labeled antibodies against myosin or tropomyosin, squashed, and the numbers of individual, mononucleated, postmitotic myoblasts with striated myofibrils counted under the fluorescence microscope (Holtzer et al., 1957; Holtzer et al., 1973). In a second series, the thoracic segments were first grown in FdUrd for 10 hours and then either right or left halves were removed from the inhibitor, washed several times, and grown for 15 hours in the presence of excess cold thymidine. These experiments were designed to dem-

6.

CELL CYCLE, CELL LINEAGES, AND CELL DIFFERENTIATION

239

onstrate that FdUrd did not kill the presumptive myoblasts held a t the GI-S interphase. The results are shown in Tables I and 11. These experiments demonstrate that: (1) presumptive myoblasts do not have the option of synthesizing myofibrillar proteins, though they do not synthesize DNA; and (2) if the synthesis of contractile proteins is to occur, the presumptive myoblast must synthesize DNA and form daughter nuclei. Experiments using Cytochalasin-B suggest that though nuclear diviTABLE I1

NUMBER OF STRIATED MYOBLASTS PER POSTERIOR DAY MYOTOME" ~

~~

A B C

FdUrd-treated and not reversed

FdUrd-treated and reversed

380 650

840 1050 1010

430

The trunks were transected into right and left halves and organ cultured in fluorodeoxyuridine (FdUrd) for 10 hours. Either the right or left half was removed, washed, and then grown in normal medium for an additional 15 hours; the other half remained in FdUrd. (1

sions are required, cytokinesis is not obligatory (Sanger e t al., 1971; Sanger and Holtzer, 1972; Holtzer e t al., 1972). 111. Aspects of Erythrogenesis I n an effort to concentrate on a less complicated differentiating system, we have been studying the primitive line of red cells derived from the yolk sac (Weintraub e t al., 1971; Campbell e t al., 1971). H b molecules first appear in morphologically distinct primitive erythroblasts in 35-hour chick embryos. These first generation erythroblasts are the progeny of precursor hematocytoblasts. The parent hematocytoblasts do not synthesize Hb. As these hematocytoblasts produce erythroblasts primarily between 35 and 65 hours of incubation, they are presumed to be a transient population. This distinguishes them from the hematocytoblasts of the adult which continue to replicate in the marrow throughout life. Whether the hematocyto'blast yielding the primitive line also gives rise to the definitive erythroblasts, or whether there are two species of hematocytoblasts both derived from a still earlier erythrogenic cell, is still unknown (Hagopian and Ingram, 1971).

240

H. HOLTZER, H. WEINTRAUB, R. MAYNE, B. MOCHAN

The first Hb-producing erythroblasts undergo six doublings (Fig. 5 ) . The mitotic cycle of these erythroblasts lengthens as ,the cells mature. During each of these divisions the average amount of Hb synthesized per unit of time is constant, decreasing somewhat during the last cell cycle. The population matures as a relatively homogeneous cohort and

10 Division

I

20 30 2

3

47

64

93

4

5

6

,

180hr Postmilolic period

F I ~ 5. . The relationship between hemoglobin (Hb) content and the division cycle during the development of the erythrocyte lineage. Smears were made from the blood of embryos between the ages of 35 hours and 9 days. The amount of Hb per metaphase cell was determined for mitotic cells in those populations younger than 6 days. This was done cytophotometrically with and without the added sensitivity of benzidine staining (Campbell et al., 1971). The amount of Hb per cell is shown for each of the six mitoses experienced by the progeny of the hematocytoblast. After each division the amount of Hb in the daughter cells is assumed to be half that of the parent. The slope of the graph between each successive division is an indication of the average amount of Hb accumulated during a division period of a given length.

each generation displays characteristic changes in biochemical and morphological properties, e.g., a decrease in RNA and non-Hb protein and an increase in nuclear condensation. By the sixth generation RNA and DNA synthesis have ceased, non-Hb protein synthesis is less than 2076 of that observed during the fourth generation, and a new erythrogenic specific histone (Neelin et al., 1964) has appeared. H b synthesis continues, however, for 2-3 more days and the cells remain in the circulation for an additional 7-8 days (for further detail, see Wilt, 1965; Hagopian and Ingram, 1971 ; Reynolds and Ingram, 1971).

6.

CELL CYCLE, CELL LINEAGES, AND CELL DIFFERENTIATION

241

It is likely that many of the changes associated with erythropoietic development are programmed into the hematocytoblast, before translation for H b begins. When might these events take place during embryogenesis? A 35-hour embryo contains approximately loe cells (Weintraub et al., 1971). Of these some 2 X 10' are hematocytoblasts of the kind that will give rise to first-generation primitive erythroblasts. Assuming a clonal origin and no selective advantage of one cell type over another, these figures are consistent with the initiation of some step in the erythrogenic lineage occurring as early as the third or fourth generation following fertilization if each of the cell cycles is quantal. Alternatively, if some of these early cleavage divisions were proliferative, the separation of the earliest erythrogenic cells from other mesenchymal cells might occur as late as the seventh or eighth generation following fertilization. Preliminary experiments (Biehl and Holtzer, unpublished observations) do, in fact, support these calculations : Disaggregated blastoderms from 15-hour embryos, after proliferating in vitro, yield typical erythroblasts. This means that some disaggregated cells from these very early stages possess the information to yield progeny capable of differentiating into recognizable red blood cells without further interaction with other embryonic cells. Recent experiments using BrdUrd have also supported the notion of a programmed hematocytoblast. When 25-hour embryos are treated with BrdUrd, H b fails to appear a t 35 hours (see also Miura and Wilt, 1971; Wenk, 1971). A 3-fold increase in BrdUrd substitution does not, however, inhibit the synthesis of H b in erythroblasts already translating for H b (Fig. 6, p. 242). This resistance extends over as many as 2-3 cell cycles. In addition, there is no detectable alteration in the divisional, morphological, or biochemical changes which characterize the sequential progression of this lineage from first to sixth generation of blood cells. Assuming that BrdUrd prevents the initiation of new developmental programs (Holtzer et al., 1973; Weintraub et al., 1972), it would appear that the changes characterizing successive generations of erythroblasts are encoded into the lineage some time before H b makes its first appearance. The total suppression of H b synthesis when BrdUrd is incorporated into the hematocytoblasts, stands in marked contrast to its lack of effect when it is incorporated into the daughter or granddaughter cells of the hematocytoblasts, the first or second generation erythroblasts. The resistance of H b synthesis to BrdUrd in erythroblasts cannot be explained in terms of a stable H b mRNA. This follows from the fact that Hh synthesis is sensitive to both actinomycin D and cordycepin and the observation that the presumed H b mRNA, a 10 S species, is synthesized in BrdUrd-treated cells (Fig. 7, p. 243). Nor can the resistance

242

H. HOLTZER, H. WEINTRAUB, R. MAYNE, B. MOCHAN

of H b synthesis be explained in terms of a number of segments of DNA that replicate a t 25 hours, but fail to do so a t 4 days. Such a species might be amplified genes for Hb. Figure 8 (p. 244) attempts to deal with this problem. Cells were treated in o m with thymidine-% from 25 hours to the end of the experiment, a t 4 days. At 35 hours, thymidine-3H was added, also until the fourth day. From day 3 to day 4? BrdUrd was included a t 70% substitution.

BrdUrd ( p g / m l )

FIG. 6. Transition during the erythropoietic lineage from bromodeoxyuridine (BrdUrd) sensitivity to BrdUrd resistance. Three milliliters of various concentrations of BrdUrd were added to embryos during the periods stated below. Cells were then collected and the percentage substitution of BrdUrd for thymidine (dThd) was determined by CsCl gradient centrifugation. The same gradients indicated that the mitotic behavior of these cells was similar to that of controls. Hb per cell was monitored cytophotometrically or as trichloroacetic acid-precipitable leucine-'H cpm incorporated into carboxymethyl cellulose-purified Hb. For the incubations between days 1 and 2, Hb per embryo was measured as OD,, units. BrdUrd from 23, A-A; 24,A-A; 3-4, .-I; 4-5, 0-0. days 1-2, 0-0;

The ratio of I4C to SH in the isolated 4-day DNA was then determined across a CsCl gradient. Any species of DNA made only during the period of BrdUrd sensitivity would contain a higher ratio of 14C to SH and under these conditions, would not have incorporated BrdUrd, and would appear in the light-light region of the gradient, along with the DNA from the less than 1% of the cells that have left the division cycle. To a limit of resolution of 0.5% of the DNA, we have not been able to detect DNA that falls into this category.

6.

CELL CYCLE, CELL LINEAGES, AND CELL DIFFERENTIATION

243

4s

.O N

260

2

.J

I

I

a u

.o

2

6

10

14

FRACTION

18

22

26

30

NUMBER

FIG.7. Synthesis of 10s mRNA in the presence of bromodeoxyuridine (BrdUrd). The resistance of erythroblasts to BrdUrd might be explained in terms of a stable mRNA for Hb. That this is probably not the case is indicated by the fact that incubations of these cells with either actinomycin D or cordycepin both lead to a rapid inhibition of Hb synthesis (50% by 4 hours). As the inhibition varies with each type of protein synthesized by these cells, and with each subunit of the Hb tetramers, i t is unlikely that the effect of these inhibitors of RNA synthesis is primarily mediated through a step common to protein synthesis in general (Weintraub and Holtzer, 1972). Further verification for the continued synthesis of Hb mRNA comes from the demonstration above that the 10s RNA reported to code for Hb (Gurdon et al., 1971) is synthesized in control and BrdUrd treated cells. Fourday control cells and 4-day cells pretreated with BrdUrd at 70% substitutions were, respectively, incubated for 3 hours in 3H-uridine and actinomycin D at concentrations which inhibit rRNA synthesis. Polysomes were isolated from the washed cells, treated with 0.5% SDS and run on sucrose gradients with marker Escherichia coli 16s and 23s RNA. x-x, Control; 0-0, bromodeoxyuridine; A-A, ODza.

It is likely that some protein, RNA, or polysaccharide made during the period of BrdUrd sensitivity offers continued resistance to BrdUrd once the synthesis of H b is initiated. At the least, these experiments indicate that the action of BrdUrd is likely to be somewhere other than a t that portion of the genome coding directly for H b mRNA. Whereas resistance of Hb-synthesis to BrdUrd was postulated to depend on the active synthesis of a macromolecular species made during the

244

H. HOLTZER, H. WEINTRAUB, R. MAYNE, B. MOCHAN

Fraction Number

FI~. 8. T o test whether the acquired resistance to bromodeoxyuridine (BrdUrd) wm secondary to the presence of a species of DNA that was replicated during the sensitive period, but not replicated during the resistant period, 25-hour embryos were treated with thymidine-“C. At 35 hours, thymidine-aH waB added and the embryo allowed to develop in the presence of both labels until day 3, when BrdUrd was added to 70% substitution. After a little more than one division, nuclear DNA was isolated and run on CsCl equilibrium gradients. Circles represent the tritium pattern. Heavy-heavy DNA is observed in fractions 5-6; heavy-light, in fractiom 9-12; light-light (L-L) DNA is usually found in fractions 13-15. The counts on the top of the gradient are consistently observed and thought to represent a DNA complex to either lipid or polysaccharide. p is shown by the crosses and represents the ratio “C:’H normalized to the average ratio in the total DNA fraction. There is no detectable increase in p across the L-L region. The limit of detection is 0.5% of the total incorporated counts. This is obtained by establishing the number of additional “C cpm needed to raise p to 110% in fraction 14. Dividing this figure by the total “C cpm gives 0.5% resolving power. The drop in the ratio over the heavy-heavy regions indicates that the fastest dividing cells in the 3-day population are descendants of cells that were not dividing when only “C was present. sensitive period, but maintained through subsequent development, the resistance of the other biochemical and morphological changes associated with erythropoiesis may not be based on such a positive mechanism. It is possible that these changes are dependent upon carefully timed decay constants. With the synthesis of the first Hb molecules there may be a shutdown of all “steady-state” determinants. The system deteriorates according to the decay constants of its various components. When different specific regulatory substances achieve particular levels during

6.

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the decay period, coupling between a set number of these components might occur. These coupled molecules might be providing, for example, the cue to turn off RNA synthesis on day 6. A major contributor to the overaIl decay process would be the dilution component introduced by cell division. Thus far experiments have focused on how information normally generated in one generation can be processed and transmitted to subsequent generations. The following two experiments describe a failure in the process of information transfer. The first is an extension of the BrdUrd experiments previously mentioned. When 25-hour embryos incorporate BrdUrd (ca. 30% substitution) into their hematocytoblasts, these replicating cells do not give rise to first generation erythroblasts. If thymidine is added 5 hours later to these embryos a t concentrations 5 times that of BrdUrd, H b appears after about a 1 day lag; however, the amount of H b is much less than would be expected. Longer exposures to BrdUrd result in an even greater amount of inhibition, even when the time for reversal is extended some 3 4 days. Some red cell precursors clearly do not recover their capacity to give rise to erythroblasts when exposed to BrdUrd under these conditions, even though the major embryonic structures present during this period do appear. Although these observations concerning the reversibility of BrdUrd-suppressed hematocytoblasts are preliminary, it seems clear that recovery from BrdUrd will prove to be a more complicated process to follow in these hematocytoblasts than would be expected on the basis of our original findings with the more terminal presumptive myoblasts (Okazaki and Holtzer, 1965 ; Bischoff and Holtzer, 1970), chondroblssts (Abbott and Holtzer, 1968), and amnion cells (Mayne et al., 1971). With regard to reversibility, the response of hematocytoblasts to BrdUrd is more like the response of primitive somites than the response of more terminal cells (Abbott et al., 1972; Mayne et al., 1972). This difference in “reversibility” of the effects of BrdUrd between early and late cells in a lineage should prove to be of considerable interest. Experiments designed to inhibit DNA synthesis with FdUrd parallel our findings with BrdUrd. If 25-hour embryos are treated in vivo with M ; 75% inhibition of DNA synthesis) for 5 hours and FdUrd M ) , the appearance of H b is delayed then reversed with thymidine by about 5 hours. Longer exposures yield longer delays, while the same exposure to higher concentrations or lower concentrations of FdUrd give respectively longer or shorter delays, During all manipulations with FdUrd, incorporation of I e ~ c i n e - ~ H into protein was unaffected, and autoradiography using labeled uridine, leucine, and thymidine showed no signs of thymine-less death. About 70% of the embryos characteristi-

246

H. HOLTZER, H. WEINTRAUB, R. MAYNE, B. MOCHAN

cally go on to hatch. The FdUrd-induced lag extends throughout the second, third, fourth, and fifth days of development. By the end of the fifth day, the difference between controls and experimental embryos becomes insignificant. I n contrast to these studies on the 25-hour hematocytoblast, a 7-hour FdUrd treatment M ; 95% inhibition of DNA synthesis) of 4-day erythroblasts already synthesizing H b shows a slight stimulation of l e ~ c i n e - ~incorporation H into Hb. These experiments suggest that there is an obligatory requirement for the synthesis of DNA if the synthesis of H b is to be initiated in the daughters of hematocytoblasts. Once the system is established and H b synthesis is underway, neither BrdUrd nor FdUrd is able to perturb it. Analogous to the BrdUrd experiments, prolonged exposure to FdUrd (10-20 hours) results in an increasing decay in the ability of these cells to be reversed by thymidine. A rather trivial explanation for this behavior is possible; for example, cell death or “poor” environmental conditions. Preliminary results indicate that these effects are probably minimal. Given these qualifications together with our observations on BrdUrd “reversibility,” the studies using FdUrd imply that the machinery required for the primary formation of H b by a red cell is basically unstable. If primitive hematocytoblasts do not effect their developmental program a t the proper time, their ability t o do so gradually declines. IV. Aspects of Chondrogenesis

Unlike the terminally differentiated myoblast or erythroblast, terminally differentiated chondroblasts cannot a t present be recognized solely by the molecules they synthesize. Myosin synthesis or hemoglobin synthesis sharply separate a myoblast or erythroblast from the parent presumptive myoblast or hematocytoblast. I n contrast, the synthesis of glycosaminoglycans, such as chondroitin sulfate has been shown to occur in a wide variety of cells (Holtzer, 1968; Holtzer and Matheson, 1970; Conrad, 1970; Mayne et al., 1971; Dorfman and Ho, 1970; Sueuki et al., 1971). The analytical techniques for detecting chondroitin suIfate, however, involve digestion of the protein component of the glycosaminoglycan complex by proteases. If this protein component were to differ in amino acid sequence, or even if there were more subtle rearrangements of the sugar components in different cell types, then the synthesis of chondroitin sulfate could involve the activities of different genes. The deposition of extracellular matrix and the unique a,-collagen (Miller and Matukas, 1969; Trelstad et al., 1970) suggest that there are specific “cartilage” genes and that these are suppressed in related mesenchymal cells. Early in vivo experiments (Holtzer and Detwiler, 1953; Watterson

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et al., 1954; Avery et al., 1955) demonstrated an inductive interaction during chondrogenesis between the spinal cord or notochord and the somites. Claims for the detection of specific inducing substances have not been verified, and most workers have suggested that the events of induction are apt to be permissive in nature rather than instructive (Holtzer, 1963, 1964, 1968; Thorp and Dorfman, 1967; Holtzer and Matheson, 1970; Ellison and Lash, 1971; Abbott et al., 1972; Holtzer and Mayne, 1973). In &TO, as well as in t h o , the difference between (a) a cluster of stage 12-13 somites alone and (b) a similar cluster of somites plus ~

- 8

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i

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0

.-

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5 0 0-3 0-6 0-9 Days in S ~ l f a t e - ~ ~ S

FIG.9. Stage 12-13 somites alone ( S cultures) or stage 12-13 somites plus a piece of notochord (NS cultures) were grown and analyzed for chondroitin sulfate as described in Abbott et al., (1972). From these results it is clear that somite cells by themselves do not synthesize the quantities of chondroitin sulfate that characterize a recognizable chondroblast. I t is also worth stressing that when 3 day old NS cultures are trypsinized, they will yield individual chondrogenic cells capable of yielding chondrogenic clones. When 3 d a y old S cultures are challenged in the same way to yield chondrogenic clones, they do not do so.

a small piece of notochord is striking. Thousands of typical chondroblasts emerge in the notochord-somite cultures, whereas not a single chondroblast emerges in cultures of somites by themselves. Figure 9 shows the amount of chondroitin sulfate produced by somites by themselves and somites plus notochord. It is still unclear whether the small amount of sulfate incorporated by somites alone is authentic chondroitin sulfate of the kind produced by frank chondroblasts, whether it is another unknown sulfated molecule, or whether it is synthesized by the fibroblasts in these cultures (Schubert and Hammerman, 1968).

