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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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 <0.002 <0.002

0.013 0.1 0.009 0.030 0.044

From Ryskov and Georgiev (1970). bpppXp, ppXp, and pXp = nucleoside tetra-, tri-, and diphosphate. a

higher in the light material; this is in good agreement with the difference in the molecular weights of the fractions (Georgiev et al., 1972a). Thus nascent molecules can be found only among giant dRNA chains, and this means that probably giant dRNA is the only primary product of dRNA synthesis. The origin of shorter chains will be discussed below.

B. CLEAVAGE OF GIANTdRNA OF

AND

TRANSLATION

MONOCISTRONIC MESSAGE

Very soon after the discovery of nuclear dRNA, RNA with similar properties was found in the cytoplasm of animal and plant cells. Georgiev et al. (1963) using low doses of actinomycin, which selectively inhibit rRNA synthesis, found labeled dRNA in the cytoplasm of animal cells after 6 hour-labeling wifh radioactive phosphate. Penman et al. (1963) found that polysomes contained some nonribosomal rapidly labeled RNA

1.

TRANSCRIPTIONAL U N I T S I N EUKARYOTIC CELLS

9

with sedimentation coefficients between 6 and 25 S. Brawerman et al. (1963) and DiGirolamo et al. (1964) demonstrated the template activity of such RNA in cell-free systems. In all these first experiments it was found that cytoplasmic rapidly labeled nonribosomal RNA had rather low sedimentation coefficients, between 6 and 25 S. Samarina ( 1964) compared the sedimentation properties of cytoplasmic and nuclear dRNA after long-term labeling in the presence of low doses of actinomycin and found that the cytoplasniic sedimented much more slowly than the nuclear dRNA (Fig. 5) with a mode in the 12 S zone. She concluded that dRNA during the transfer from the I

I

300

I00

Fraction No

FIG.5. Sedimentation properties of nuclear and cytoplasmic dRNA’s isolated from rat liver cells labeled for 6 hours in the presence of low actinomycin. 0-0, Nuclear dRNA; 0-0, Cytoplasmic dRNA. Data of Samarina et al. (196513).

nucleus to cytoplasm is cleaved to rather short chains. Thus the giant dRNA was considered to be a precursor to the shorter mRNAs. Later a number of authors studied the sedimentation properties of cytoplasmic dRNA and found in most cases coefficients in the 6-25 S zone with maximum in 12-18 S region (Munro and Korner, 1964; Scherrer and Marcaud, 1968; Brown and Gurdon, 1966; Tsanev, 1968). Only a few exceptions to this were observed. Thus in the case of brain and of fish embryo (Vesco and Giuditta, 1967; Ovchinnikov et al., 1969), heavy dRNA was also found in cytoplasm; although it seems very likely that in these cases the cytoplasm was contaminated either by leaked nuclear material (Weinberg et al., 1969), or by RNA released from the nuclei during mitosis (Neifakh et al., 1971; Neifakh and Kostomarova, 1971). Indeed, if the cytoplasm is isolated carefully enough