248

H. HOLTZER, H. WEINTRAUB, R. MAYNE, B. MOCHAN

The following experiments attempt to probe the nature of the transition of precursor cells from the sclerotome compartment to the frank chondroblast compartment. Matrix-accumulating chondroblasts clearly divide in developing cartilage (Cohen and Berill, 1936; Abbott and Holtzer, 1966; Seegmiller et al., 1971), but whether this represents part of a “program” of ordered divisions as postulated for the myogenic and erythrogenic lineage, has yet to be determined. To examine the possible relationship between the mitotic cycle and differentiation, the effects of BrdUrd on somitic chondrogenesis have been investigated. The effect of this analog on cultures of mature chondrocytes has been reviewed elsewhere (Holtzer and Abbott, 1968; Lasher, 1971). At moderate doses (ca. 3.5 x M ) replicating chondroblasts assume a fibroblastic morphology and cease to synthesize or accumulate large amounts of chondroitin sulfate (Schulte-Holthausen et al., 1969). At these doses BrdUrd does not markedly suppress cell replication, nor affect the activities of many enzymes. These differential effects of BrdUrd are likely to be due to incorporation into DNA, although the profound influence of BrdUrd on the morphology of cultured chondrocytes has raised the question of a direct effect on the synthesis of cell surface components (Abbott and Holtzer, 1968; Holtzer and Abbott, 1968). Stage 17-18 chick somites were cultured either with or without notochord and exposed to BrdUrd for 3-day periods from 0 to 3, 1 to 4, 2 t o 5, and 3 to 6 days. BrdUrd was then removed from the medium and replaced by an equal concentration of thymidine. Cultures were examined for cartilage development 10-14 days later. I n control cultures, extracellular matrix could first be detected, using staining techniques, by day 5. Cultures exposed to BrdUrd either on days 0 to 3 or 1 to 4 failed to deposit matrix, whereas exposure on days 2 to 5 or 3 to 6 resulted in detectable cartilage matrix (Abbott et al., 1972). This result was confirmed by continuous labeling with gluc~samine-~H on days 1 to 3, 4 to 6, and 9 to 11 in cultures exposed to BrdUrd from day 0 to 3. Glycosaminoglycans were isolated after digestion with pronase, followed by extensive dialysis, and then fractionation on strips of cellulose acetate by high voltage electrophoresis (Nameroff and Holtzer, 1967). The separations obtained in comparison to standards of hyaluronic acid and chondroitin sulfate are shown in Fig. 10. In control cultures labeled for days 1 to 3, most of the label migrates either before, or as a peak corresponding in position to, a standard of hyaluronic acid. I n some, but not all experiments, some label could be observed migrating beyond this region. From day 4 to day 6, a peak migrating in the region of

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CELL LINEAGES,

CELL CYCLE,

AND CELL DIFFERENTIATION

249

a standard of chondroitin sulfate was present, and from day 9 to day 11 it formed the major component. For cultures exposed to BrdUrd on days 0 to 3, and analyzed on days 1 to 3, 4 to 6, or 9 to 11, little effect could be observed either on label migrating prior to hyaluronic acid or the hyaluronic acid peak itself. Chondroitin sulfate, however,

4-6dayr

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4-6 days

BrdUrd

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-2

0

2

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6

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FIG. 10. Separation by high voltage electrophoresis of the glycosaminoglycan fraction obtained from notochord-somite cultures exposed to gluco~amine-~H on days 1 to 3, 4 to 6, or 9 to 11. Cultures were exposed to either BrdUrd (10 pg/ml) or thymidine (10 pg/ml) from days 0 to 3. Electrophoresis was carried out on cellulose acetate strips in pyridinium formate buffer (pH 3.0, 500 V, 120 minutes, 0°C). HA, hyaluronic acid ; CSA, chondroitin sulfate.

did not appear even by 9-11 days. Analysis of the g1ucosamine:galactosamine ratios of the peaks, their susceptibility to testicular hyaluronidase and chondroitinase AC, have confirmed that the peaks are largely hyaluronic acid and chondroitin sulfate (Abbott et al., 1972). The material which migrates prior to hyaluronic acid is probably “glycoprotein,” which is resistant to attack by pronase. From these analyses, it appears that BrdUrd specifically interferes with the appearance of chondroitin sulfate, while leaving hyaluronic acid and “glycoprotein” synthesis relatively unaffected. BrdUrd does not inhibit all glycosamino-

250

€I HOLTZER, . H. WEINTRAUB, R. MAYNE, B. MOCHAN

glycan synthesis, or interfere in any gross way with overall glucosamine metabolism. More recent experiments have focused on the effects of BrdUrd on still earlier somite cells. Stage 16 somites, synthesizing little if any chondroitin sulfate, have been exposed to BrdUrd and then challenged to yield chondrogenic clones. Though replicating for many generations in normal medium, the progeny of BrdUrd-suppressed somite cells do not yield frank chondroblasts. Synthesis of hyaluronic acid before chondroitin sulfate confirms the results of Kvist and Finnegan (1970) for chick somites, and has also been shown to occur in the regenerating newt limb (Toole and Gross, 1971) and in the development of the chick cornea (Toole and Trelstad, 1971). The latter authors have suggested that hyaluronic acid may provide a suitable substratum for cell migrations during morphogenesis. An alternate explanation also seems possible-namely, that in order to initiate synthesis of chondroitin sulfate, cells must have already commenced hyaluronic acid synthesis. If this is so, then the concept begins to emerge of a program a t the biochemical level of specific synthesis leading t o frank chondrogenesis. I n the same way that the hematocytoblast is sensitive to BrdUrd, whereas subsequent generations of cells are not, then one sensitive event during chondrogenesis appears to be the initiation of chondroitin sulfate synthesis. Snch a conclusion does not necessarily detract from the effects of BrdUrd on cultures of mature chondrocytes. In these cells reinitiation of chondroitin sulfate synthesis may well occur during each round of DNA synthesis. It might then be argued that BrdUrd suppresses not only initiation, but also reinitiation. The prediction would then be that exposure of the terminally differentiated chondrocyte to BrdUrd would result in a cell in which the predominant glycosaminoglycan synthesized would be hyaluronic acid (see Mayne et al., 1973). The nature of the inductive interaction between spinal cord and notochord and embryonic somite cells is still largely unknown (Holtzer, 1963, 1964, 1968; Holtzer and Matheson, 1970; Mayne et al., 1973). Most investigators now agree that there is no evidence that an “inducing” molecule released by the spinal cord or notochord directly stimulates virginal somite cells t o synthesize chondroitin sulfate. On the contrary, it is clear that the induced somite or scleratome cell itself does not transform into a frank chondroblast. The capacity to differentiate into a frank condroblast is a property displayed only by the progeny of the induced scleratome cell. That the induced cell is obligated to synthesize DNA and undergo a quanta1 cell cycle if it is to yield descendents that will develop into chondroblasts is shown by experiments which block DNA synthesis. Induced scleratome cells reared in FdUrd, Ara-C, or hydroxyurea fail to develop into chondroblasts. I n these treated cultures, the postmitotic

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myogenic cells, the notochord cells, and the fibroblasts differentiate normally, but chondroblasts fail to appear. The primary action of the inducing tissues in this system, and other embryonic induction systems, may be to control mitotic activity in the responding cells. This means that the inductive event by way of a quantal cell cycle allows the next stage of the genetic program for that lineage to be expressed. I n brief, the effect of BrdUrd and inhibitors of DNA synthesis on induced somite cells is very similar to their effect on myogenic beta cells and hematocytoblasts. These subtle differences in response of presumptive myoblasts, erythroblasts, and chondroblasts, and the response of their antecedent cells in the myogenic, erythrogenic, and chondrogenic lineage should be probed further. V. Discussion We should like to discuss these experiments in terms of our general model for cell speciation based on the central roles of DNA synthesis and the cell cycle. The data for this model is admittedly meager; however, it forms the basis for our future experiments, and will, it is hoped, stimulate others to design experiments to test the theory. I n particular we should like to deal specifically with the type of mechanisms that may be mediating those events which we have categorized under “quantal” and “proliferative” mitoses. Two phases of differentiation can be recognized. The first involves the rapid commitment of cells to their respective lineages during early cleavage stages, and in this regard there is no basic difference between regulative and mosaic systems (Holtzer, 1963, 1970a). The end product of the early cleavage stages is the “definitive stem cell.” The definitive stem cell retains the capacity to yield daughter cells that are either replicate stem cells or terminally differentiated cells. The second phase of differentiation involves the biological conditions either in vivo or in vitro which manipulate these stable stem cells for growth, for morphogenesis and for maintaining populations which turn over. During early cleavage these two phases are usually separable temporally. It is likely that they will prove to be separable mechanistically as well. The fact that a particular presumptive myoblast may have the option to undergo a quanta1 cell cycle or a proliferative cell cycle, does not mean that its ancestors in the myogenic lineage had that same option. We think of the presumptive myoblast as a “time independent” cell and attribute stem cell properties to it. Its ancestors, the so-called myogenic beta cells, are “time dependent.” What distinguishes these two aspects of the differentiation problem? The differences, as we have indicated, relate to time and how cells sense time. A definitive stem cell must last the lifetime of the organism. It

252

H. HOLTZER, H. WEINTRAUB, R. MAYNE, B. MOCHAN

must therefore be a system of molecules whose stability is independent of time. Some function of decay must be balanced by some function of synthesis. Although a hematopoietic stem cell derived from the bone marrow has the potential to differentiate into a number of different types of blood cells, its potential to yield a variety of cell types is limited in comparison to its mesenchymal ancestors. These mesenchymal ancestors-and their ancestors-cease to exist after a short lifetime during cleavage. They represent what we would term the “time-dependent” phase of the differentiation problem. The myogenic beta cells or the yolk sac hematocytoblasts and their precursors, or the sclerotomal cells, fall into this category. Their presence is transient, and they function as mediators of those time-dependent processes that give rise to the different types of stable, definitive stem cells. How might time be incorporated into development? For biological systems, time has its basis in rate constants. As these constants are directly or indirectly determined by base sequences in the DNA, it is to be emphasized that timing mechanisms can be built into the genome by evolution. In addition, the local milieu of the egg, morula, or blastula cytoplasm must also contribute to the overall timing of a particular reaction (Gurdon, 1970). These reactions might include, for example, the transient decay of a protein-DNA complex or the denaturation of a specific segment of the genome, not to mention the more apparent reactions associated with degradation of protein and RNA and formation of the various cellular synthetic complexes. The most unique form of time dependence, however, is probably that offered by cell division and DNA synthesis and the kinds of ancillary events that can occur in a GI, S, or G, cytoplasm. Whereas many cellular events tend to be continuous functions, or probabilistic functions describable by a continuous probability curve, changes mediated by cell division can be discontinuous, the numbers of molecules halving with each division. Likewise, the functions associated with DNA synthesis can also be discontinuous, such as replication-dependent RNA synthesis and the ratio of DNA to DNA-binding proteins doubling some time during S. Although it is impossible a t present to isolate a particular reaction and identify its time dependence with a particular event during cell speciation, it is to be emphasized that since such events do occur in biological systems, it is reasonable to assume that the process of selection has used them also to generate the time-dependent events channeling differentiation. As the expression of a given phenotype is likely to be a function of many genes working in concert, a given phenotype might never be expressed if one of several required time-dependent events is inhibited, even transiently. The control of the synthesis of p-galactosidase in E .

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coli requires 4 or 5 gene products which are relatively specific for the proper funotioning of the lac operon (decrombrugghe et al., 1971). It is likely that the number of these “helper” genes have increased with eukaryotes. Thus the ability to translate for myosin, and the acquisition of the potential to give rise to a cell that will translate for myosin both require the functioning of different LLbatteries”of gene products (Britten and Davidson, 1969). I n the three systems described in this paper, we have cited experiments in which the appearance of luxury gene products is blocked by perturbing DNA synthesis. By interrupting the normal pattern of DNA synthesis either by BrdUrd or by inhibiting DNA synthesis, some function necessary for the emergence of terminally differentiated myogenic, erythrogenic, or chondrogenic cells was inhibited. I n our previous work with BrdUrd, we have always stressed that after 3-5 divisions the suppressed cells yield normal progeny : BrdUrd-suppressed presumptive myoblasts or BrdUrd-suppressed chondroblasts (definitive stem cells) do yield normal functioning myoblasts and chondroblasts. If, however, BrdUrd-suppressed earlier precursor cells such as myogenic beta, or primitive yolk sac hematocytoblasts or sclerotome cells do differ from the definitive stem cells with respect to “reversibility,” then a distinction will have been made between time-dependent and time-independent events. Indeed, the experiments cited here imply an association of the “time-independent” stages of differentiation with stem cells and the “time-dependent” stages with earlier precursors. The experiments with BrdUrd suggest that we may be dealing with an agent which will interfere in a unique manner with differentiation. The observation that BrdUrd did not cancel commitment of the definitive stem cells argued that BrdUrd does not block the synthesis of all luxury molecules in all cells (Holtzer and Abbott, 1968). The demonstration that BrdUrd does not block Hb-synthesis in erythroblasts, or collagen synthesis in amnion cells (Mayne et al., 19731, or antibody-like proteins in myeloma cells (Baglioni, personal communication) suggests BrdUrd does something other than simply blocking terminal expression of certain luxury molecules. More subtle explanations are required, and we now suggest that BrdUrd is able to inhibit the initiation of new synthetic activities in a lineage. How it does so remains unclear. If it is presumed that BrdUrd acts as a consequence of its incorporation into DNA-and all current evidence supports this view-then during initiation of a new synthetic activity some factor must be present which can distinguish bromouracil-substitution from thymine. Concomitant with all observations with BrdUrd are changes in t,he properties of the cell surface, and it seems not unlikely that part of the paradoxical effects of BrdUrd might stem from changes in the cell surface brought about by the bromouracil-DNA.

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Last, it will be interesting to learn more about the properties of the early precursor cells that are suppressed by BrdUrd and if, indeed, they do represent a stage of differentiation during which time-dependent events irreversibly dictate the playing out of the developmental program. Most current models of differentiation assume that repressor or derepressor sites on the chromosomes are available throughout the mitotic cycle. The experiments reviewed in this report stress that this is probably not the case. Rather it is proposed that the transmission of an ongoing synthetic program to daughter cells, associated with proliferative cell cycles, or the reprogramming associated with quanta1 cell cycles, may be coupled to specific phases of the mitotic cycle and to specific cell cycles. REFERENCES Abbott, J., and Holtzer, H. (1966). Amer. Zool. 6, 548. Abbott, J., and Holtzer, H. (1968). Proc. N a t . Acad. Sci. U.S. 59, 1144. Abbott, J., Mayne, R., and Holtzer, H. (1972). Develop. Biol. 28, 430. Allen, E., and Pepe, F. (1966). Amer. J . Anat. 116, 115. Awry, G., Chow, M., and Holtzer, H. (1955). J . E x p . Zool. 132, 109. Baglioni, R. Personal communication. Biehl, J., and Holtzer, H. Unpublished data. Bischoff, R., (1970). In “Regeneration of Striated Muscle” ( A . Maure, S. Shafiq, and A. Milhorat, eds.), p. 218. Excerpta Med. Found., Amsterdam. Bischoff, R. (1971). Exp. Cell Res. 66, 224. Bischoff, R., and Holtzer, H. (1968). J . Cell Biol. 36, 111. Bischoff, R., and Holtzer, H. (1969). J . Cell Biol. 41, 188. Bischoff, R., and Holtzer, H. (1970). J . Cell Biol. 44, 134. Britten, R., and Davidson, E. (1969). Science 165, 349. Campbell, G., Weintraub, H., and Holtzer, H. (1971). J . Cell B i d . 50, 669. Chacko, S., Holtzer, S., and Holtzer, H. (1969a). Biochem. Biophys. Res. Commzin. 34, 183. Chacko, S., Abbott, J., Holtzer, S.,and Holtzer, H. (196913). J . E x p . M e d . 130, 417.

Cohen, A., and Berill, N. J. (1936). J . Morphol60, 243. Coleman, A., Coleman, J., Kankel, D., and Werner, I. (1970). E z p . Cell. Res. 59, 319.

Coleman, J., and Coleman. A. (1968). J . Comp. Physiol. 72, 19. Conrad, G. W., (1970). Develop. Biol. 21, 611. Coon, H. G. (1966). Proc. Nut. Acad. Sci. U.S. 55, 66. decrombrugghe, B., Chen, F., Gottesman, M., Pastan, I., Varmus, H. E., Emmes, M., and Perlman, R. L. (1971). Nature (London) 230, 37. Dienstman, S., and Holtzer, H. Unpublished data. Dingle, A,, and Fulton, C. (1966). J . Cell Biol. 31, 43. Dorfman, A., and Ho, P-L. (1970). Proc. Nut. Acad. Sci. U S . 66, 495. Ellison, M. L., and Lash, J. (1971). Develop. Biol.

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Holtzer, H.,Sanger, J.,and Ishikawa, H. (1972). Cold Spring Harbor Symp. Q t i n n t . B i d . Holtzer, H., Weintraub, H., and Biehl, J. (1973). In. “FEBS Symposium on Cell Differentiation’’ (A. Monroy and R. Tsonev, eds.). Academic Press, New York. Ishikawa, H., Bischoff, R., and Holtzer, H. (1968). J . Cell Biol. 38, 538. Ishikawa, H., Bischoff, R., and Holtzer, H. (1969). J . Cell Biol. 43, 312. Konijn, T., nan de Meene, T., Bonner, J., and Barkly, D. (1970). Proc. Nut. Acad. Sci. U S . 58, 1152. Kvist, T. N., and Finnegan. C. V. (1970). f. Exp. 2001. 175, 241. Lasher, R. (1971). In “Developmental Aspects of the Cell Cycle” (I. L. Cameron, G. M. Padilla, A. M. Zimmerman, cds.), p. 223. Academic Press, New York. Levi-Montalcini, R. (1963). In “The Nature of Biological Diversity” (J. Allen. ed.), p. 238. McGraw-Hill. New York. Lillie, F. (1902). Arch. Milirosk. Anal. E,1/tc~ic~ln,tgsmech. 14, 477. Mayne, R., Sanger, J. W,, and Holtzer, H. (1971). Develop. Biol. 25, 547. Mayne, R., Abbott. J., and Holtzer. H. (1972). Exp. Cell Res. (in press). Mayne, R., Schiltz, J., and Holtzer, H. (1973). In “Biology of the Fibroblast” (J. Pikknrainen, ed.). Academic Press. Kew York. Miller, E. J., and Matukas, V. J . (1969). Proc. N n l . Acnd. Sci. (IS.64, 1264. Miura, Y., and Wilt, F. H. (1971). J. Cell B i d . 48, 523. Nameroff, M., and Holtzer, H. (1967). Dezlelop. B i d . 16, 250.

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Neelin, T. M., Callahan, P. X., Lamb, D. C., and Murray, K. (1964). Con. J. Biochem. Physiol. 42, 1743. Okazaki, K., and Holtzer, H. (1965). J . Histochem. Cytochem. 13, 726. Okazaki, K., and Holtzer, H. (1966). Proc. Nat. Acad. Sci. U S . 56, 1484. Prasad, K. (1971). Nature New Biol. 228, 997. Pryzbalski, R., and Blumberg, J. (1966). Lab. Invest. 15,863. Pujara, C., and Whitmore, H. (1970). Cell Tissue Kinel. 3, 99. Reynolds, L., and Ingram, V. (1971). J. Cell Biol. 51, 433. Rudnick, D. (1948). Ann. N . Y. Acad. Sci. 49, 761. Sanger, J., and Holtzer, H. (1972). Proc. Nal. Acad. Sci. 69, 253. Sanger, J., Holtzer, S., and Holtzer, H. (1971). Nature, New Biol. 4, 121. Sarkar, P., and Moscona, A. (1971). Proc. N a t . Acnd. Sci. 68, 2308. Schneiderman, H., and Bryant, P. J. (1971). Nature (London) 234, 187. Schubert, M., and Hammerman, D. (1968). “A Primer on Connective Tissue Chemistry.” Lee & Febiger, Philadelphia, Pennsylvania. Schubert, D., and Jacob, F. (1970). Proc. N u t . Acad. Sci. U.S. 67, 247. Schulte-Holthausen, H., Chacko, S., Davidson, E. A., and Holtzer, H. (1969). Proc. N a t . Acad. Sci. U.S. 63, 864. Seegmiller, R., Fraser, F. C., and Sheldon, H. (1971). J . CeEl Biol. 48, 580. Sreter, F., Gergely, H., Holtzer, S., and Holtzer, H. (1972). J. Cell Biol. (in press). Stockdale, F., and Holtzer, H. (1961). E z p . Cell Res. 24, 508. Stockdale, F., Okazaki, K., Nameroff, M., and Holtzer, H. (1964). Science 146, 533. Suzuki, S., Kojima, K., and Utsami, K. (1971). Biochim. Biophys. Acta 222, 240. Thorp, F. K., and Dorfman, A. (1967). Curr. Top. Develop. Biol. 2, 151-190. Tomkins, G. M., Gelehrter, T. D., Crammer, D., Martin, D., Jr., and Samuels, H. H. (1969). Science 166, 1474. Toole, B. P., and Gross, J. (1971). D e v d o p . Biol. 25, 57. Toole, B. P., and Trelstad, R. L. (1971). Develop. Biol.26, 28. Trelstad, R. L., Kang, A. H., Igarashi, S., and Gross, J. (1970). Biochemistry 9, 4993. Watterson, R. L., Fowler, I., and Fowler, B. J . (1954). Amer. J. Anat. 95, 337. Weintraub, H., and Holtzer, H. (1972). J. Mol. Biol. 66, 13. Weintraub, H., Campbell, G., and Holtzer, H. (1971). J. Cell Biol. 50, 652. Weintraub, H., Campbell, G., and Holtzer, H. (1972). J . Mol. Biol. (in press). Wenk, M. (1971). Anat. Rec. 169, 453. Wilt, F. H. (1965). J. Mol. Biol. 12, 331.

CHAPTER 7

STUDIES ON THE DEVELOPMENT OF IMMUNITY: THE RESPONSE TO SHEEP RED BLOOD CELLS Robert Auerbach DEPARTMENT O F ZOOLOGY, UNIVERSITY OF WISCONSIN, MADISON, WISCONSIN

I. Introduction., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Ontogeny of Responsiveness.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Phylogenetic Considerations, . . . . IV. Cell Interactions during the Response t A. Thymus and Bone Marrow.. . . . . . . . . . . ............. B. Macrophages and Adherent Cells.. . V. Ontogeny of Cells Responding to Sheep Red Blood Cells (SRBC) A. “T” Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. “B” and “A” C ......... VI. Immunological Tolerance to S R B C . . . . . . . . . . . . . . . . . . . . . . . . . VII. Ontogeny of Antibody Variability, . . . . . . . . . . . . . . VIII. General Considerations. . . . . . . . . . . . . . . . . . . . . . . . . References, . , , . , . , ..........

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I. Introduction Progress in immunology over the last few years has been almost overwhelming, but fortunately in the past year several major publications have admirably pulled together many aspects of immunological research. The Proceedings of the First International Congress of Immunology, held in August, 1971, have been published in a single volume, combining vast numbers of individual symposium papers with succinct summaries of 80 separate workshops (Amos, 1971 1 . As a result, an almost complete survey of the state of thinking in immunobiology is available at this time. The publication in the past year of papers presented at a week-long conference on cellular differentiation in immunity (Sterzl and Riha, 1971) and of two conferences specifically devoted to cell interactions during immunity (Makela et al., 1971; s. Cohen et al., 1971), as well as of two superbly organized and documented monographs-one on the cellular aspects of immunology in general (Nossal and Ada, 1971) and one specifically devoted t o fetal and neonatal immunology (Solomon, 1971)-permit the author the privilege of choosing for discussion and review only a small microcosm of the immunological universe without fear that the reader would be left without ready access to other studies or approaches. 257

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A number of immunological systems have been analyzed extensively, but from a developmental standpoint two of these have figured most prominently. For cellular immunity the graft-versus-host reaction has been of primary importance. Largely founded on the fundamental studies of Simonsen (1962), recent emphasis has been on the maturation of immunocompetent cells of the liver in vitro (Umiel et al., 1968; Umiel, 1971a) and in transfer systems (Umiel, 1971a,b; Tyan and Cole, 1963; Bortin and Soltzstein, 1969), on ontogeny of spleen, thymus, and bone marrow immunocompetence in vitro (Auerbach, 1966; Auerbach and Globerson, 1966) and on maturation of thymus cells both in vitro (Auerbach, 1966; Ritter, 1971) and in transfer systems (Fidler et al., 1972). Phylogenetic studies of interest have also been reported especially for chickens (Solomon, 1961; Solomon and Tucker, 1963; cf. Solomon, 1971, Chapter 10; Seto, 1967), as have studies demonstrating the requirement for cell interactions (Auerbach, 1966; Globerson and Auerbach, 1967; Cantor and Asofsky, 1970; Asofsky et al., 1971). In spite of these newer studies, however, a review of the developmental aspects of cellular immunity seems relatively less urgent, and will not be attempted in this paper. While numerous systems involving defined hapten-carrier combinations and bacterial, viral, and cellular antigens have been studied, the most significant developmental information relating to humoral immunity has come from studies carried out with heterologous erythrocytes, usually sheep red blood cells (SRBC), as antigen and with the mouse as experimental animal. The reasons for this are primarily technical: SRBC are a potent antigenic material ; the serum response can be readily measured both by hemolysis in the presence of complement and by hemagglutination; individual antihody-forming cells can be detected by a plaque-forming cell assay (Jerne and Nordin, 1963; Ingraham and Bussard, 1964) that can distinguish between 19 S and 7 S antibody-forming cells (Sterzl and Riha, 1965) ; antigens with varying degrees of crossreactivity are available for controls, in vitro methods exist for obtaining responses to SRBC both in organ cultures (Globerson and Auerbach, 1965, 1967), and the application of more sophisticated methods involving, for example, cell fractionation (Shortman et al., 1970; Gorczynski et al., 1971a; Haskill and Marbrook, 1971) or serial transfer with intervening treatment with antisera (Nossal e t al., 19711 can be readily carried out. Moreover, such classical features as sensitivity to induction of tolerance (Friedman, 19651, feedback regulation by antiserum (Rowley and Fitch, 1969), thymus dependency (Humphrey et al, 1964), and genetic variability in responsiveness (Biozzi et al., 1971 ; Click e t al., 1972) are all applicable to the SRBC system.

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Needless to say, a bias in favor of the SRBC system is admitted, since the author has worked extensively with SRBC responses. II. Ontogeny of Responsiveness

A number of studies have been carried out in the attempt to pinpoint the precise time when the young mouse is capable of responding to an injection of sheep red blood cells. While earlier experiments examined serum antibody levels, the plaque-forming cell (PFC) assay proved more sensitive and meaningful for developmental studies (cf. review in Solomon, 1971, p. 289). hlost of these studies, moreover, have concentrated on PFC development in the spleen, for it is in the spleen that the major population of antibody-forming cells can be detected. P F C in lymph nodes (Playfair, 1968; Battisto et al., 1971) and bone marrow (Saunders and Schwartzendruber, 1970) are severely limited in number. The results have been quite variable, although in general immune responsiveness to SRBC is detectable only if antigen is injected 3-4 days after birth. Playfair (1968) found that BALB/C mice could respond when immunized 3 days after birth (but cf. Alter, 1969), while C57BL mice failed to respond when immunized prior to 7 days of age. Other investigators (Hechtel et al., 1965a,b; Takeya and Nomoto, 1967; Argyris, 1968; reported widely ranging initiation times for immune reactivity against SRBC by strains SL, AKR, Ha/ICR, CBA and C,H, the onset of responsiveness never beginning before 4 days after birth. An exceptional finding was made with NZB mice (Playfair, 1968) which could respond to SRBC already 1 day after birth. Interpretation of this finding should, however, take cognizance of the fact that NZB mice are distinguished by severe manifestations of immunopathology in later life, including aberrant thymus function. While the route of injection and dose of antigen can make a difference in the number of antibody-forming cells produced, there does not appear to be a shift in the observed date of onset of responsiveness for a given strain. Even the injection of adjuvants, while enhancing the response of young mice to SRBC (Hechtel et al., 1965b), does not shift the initial day of responsiveness, Moreover, a reasonably uniform response to SRBC is achieved over a wide range of antigenic doses above the minimum needed to achieve optimal resppnse (Playfair, 1968; Dietrich, 1966). A new and highly suggestive discovery was fortuitously made by Shalaby (1972; Shalaby and Auerbach, 1972). Working with BDF, mice (C57BL/6 X DBA/2), Shalaby discovered that in young mice two injections of antigen (on day 2 and day 4) could lead to a response 5 days after the first injection of antigen (day 7) while the same total dose

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of antigen injected singly (day 2 or day 4) did not provoke a response as measured a t day 7. That the response indeed included a specific requirement for antigen on both days was shown by use of a series of non-cross-reactive (guinea pig, chicken, horse) or cross-reactive (goat) erythrocyte injections (Shalaby and Auerbach, 1972). When non-crossreacting erythrocytes were used in place of SRBC either on day 2 or day 4, no detectable response to eit,her antigen was seen on day 7. On the other hand, when combinations of goat and sheep erythrocytes were used in the injection schedule, detectable responses to both antigens resulted. I n this instance, however, the response in reciprocal experiments also reflected specificity: The PFC response was always greater to that antigen injected on day 4. This observation is readily interpreted on the basis of cell interactions in the response to SRBC (see below). In earlier studies, Playfair (1968) had suggested that prior to a typical response pattern mice could give an exceedingly weak reaction to SRBC, with no obvious peak day of response. The results of Shalaby, however, indicate that a typical response curve can be obtained with the injection protocol he developed, although there is a delay of 1-2 days in the day of peak PFC (Shalaby, 1972). While Playfair (1968) detected no background PFC in unimmunized mice, using the Jerne (Jerne and Nordin, 1963) assay system, Shalahy did observe background PFC by the use of the more sensitive liquid monolayer assay system developed by Cunningham and Szenberg (1968). It may well be that Play fair observed an increased production or release by such cells, responding to antigen without a concomitant initiation of cell division, as suggested by the work of Saunder and Swartzendruber (1970). Several efforts have been made to correlate the ontogeny of the response by in vivo and in vitro methods, Using the original organ culture methods developed by Globerson and Auerbach (1965, 1966) , Alter (1969) found that BALB/C mouse spleen could respond in vitro in a manner analogous t o in vivo experiments. She demonstrated that spleen fragments from 5-day-old animals could produce agglutinins in a small percentage of cultures, and that the incidence of positive cultures increased with explants from successively older animals, a normal level being reached only a t about 2 months of age (cf. Makinodan and Albright, 1962). Fidler et al. (1972) have used cell suspension cultures (Mishell and Dutton, 1966, 1967) to examine spleen maturation. With this technique, to date it has not been possible to demonstrate responsiveness prior to 2 weeks after birth. Since, however, organ fragments are competent a t an earlier date (Alter, 1969; cf. also Saunders and King, 1966), one must assume that less than optimal conditions exist in the suspension culture systems. An assessment of concentrations of various

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cell components (see below) might well indicate what experimentaI manipulations could be used to encourage a response in suspension culture systems with young spleen cells. 111. Phylogenetic Considerations

If one were willing to apply the adage that ontogeny repeats phylogeny to immunology, one could not help but observe that the recognition of self from not-self, of foreign from not-foreign, occurs, phylogenetically, in single-celled organisms, in prokaryotes as well as eukaryotes, as it does ontogenetically beginning with egg-sperm recognition and interaction. If only for this reason, one would need to question the concepts of adaptive immunity and of the origin of surveillance mechanisms described by Burnet (1970a,b), who argues for a gradual evolutionary rise in immunocompetence during vertebrate phylogeny. While phylogenetic studies have included a large variety of species, primarily vertebrate, most research has centered on cellular immunity, with special emphasis on transplantation, These studies will not be reviewed here, and the reader is referred to a recent symposium which includes many of the current studies in this area (Hildemann and Cooper, 1971; cf. also Amos, 1971, Workshop No. 33). There are a number of fascinating observations on response to sheep erythrocytes in invertebrates (Amos, 1971, Workshop No. 33), but the information is not yet sufficiently detailed to permit critical evaluation and clearly there is no developmental information a t all. On the other hand, virtually all vertebrates tested have shown a competence to react to heterologous erythrocytes, and considerable developmental information has become available for a large variety of species. One of the best correlative studies has been carried out in lizards by Muthukkaruppan and his students (Muthukkaruppan et al., 1970; Kanakambika, 1971; Kanakambika and Muthukkaruppan, 1972a,b,c). Examining the immunological competence in the newly hatched lizard Calotes versicotor, Kanakambika and Muthukkaruppan (1972a) found that the response of the l-day-old lizard was quite comparable to that of the adult, both in terms of serum titers and as studied by assay of PFC. It is interesting that in the lizard, even in the adult, the spleen seems to be the only major organ of antibody production (Kanakambika and Muthukkaruppan, 1972c), and this may explain the early detectable splenic response. In contrast, development of immunity in the turtle occurs only several months after hatching (Sidky and Auerbach, 1968; Borysenko, 1970). The most extensive studies on ontogeny of response to SRBC in

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amphibians have been those of DuPasquier (1965, 1970) working with Randae. In a superbly designed series of experiments, he correlated variou6 aspects of immune reactivity during development and demonstrated that tadpole stages are already capable of producing humoral immunity to SRBC. In Xenopus, Auerbach and Ruben (1970) used in witro methods to determine immunocompetence to SRBC. The earliest detectable response was obtained at time of metamorphosis. The development of responsiveness to SRBC in chickens has been studied by several investigators (Solomon, 1968; Abramoff and Brien, 1968; Moticka and Van Alten, 1971; Set0 and Henderson, 1968; cf. Solomon, 1971, for discussion of earlier work). The general pattern of maturation appears similar to that seen in the mouse, with immunocompetence rising gradually during the first week following hatching. It should be emphasized that the variety of “strains” and of assay procedures makes comparisons between studies difficult. Work of Fredericksen (1971, 197’2) using inbred chickens and a combination of in vitro and in vivo methods is in general agreement with earlier findings. Interesting, however, is the finding that a double injection schedule, similar to that employed by Shalaby (cf. above) may be equally significant for the developing posthatching chick. In vivo immunization, followed 2 days later by in vitro explantation and addition of fresh antigen, appears to induce a significant increase in the number of antibody-forming cells detected. Many studies have been carried out with species other than the mouse, but they have been too scattered to provide additional insight into maturational events associated specifically with the response to SRBC (for summary, see Solomon, 1971). It might be well, at any rate, to caution that even two closely related animals, such as mouse and rat, may differ drastically not only because of developmental differences, but because the response to SRBC is thymus dependent in the mouse, but thymus independent in the rat (Steward, 1971). According to Silverstein (cf. Silverstein and Prendergast, 1970), the rhesus monkey can produce good responses to SRBC in mid-gestation; our own in witro studies (Alter and Auerbach, 1969) confirm this observation: spleen fragments obtained from 75-day-old rhesus embryos were unable to produce agglutinins to SRBC in witro, while explants from 100-day-old embryos were fully competent. In evaluating all the experiments with SRBC one should not lose sight of the possibility that various environmental cues alter the pattern of ontogenesis, including exposure to tolerogens (see below) or to various bacterial and viral cross-reacting antigens. A germfree environment may modify immune responsiveness (Kim et al., 1967; Sterzl et al., 1971),

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although in mice effect appears to be minimal (Auerbach, 1 9 7 2 ~ )Clearly, . the hormonal environment also plays a critical role (Pierpaoli et al., 1971; Baroni et al., 1971). As a general comment it should be pointed out that many comparisons between mammalian species with respect to “early” or “late” response to SRBC have been made on the basis of some fixed point, such as “gestational age,” or “neonatal period.’’ A neonatal dog (Jacoby et al., 1969) is not biologically a t the same stage of development as a neonatal mouse; nor is mid-gestation the same in the sheep (Silverstein et al., 1966) as in the opossum (Block, 1964). A useful summary table of the development of lymphoid systems of various species has recently been made (cf. Sterzl and Riha, 1971, p. 785), which a t least suggests a more reasonable basis for making comparisons. An excellent comparison has also been recently presented‘by Solomon (1971, Chapter 13).