10

G. P. GEORGIEV

so as to avoid damage of nuclei, no giant dRNA is detected (Stevenin et al., 1969; Scherrer et al., 1970). I n all the above-mentioned papers cytoplasmic dRNA was equated with mRNA. However, this is not always true. I n fact, a significant part of nonribosomal rapidly labeled RNA in the cytoplasm is not combined with ribosomes. It may correspond to the RNA of informosomes (Spirin et al., 1964; Spirin, 1969) or to the material leaked from the nucleus during the isolation procedure (Perry and Kelley, 1968; Weinberg et al., 1969; Olsnes, 1970; Scherrer et al., 1970). This material may have rather high sedimentation coefficients and sediment in the same region as polysomes. For this reason, the polysomes of cells producing only few types of proteins were examined. The simplest example is the reticulocyte which synthesizes almost exclusively hemoglobin. Hemoglobin is synthesized on polysomes containing 5-6 ribosomes. The analysis of the RNA isolated from polysomes yield the peaks of ribosomal RNA, 28 S, 18 S, and 5 S, the 4 S tRNA, and in addition a 9 S RNA. The latter was purified and found to have template activity in a cell-free system (Chantrenne et al., 1967; Huea et al., 1967; Labrie, 1969). Moreover, recently synthesis of hemoglobin has been obtained using this RNA fraction as a template in a cell-free system (Lingrel, 1971). Finally the injection of 9 S RNA into the amphibian eggs leads to the induction of hemoglobin synthesis in the cytoplasm of these cells (Gurdon et al., 1971). This is unequivocal evidence in favor of the messenger nature of 9 S RNA. The molecular weight of 9 S RNA is about 0.17 X lo6 (R. Williamson et al., 1971). Thus it can code for not more than one polypeptide chain with a molecular weight of 15,000-17,000 (the molecular weight of a single hemoglobin chain). Thus in the case of hemoglobin synthesis a monocistronic messenger is translated in the cytoplasm. It is important to note that 9 S RNA with the same properties can be isolated not only from polysomes, but also from whole cells. For this reason its low molecular weight cannot be a result of RNase action during the preparation of polysomes. Very similar, although not so complete analysis has been done with a number of other systems of protein synthesis such as the synthesis of H and L chains of antibodies; the synthesis of the embryonic muscle proteins, actin, myosin, and tropomyosin ; the synthesis of protamine and histones (Becker and Rich, 1966; Borun et al., 1967; Heywood et al., 1967; Kuechler and Rich, 1969; Ling et al., 1969; A. R. Williamson and Askonas, 1967). I n all cases the chain length of the mRNA present in polysomes corresponds roughly to the size of a monocistronic message for a given protein. The only exception is the synthesis of poliovirus proteins on the template of viral RNA. I n this case a giant RNA (about

1.

TRANSCRIPTIONAL UNITS I N EUKARYOTIC CELLS

11

2 X lo6 daltons) is translated and giant polysomes containing 6 6 8 0 ribosomes are formed. However, even in this case viral RNA seems to be pseudomonocistronic. Only one large polypeptide chain is synthesized which is then cut into shorter chains by specific proteases (Summers and Maizel, 1968). I n no case has the synthesis of more than one polypeptide chain on the messenger RNA been found in animal cells. Thus since giant dRNA is the only primary product of transcription (see above) and cytoplasmic mRNA is of a lower molecular weight than newly formed nuclear dRNA, one can conclude that cytoplasmic mRNA is formed from the latter by cleavage. To further test this suggestion, competitive hybridization experiments were carried out. It was found that cytoplasmic dRNA competed by about 2640% with total nuclear dRNA whereas total nuclear dRNA inhibited completely the hybridization of cytoplasmic dRNA with DNA (Georgiev, 1966; Arion and Georgiev, 1967 ; Shearer and McCarthy, 1967). Thus the base sequences in cytoplasmic mRNA are also all present in nuclear dRNA ; this indicates a precursor-product relationship between nuclear and cytoplasmic dRNA’s. The next question is whether or not there is also sequence homology between giant nuclear dRNA and the low molecular weight cytoplasmic dRNA. The answer is “yes.” Arion and Georgiev (1967) demonstrated that cytoplasmic RNA competed to some extent with the giant nuclear dRNA in Ehrlich ascites carcinoma cells. Later this result was confirmed in other systems (erythroblasts and HeLa cells) (Gazaryan and Kirjanov, 1968; Scherrer and Marcaud, 1968; Soeiro and Darnell, 1970; Scherrer et al., 1970). The crucial experiment in this respect is the competition of nonlabeled giant nuclear dRNA against cytoplasmic mRNA in the hybridization reaction with DNA. Such experiments were performed by Scherrer (1971) with HeLa cells and by Georgiev et al. (1972a) with rat liver cells. Complete inhibition of cytoplasmic mRNA binding was achieved (Fig. 6 ) . Lindberg and Darnel1 (1970) obtained similar results using cells transformed by SV40 virus. I n the nucleus, virus-specific RNA was found in the heavy fraction; in the cytoplasm, in the light one. These experiments may be taken as proof that cytoplasmic mRNA is formed by cleavage from the giant precursor molecules synthesized in cell nucleus. Where does this cleavage take place? It was pointed out above that R significant part of nuclear dRNA is recovered in -18 S zone after sucrose gradient ultracentrifugation. This fraction contains almost no radioactivity after short pulses, but after longer incubations, its specific activity becomes equal to that of heavy dRNA. Cytoplasmic RNA competes with this relatively low-molecular weight nuclear dRNA more