IV. Cell interactions during the Response to SRBC A. THYMUS AND BONEATARROW Although cell interactions had long been implicated in various aspects of the development of immunoconipetence (see, for review, Auerbach, 1967, 1971b), the finding that several cell types appeared to collaborate during a n immune response has had far-reaching developmental implications. While several in vitro systems had indicated that such cell interactions probably were needed (cf. Auerbach, 1967; Saunders and King, 1966), the first clear indication of specific cell collaboration came with the in v i m studies of Claman and co-workers (Claman et al., 1966; Claman and Chaperon, 1969), who demonstrated a synergistic effect of thymus and bone marrow cells during restoration of immune responsiveness to SRBC after lethal irradiation of mice. A clearer delineation of the roles played by thymus and marrow cells was presented by the work of Miller and his colleagues (see review by J. F. A. P . Miller et al., 1971), who, by use of complex serial transfer experiments, chromosome markers, and H-2 alloantigenic markers demonstrated convincingly that both thymus and bone marrow contributed cells that specifically recognized the SRBC antigens. The thymus-derived (“T”) cell, termed antigen-reactive in these studies was activated (“educated”) first, but the bone marrow-derived (“B”) cell, termed antigensensitive was the actual precursor of the definitive antibody-forming cell (Mitchell and Miller, 1968a). As can be seen from the exhaustive studies which followed the initial experimental observations, “T” and “B” cell interaction represents a complex and as yet not well understood series of events (Makela et

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al., 1971; S. Cohen et al., 1971; Amos, 1971, Workshop Nos. 25a and 25b). One is reminded here of the history of studies of tissue interactions of the first half of this century in which, also, a series of overtly simple observations gave rise to a body of literature too complex and too contradictory to be readily interpreted. A few general conclusions from the “T”and “B” cell interaction studies, as they apply to the SRBC system, will be restated here, but the reader should consult the abovecited compendia for details. 1. The specificity ascribed to both “T” and “B” cells by the work of Miller and his associates was initially based on the use of non-crossreactive erythrocyte antigen studies: both the thymic and bone marrow components of the system required exposure to the same antigen. However, since SRBC is a complex “antigen” there is no evidence that the “T” and “B” cell populations see the same determinants. Thus the SRBC system may not be different from the hapten-carrier systems so well analyzed by Mitchison (1971; Mitchison et al., 1971) Taylor (1969), and Rajewsky (Rajewsky and Pohlit, 1971). It may well be that “T” and “B” cell populations see carrier and hapten determinants, respectively. From an ontogenic standpoint this interpretation may be useful in evaluating studies on the rise of immunocompetence to SRBC (Shalaby and Auerbach, 1972; Chiscon and Golub, 1972; see Section 11). 2. Radiation sensitivity of both “T” and ILB”cells collaborating in the response to SRBC was originally described (R. E. Anderson et al., 1972; Claman, 1971), but has now been questioned in some instances (Katz et al., 1970; Kettman and Dutton, 1971). Since all collaboration experiments rely for demonstration on a critical ratio of “T” to “B” cells, however, demonstrated radiation sensitivity may reflect an effect on cell division leading to inadequate cell numbers, rather than simply destruction by irradiation. Moreover, radiation sensitivity of ‘IT”cells is based primarily on in vivo studies, where it is not clear whether injection or irradiated cells leads to similar localization patterns of “T” cells (cf. Yoffey and Courtice, 1970, Chapters 9 and 10; Metcalf, 1970) as seen when normal “T”cells are injected. Also, embryonic stem cells of the thymus are highly radiation resistant, in contrast to more mature thymic cells (Auerbach and Kubai, 1972), so that regeneration with time cannot be excluded. 3. Although the literature refers to “T” and “B” cells with little emphasis on the state of differentiation of these cells, it should be kept in mind that it is developmental derivatives of these cells, not the original cells, that provide information on cell collaboration. The “T” cell from the thymus, functional as .collaborator in the SRBC system, is

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derived from a minority component of thymus lymphoid cells that differs from most thymus lymphocytes in concentration and distribution of surface antigens (cf. Raff and Cantor, 1971; Schlesinger, 1970) and cortisone sensitivity (Claman et al., 1971; Segal et al., 1972). The “T” cell from the thymus may not be the same as the “T” cell from the thoracic duct (Mitchell and Miller, 1968a,b) and the splenic environment as well as antigen are essential components of “T” cell maturation or stimulation since it is only after passage through the spleen in the presence of antigen that “T” cells can function in vitro (Dutton et al., 1971; Hartmann, 1970, 1971 ; Globerson and Feldman, 1969). The developmental history of the “B” cell is yet more complex. I n the mouse the assumption that the bone marrow is in fact a bursalike organ (cf. discussion in Davies et al., 1971) is negated by the inability to demonstrate “B-T” collaboration in the chicken by the use of cells obtained from the bursa of Fabricius. The bone marrow “B” cell precursor of the SRBC system is also a minority component, its function in vitro requires prior maturation in vivo (Hartmann, 1970, 1971), and the in vivo maturation proceeds simultaneously with differentiation of erythroid and granuloid cells (Metcalf, 1970; Trentin et al., 1971; Hanna et al., 1971 ; cf. Gordon, 1970). 4. Whether “T” and “B” cells can both be rendered tolerant has been debated extensively (Playfair, 1969; Playfair and Purves, 1971 ; J. F. A. P. Miller and Mitchell, 1970; J. F. A. P. Miller et al., 1971; cf. Landy and Braun, 1969). Recent evidence from other systems suggests that both “T” and “B” cells are subject to “hot-pulse” killing by labeled antigen (J. F. A. P. Miller et al., 1971 ; Basten et al., 1971). Again, extrapolating from other systems (Weigle, 1971; Weigle et al., 1971), one suspects that tolerance in “T” cell populations may be more readily produced and be longer lasting than in ‘(B” cells. The fact that the response to SRBC can occur entirely without “T” cells under appropriate experimental conditions (see below) complicates interpretation of tolerance experiments. Tolerance to SRBC will be discussed more fully below (Section VI) . 5. Although “T” cells are normally needed for response to SRBC in the mouse, there are now many ways of bypassing the requirement for these cells, both in viva and in vitro, or for drastically altering their responsiveness. With appropriate dose of antigen (Playfair and Purves, 1971) or by “solubilization” of antigen with sonication, lysis and centrifugation (Palmer, 1972) “B” cells can be triggered to proliferation and production of PFC directly. Moreover, certain nutritional factors (e.g., lots of fetal calf serum, conditioned medium) can substitute for the in vitro requirement for “T” cells (Byrd, 1971). “T” cells can

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be stimulated by poly (A-U) (Cone and Johnson, 1972), or lipopolysaccharide (Anderson et al., 1972) to function more efficiently and low responding strains of mice similarly are converted to high responders (Braun, 1971). None of these studies negate the demonstration of specificity of “T” cells in cell collaboration studies, but rather the studies encourage the interpretation that “T” cell function may be to amplify or to change the threshold of responsiveness, rather than to be instructive. The parallel t o studies of the chemical isolation of evocators in inductive systems is obvious (cf. Ellison and Lash, 1971; A. M. Cohen and Hay, 1971 ; Auerbach, 1971b).

B. MACROPHAGES AND ADHERENT CELLS When we first tried to compare organ culture and suspension culture methods for obtaining in vitro responses to SRBC, we were struck by the observation that cell suspension cultures, in fact, remained so only for a few hours: they aggregated in the type-specific manner so well known to embryologists (Townes and Holtfreter, 1955; cf. review in Moscona, 1965). Mosier (1969; Mosier and Coppelson, 1968) had already demonstrated a need for three cell types in the in vitro immune reaction to SRBC-two nonadherent cells (presumably “B” and ‘IT” type lymphocytes) and an adherent cell or macrophage (cf. also Ford e t al., 1966). Our own observations (Auerbach, 1971b) suggested that the adherent cell requirement was in many ways a typical “mesenchyme” requirement seen in other inductive systems involving two nonmesenchyma1 epithelial tissues [e.g., lens induction (Muthukkaruppan, 1965) 1. For purposes of the present discussion, we will adopt the terminology for the adherent cell population as an “A” cell; its relation to the “M” or macrophage cell, however, will be considered briefly (cf. Nelson, 1969). Operationally, the “A” cell is obtained by plating a spleen cell suspension in a dish and after brief incubation removing the nonadherent (lymphoid and dividing) cells. Conversely, suspensions deficient in “A” cells can also be produced by passing a spleen cell suspension through glass wool, or serum-coated glass beads; the cells that are not retained tend to be nonadherent (“T” and “B”) cells. In vitro systems require critical concentrations of both “A” and non-“A” cells (Mosier and Coppelson, 1968; Virolainen et al., 1971; Cosenza and Leserman, 1972; Cosenza et al., 1971). Morphologically, the “A” cells appear as macrophages, and they are capable of both phagocytosis and antigen-binding. Most studies agree that “A” cell function in vitro is nonspecific, that “A” cells are radiation resistant, and that supernatants from “A” cells can substitute for the

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requirement for “A” cells altogether (cf. Dutton e t al., 1971; Amos, 1971, Workshop No. 481. What makes interpretation even of these points of overt agreement difficult, however, is the fact that in no instance have there been “clean” preparations of a single cell type, and the in vitro systems may well require nonspecific, nutritional factors in addition to some specific contribution. Again, one is reminded of the induction experiments in which mesenchyme can be replaced by “factors” ranging from particle fraction (Rutter et al., 1963) to collagen (Konigsberg and Hauschka, 1965), without negating the specific effect of mesenchyme as well (cf. discussion by Unsworth, 1972; Auerbach, 1972a). When one extrapolates from the results obtained with solubilized antigen (Palmer, 1972), then one sees that one function of macrophages which may be carried out by “A” cells could be to produce an antigen which is less dependent on “T” cell function. Since “T” cells are notoriously difficult to maintain in culture, such a function could be of major importance in vitro. Again, “T” cells appear to produce substances toxic to other spleen cells in vitro: the “A” cells may function in detoxification of “T”cell products. Yet other phagocytic cell functions can be readily imagined, such as removal of tolerogens (H. Anderson, 1971), destruction of inhibiting antibody (Rowley and Fitch, 1969), presentation of antigen (Mitchison, 1971), or simply provision of a substrate for attachment of nonadherent cells (Auerbach, 1971b). All these “A” cell activities, moreover, may be operative, without necessarily implying that no specific “A” cell reaction to antigen also occurs. The latter is certainly suggested by experiments with RNA extracts obtained from peritoneal macrophages (E. P. Cohen and Raska, 1968; Mosier and Cohen, 1968). A major problem faced in the study of the requirement for “A” or “M” cells is the experimental difficulty of obtaining in vivo systems deficient in these cells (Gorczynski et al., 1971a,b). To some extent irradiation will destroy a t least some of these cells, and addition of peritoneal macrophages to injected lymphoid cells appears to enhance immune recovery after lethal irradiation (Kennedy et al., 1970). The suggestion that incompetence of newborn mice to respond to SRBC is due to a deficiency in macrophages (Argyris, 1968) is of importance, but unfortunately those experiments were carried out with only partially purified peritoneal cells rather than tissue culture-passaged “A”. cells ; because of the absence of markers, the inclusion of immunocompetent lymphoid cells was possible. Experiments of Fidler et al. (1972) suggest that the “A” cell component of the in vitro culture system is already existent in spleens of newborn mice.

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V. Ontogeny of Cells Responding to Sheep Red Blood Cells A. “T” CELLS Since the newborn mouse is not yet competent to react to SRBC, but becomes competent in the next several days (cf. Section 11), one may now reexamine the ontogenic pattern of “T” cell formation from a fmctional standpoint. Experiments of Chiscon and Golub ( 1972) suggest that the newborn mouse already has “T” cell function as judged by transfer experiments into irradiated recipients, but these experiments do not exclude the possible maturation of “T”cells after transplantation. Conversely, the in vitro experiments performed in the same laboratory (Fidler et al., 1972) suggest that “T”cell function arises much later, but these experiments are subject to the technical criticism that negative experiments with cell suspension systems are difficult to interpret. The work of Shalaby (1972; Shalaby and Auerbach, 1972) suggests that possibility that “T” cell maturation occurs shortly after birth, and permits the possibility that “T” cell mobilization (cf. also Sprent et al., 1971), is responsible for the acceleration of immune response to SRBC observed in these experiments. Much progress with analysis of the “T” cell functions has been made recently through the use of specific antisera directed against thymus cell surface antigens, especially anti-TL, which is uniquely thymus specific, and anti-0 which is believed to detect thymus-derived cells in lymph node, spleen, and bone marrow as well as thymus cells themselves (see Boyse and Old, 1969; Reif and Allen, 1964; Raff, 1971). Unfortunately, although these antigenic markers are useful for selective cell destruction (see 8. Cohen e t al., 1971; Makela et al., 1971) and for identification of cell source (Owen and Raff, 1970), they tell us little about the ontogeny of immunocompetence. Both T L and 6 antigenicity is expressed in early embryonic thymus cells (Raff, 1971). Moreover, since virtually all thymus cells are &positive, identification of the 5% subpopulation of cortisone-resistant, collaboration-capable thymus cells (Claman et al., 1971) cannot be made with accuracy; and the fact that the cells which do collaborate are T L negative makes the T L marker of little use in tracing the steps leading to functional maturation. Although originally all thymic lymphocytes were believed to be of epithelial origin (Auerbach, 1961 ; cf. Auerbach, 1967), subsequent work (Moore and Owen, 1967, Owen and Ritter, 1969) left little doubt that the major source of thymic lymphoid precursors was the yolk sac. Most recently, however, new problems concerning “T”cell origins have arisen : 1. Since the functional “T”cell in the thymus is cortisone resistant

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(Claman, 1971) whereas the stem cells appear to be cortisone sensitive, we may be examining not two stages of the same cell lineage, but rather two separate stem lines. The former interpretation is favored by the observation that although the Giemsa-positive stem cell of Moore and Owen (1967) can be destroyed by cortisone in vitro, thymus lymphoid cells subsequently still form in such cultures (Sidky, 1968). 2. I n amphibian embryos, transplantation of prethymic regions into cytologically distinguishable hosts indicates self-differentiation of grafted tissue (cited in Amos, 1971, Workshop No. 9 ) . 3.’Even within the 5% subpopulation of “T” cells that is T L negative and cortisone resistant (discussed above), there are subpopulations as indicated by the finding that the cells involved in induction of a graftversus-host reaction are more sensitive to treatment with hydrocortisone than are cells collaborating in humoral immunity; the latter may even be enhanced in some instances (Segal et al., 1972; J. J. Cohen et al., 1970). 4. The assumption that @-positive cells must be thymus derived, no matter whether they are found, ultimately, in the bone marrow or spleen or lymphatic circulation, is based largely on circumstantial and circuitous reasoning: Why, for example, do %ude” mice, thymusless, have some &positive cells (Raff, 1971), and why can thymosin, an extract of the thymus, increase the number of 6 positive cells (Bach et al., 1971)? 5 . Antigenic modulation has been clearly demonstrated for the TL antigen (Old et al., 1968) and modulation is suggested for other thymus antigens as well as judged by the effect of thymosin on the numbers of &positive cells (Amos, 1971, Workshop No. 55; Bach and Dardenne, 1972a,b). B. “B”

AND

“A” CELLS

While all transfer experiments suggest that the original stem cell of the antibody-synthesizing systems is a yolk sac cell, and that such stem cells can be found variously in the embryonic liver and then the bone marrow, little is known about the ontogenic pattern of individual, antigen-sensitive “B” cells. When embryonic liver cells are injected into lethally irradiated animals where they subsequently function in response to SRBC (Tyan and Herzenberg, 1968; Tyan et al., 1969; Chiscon and Golub, 1972), there has not been any indication as to the time of actual maturation. No experiments have yet been carried out to examine antigen-binding, tolerization or hot-pulse elimination of embryonic yolk sac or liver cells, so that the ontogeny of antigen sensitivity is simply not known. I n contrast to the thymic antigens, the only useful “B” cell antigens are the immunoglobulins themselves (Takahashi et al., 1971)

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and these arise in detectable amounts only shortly after birth. The coincidence between the timing of surface immunoglobulin appearance both in the bursa of Fabricius and the liver with the first detectable responsiveness to antigen should not be overlooked. It is the more significant since neither in the bursa nor in the liver has antibody been detected (Warner et al., 1969; Kincade and Cooper, 1971). No meaningful studies have been reported on the ontogeny of the “A” cells involved in the response to SRBC. This is readily explained by the fact that the “A” cell appears to have so many functions (cf. Section IV,B) that the results of any experiment become difficult to interpret. Fidler et al. (1972) demonstrated indeed that “young” adherent cells could participate with adult nonadherent cells in a cell suspension culture system, but the source of the “young” cells was 6-day-old spleen known by other tests already to be immunocompetent. The phagocytic activity of embryonic cells is well known, but the role of phagocytic cells in the SRBC system reniains open to speculation. A major problem in interpretation of studies with embryonic adherent cells is that embryonic cells or extracts increase immune reactivity following irradiation (cf. Taliaferro et al., 1964), enhance the immune response in vitro (Globerson and Auerbach, 1966), and increase the number of P F C in thoracic duct cell restituted irradiated animals (Auerbach, 1 9 7 2 ~ )More. over, virtually all embryonic cells are adherent cells, making distinctions on the basis of adhesion to glass meaningless. VI. Immunological Tolerance to SRBC

Tolerance to SRBC can be induced both in neonatal (Friedman, 1965) and adult (Friedman, 1969) animals by a prolonged injection schedule with massive doses of sheep red blood cells. More generally used has been the induction of tolerance by the combination treatment with antigen and cytotoxic agents such as cyclophosphamide (e.g. Frish and Davies, 1966; Aisenberg, 1967; Dietrich and Dukor, 1967; Playfair, 1969; Schwartz, 1965; Landy and Braun, 1969). Recently, H. Anderson (1971 ; H. Anderson et al., 1972) has demonstrated that the membrane-free hemolysate of sheep red blood cells is nonantigenic to mice, as measured by the induction of PFC, and, on the other hand, renders them partially tolerant to a subsequent injection of intact SRBC (15% of normal response). By this method, tolerance to SRBC can apparently be achieved in a manner analogous to the tolerance induced by solubilized protein antigens (see Weigle et al., 1971, for review), Partially similar results were obtained by Fetherstonhaugh (1970) with butanol-extracted membranes; in these

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studies however, single injections of the material was clearly immunogenic, tolerance only being achieved with massive multiple injections, Immunogenicity was also observed with other extraction procedures (Palmer, 1972; Waterstone, 1970). I n contrast to the drug-dependent tolerance induction systems which cause both specific cell death and general lymphopenia (Aisenberg, 1967 ; Marbrook and Baguley, 1971; Winkelstein et al., 1971) induction of tolerance with solubilized antigen appears to have no obvious effect on lymphoid cells and may well represent a more biologically acceptable model for tolerance induction during development. Transfer experiments utilizing cells from animals made tolerant to SRBC by this new method have not yet been carried out; judging from the successful dissection of the effects of “B” and “T” cells after tolerization by solubilized protein antigens (Weigle et al., 1971), such experiments should provide valuable information on the mechanism of induction of tolerance to SRBC. A major question in tolerance studies has been to determine whether there is a “tolerant cell,” or whether tolerance is simply the elimination of cells competent to react to a given antigen. When cyclophosphamideinduced tolerant spleens are placed in culture, they fail to recover responsiveness to SRBC (H. Anderson et al., 1972), and thymus or marrow cells from tolerant animals are incompetent to participate collaboratively in the response to this antigen (Playfair and Purves, 1971; J. F. A. P. MilIer and Mitchell, 1970; cf. Landy and Braun, 1969). When spleens from animals tolerized by sheep red blood cell hemolysate are placed in culture, however, they can recover to a considerable extent their responsiveness to SRBC (H. Anderson et al., 1972). That this is not simply due to proliferation of unaffected immunocompetent cells in a partially tolerant spleen was shown by the use of drug-induced tolerization following hemolysate-induced tolerization: even after elimination with cyclophosphamide of all cells still immunoresponsive after prior hemolysate treatment, spleen explants were capable of partial recovery in vitro. Since all three cell types involved in the SRBC response must presumably be active in vitro, it seems clear that tolerant cells do exist. Viewed in developmental context, it seems reasonable to suggest that during the early maturational events leading to immunocompetence, antigens may be processed nonimmunologically, the partially solubilized or digested antigens acting as tolerogens. Such induced tolerance would act as a block to phenotypic expression rather than lead to destruction of the affected cell. Tolerance would be maintained indefinitely in the presence of antigen since because of the block, tolerogenic products would continue to be produced by alternatives to immunological elimination. It would be tempting to speculate more broadly concerning the mecha-

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nism by which tolerance is established during development (cf. Landy and Braun, 1969). Especially intriguing is the finding that blocking agents (antibodies?) can be demonstrated in chimeric (allophenic) mice (Phillips et nl., 1971). On the other hand, to what extent this type of serum blocking material is involved in tolerance to SRBC has not yet been determined. VII. Ontogeny of Antibody Variability Of all the problems faced by the developmental biologist interested in immunological systems, the questions concerning the origin of antibody diversity seem the most fascinating. Clearly, a discussion of all the theoretical considerations is beyond the scope of this review (cf. Bretscher and Cohn, 1968; Edelman and Gally, 1969; Pink et al., 1971; Hood, 1971; Jerne, 1971; Smithies, 1970; cf. Amos, 1971). Whether antibody variability comes through selective activation of genes, by mutational or recombinational events after fertilization, by some other epigenetic mechanism, or by a combination of these is unknown. With the increasing acceptability of the notion that v genes and c genes can collaborate in the synthesis of a single polypeptide chain, moreover, most of the previous constraints on the genesis of variability have been removed. Whatever is the basic mechanism of the generation of diversity, two observations made in the SRBC system need explanation: 1. The timing of responsiveness to SRBC is rigidly predictable for a given strain of mouse under controlled experimental conditions (Section 11). This implies that either the origin of antibody variability is not random, or-and this would seem more likely-that events subsequent to the generation of diversity trigger replication, maturation, and synthesis of specific antigen-sensitive cells. 2. The number of cells that can respond to SRBC is exceedingly high if one accepts the view, generally held, that individual cells synthesize only antibody to a single antigenic determinant. Using in witro systems, the minimum number of cells needed to produce a ready response to SRBC is now fewer than 1W spleen cells in the mouse (Auerbach, 1971b; see also Haskill and Marbrook, 1971). Of this number, moreover, only a fraction are “B” cells, and the effieiency of the culture and detection systems is certainly not absolute. The frequency of antigen-specific rosette-forming cells in the mouse is approximately 1/1 o o o (Biozzi et al., 1971). I n Xenopus as few as 104 cells give a good in vitro response to SRBC (Auerbach and Ruben, 1970), and in R a m embryonic spleens of as few as 1000 cells give measurable responses (DuPasquier, 1970). One could argue that the response to heterologous

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erythrocytes is a form of “cellular immunity”; this might serve to explain the high incidence of rosette-forming cells, especially if these are, in part, “T”-cell rosettes (Bach and Dardenne, 1972a,b), the PFC test certainly measures humoral antibody, and antigenic and allotypic markers point to a “B” cell origin of the antibody-producing cells (Section I V ) .

VIII. General Considerations At present, more than a t any time in the past, the problems of the immunologist and the developmental biologist are fundamentally the same (cf. Viza and Harris, 1972). Each of these workers is attempting to determine the series of genetic controls and molecular events that permit phenotypic expression ; each is studying the triggers that encourage clonal proliferation of appropriate cells ; and each is attempting to unravel the problems of cell interactions, affinities, and migrations. Not long ago, it seemed almost ludicrous that immunologists were publishing literally hundreds of papers on cell interactions in immunity, without having even peripheral knowledge of the vast literature of cell interactions in other developmental systems. Moreover, the cellular events during the development of immunity, as well as the series of differentiative steps occurring during response to antigen, seemed so like other embryonic processes that analogies could readily and profitably be drawn (Auerbach, 1962, 1971a). But it is equally apparent that the progress in immunology of the past few years has been phenomenal, and that findings in immunology should have critical impact on investigations in other developmental systems. The availability of chemical identification of the significant molecules, of monoclonal tumor lines, of allotypic and idiotypic markers, of monospecific antisera, of chemically pure haptens, of quantifiable in vitro assays, of genetic variants-virtually all the tools that the developmental biologist always pleads for are available. Moreover, the response to antigen includes gene activation, proliferation, cell differentiation, cell interactions, cell migrations, feedback niechanisms-and the impact of immunological research on our understanding of these processes is already profound. If each clone of cells responding to antigen is unique (Askonas et al., 1970), are we not obliged to ask whether similar uniqueness holds for clones of pigment cells, nerve cells, or cartilage cells as well? Will we find that the complexities of the immune system apply equally well to the development of patterns as seen, for example, within the epidermis (Bernfield and Wessells, 1970) ? Do the cell surface interactions between immunoglobulin and antigen (Taylor et al., 1971) provide the necessary

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model for explanation of the surface events associated with fertilization and inductive interaction? ACKNOWLEDGMENT

I would like to emphasize that various students in my laboratory have played a significant part in helping me to develop the ideas presented in this paper. These include Heidi Anderson, Ashim Chakravarty, Tom Fredericksen, Carol Landahl, Refaat Shalaby, and Tehila Umiel. I n addition, I wish to thank Louis Kubai and Joan Roethle for their excellent technical assistance as well as their intellectual participation in the original work included in this report. Research work reported in this paper was supported in part by research grants GB 6637X from the National Science Foundation and C 5281 from the National Cancer Institute. REFERENCES Abramoff, P., and Brien, N. B. (1968). J . Immunol. 100, 1210. Aisenberg, A. C. (1967). J. E z p . Med. 125, 833. Alter, B. (1969). M. S. Thesis, University of Wisconsin, Madison. Alter, B., and Auerbach, R. (1969). Unpublished studies. Amos, B., ed. (1971). “Progress in Immunology.” Academic Press, New York. Anderson, H. (1971). M. S. Dissertation, University of Wisconsin, Madison. Anderson, H., Roethle, J., and Auerbach, R. (1972). Unpublished data. Anderson, H., Roethle, J., and Auerbach, R. (1972). I n preparation. Anderson, R. E., Sprent, J., and Miller, J. F. A. P. (1972). J. Ezp. Med. 135, 711. Andersson, J., Sjoberg, O., and Moller, G. (1972). Eur. J. Immunol. (in press). Argyris, B. F. (1968). J. Exp. M e d . 128, 459. Askonas, B. A., Williamson, A. R., and Wright, B. E. G. (1970). Proc. N a t . Acad. Sci. U.S. 67, 1398. Asofsky, R., Cantor, H., and Tigelaar, R. E. (1971). In “Progress in Immunology” (B. Amos, ed.), p. 369. Academic Press, New York. Auerbach, R. (1961). Develop. Biol. 3, 336. Auerbach, R. (1962). J. Cell. Comp. Phys. 60, Suppl. 1, 159. and Clinical Studies” Auerbach, R. (1966). In “The Thymus-Experimental (G. E. W. Wolstenholme and R. R. Porter, eds.), p. 39. Churchill, London. Auerbach, R. (1967). Develop. Biol., Suppl. 1, 254. Auerbach, R. (1971a). In “Developmental Aspects of Antibody Formation and Structure” (J. Steral and I. Riha, eds.), 2nd ed., Vol. 1, p. 23. Academic Press, New York. Auerbach, R. (1971b). In “Cell Interactions and Receptor Antibodies in Immune Responses” (0. Makela, A. Cross, and T. U. Kosunen, eds.), p. 393. Academic Press, New York. Auerbach, R. (1972a). Develop. Biol. (in press). Auerbach, R. (1972b). In “The Dynamic Structure of Cell Membranes” (H. Fischer and D. Hoelz-Wallach, eds.), p. 37. Springer-Verlag, Berlin and New York. Auerbach, R. (1972~).Unpublished experiments. Auerbach, R., and Globerson, A. (1966). Exp. Cell Res. 42, 31. Auerbach, R., and Kubai, L. (1972). Submitted for publication. Auerbach, R., and Ruben, L. N. (1970). J. Immunol. 104, 1242. Bach, J.-F., and Dardenne, M. (1972a). Cell. Immunol. 3, 1.

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AUTHOR INDEX Numbers in italics refer to t,he pages on which the complete references are listed. Ashman, R. F., 205, 225 Ashworth, J. M., 161, 190 Askonas, B. A., 10, 60, 273, 274 Asofsky, R., 258, 274, 275 Asriyan, I. S., 18, 58 Attnrdi, B., 4, 5 , 53, 174, 188 Attnrdi, G., 4, 5, 8, 53, 65, 174, 188 Aubcrt, J.-P., 92, 96, 97, 98, 102, 106, 114, 115, 120, 123 Auerbach, R., 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 270, 271, 272, 273, 274, 276, 279 Autissier, F., 103, 120 Avakyan, E. R., 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20,33, 36,55,67, 68 Avery, G., 247, 254 ATila, J., 93, 180 Axelrod, A. E., 171, 191 Azarnia, R., 210, 225

Abbott, J., 128, 188, 235, 245, 247, 248, 249,250, 253,254, 255 Abramoff, P., 262, 274 Abramova, N. B., 9, 57 Abrosimova-Omeljanchic, N. M., 32, 59 Ada, G. L., 257, 273 Adams, W., 34, 54 Adesnik, M., 174, 187 Adler, H. I., 101, 120 Afanasieva, T. P., 2, 56 Aidley, D. J., 215, 225 Aisenberg, A. C., 270, 271, 214 Aitkhozhin, M. A,, 9, 10, 18, 57, 59 Aitkhozhina, N. A., 18, 20, 56, 58 Akaan-Penttila, E., 266, 679 Albright, J., 260, 277 Allen, E., 237, 264 Allen, Joan M. V., 268, 278 Allfrey, V. G., 4, 47, 63, 56, 58, 69 B Alter, B., 258, 259, 260, 262, 274 Rach, J.-F., 269, 273, 274, 876 Amaldi, F., 8, 65 Bacli, M. L., 91, 96, 120 Ambrose, E. J., 255 Amos, B., 257, 261, 264, 267, 269, 272, Badenhausen, S., 212, 227 Baev, A. A,, 32, 69 274 Ananieva, L. N., 5, 9, 16, 21, 23, 24, 53, Baglioni, C., 36, 53, 185, 188, 253, 254 Bagroba, A. M., 9, 57 55, 56, 68 Baguley, B. C.. 271, 277 Anderson, H , 267, 270, 271, 274 Baker, R F., 3, 57 Anderson, R. E., 264, 274 Balassn, G., 90, 91, 93, 96, 97, 98, 101, Andersson, E., 195, 228 107, 108, 120, 123, 124 Andersson, J., 266, 274, 277 Raldwin, K. M., 198, 204, 225 Apirion, D., 3, 56 I3alsaino, J., 185, 139 Arggris, B. F., 259, 274 Arion, V. Ya., 4, 6, 11, 12, 13, 14, 30, Baltimore. D., 52, 53, 188 Barkly, D., 256 31, 39, 53, 57 Barnett, H. L., 87, 122 Armelin, H. A,, 185, IS9 Baroni, C. P., 263, 275 Armentrout, S. A., 18, 59 Barr, H., 35,36,53 Armstrong, W. D., 258, 279 Barr, L., 196, 201. 225 Arnaud, M.. 63, 70, 71, 72, 82 Bnsten, A,, 263, 265, 275, 277 Aronson, A. I., 96, 117, 118,120 Rattisto. J. R., 259, 275 Arrighi, F. E., 21, 53 Bautz, E. K. F., 2, 54 Asada, Y., 203, 205. 225, 2 3 Beaman Cabrera, T., 117, 126 Ashburner, M., 130, 188 28 1

282

AUTHOR INDEX

Beatty, B. R., 40, 67, 182, 189 Beaufils, A.-M., 104, 121 Becker, I., 8, 67 Becker, M., 10, 69 Becker, Y . , 171, 190 Beckwith, G., 1, 2, 66 Beermann, W., 41, 43,63 Begg, K. J., 102, 104, 105,121 Behforouz, N. C., 94, 121 Bekhor, J., 23, 63 Belitsina, N. V., 10, 18, 69 Bell, E., 171, 190 Benacerraf, B., 264, 277 Benck, L., 258, 276 Benjamin, T. L., 49, 63 Bennet, G., 140, 188 Bennett, M. V. L., 203, 205, 206, 211, 226, 227 Berg, W. E., 150, 188 Bergendahl, J., 21, 63 Berger, E., 136, 142, 144, 145, 146, 147, 148, 151, 153, 184, 188 Berger, W., 196, 226 Berill, N. J., 248, 264 Bernardi, A., 32, 63 Bernardi, G., 32, 63 Bernardini, A., 52, 69 Berne, R. M., 195, 228 Bernfield, M. R., 273, 276 Bernhard, W., 20, 67 Bernlohr, R. W., 94, 115, 120,124 Bernstein, P., 69, 82 Bertoli, G., 263, 276 Besson, J., 38, 63 Biehl, J., 231, 233,235, 238, 241,264, 266 Biezunski, N., 9, ti4 Billingham, R. E., 276 Biozzi, G., 258, 272, 276 Birnboim, C., 4, 5, 69 Birnstiel, M. L., 16, 17, 40, 41, 44, 63, 66 Bischoff, R., 233, 234, 235, 237, 245, 264, 266

Bishop, J. O., 21, 23, 36, 63, 67, 185, 188 Bistrova, T. F., 9, 67 Bjoraker, B., 34, 66 Block, M., 263, 276 Blondel, B., 220, 226 Blumberg, J., 237,266 Bock, R. M., 34, 66 Bogdanova, S. L., 2, 66

Bonner, J., 23, 47, 63, 66, 266 Borek, C., 210, 226 Borek, F., 259, 876 Bortin, M. M., 258, 276 Borun, T.W., 10, 64 Borysenko, M., 261, 276 Bossert, W. H., 127, 189 Bouthillier, Y., 258, 272, 276 Boyse, E. A., 268, 269, 276, 278, 279 Bradley, S. G., 262, 277 Bradshaw, W. S., 153, 154, 155, 156, 157, 158, 186, 190

Brandhorst, B. P., 171, 188 Braun, W., 259, 265, 266, 270, 271, 272, 276, 276, 277 Brawerman, G., 4, 9, 37, 64, 66, 67 Brearley, I., 70, 83 Bremer, H., 32, 67 Brenner, S., 104, 122 Bresch, C., 70, 82 Bretscher, P. A., 272, 276 Brien, N. B., 262, 274 Brightman, M. W., 201, 202, 226 Britten, R. J., 21, 22, 26, 27, 64, 253, 264 Brown, D. D., 9, 39, 64, 68, 175, 180, 181, 184, 188, 190

Brown, W. C., 116, 120 Bruskov, V. I., 19, 68 Bryant, P. J., 232, 266 Burden, L., 113, 125 Burgess, R. R., 2, 64 Burka, E. R., 171,189 Burnet, F. M., 261, 276 Burny, A., 10, 52, 64, 66,69 Burrows, M. T., 212, 226 Busch, H., 39, 64 Busci, R. A,, 259, 876 Bussard, A. E., 258, 277 Byrd, W., 265, 276 Byrt, P., 276

C Callahan, P. X., 240, 266 Callan, H. G., 39, 64 Calvin, J., 63, 73, 83 Cami, B., 91, I22 Campbell, G. LeM., 158, 159, 188, 191, 239, 240, 241, 264. 266 Campbell, P., 265, 267, 276 Canellakis, E. S., 37, 66

283

AUTHOR INDEX

Cantor, H., 258, 265, 274, 276, 278 Caramela, M. G., 37, 64 Carroll, A,, 47, 66 Carter, B. L. A., 67, 78, 83 Carter, R. L., 265, 276 Cashel, M., 91, 93, 94, 95, 120, 121, 122 Cavanaugh, M. W., 213, 226 Celikkol, E., 94, 123 Chacko, S., 235, 248, 264, 266 Chambon, P., 38, 64, 94, 120 Chan, E., 265, 267, 276 Chan, L., 104, 123 Chantrenne, H., 10, 64 Chaperon, E. A , , 263, 276 Chapman, G. B., 108, 123 Chappelle, E. W., 95, 110, 111, 112, 121, 122

Chasin, L. A., 91, 96, 118, 120 Cheers, C., 263, 265, 277 Chen, A. W.-C., 73, 82 Chen, F., 253, 264 Cheneval, J. P., 220, 226 Cheng, T-Y., 17, 44, 67 Chezzi, C., 10, 11, 13, 16, 68 Chiller, J. M., 265, 270, 271, 280 Chipchase, M., 16,63 Chiscon, M. O., 258, 260, 264, 267, 268, 270, 276, 276 Choi, Y. C., 39, 64 Chow, M., 247, 264 Church, R. B., 21, 25, 64 Claman, K. N., 263, 264, 265, 268, 269, 276