12

G. P. GEORGIEV Amount of competitor, pg

0 200 400

700

100

1

w .-.U

-u

F

0

100 200 300 400 5a Ratio : competitor/nuclear dRNA

Fro. 6. The competitive hybridizntion of nuclear and cytoplasmic dRNA with DNA from rat liver (Data of Georgiev et al., 1972a). (A) Labeled polysomal dRNA in the presence of nonlabeled heavy nuclear dRNA. (B) Curve 1: labeled heavy nuclear dRNA in the presence of nonlabeled polysomal RNA. Curve 2 : labeled heavy nuclear dRNA in the presence of nonlabeled total cellular RNA.

efficiently than with giant dRNA (Arion and Georgiev, 1967). One can conclude that the cleavage of precursor molecules into shorter chains takes place inside the cell nucleus.

C. NONCONSERVATIVE TRANSFORMATION OF GIANTPRECURSORS On the basis of a number of experiments on the kinetics of nuclear and cytoplasmic RNA labeling, Harris (1962) suggested that a significant part, if not all, of newly synthesized nuclear RNA is degraded without being transferred into the cytoplasm (see also Harris, 1963b). I n 1963, Scherrer et al. followed the fate of newly synthesized RNA under conditions in which RNA synthesis was completely blocked by actinomycin. In these conditions, about 30% of newly formed RNA was degraded. In a similar experiment Harris (1963) and collaborators observed more extensive degradation (about two-thirds) . Gvozdev and Tikhonov (1964) demonstrated the degradation of dRNA not only in actinomycin, but also during chase incubation without isotope. Lerman

1.

TRANSCRIPTIONAL UNITS I N EUKARYOTIC CELLS

13

et al. (1965), using the hot phenol fractionation technique, observed the degradation of newly formed rRNA. The question then arises whether only the excess of overproduced RNA is degraded or whether two different kinds of sequences are present in the cell nucleus-one being the true mRNA and the other the RNA to be degraded. To answer this question, competitive hybridization experiments have been done (Georgiev, 1966; Arion and Georgiev, 1967; Shearer and McCarthy, 1967). Should only an overproduction of the same RNA take place then one could expect that the base sequences of newly formed nuclear RNA and of cytoplasmic RNA should be the same ; and cytoplasmic RNA should compete completely with labeled nuclear RNA. However, this is not the case. Actually, even in the presence of a large excess of cytoplasmic RNA, the hybridization of nuclear dRNA is decreased by no more than 5070 (usually about 30%). And no further decrease could be obtained by increasing the amount of cytoplasmic RNA. In control experiments the total cellular RNA completely inhibited the hybridization of nuclear dRNA with DNA. Thus it is clear that in addition to base sequences contained in cytoplasmic mRNA, nuclear dRNA contains sequences that never reach the cytoplasm. Therefore it appears that two kinds of dRNA are present in the cell nucleus: dRNA, or the precursor of true polysomal mRNA; and dRNA,, which never leaves the nucleus; and these two kinds of RNA contain different base sequences; i.e., they are synthesized on different sites of DNA. It is important (see below) that the proportion of dRNA that can be hybridized to excess DNA is significantly higher than that of cytoplasmic mRNA (Arion and Georgiev, 1967). On the other hand, it was found in experiments 011 the saturation of DNA with increasing amounts of RNA, that nuclear RNA saturates about 5-10 times more DNA than cytoplasmic mRNA (Shearer and McCarthy, 1967; Scherrer and Marcaud, 1968; Scherrer et al., 1970). For example, nuclear RNA from L cells and erythroblasts saturated about 5% of the cell DNA. For the cytoplasm corresponding figures are 1% and 0.5%, respectively. Thus nuclear dRNA, is homologous to a larger number of DNA sites than dRNA,. In sucrose gradients, dRNA, and dRNA, are distributed differently. The heavy region of density gradient is usually enriched in dRNA, while more dRNA, is found in the light zone, as shown in rat liver and Ehrlich ascites carcinoma cells (Arion and Georgiev, 1967 ; Georgiev et al., 1972a). On the other hand, in HeLa cells the enrichment of light fraction with dRNA, is less pronounced (Scherrer et al., 1970). The significance of these differences will be discussed.