Clark, D. J., 102, 120 Clark, W. R., 153, 154, 155, 156, 157, 158, 186, 190 Clayton, R. M., 159, 188 Click, R. E., 258, 276 Cochrane, V. W., 87, 120 Cohen, A., 101, 120, 248, 264 Cohen, A. M., 266, 276 Cohen, E. P., 267, 276, 277 Cohen, I. R., 265, 269, 278 Cohen, J. H., 159, 191 Cohen, J. J., 265, 268, 269, 276 Cohen, S., 257, 264, 268, 276 Cohn, M., 272, 276 Cole, L. J., 258, 279 Cole, R. M., 91, 94, 99, 117; 121 Coleman, A., 234, 235, 264

Coleman, J., 234, 235, 264 Comings, D. E., 46, 64 Cone, R. E., 266, 276 Conrad, G. W., 246, 264 Conti, S. F., 64, 65, 66, 67, 68, 69, 82, 83 Coon, H. G., 264 Cooper, E. L., 261, 276 Cooper, M. D., 270, 277 Cooper, S., 102, 104, 120, 121 Copeland, J. C., 97, 109, 120 Coppleson, L. W., 266, 27b Cosenza, H., 266, 276 Costerton, J. W. F., 222, 228 Courtice, F. C., 264, 280 Coutelle, C., 5, 6, 7, 8, 11, 12, 13, 33, 34, 35, 36, 64, 66 Cozzone, A,, 156, 159, 190 Craddock, C. G., 271, 280 Craig, E., 174, 190 Crick, F., 42,48, 49,224,226 Crill, W. E., 196, 198, 203, 204, 220,228 Crippa, M., 24, 47, 64, 172, 188 Croes, A. F., 63, 65, 73, 78, 82 Cross, A,, 257, 263, 268, 277 Cudkowicz, G., 257, 264, 268, 276 Culotti, J., 78, 89 Cummins, J. E., 149, 189 Cunningham, J. A., 260, 276 Cuzin, F., 104, 122

D Dalen, H., 211, 227 Dane, B , 203, 228 Daneholt, B., 16, 41, 43, 44, 64 Dardenne, M., 269, 273, 274, 276 Darland, G. K., 73, 82 Darnell, J. E., 4, 5, 8, 11, 12, 32, 37, 52, 64, 66, 67, 68, 69, 171, 175, 183, 188, 190

Das, M. R., 52, 69 Davidson, E. A., 248, 266 Davidson, E. H., 21, 26, 27, 64, 172, 188, 253, 264 Davies, A. J. S., 265, 276 Davies, G. H., 270, 276 Dawes, I. W., 82, 97, 107, 121, 124 Dawid, I. B., 184, 188 Decreusefond, C., 258, 272, 276 de Crombrugghe, B., 253, 264

284

AUTHOR INDEX

De Haan, R. L., 207, 208, 209, 213, 214. 215, 216, 217, 220, 221, 226, 228 Dehm, P., 140, 188 DeKloet, S. R., 4, 68 De la Chapelle, A,, 158, 172, 181, 188 De Lange, R. L., 142, 190 Deleze, J., 203, 226 del Valle, M. R., 118, 120 Dennis, R. A,, 263, 277 de Petris, S., 273, 279 Desneulle, P., 156, 159,190 Detwiler, S. R., 246, 266 Deutscher, M. P., 88, 94, 117, 120, 126 Dewey, M. M., 201, 226 Diener, E., 258, 279 Dienstman, S., 233, 264 Diesterhaft, M. D., 92, 94, 95, 114, 115, 121, 123 Diet.rich, F. M., 259, 270, 276 Di Girolamo, A., 64 Dingle, A., 264 Dishon, T., 259, 276 Dixon, G. H., 10, 66 Dixon, J. S.,210, 226 Doi, R. H., 93, 96, 98, 121, 122, 123, 124 Donachie, W. D., 102, 104, 105, 121 Dorfrnan, A,, 246, 247, 264, 256 Dori, R. H., 61, 83 Dowben, R. M., 10, 66 Draper, M. H., 203, 226 Dreifuss, J. J., 204, 226 Dring, G. J., 90, 121 Dubnoff, J., 7, 67 Duffus, W. P. H., 273, 279 Dukor, P., 270, 276 Dulbecco, R., 49, 52, 64, 67, 68, 69 Dunham, P. B., 211, 226 Durn, J. J., 2, 64 DuPasquier, L., 262, 272, 276 Dutton, R. W., 260, 264, 265, 267, 276, 2YY

E Eason, R., 10, 36, 60 East, J., 258, 276 Easty, G . C., 266 Ebertfi, C. J., 194, 226 Eckhart, W., 54

Edelnian, J., 272, 176 Edmonds, M., 37, 64 Edstrorn, J. E., 16, 41, 43, 44, 54 Edwards, C., 205, 228 Egel, R., 70, 82 Egyhazy, E., 16, 41, 43, 44, 64 Eisenstadt, J., 9, 64 Ellem, K. A. O., 4, 64 Ellison, M. L., 247, 264, 256, 266, 276 Elmerich, C., 115,121 Emmes, M., 253, 264 Engels, F. M., 65, 8.2 Ericsson, J. L., 140, 168 Esposito, M. S., 63, 68, 69, 70, 71, 72, 82 Esposito, R. E., 63, 68, 69, 70, 71, 72, s2 Evans, W. H., 142, 190

F Fabris, N., 263, 278 Falzone, J. A., 4, 68 Fambrough, D., 209, 212, 226, 227 Fan, H., 16, 32, 39, 57 Fantoni, A , , 158, 172, 181, 188 Farashyan, V. R., 35,37,38,63, 68 Farley, B., 213, 226 Farquhar, M. G., 202, 226 Faulkner, R., 47, 53 Fawcett, D. W., 196, 198, 201, 226, 227 Feder, N., 130, 189, 211, 227 Feldrnan, M., 265, 269, 276, 278 Felsted, R. L., 142, 143, 144, 145, 146, 188 Fetherstonhaugh, P., 270, 276 Fidler, J. M., 258, 260, 267, 268, 270, 276 Fielding, P., 103, 124 Filloux, B., 220, 226 Finck, H., 237, 238, 266 Fink, G. R., 70, 83 Fink, K., 34, 64 Finnegan, C. V., 250, 266 Fischman, D. A., 212, 214, 226, 228, 233, 255 Fishhach, G. D., 212, 226 Fishbach, M., 269, 276 Fisher, W. D., 101, 120 Fitch, F. W., 258, 267, 278

AUTHOH INDEX

Fitz-James, P. C., 88, 92, 93, 96, 98, 99, 101, 116, 117, 119, 120, 121, 124 Flamm, W. G., 22, 54 Fleischmajer, R., 276 Florence, J., 206, 227 Flower, N. E., 204, 226 Fogel, S., 70, 83 Ford, W. L., 266, 276 Forro, J. R., 94, 124 Forssniann, W. G., 204, 225 Fortnagel, P., 95, 96, 110, 112, 121 Fortnagel, U., 112, 113, 121 Fowell, R. R., 62, 63, 73, 82 Fowler, B. J., 246, 2556 Fowler, I., 246, 256 Fox, C. F. F., 103, 124 Fraser, F . C., 248, 256 Fredericksen, T. L., 262, 276 Freed, J. J., 17, 44, 57 Freese, E., 91. 92, 94, 95, 96. 97, 99, 101, 110, 111, 112, 113, 114, 115, 117, 118, 119,120, 181, 122,125,124 Freese, E. B., 91, 94, 99, 112, 114, 115, 117, 121 Freese, P. K., 61, S3, 96, 122 Frehel, C., 99, 104, 119, 121 Frenster, J. H., 24, 46, 54 Friedman, H., 258, 270, 27G Frink, N., 69, 70, 71, 82 Frish, A. W., 270, 276 Fry, B. J., 149, 188 Fuchs, F., 2, GO Fudenberg, H. H., 272, 27s Fudjinaga, K., 49, 54 Fukada, T., 5 , GO Fukuda, A., 96, 121 Fulton, C.. 254, 255 Furmanski, P., 229, 256 Furshpan. E. J., 204, 209, 210, 211, 212: 223, 226, 227

G Gage, P., 185, 18s Gall, J. G., 22, 67 Gallagher, M. I., 265, 279 Gallant, J., 93, 122 Galliers, E., 94, 118, 119, 161 Gally, W. E., 272, 276 Gardner, R.. 94, 121

255

Gazarysn, K. G., 5, 11, 64 Gelehrter, T. D., 173, 191, 229, 266 Georgiev, G. P., 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 23, 24, 26, 27, 28, 30, 33, 34, 35, 36, 37, 38, 44, 45, 46, 51, 63, 54, 55, 5G, 67, 58 Gergely, H., 235, 256 Gerhardt, P., 64, 83, 117, 121, 122 Gerscherson, L. E., 213, 226 Gihbs, P. R., 269, 279 Gilbert, W.. 48, 66 Gilmour, R. S., 23, 24, 46, 47, 55, 67 Gilula, N. B., 204, 210, 211, 220, 226, 927 Gilvarg, C., 91, 96, 98, 99, 104, 120, 121, 1.22, 123

Girard, M.. 4, 5, 59, 188 Girardier, L., 204, 220, 225, 226 Cissinger, F., 38, 64 Giuditta, A., 9, 69 Glaser, L., 104, 122 Glaumann, H., 140, 188 Glenn, A. R., 95, 121 Glickman, G., 37, 58 Globerson, A., 258, 260, 265, 270, 274, 676, 279 Gniazdowski, M., 3&,54 Godlewski, E., 194, 226 Goidl, E. A,, 264, 277 Goldsmith, W. R., 181, 18s Goldsmith, M. R., 172, 190 Goldstein, A,, 269, 276 Golub, E. S., 258, 260, 264, 267, 268, 269, 270,275, 276 Goodenough, D. A , , 202, 222, 226 Gorczynski, R. M., 258, 267, 276 Gordon, A. M., 203, 228 Gordon, A. S., 265, 276 Gordon, R. A., 117, 123 Gorlenko. Z. M., 2, 5G Gorowski, M. A,, 46, 66 Goshima, K., 213, 220, 226 Gottesman, M., 253, 254 Could, G. W., 90, 92, 95, 121 Gowans, J. L., 266, 276 Crammer, D., 229, 25G Granboulan, N., 5, 10, 11, 13, 16, 66, 65

Granner, D., 173, 191 Gray, B. H., 94, 120

286

AUTHOR INDEX

Gray, R., 70,81 Green, E. W., 103, 121 Green, M. M., 43, 49, 64, 66 Greenberg, H., 183, 188 Greenberg, J. R., 17, 44, 67 Griesemer, R. A., 263, 277 Grobstein, C., 267, 278 Gros, D., 204, 226 Grosclaude, J., 10, 11, 13, 16, 68 Gross, J., 246, 250, 266 Gross, P. R., 149, 172, 188 Grossbach, U.,41, 66 Guha, A., 269, 276 Gurdon, J. B., 9, 10, 64, 66, 172, 188, 232, 243, 252, 266 Gustafson, T., 206, 226 Guth, E., 64, 65, 66, 67, 68, 69, 82 Gvozdev, V. A., 12, 66

H Habel, K., 50, 66 Haber, J. E., 74, 77, 78, 79, 81, 83 Habircht, G. H., 265, 270, 271, 280 Hackney, J. D., 213, 227 Hadorn, E., 86, 121 Haggmark, A., 101, 124 Hagopian, H., 239, 240, 266 Halvorson, H. O., 63, 67, 70, 71, 72, 73, 74, 77, 78, 79, 81, 82, 83, 88, 91, 94, 110, 112, 121, 122, 123 Hamkalo, B. A., 40, 67, 69, 182, 189 Hammerman, D., 247, 266 Hampton, M., 94, 121 Hanna, M. G., Jr., 265, 276 Hanson, R. S., 61, 73, 83, 88, 95, 110, 112, 191, 124 Harary, I., 213, 226 Hardigree, A. A., 101, 120 Hare, J. D., 50, 66 Harris, R., 273, 279 Harris, H., 12, 66 Harrison, J. S., 74, 83 Hartmann, K-U., 265, 976 Hartwell, L. H., 78, 83 Hashimoto, J., 64,65, 66, 67, 68,69, 82 Hashimoto, T., 64, 83 Haskill, J. S., 258, 272, 276

Hatlen, L. I., 8, 66 Hauschka, S. D., 267, 277 Hawker, L. E., 87, 121 Hay, E. D., 205, 207, 208, 226, 228, 266, 276

Hayry, P., 266, 279 Hechtel, M., 259, 276 Heidena, J., 47, 66 Heidenhain, M., 194, 226 Helmstetter, C. E., 102, 104, 120, 1.21 Hellstrom, I., 280 Hellstrom, K. E., 280 Henderson, W. G., 262, 278 Hendler, R. W., 150, 188 Hennig, I., 22, 66 Hennig, W., 22, 66 Henshaw, E. C., 18, 64, 66 Heppner, D. B., 196, 226 Hermann, H., 158, 188 Hermoso, J. M., 93, 120 Hershberg, R. A., 210, 227 Herzenberg, L. A,, 269, 279 Heywood, S. M., 10, 65, 158, 188 Hiatt, H. H., 4, 64, 66 Hierowski, M., 96, 121 Higashino, S., 210, 286 Higgins, M. L., 101, 102, 104, 106, 109, 182 Hildemann, W. H., 261, 276 Hill, F. F., 103, 123 Hill, S. E., 196, 227 HirBkow, R., 198, 202, 204, 214, 215, 220, 221, 226, 226 Hirota, Y., 102, 104, 123 Hirst, J., 265, 267, 276 Hitchins, A. D., 99, 101, 122 Hixmn, H. F., Jr., 142, 143, 188 Ho, P-L., 246, 264 Hoch, J. A., 88, 91, 109, 129 Hodgkin, A. L., 194, 226 Hoffmann, M., 265, 267, 276 Hoffmann-Ostenhof, O., 73, 83 Holme, T., 101, 124 Holt, S.C., 88, 109, 122 Holtfreter, J., 266, 279 Holtzer, H., 128, 158, 159, 186, 188, 191, 229, 230, 231, 233, 234, 235, 237, 238, 239, 240, 241, 243, 245, 246, 247, 248, a49, 250, 251, 253, 264, 265, 266 Holtzer, S., 235, 239, 250, 264, 266

287

AUTHOR INDEX

Hood, L. E., 272, 276 Hori, N., 205, 226 Horn, D., 96, 117, 120 Hoshiko, T., 195, 228 Hough, B. R., 21, 54, 172, 188 Hruska, J. F., 142, 189 Hsu, T. S., 21, 63 Huang, M. I. H., 4, 5 , 53 Huang, R. C., 23, 47, 55 Hudspeth, A. J., 203, 204, 210, 226,227 Huez, G., 10, 55 Humphrey, J . H., 258, 276 Humphreys, T., 161, 171, 172, 188,189 Hunsley, J., 172, 190 Hunt, T., 172, 181, 189 Hunter, J. R., 95, 123 Hunter, T., 172, 181, 189 Hurwitz, J., 7, 33, 67 Hussey, C., 93,122 Hwang, McI. H., 174, 188 Hyde, A., 220, 226

Idriss, J . M., 94, 122 Igarashi, S., 246. 256 Ilyin, Yu. V., 46, 56 Imamoto, F., 2, 67 Ingraham, J. S., 258, 277 Ingram, V., 239,240,265,256 Ionesco, H., 89, 90, 91, 96, 98, 107, 114, 122, 123 Ippen, K., 1, 2, 65 Irlin, I. S., 52, 55 Ishikawa, H., 233, 237, 239, 265 Ito, S.,205, 206, 226

J Jacob, F., 1, 2, 3, 55, 56, 102, 103, 104, 109, 122, 123, 229, 234, 256 Jacob, M., LO, 59 Jacobson, K. B., 4, 59 Jacoby, R. O., 263, Jacquet, M., 174, 189 Jamakosmanovic, A,, 210, 226 James, D. W., 212, 226 Jamieson, J . D., 139, 140, 189, 191 Jaroslow, B. N., 270, 279 Jayaraman, K., 97, 122

Jelinek, W., 38, 56 Jenkins, V. K., 265, 279 Jennings, R. B., 194, 228 Jerne, N. K., 258,260,272,277 Johnson, A. G., 266, 276 Johnson, E. A., 195, 198, 204,226, 228 Johnson, K. E., 206, 226 Johnson, R. G., 210, 211, 226 Jolly, M. S., 142, 143, 188 Jones, K. W., 17,22,40, 44,53,66 Jordan, H. E., 194, 226 Jordansky, A. B., 41, 56 Judd, B. H., 43, 66, 68 Juhasz, P., 23, 56

K Kadota, K., 212, 226 Kadowaki, K., 70, 74, 83 Kafatos, F. C., 127, 129, 130, 131, 139, 140, 141, 142, 143, 144, 145, 147, 148, 149, 151, 152, 153, 160, 162, 163, 164, 165, 166, 168, 171, 188, 189, 190, 191 Kambysellis, M. P., 160, 189 Kamiyama, A., 195, 226 Kanakambika, P., 261, 277, 278 Ksnaseki, T., 212, 226 Kang, A. H., 246, 256 Kankel, D., 234, 254 Kanno, Y., 205, 210, 211,225, 226, 227 Kara. J., 50, 56 Karavanov, A. A., 41, 56 Karnovsky, M. J., 201, 202, 227 Karp, D. F., 110, 117, 122 Karrer, H. E., 198, 201, 202, 226 Kasper, C. B., 142, 190 Katz, D. H., 264, 277 Katz, M., 50, 59 Katzenellenbogen, B. S., 131, 143, Kaufman, T. C . , 43, 6G, 58 Kavaler, F., 195, 286 Kawada, Y., 5 , 60 Kay, D., 82, 97, 107, 121, 124 Kayibands, B., 19, 57 Kedes, L. H., 41, 56 Kedinger, C., 38, 54 Kelley, D. E., 10, 17, 18, 44, 67, 190

Kelln, R. A., 83, 96, 122

136, 146, 161, 172,

189

171,

288

AUTHOR INDEX

Kelly, D. E., 201, 226 Kemp, J. D., 153, 154, 155, 156, 157, 158, 186, 190 Kennedy, J. C., 267, 277 Kennell, D., 174, 190 Kenyon, K., 212, 227 Kepes, A., 103, 120, 174, 189 Kerjan, P., 93, 112 Kettman, I., 264, 277 Kettman, J., 265, 267, 276 Keydar, J., 52, 69 Kkzdy, F. J., 142, 189 Khesin, R. B., 2, 66' Kholodenko, L. V., 20, 68 Kidwai, J. R., 49, 68 Kim, J. H., 269, 278 Kim, Y. B., 262, 277 Kincade, P. W., 270, 277 Xing, D. W., 260, 263, 278 Kiortsis, V., 139, 140, 141, 189 Kirjanov, G. I., 11, 64 Kit, S., 21, 66 Kjeldgaard, N. O., 92, 122 Klebs, G., 87, 122 Kleinsmith, L. J., 47, 66 Klofat, W., 91, 94, 95, 99, 110, 111, 112, 117, 118, 119, 121, 122 Kobyashi, Y., 94, 122 Kohne, D. E., 21, 22, 64 Kojimba, K., 246, 266 Kolodny, G. M., 211, 266 Kolsch, E., 32, 69 Kominek, L. A., 110, 122 Konigsberg, I. R., 212, 227, 267, $7'7 Konijn, T., 266 Konings, W. N., 110,112,120,122,123 Korenjako, A. I., 32, 69 Kornberg, A., 88, 94, 96, 117, 120, 121, 122, 124 Kornberg, R. D., 103, 122 Korner, A., 9, 67 Kostomarova, A. A,, 9, 67 Kosunen, T. U., 257, 263, 268, 277 Kozlov, I'u, V., 21, 23, 45, 46, 63, 66, 66 Kramer, K. J., 142, 144, 145,188,189 Kretschmer, S., 97, 101, 102, 107, 108, 122 Krichevskaya, A. A., 19, 66, 68 Kriebel, M. E., 204, 226'

Xubai, L., 264, 274 Kuechler, E., 10, 66 Kuhn, A., 131,189 Kulguskin, V., 18, 66 Kung, G., 23, 65 Kurylo-Borowska, Z., 96, 121, 122 Kuwano, M., 3, 66 Kvist, T. N., 250, 266

L Labrie, F., 10, 66 Laing, R., 17, GO Lamb, D. C., 240, 266 Lambert, B., 16, 41, 43, 44, 64 Lamnek-Hirsch, I., 103, 123 Landman, O., 102, 123 Landon, M., 142, 190 Landy, M., 265, 270, 271, 272, 277 Lane, B. G., 34, 69 Lane, C. D., 10,66, 172,188, 243,266 Lang, D. R., 110, 117, % 21' Langan, T. A., 46, 47, 66 Lnnyi, J. K., 94, 122 Lanyon, G., 10, 36, GO Lara, F. J. S., 185, 189 Lash, J. W., 247, 264, 266, 276 Lasher, R., 248, 266 Laskowski, M., Jr., 142, 143, 188 Latham, H., 4, 12, 68 Law, J. H., 110, 124, 142, 143, 144, 145, 146, 189 Lawrence, J. S., 271, 280 Lazzarini, R. A., 92, 93, 162 Leadbetter, E. R., 88, 109, 122 Leak, L. V., 204, 226 Leanz, G. F., 117, 12%' Lechel, K., 2, 60 Lederberg, J., 94, 122 Lee, C. S., 40, 69 Lee, S.Y., 37, 66, 67 Lehmkuhl, D., 213, 219, 226, 227 Leighton, T. J., 61, 83, 96, 122, 123 Lennox, E. S., 209, 210, 227, 267, 277 Lentz, T. L., 207, 227 Leppla, S.H., 34, 66 Lennan, M. I., 4, 5, 7, 9, 12, 14, 16, 24, 66, 66, 68 Leserman, L. D., 266, 276 Lesley, J., 265, 267, 276

AUTHOR INDEX

Leuchars, E., 265, 275 Levi-Montalcini, R., 229, 255 Levine, M. A. ,265, 268, 275 Levinthal, C., 174, 187 Lewis, H., 258, 278 Lewis, J. C., 96, 121 Lewis, M. R., 212, 213, I27 Lewis, W. H., 194, 227 Lieb, W. R., 224, 227 Lieberman, M., 195, 226 Lillie, F., 255 Lilly, V. G., 87, 122 Lim, L., 37, 56 Limborska, S.A., 23, 56 Lin, T. P.. 206, 227 Lindberg, U., 11, 52, 56 Ling, V., 10, 56 Lingrel, J. B., 10, 35, 36, 53, 56 Lipton, B., 212, 227 Littlewood, R., 70, 83 Loening, U. E., 17, 40, 44, 53, 56, 59 Loewenstein, W. R., 204, 205, 206, 210, 211, 223, 225, 226, 227, 228 Lorber, V., 202, 204, 222, 827 Losick, R., 2, 56, 93, 94, 122, 124 Lubin, M., 229, 255 Lukanidin, E. M., 18, 19, 20,56,57, 58 Lundgren, D. G., 108, 110, 117, 122, 123 Lundquist, P. G., 101, 124 Luther, S. W., 25, 54 Lvova, T. N., 32, 59

M Maal@e,O., 92, 122 McCallum, M., 22, 54 McCarthy, B. J., 11, 13, 21, 25, 54, 58 McCarthy, M., 265, 267, 276 McCluskey, R. T., 257, 264, 268, 275 MeConnell. H. M., 103, 12-7 McConnick, N. G., 94, 121, 122 McCullagh, P. J., 266, 276 McGarm, M. P., 265, 279 Mach, B., 96, 122 McIntire, K. R., 269, 279 McNutt, N. S., 196, 201, 202, 203, 210, 222, 225, 227 Macpherson, I.. 50, 57 MacWilliams, H. K., 127, 189 Maio, J. J., 21, 67

289

Maitra, U., 7, 33, 67 Maizel, J. V., 188 Maizel, L., 11, 69 Makela, O., 257, 263, 268, 277 Mskinodan, T., 260, 277 Manasek, F. J., 207,208,2%? Mandel, J. L., 38, 54 Mandel, L., 262, 279 Mandel, M., 21, 53 Mandel, P., 10, 59 Mandelstam, J., 82, 88. 91, 95, 96, 97, 107, 113, 118, 121, 122, 124 Manickavel, V., 261, 278 Mantieva, V. L., 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 19, 20, 31, 33, 36, 37, 39, 53, 55, 57, 58

Marbaix, G., 10, 54, 55, 172, 188, 243, 265

Marbrook, J., 258, 271, 272, 276, I77 Marcaud. L , 4, 5, 9, 11, 13,24, 25, 58 Marchis-Mouren, G., 156, 159, 1.90 Marchok, A. C., 158, 188 Mark, G. E , 213, 214, 227 Markert, C. L., 42 Markland, F. S., 142, 190 Marks, P. A., 158, 171, 172, 181, 188, 189

Marrs, B. L., 186. 189 Marshall, J., 237, 238, 255 Martin, D. N., Jr., 48, 59, 173, 191, 229, 256

Martin, D. T. M., 102, 121 Martin, R. G., 1, 2, 50. 57 Martin. W. J., 272, 678 Martinez-Palomo, A,, 202, 203, 204, 227 Masrri, Y . , 206, 227 Matheson. D. W., 229,246, 247, 250,255 Mathews, J., 91, 122 Matsuda, K., 195, 226 Matsui, Y., 210, 226 Matsuura, S., 156, 157, 158, 180, 181, 189, 190

Matter. A., 220, 226 Matukas, V. J., 246, 255 Matz, L. L.. 117. 122 Mauck, J., 104, 123 Mayall, B. H., 158, IS8 Mayne, R., 235, 245, 246, 247, 248, 249, 250, 253, 264, 255 Mazur, G., 136, 189

290

AUTHOR INDEX

Medlin, J., 262, 279 Meithlac, M., 38,64 Melli, M., 21, 23, 67 Melnick, J. L., 50, 67 Mendecki, T., 37, 66, 67 Mendez, R., 202, 203, 204, 227 Meneghini, R., 185, 189 Metcalf, D., 264, 265, 277 Michalke, H., 32, 67 Michalke, W., 211, 227 Michel, J. F., 91, 96, 122, 123 Mikelsaar, U., 46, 66 Miller, E. J., 246, 266 Miller, G. H., 1, 2, 66 Miller, J. F. A. P., 263, 264, 265, 268, 271, 274, 276, 277, 279 Miller, J. J., 63, 73, 74, 82, 83 Miller, 0. L., Jr., 2, 40,67, 182, 189 Miller, R. G., 258, 267, 276, 277 Millet, J., 96, 120, 123 Mindich, L., 103, 123 Mirsa, D. N., 40, 69 Mirsky, A. E., 4, 47, 63, 66, 68 Mishell, R. I., 260, 265, 267, 276, 277 Mitchell, G. F., 263, 265, 268, 271, 277, 279

Mitchison, J. M., 149, 189 Mitchison, N . A., 264, 267, 277 Miura, Y., 241, 266 Mizutani, S., 52, 69 Moens, P. B., 64, 65, 83 Moller, G., 266, 274, 277 Molnar, J., 18, 19, 20, 68 Monneron, A., 20, 67 Monod, J., 1, 2, 3, 66, 66 Monro, R. E., 93, 123 Monteal, J., 97, 122 Montjar, M., 171, 191 Moore, D. H., 195, 227 Moore, M. A. S., 268, 269, 27Y Moore, P. B., 163, 167, 168, 189 Morel, C., 10, 11, 13, 16, 19, 67, 68 Morikawa, N., 2, 67 Morimoto, T., 156, 157, 158, 180, 181, 189, 190

Morrison, M., 10, 36, 60 Morse, D. E., 3, 67 Moscona, A., 229, 256 Moscona, A. A,, 161, 171, 190, 206, 212, 214, 226, 227, 228, 266, 277

Moscona, M. H., 161, 171, 190 Mosier, D. E., 266, 267, 277 Mosteller, R. D., 3, 67, 174, 183, 190 Moticka, E. J., 262, %77 Moule, Y., 20, 67 Mouton, D., 258, 272, 276 Mueller, P., 97, 123 MuIler-Hill, B., 48, 66 Muir, A. R., 195, 198, 201, 227 Muller, G., 70, 82 Mundkur, B., 64, 76, 83 Munro, A. J., 9,67, 172, 181,189 Murray, K., 240, 266 Murray, M. R., 212, 228 Murrell, W. G., 88, 94, 110, 117, 123 Muthukkaruppan, VR., 261, 266, 277, 27s Mya-Tu, M., 203, 226

Nagata, S., 156, 157, 158, 180, 181, 189, 190

Nakata, H. M., 110, 123 Nakazoto, H., 37, 64 Nameroff, M., 234, 248, 266, 266 nan de Meene, T., 255 Nardi, J., 134, 181, 190 Naylor, H. B., 64, 83 Neale, E. K., 108, 123 Neelin, T. M., 240, 266 Neifakh, A. A,, 9, 67 Nelson, D. L., 88, 94, 117, 122 Nelson, D. S., 266, 278 Nemer, M., 172, 190 Netlesheim, P., 265, 276 Niessing, J., 30, 67 Noll, H., 135, 172, 181, 190 Nomoto, K., 259, 279 Nordin, A. A,, 258, 260, 262, 877, 270 Nossal, G. J. V., 257, 258, 278

Oda, K., 49, 67 Oh, Y. K., 114, 115, 121 Ohye, D. F., 117, 123 Oishi, M., 97, 123 Oishi, S., 79, 83 Oka, T., 126, 153, 156, 158. 159, 190

29 1

AUTHOR INDEX

Okazaki, K., 233, 234, 245, 256 O'Lague, P., 211, 227 Old, L. J., 268, 269, 275, 278, 279 Olsnes, S., 10, 19, 67 Osterhout, W. J. V., 196, 227 Otsuki, E., 181, 190 Oura, H., 135, 172, 181, 190 Ovary, Z., 270, 280 Ovchinnikov, L. P., 9, 67 Overath, P., 103, 123 Overton, J., 206, 227 Owen, J. J. T., 268, 269, 177, 278

P Palade, G. E., 139, 140, 189, 191, 202, "5

Palm, P., 2, GO Palmer, J., 265, 267, 271, 278 Palmer, J. F., 205, 211, 227, 228 Palmiter, R. D., 153, 155, 156, 157, 158, 172, 173, 181, 190 Panek, A,, 74, 83 Papaconstantinou, J., 172, 190 Pappas, G. D., 203, 205, 211, 227 Pardee, A. R., 102, 109, 122 Pardue, M. L., 22, 57 Parnas, H., 4, 5, 53, 174, 188 Parrott, D. M. V., 258, 276 Parshall, C. J., 263, 279 Pasanen, W., 266, 279 Pasero, L., 156, 159, 190 Pastan, I., 253, 254 Paul, J., 10, 23. 24, 36, 46, 47, 55, 57, GO, 186, 190

Paul, M., 172, 190 Paul, W. E., 264, 277 Paulton, R. J. L., 102, 104, 123 Paulus, H., 97, 122 Payton, B. W., 211, 227 Peniberton, R., 36, 53, 185, 188 Penman, S., 8, 9, 10, 16, 17, 32, 38, 67, 59, GO, 171, 183, 188, 190 Pepe, F., 237, 254 Perlman, R. L., 253, 264 Perlman, S., 16, 32, 39, 57 Perry, E. T., 267, 277 Perry, R. P., 4, 10, 17, 18, 37, 44, 67, 171, 190

Pesando, P. D., 263, 275

Peters, L. C., 265, 276' Peterson, J. A,, 61,73, 83, 88, 110,121 Philipson, L., 37, 58 Phillips, R. A., 258, 267, 272, 276, 277, 278 Picciolo, G., 110, 111, 122 Pierpaoli, W., 263, 278 Pierucci, O., 102. 121 Pink, R., 272, 278 Pinto de Silva, P., 220, 227 Pitel, D. W., 98, 99, 104, 123 Playfair, J. H. L., 259, 260, 265, 270, 271, 278 Plonsey, R., 196, 626 Pogo, B. G. T., 47, 58 Pohlit, H., 264, 278 Pontefract, R. D., 64, 83 Porter, K. R., 212, 227 Potter, D. D., 204, 209, 210, 211, 212, 223, 22G, 227 Powell, J. F., 92, 95, 123 Prasad, C., 92, 94, 95, 114, 115, 121, 123 Prasad, K., 229, 256 Pratt, I., 117, 124 Prendergast, R. A., 262, 279 Pringle, J. R., 165, 190 Prockop, D. J., 140, 188 Prokoshkin, B. D., 5, 64 Pryzybaski, R., 237, 256 Ptashne, M., 48, 58 Pujara, C., 235, 256 Purdom, I., 40, 53 Purves, E. C., 265, 271, 278 Pye, J., 265, 276

R Rabussay, D., 2, GO Raff, M. C., 265, 268, 269. 273, 278, 279 Raidt, D. J., 265, 267, 278 Rajewsky, K., 264, 277, 278 Ramaley, R . F., 113, 123 Ranvier, L., 194, 2.27 Rao, K. V., 23, 57 Rapp, F., 50, 57, 59 Rapport, E., 64, 65, 83 Rash, J. E., 209, 212, 225, 227 Raska, K., Jr., 267, 275 Raynes. D. G., 202, 204, 222, 227 Reboud, J. P., 156, 159, 190 Reder, R. H., 39, 58

292

AUTHOR INDEX

Redman, C. M., 128, 190 Reese, T. S., 201, 202, 211, 226, 227 Reeves, 0. R., 210, 211, 226 R.egier, J., 149, 165, 166, 190 Reich, E., 171, 190 Reich, J., 139, 161, 162, 171, 189 Reid, B., 78, 83 Reif, A. E., 268, 278 Remsen, C. C., 108, 123 Revel, J. P., 201, 202, 203, 204, 205, 207, 210, 222, 226, 227, 228 Revelas, E., 102, 121 Reynolds, L., 240, 266 Ribi, E., 117, 121 Rich, A., 10, 63, 66, 66 Richardson, M., 23, 67 Rifkind, R. A., 158, 172, 188 Riha, I., 257, 258, 263, 279 Ringborg, U., 16, 41, 43, 44, 64 Ritter, M. A , , 258, 268, 278 Robbins, E., 10, 64 Robbins, N., 212, 227 Roberts, C., 68, 83 Robertson, F. R., 22. 66 Robertson, J. D., 202, 227' Roblin, R., 33, 55 Roethle, J., 270,271,274 Roman, H., 82, 83 Ronzio, R. A., 153, 154, 155, 156, 157, 158, 186, 190 Rosbash, M., 16, 32, 39, 67 Rose, B., 204, 206, 228 Rose, J. K , 174, 183, 190 Roseman, S., 118, 123 Rosenquist, G. C., 207, 209, 228 Roth, R., 63, 70, 73, 74, 78,83, 161,190 Rowley, D. A., 258, 266, 267, 276, 278 Ruben, L. N., 262, 272, 274 Rubin, H., 211, 227 Rudin, D. O., 97, 123 Rudnick, D., 238, 266 Ruska, H., 195, 227 Russell, P., 258, Z79 Rctter, W. J., 153, 154, 155, 156, 157, 158, 186, 190, 267, 278 Ryskov, A. P., 5, 6, 7, 8, 11, 12, 13, 21, 23, 33, 34, 35, 36, 37, 38, 63, 64, 66, 68 Ryter, A., 89, 90, 92, 96, 97, 98, 99, 102, 103, 104, 106, 107, 108, 109, 119, 120, 111, 123

S Sabatini, D. D., 128, 190 Sachs, H. G., 214, 216, 217, 220, Z28 Sadoff, H. L., 61, 83, 94, 95, 96, 123 Saenz, N., 161, 171, 190 Salas, M., 93, 120 Samarina, 0. P., 4, 5 , 7, 9, 14, 16, 18, 19, 20, 21, 24, 52, 66, 68 Sambrook, J., 49, 68 Samis, H. F., 4, 65 Samuels, H. H., 173, 190, 229, 266 Sanders, T. G., 153, 154, 155, 156, 157, 158, 186, 191 Snndo, N., 79, 83 Sanger, J. W., 230, 233, 235, 237, 239, 245, 246, 265, 266 Santo, L. M., 96, 123 Sarkar, P., 239, 266 Sasaki, Y., 194, 203, 228 Satir, P., 204, 226 Sauer, G , 49, 68 Saunders, G. C., 259, 260, 263, 178 Saunders, J. W., 205, 228 Scnife, J., 1, 2, 66 Schaechter, M., 103, 121 Schneffer, P., 61, 73, 83, 87, 88, 89, 90, 91, 92, 95, 96, 97, 98, 102, 106, 107, 114, 120, 122, 123, 124 Scharff, M. D., 10, 64 Scherrer, K., 4, 5, 8, 9, 10, 11, 12, 13, 16, 19, 24, 25, 38, 66, 67, 68, 171, 190 Schiltz, J., 250, 253, 266 Schimke, R. T., 126, 147, 153, 156, 158, 159, 173, 176, 190 Schlesinger, M., 265, 278 Schlessinger. D., 3, 66, 171, 189 Schlom, J., 52, 69 Schrnitt, R., 95, 96, 97, 110, 112, 121, 123 Schneiderman, H., 232, 266 Schreiber, G., 172, 190 Schrevel, J., 204, 226 Schubert, D., 229, 234, 247, 666 Schubert, M., 247, 266 Schulte-Holthausen, H., 248, 266 Schurnan, D., 229, 266 Schuppe, N. G., 5 , 64 Schwartz, R. S., 270, 278 Schwartz, T., 174, 190 Scott, R. B., 171, 190