14

Q. P. QEORGIEV

D. PROCESSING OF dRNA The results of preceding sections may be interpreted to indicate that the giant dRNA synthesized in the cell nucleus is cleaved into shorter chains by specific ribonucleases (endo- and exonucleases) . About onehalf to three-fourths of these chains (dRNA,) are then rapidly degraded to acid-soluble mono- or oligonucleotides. As a result only dRNA, chains survive and are then transferred into the cytoplasm. If the degradation of dRNA, is faster than the cleavage process, an enrichment of light fraction with dRNA, should take place; if it is slower, this enrichment might be absent. This may explain some differences in results obtained with different types of cells. The rate of the processing and of its different steps may vary. The question arises whether dRNA, and dRNA, are parts of the same or of different giant molecules. To answer this question, the changes of RNA properties in chase experiments have been investigated (Mantieva et al., 1971). High concentrations of actinomycin completely inhibit RNA synthesis, and this allows one to follow the fate of RNA synthesized during a pulse labeling experiment. High doses of actinomycin usually also inhibit the RNA transport from the nucleus to cytoplasm (Georgiev e t al., 1963), and the distribution of RNA between different fractions obtained by hot phenol fractionation remains unchanged (Georgiev et al., 1963; Lerman e t al., 1965; Arion et at., 1967). The fraction isolated a t 65O (temperature interval 55-65OC) is rather stable; on the other hand the 85O fraction (temperature interval 65-85OC), which contains the main part of rapidly labeled dRNA (about 70-80%), is degraded after the actinomycin treatment. The extent of degradation may vary from 3&40 to 8&90% since actinomycin interferes also with the processing of RNA. Although in general the actinomycin chase is not a very reproducible procedure, only experiments in which about 50-70% of RNA in the 85OC fraction were degraded were selected; indeed, in this case no redistribution of material between fractions takes place (Mantieva et al., 1971). The degradation of about two-thirds of the dRNA in the 85OC fraction (with a corresponding decrease of total radioactivity and of total amount of RNA in the fraction) is accompanied by a decrease in the molecular weight of the RNA. The molecular weight of RNA in the peak, calculated on the basis of Spirin (1963) equation, is about three times higher after a pulse than after a chase incubation (Fig. 7 ) . It was pointed out above that the UV and radioactivity curves in the 85OC fraction coincided after pulse labeling and the specific activity is not changed after a chase in actinomycin. The decrease in molecular

1.

TRANSCRIPTIONAL UNITS I N EUKARYOTIC CELLS

15

weight of the RNA is proportional to the extent of degradation. Presumably two-thirds of the length of the original giant precursor molecules is broken down to acid-soluble products. Other explanations cannot be excluded a t present, but they seem to be less probable.

f.5

4 .O m?I

0 0.5 n r, + ..-+>

::

.-

Ba

3.0

2.0 4.0

I

I

I

40

20

Fraction No.

RG.7. Sedimentation properties of nuclear dRNA (6545°C fraction) isolated from rat liver (curves a, b) or Ehrlich carcinoma cells (c, d) after pulse labeling (a, c) and actinomycin chase (b, d ) . From Mantieva et al. (1971). I n hybridization experiments, cytoplasmic polysomal RNA (containing true mRNA) competes with “chase” dRNA much more effectively than with “pulse” dRNA (Fig. 8 ) . This means that during the chase in actinomycin a selective degradation of dRNA, takes place, and in this respect the actinomycin chase is similar to the natural process of selective degradation of dRNA, sequences. The “chase” 85OC dRNA is enriched in dRNA,. One can conclude that giant precursor molecules contain both kinds of sequences, dRNA, and dRNA,. The degradation of dRNA, sequences to acid-soluble products should lead to a decrease in molecular weight and to an accumulation of dRNA, sequences in the product that actually is formed. These results were obtained with rat liver and Ehrlich carcinoma cells. Sometimes, in particular with

16

0. P. GEORGIEV

HeLa cells and Drosophila salivary glands (Daneholt et al., 1969a,b; Penman et al., 1970), actonomycin chase experiments do not give such clear results. The distribution of label in sucrose gradients is not changed. To avoid the possible artifacts produced by the action of actinomycin, Scherrer et al. (1970) determined the sedimentation properties of dRNA from erythroblast cells incubated in nonlabeled precursors after pulse labeling (cold chase). It was found that during the chase the molecular weight of dRNA is decreased in agreement with results above. P-RNA added I

50

100

50

0.5

4.0tng

P-RNA added

FIG.8. Competition of polysomal RNA (P-RNA) with nuclear dRNA isolated from rat liver (A) and Ehrlich ascites carcinoma cells (B) after pulse labeling and actinomycin chase. 0-0, Pulse dRNA; 0-0, chase dDNA. From Mantieva et al. (1971).

The results show that the processing of nuclear dRNA occurs entirely in the cell nucleus, as indicated by the accumulation of rather low molecular weight dRNA enriched in dRNAl (true mRNA) after long-term incubation or in chase conditions (cold or actinomycin chase) (Samarina et al., 1965b; Matieva et al., 1971; Scherrer et al., 1970). Also, no differences can be found between the base sequences of mRNA in polysomes and in free cytoplasmic mRNP particles (informosomes) by competitive hybridization (Scherrer et al., 1970). Thus the processing is completed inside the nucleus and does not take place in the cytoplasm. The general conclusion can be drawn that newly formed nuclear dRNA is synthesized in the form of giant precursors containing sequences

1.

TRANSCRIPTIONAL UNITS I N EUKARYOTIC CELLS

17

corresponding to the true mRNA and to dRNA which is degraded inside the nucleus.

E. THESYNTHESISAND PROCESSING OF RIBOSOMAL RNA PRECURSOR The formation of ribosomal RNA follows the same main steps as the formation of mRNA. The primary product of transcription is a giant ribosomal RNA precursor, or 45 S rRNA. This RNA was discovered by Perry (1962), who showed that in the presence of a low dose of actinomycin the inhibition of synthesis of 45 S RNA with a high GC content is followed by the inhibition of cytoplasmic ribosomal RNA formation. On the basis of kinetics and competitive hybridization experiments, fingerprinting, and methylation (see reviews, Perry, 1967 ; Loening, 1970 ; Birnstiel et al., 1971), the conclusion has been drawn that 45 S rRNA contains one 28 S rRNA and one 18 S rRNA. The sum of the molecular weights of 28 S and 18 S rRNA is 2.25 x lo6,half of the molecular weight of 45 S rRNA. Thus about half the sequences of precursor corresponds to unstable rRNA that is degraded inside the cell nucleus. The size of degraded part is different in different species (Perry et al., 1970; Loening et al., 1969). The situation is very similar to that we met in the case of dRNA: the giant polycistronic precursor is present; it is cleaved to shorter chains, and part of the sequences represented in precursor is degraded. The details of processing steps have been established mainly with the aid of the gel polyacrylamide electrophoresis technique, which allowed the demonstration of minor intermediates. The result may be described by the following scheme (Weinberg et al., 1967; Willems et al., 1968; Weinberg and Penman, 1970) : 45

S -+

41

S -+ 35 S + 32 S + 28 S

\

20

S-

18 S

All intermediates except 32 S rRNA are present only in trace quantities. However, the existence of discrete RNA intermediates allows one to suggest that the endonuclease attack in some specific points precedes the exonuclease attack. Thus, first some blocks are split off from the precursor and then part of them are degraded to acid-soluble compounds. Therefore the transcriptional units for ribosomal RNA synthesis (R operons) consist of sequences encoding ribosomal RNA and sequences which do not participate in the produltion of stable functional molecules. Although in the case of dRNA the presence of both kinds of sequences in the same precursor molecule is not proved unequivocally, nevertheless

18

0. P. GEORGIEV

many data are in favor of this suggestion (see above). The nature of the nuclear specific sequences remains obscure. On the other hand, it has to be understood if one is t o understand the operon structure. A number of different hypotheses have been suggested, but before their analysis other data will be considered that help to distinguish different models.