AUTHOR INDEX

Scott, T. M., 204, 228 Srb'wtian, J., 78, 53 Seegmiller, R., 248, 256 Sepal, S.. 265, 269, 275 Sekeris, C. E., 30, 57 Selmnn, K., 130, 131, 135, 190 Seraydarian, M., 213, 226 Setlow, R . B., 104, 124 Seto. F., 258. 262, 27s Shaffer, B., 70, 53' Shalnhy, M. R. Y., 259, 260, 264, 268, 279 Shannon, M. P., 43,55 Shaw. A. R., 272,275 Shearer, R. W., 11, 13, 55' Sheffield, J. B., 206, d2S Sheldon, H.. 248, 256 Shemjakin, M. F.. 2, 56 Slren, M . W., 43, 56 Sliepherd, J., 190 Sheridan. J. D., 204, 205, 206, 209, 210, 211, 226, 225' Sheridan, J. W., 4, 54 Sherman, F., 82, 53 Sheu,, C., 112, 123 Shildkraut, G. L., 21, 57 Shimada. Y., 212. 228 Shockman, G. D., 101, 102, 104, 106, 109, 112 Shorenst,ein. R. G., 2, 56, 93, 122 Shortman, K. D., 258, 279 Sihat,ani, A,, 4, 55 Sidky, Y., 261, 269, 279 Siewert, G., 96, 124 Silverstein, A. M., 262, 263, 279 Sinla, P., 262, 279 Simonsen. M., 258, $79 Sjoherg, O., 266, 274, 277 Sjostrand, F. S., 195, 228 Sla.ck, C., 205, 211, 267, 225 Slepecky, R. A,, 99,101,110, 122. 124 Smirnov, M. N., 4, 7, 14, 56 Smith, E. L., 142, 190 Smith, K. D., 23, 55 Smith. L. K., 46, 56 Smithies, O., 272, 279 Snider, I. J., 74, S3 Soeiro, R., 4, 5, 11, 58, 59, 175, 183, 190 Sogin, S. J., 74, 77, 83 Solomon, J. B., 257, 258, 259, 262, 263, 279

293

Soltzstrin, E. C., 258, 275 Sommrr, J. R., 198, 204, 22s Sonenshein, A. L., 2, 56, 93, 94, 122, 124 Sorkin, E., 263. 27s Soutlirrn, E. M., 21, 22, 59 Spsnswick, R. M.. 222, 225 Speirs, J., 16, 40, 53 Speirs, R. S.,265, 219 Sperrlakis, N., 195, 213, 219, 226, 227,, 225 Spiegelman, S., 52, 59 Spirn, A. W., 194, 195, 196, 198, 204, 226, 22s Spirin, A. S., 4, 9, 10, 14, 18, 57, 59 Spitzer, N . C., 222, 228 Spizizrn, J., 91, 122 Spohr. G., 10. 11, 13, 16, 5s Sprrnt, J., 263, 264, 265, 268, 2?4, 277, 219 Spudich, J. A , , 88, 94, 96, 117, 122, 124 Sreter, F., 235, 566 Srinivasan, P. R., 49, 55 Srinivasan, V. R., 112, 121 St.nehelin, T.. 135, 172, 181, 190 Stalsberg, H., 207, 208, 209, 225 Starlinger, P., 32, 59 Steele. K. B.. 194, 826 Stein, H., 22, 55 Stein, W. D.. 224, 227 St.einbach, A , , 210. 211, 226 Sterlini. J . M., 91, 95, 118,122, 124 Sterzl, J., 257, 258, 262, 263, 279 Stevenin. J., 10, 59 Steward, J. P., 262, 279 Stiffel, C., 258, 272, 275 Stocktiale, F.! 234. 256 Stocken, L. A., 47, 59 Stockert, E., 269, 275 Stoerkenius, W., 222, 226 St.rrtnge, R. E., 92, 95, 123 Strasser, F. F., 213. 214, 227 Strominger, J . L., 96, 117, 124 Sueoka, N., 21, 5.9, 97, 123 Sugae, K., 113, 124 Summers, D. F., 11, 59, 188 Sussmnn, M., 161, 190 Suto, T., 79, 53 Sut.ton, W. D., 22, 59 Suzuki, S., 246, 256 Suzuki. Y., 175, 180, 181, 185, 188, 190 Swanson, A., 91, 121 Swartzendruber, D., 259, 260, 278

294

AUTHOR INDEX

Swift, H., 46, 69 Szenberg, A., 260, 276 Szulmajster, J., 91, 93, 95, 96, 110, 118, 120, 122, 124

T Taber, H., 101, 110, 112, 184 Takahashi, I., 91, 124 Takahashi, T., 269, 279 Takanami, M., 8, 69 Takeya, K., 259, 279 Taliaferro, L. G., 270, 279 Taliaferro, W. H., 270, 279 Tamaoki, T., 34, 69 Tanaka, I., 194, 203, 228 Tarr, M., 195, 228 Tartakoff, A. M., 142, 189 Tartof, K. D., 17, 44, 67 Tashiro, Y., 156, 157, 158, 180, 181, 189, 190

Tatarskaya, R. I., 32, 69 Tatum, E. L., 96, 122 Tauro, P., 67, 8s Taylor, R. B., 264, 273, 677, 279 Teeter, E., 4, 69 Temin, H. M., 52, 69 Teng, C. S., 47, 69 Teng, C. T., 47, 69 Terskich, V. V., 12, 13, 14, 66 Tevethia, S. S., 50, 69 Thomas, C. A,, Jr., 40, 67, 69, 182, 184, 189, 190

Thompson, E. B., 173, 191 Thorbecke, J., 270, 280 Thorp, F. K., 247, 266 Tigelaar, R. E., 258, 974 Tikhonov, V. H., 12, 66 Tille, J., 195, 203, 218 Tipper, D. J., 117, 124 Tlaskalova, H., 262, 279 Tobias, C., 211, 627 Tomkins, G. M., 48, 69, 173, 191, 229, 256

Tonegawa, S., 52, 69 Tonomura, Y., 213, 220, 226 Toole, B. P., 250, 266 Townes, P. L., 266, 279 Trakatellis, A. C., 171, 191 Travers, A. A., 2, 64

Travnicek, M., 52, 69 Trelstnd, R. L., 205, 207, 228, 246, 250, 266

Tremaine, J. H., 63, 73, 83 Trentin, J. J., 265, 679 Tressman, R. L., 212, 22'6 Trevelyan, W. E., 74, 83 Trevithick, J. R., 10, 66 Trinkaus, J. P., 205, 206, 207, 226, 227 Triplett, R. F., 263, 276 Tsanev, R., 9, 69 Tsukagoshi, N., 103, 124 Tucker, D. F., 258, 279 Tumanyan, V. G., 5,9, 16,24,68 Tuominen, F. W., 115, 124 Tupper, J., 205, 228 Tushinsky, R. J., 32, 37, 64 Tyan, M. L., 258,269,279 Tyndall, R. L., 4, 69

U Udem, S. A., 75, 76,85 Uhr, J. W., 140, 191, 263, 270, 280 Umiel, T. H., 258, 279 Unsworth, B., 267, 279 Utsami, K., 246, 266

V Van Alten, P. J., 262, 277 Van Breeman, V. L., 195, 228 Van Der Kloot, W. G., 203, 628 Vann, D., 265, 267, 276 van Tubergen, R. P., 104, 124 Varmus, H. E., 253, 264 Varshansky, A. Ya., 46, 66 Vaughan, M. H., 37, 64, 175, 183, 190 Veeraraghavan, K., 261, 278 Veneroni, G., 212, 228 Vesco, C., 9, 10, 69 Vinter, V., 98, 117, 124 Vinuela, E., 93, 120 Virolainen, M., 266, 679 Viza, D., 273, 279 Vladimirzeva, E. A., 12, 13,14,66

W Wagner, E., 17, 60 WBhren, A,, 101, 124

AUTHOR INDEX

295

Winkelstein, A., 271, 280 Waites, W. M., 107, 113, 122, 12.4 Winslow, R. M., 92, 122 Walker, P. M. B., 22, 54, 59 Woese, C. R., 94, 98, 12.4 Wall, R., 32, 37, 52, 64, 58, 59 Wolf, H. H., 214, 215, 228 Wallis, V., 265, 275 Wolf, N. S., 265, 279 Walter, G., 52, 59 Wolpert, L., 159,191, 210,223,224,228 Wang, A-C., 272, 278 Warner, J. R., 4, 5, 59, 75, 76, 83, 175, Wood, D. A , , 107, 124 Woodard, J., 46,55 183, 190 Warner, N. L., 258, 265, 270, 275,. 278, Woodbury, J. W., 196, 198, 203, 204, 220, 228 280 Woodland, H. R., 10, 55, 172, 188, 243, Warren, R. A. J., 83, 96, 122 255 Warren, S. C.,’95, 107, 124 Woodward, D. J., 205, 628 Warth, A. D., 117, 124 Wrenn, J. T., 155, 156, 157, 158, 190 Wartman, W. B., 194, 228 Wright, B. E. G., 273, 274 Waterstone, H. R., 271, 250 Wulff, V. J., 4, 58 Watson, D. W., 262, 277 Wyatt, G. R., 130, 191 Watson, K., 52, 59 Watterson, R. L., 246, 256 Wegemann, T. G., 272, 278, 280 Y Weidmann, S., 194, 195, 196, 198. 203, 204, 228 Yagisawn, M., 79, 85 Weigle, U’. O., 265, 270, 271, 280 Yamakawa, T., 93, 124 Weil, R., 50, 56 Yamamoto, T., 91, 98, 101, 107, 108, 120, Weinberg, R. A,, 9, 10, 16, 17, 32, 38, 57, 124 59 Yamamoto, T. J., 204, 228 Weinstein, R. S., 196, 202, 203, 210, 222, Yanagita, T.. 79, 83 227 Yanofsky, C., 3, 57, 174, 183, 186, 189, Weintraub, H., 158, 159, 188, 191, 231, 190 233, 238, 239, 240, 241, 243, 254, 255, Yasmineh, W. G., 22, GO 256 Yee, A. G., 203, 204, 210, 227 Weisberger, A. S., 18, 59 Yehle, C. O., 93, 124 Wenk, M., 241, 256 Yoffey, J. M., 264, 250 Werner, I., 234, 254 Yokoyama, H. O., 194, 228 Werner, M., 194, 228 Yonezawa. T., 212, 627 Wessells, N. K., 159, 171, 191, 267, 273, Yoshikawa, H., 97, 123 1276, 278 Yoshikawa, M., (also YoshikawaWestphal, H., 49, 68, 59 Fukada, M.), 5, GO Wettstein, F. O., 135, 172, 181, 190 Young, I. E., 88, 92, 93, 98, 99, 101, 107, White, A., 269, 275 119,121, 124 Whitefield, C., 23, 67 Young, F. E., 116, 120 Whitehouse, H. L. K., 39, 59 Yousten, A. A., 61, 73, 83, 88, 110, 112, Whitmore, H., 235, 258 l g l , 124 Wigglesworth, V. B., 159, 191 Yunis, J. J., 22, 60 Willems, M., 17, 59, GO Williams, C. M., 129, 130, 160, 189, 191 Z Williamson, A. R., 10, GO, 273, 274 Williamson, J., 104, 123 Williamson, R., 10, 19, 36, 57, 60 Zagury, D., 140, 191 Wilt, F. H., 171, 191, 240, 241, 255, 256 Zillig, W., 2, 60 Winge, g., 68, 83 Zylber, E., 16, 32, 39, 57

SUBJECT INDEX A “A” cells, response to sheep red blood cells, ontogeny of, 269-270 Adherent cells, reaction to sheep red blood cells, 266267 Antiobiotics, formation of, during bacilli sporulation, 96-97 Ascus, yeast sporulation from vegetative growth of, 62-63 Autoradiography, of zymogen synthesis, 139-1 41 Auxotrophic bacilli, definition of, 87

B “B” cells, response to sheep red blood cells, ontogeny of, 269-270 Bacilli sporulation, 85-124 antibiotic formation during, 9697 cell wall synthesis during, 98-99 “committment” to, 117-120 double-membrane type, 119-120 single-membrane type, 118-119 DNA synthesis during, 97-98 energy production during, 110-113 enzymes active during, 95-97 later spore development during, 11&-117 membrane synthesis during, 99-109 asymmetric septation, 106 normal cell division, 101-106 morphology and genetics of, 88-91 onset of, conditions necessary for, 91-113 pleiotrophic conditions for, 91 protein synthesis during, 94-97 RNA synthesis during, 93-94 suppression of, 114-116 synthesis of cytopIasmic polymers in, 92-98 Bone marrow, reaction to sheep red blood cells, 263-266

C Cacogenic bacilli, definition of, 87-88 Cascade regulation hypothesis, for transcriptional units of eukaryotic cells, 24-25 Cell coupling in developing systems, 193 in early embryo, 205-209 in early development, 205-207 in precardinc mesoderm and heart, 207-209 in mature cardiac tissue, 194-205 electrotonic type, 194-196 heart-cell contacts, 196-203 nexus as pabh for, 203-205 synchronization and, 213-222 tissue culture studies on, 209-222 electrical communication studies, 209-213 Cell cycle, 229-256 Cell differentiation, 229-256 in chondrogenesis, 246-251 in erythrogenesis, 239-246 in myogcnesis, 232-239 Cell lineages, 229-256 Cell transformation, by oncogenic viruses, possible mechanism, 49-53 Cell wall, synthesis of, during bacilli sporulation, 98-99 Chondrogenesis, cell differentiation in, 246-251 Cocoonase enzymology of, 142-144 production of, morphological studies, 129-14 1 zymogen of, 144-145 cells for, in silk moths, 125-141 See also Zymogen Crick model of transcriptional unit, 48-49

D DNA, synthesis of, during bacilli sporulation, 97-98 296

297

SUBJECT INDEX

E Enzymes, active during barill] sporulation, 95-97 Erythrocytes, from sheep, see Sheep red blood cells Erythrogenesis, cell differentiation in, 239-246 Bscherichia coli, lnc operon structure in, 2 Eukaryotic cells genome of organization, 21-22 tandem repetitions, 39-42 operons of, genetic aspects of structure, 42-44 transcriptional units in, 1-60 Exonuclease. effects on dRNA. 32

Galea, of butterflies and moths, 129 differentiation-specific product of, 142-145 morphology of, 130-133 Genome, of eukaryotic cells, organization of, 21-22 Georgiev transcriptional unit model for eukaryotic cells, 27-39

H Heart, cultured cells of, elertrical communication and, 212-213 Heart-cell paradigm, 193-228 Histoncs, ns inhibitors of transcription, 45-47

I Immunity, development of, 257-280

1 lac operon, of E. coli, 2 “Luxurs’’ molecules, in cell, 232

M Macrophages, reaction to sheep red blood cells, 266267 Mutations affecting sliorulation, 66-72 in bacilli, nomenclature for, 87-88 Myogenesis, cell differentiation in, 232-239

0 Oncogenic viruses, cell transformation by, possible mechanism, 49-53 Operator, of transcriptional unit, 2

P Promoter. of transcriptional unit, 2 Pro tein synthesis, during bacilli sporulation, 94-97 Protogenic bacilli, definition of, 87 Prototrophic bacilli, definition of, 87

Rrpressors of transcription, nonhistone proteins as, 4 7 4 8 RNA “heterogeneous nuclear’’ type, 4 “mcssengerlike,” 4 synthesis of control, 2 during bacilli sporulation, 9%94 dRNA base sequence analysis of, 37-38 end analyses of, 33-36 exonurlrus- effects on, 32 giant, from transcription in eukaryotic cells, 3-24 cleavage of, 8-12 processing, 14-17 prerursors, transformation, 12-13 transport mechanism, 1%21 ribosomal operon structure, 38-39 UV rndiation effects on, 31-32 inRNA, of cocoonase zymogen stability of, 171-173 synthesis rate, 178-185

298

SUBJECT INDEX

rRNA, precursor, synthesis and processing of, 17-18

s

patterns of operation of, 3-24 promoter of, 2 regulation of, 44-49 regulation of termination in, 48 structural genes of, 2

Saccharomyces cerevisiae, life cycle of,

U

62-63

Sheep red blood cells induction of tolerance to, 270-272 response to, in development of immunity, 257-280 cell interactions during, 263-267 ontogeny of antibody variability in,

UV radiation, effects on dRNA, 31-32 V Viruses, oncogenic, see Oncogenic viruses

272-273

ontogeny of cells responding to, 268-270

ontogeny of responsiveness, 259-261 phylogenetic aspects, 261-263 Silk moths cocoonase zymogen cells of, 125-191 escape of, from cocoon, 129 Sporulation of bacilli, see Bacilli sporulation of yeast, see Yeast sporulation Structural genes, of transcriptional unit, 2

Yeast sporulation, 61-83 biochemical events specific to, 72-77 cell cycle dependency of, 77-82 morphological changes during, 6 4 4 6 mutations affecting, 66-72 regulation of, 61-83 from vegetative growth to ascus, 62-63

Z

T “T” cells, response to sheep red blood cells, ontogeny of, 268-269 Thymus, reation to sheep red blood cells, 263-266

Transcriptional units, in eukaryotic cells, 1-40

cell transformation by oncogenic viruses and, 49-53 Crick model of, 48-49 dRNA from action of, 3-8 histones as inhibitors of, 4 5 4 7 inhibition of, from repetitive base sequences, 23-24 models of, 24-44 activator RNA, 2 6 2 7 cascade regulation hypothesis, 24-25 Georgiev model, 27-39 operator of, 2 parts of, 1-2

Zymogen cells, for cocoonase, in silk moths, 125-191

mRNA of stability of, 171-173 implications, 173-178 synthesis rate, 178-185 synthesis of, 147-149 autoradiographic studies on, 139-141 cell differentiation and, 153-154 extrusion of, 135-139 methods used in study of, 149-153 in phase 11, 161-171 preparation for, 134-135 quantitation, 145-159 rates of, 140-141 transition points in, 159-161 transport of autoradiography of, 139-141 kinetics. 139-140