F. ON THE MECHANISM OF dRNA TRANSPORT The question is how and in what form dRNA is processed and transferred from the sites of synthesis in chromosomes to the sites of functioning in polysomes. T o answer this question, it is necessary to look at the complexes containing mRNA during the main steps of its formation. At present three kinds of ribonucleoprotein complexes containing dRNA have been described. These are free cytoplasmic dRNA containing ribonucleoproteins or informosomes (Spirin et al., 1964; Spirin, 1969) polysome-bound mRNA proteins (Weisberger and Armentrout, 1966; Henshaw, 1968; Perry and Kelley, 1968) and nuclear particles containing dRNA (Samarina et al., 1965a; Georgiev and Samarina, 1971). Almost all nuclear dRNA may be recovered in the latter type of particles. They may be easily extracted from the isolated cell nuclei and purified by ultracentrifugation in sucrose density gradients. This allows a detailed study of the structure of the complexes. It was shown that the nuclear particle consists of a long dRNA chain which is combined with specific protein macroglobular particles or informofers (Samarina et al., 1968). The informofers are aggregates of relatively high molecular weight (1 x loGto 2 x loo) consisting of a number of subunits. Such large complexes corresponding to native structures may be isolated by using the RNase inhibitor from rat liver supernatant during the extraction. However, if the RNase inhibitor is omitted from the extraction solution or a low dose of RNase is added, large complexes are degraded and radioactive material is converted quantitatively into 30 S particles. These particles correspond to monomers of the above-mentioned giant polysomelike particles ; they are the complexes of single informofers and dRNA chains about 2 X lo5 daltons (Samarina et al., 1968). The treatment of 30 S particles with 2 M NaCl leads to the removal of dRNA without destruction of informofers. The latter are discovered in the form of high molecular weight protein particles (Lukanidin et al., 1971a) ; 30 S particles were dissociated by urea into RNA and protein subunits, and the latter were analyzed by means of polyacrylamide gel electrophoresis. If S-S bonds were reduced with mercaptoethanol, only one kind of polypeptide chain was found. The molecu-

1.

TRANSCRIPTIONAL UNITS I N EUKARI'OTIC

CELLS

19

lar weight of the subunit is about 40,000 (Krichevskaya and Georgiev, 1969). Although a t this moment it is impossible to exclude that some minor protein components are also present in informofers, their content cannot be more than 5% of the total protein of the informofer. It is unclear now what is the relation of these nuclear particles containing dRNA to cytoplasmic particles-the informosomes. The solution of this question awaits the isolat,ion of cytoplasmic particles in a pure state. I n the case of polysomal mRNA protein complex, it was found that protein moiety is quite different from that of nuclear particles (Lukanidin et al., 1971b; Olsnes, 1971; Morel e t al., 1971). It is important to note that nuclear complexes containing dRNA consist of informofers and dRNA only. These complexes do not contain DNA. No ribosomes were found. Moreover, attempts to obtain complexes between ribosomes and nuclear particles or to induce protein synthesis with the particles were unsuccessful, although the RNA isolated from the particles stimulated amino acid incorporation in cell-free systems (Samarina et al., 1967a,b; Lukanidin, 1969). This means that the nuclear complexes are not engaged in the protein synthesis and dRNA combined with informofers is not translated. To analyze the question about the fate and the role of different dRNA classes in the nucleus, one should know which kinds of dRNA are combined with informofers. As was pointed out above, almost all nuclear dRNA ( 2 8 0 % ) may be recovered in the form of complexes with informofers. Moreover, by incubating nuclei a t 37OC it is possible to extract practically all dRNA from the nuclei, and this dRNA is again combined with informofers only (Lukanidin, 1969). I n other experiments RNA was isolated from the nuclear complexes, from total nuclei, and from the cytoplasm and studied by competition-hybridization experiments. The complete inhibition of hybridization by excess of competitor RNA was achieved in the case of the following pairs:'RNA of nuclear complexes (competitor) against total nuclear dRNA (Iabeled) or against cytoplasmic RNA; total nuclear dRNA against dRNA of nuclear complexes; total RNA of the cell against RNA of nuclear complexes. On the other hand, in the pair cytoplasmic (or polysomal) RNA against RNA of nuclear complexes only partial competition (about 30-40%) was observed (Samarina et al., 1967a; Mantieva et al., 1969). Thus, both kinds of dRNA-dRNA, (a precursor of mRNA) and dRNA, (the unstable nuclear dRNA)-are complexed with informofers. It seems very probable that informofers are engaged in the removal of newly formed dRNA from the chromosomal template. There is indirect evidence in favor of this idea. In particular, it is possible to obtain the formation of similar particles in a cell-free system, using isolated

20

G. P. GEORGIEV

nuclei synthesizing RNA from labeled nucleoside triphosphates. All RNA liberated in the medium is combined with informofers (Georgiev and Samarina, 1969; Samarina et al., 1972). Thus immediately after it is formed, dRNA is combined with informofers. The next step is the processing of dRNA inside the cell nucleus. It is possible to follow the processing of the dRNA by studying distribution of nuclear complexes isolated in the presence of RNase inhibitor as there is proportionality between the size of dRNA and the size of complexes containing this dRNA. It was found that after pulse labeling most of the labeled material is recovered in a heavy zone that contains the complexes of 5-10 or more informofers. Such complexes contain RNA with molecular weights of about 1 to 2 X los or more. This figure is somewhat underestimated, as during the isolation of complexes even in the presence of RNase inhibitor some slight degradation takes place, and the very long dRNA chains are especially sensitive to it. However, after long-term labeling or after actinomycin treatment the complexes contain predominantly only 2-4 informofers corresponding to dRNA of molecular weight 0.4 to 0.8 X lo6 (Mantieva et al., 1969). Thus both the precursor and its product are combined with informofers, and processing occurs in the complex. dRNA, is broken down at this stage and is therefore probably not translated. The decay of dRNA, without being translated is one of the important conclusions from the analysis of dRNA transport. The final step of dRNA transport is the transfer of dRNA, to the nuclear membrane and then to the cytoplasm. Although the mechanism of this process is unclear, one can suggest that during this step dRNA remains complexed with informofers. This idea is supported by some electron microscopic observations (Monneron and Bernhard, 1969) indicating the very similar structure of dRNP particles in chromosomes, in nuclear sap, and near the nuclear membrane. Only during the passage through the nuclear membrane does their morphology change sharply. At the same time, electron microscopy confirmed the nonartificial nature of particles isolated in biochemical experiments. I n particular, Monneron and Bernhard (1969) and Monneron and Moule (1968) observed ribonucleoproteins as chains of globular particles 200 A in diameter, identical in morphology with the nuclear particles isolated by Samarina et al. (1968). All these data allow one to suggest that informofers combine with the growing dRNA chain, thus liberating the chain from the complex with template. The chains of informofers with dRNA distributed on their surface are formed. The RNA in this form may be easily attacked by processing enzymes : endo- and exonucleases destroying dRNA, sequences that are not translated. The particles containing mRNA are

1.

TRANSCRIPTIONAL UNITS I N EUKARYOTIC CELLS

21

transferred to the membrane, and then mRNA moves into the cytoplasm (Georgiev and Samarina, 1971). G. SOMEPECULIARITIES OF THE ORGANIZATION OF IN THE EUKARYOTIC CELLS

THE

GENOME

The genome of eukaryotic cells is distinguished from that of prokaryotes in two respects. First, eukaryotic genome is 100-1000 times larger than the prokaryotic and, second, it contains so-called reiterated DNA base sequences (Britten and Kohne, 1968). The existence of the latter was discovered in experiments on DNA renaturation (Britten and Kohne, 1968) and confirmed in DNA-RNA hybridization experiments (Ananieva et al., 1968; Church and McCarthy, 1968; Melli and Bishop, 1969). The fraction of RNA readily hybridized with DNA with a velocity much higher than could be expected. The rest of the RNA hybridized very slowly. T o characterize the degree of reiteration, Britten and Kohne (1968) proposed to use the value Cot (Co, original concentration of DNA; t, time of annealing in some standard conditions), which is necessary to . DNA of phage T4 renaturates to obtain 50% renaturation ( C o t % )Thus 50% a t Cot = 0.2 mole/liter x second; Escherichia coli DNA a t Cot = 10. Knowing the Cot, and the size of the genome, one can roughly estimate the number and the length of reiterated base sequences. The practical conclusion from these experiments is that, in usual conditions of RNADNA hybridization, only copies from reiterated base sequences are hybridized with DNA (Ananieva et al., 1968). To obtain the hybridization of RNA, synthesized on the unique DNA base sequences, one should use a very high concentration of RNA (or DNA), about 3-10 mg/ml, and/or long incubation time (weeks or months), Such techniques are elaborated now (Davidson and Hough, 1969), but they have not yet been widely used. It is well known that in almost all eukaryotes so-called nuclear satellite DNA’s are present. These are special DNA classes that can be separated from the rest of DNA (main component) by CsCl density gradient ultracentrifugation because of the difference in base composition. Satellites are very homogeneous in respect to base sequences. They comprise from 1% to 30% of the whole DNA and usually contain from lo5 to lo6 repetitive sequences per genome (Kit, 1961 ; Sueoka, 1961 ; Arrighi et al., 1970). The length of the repetitive sequence in satellite is of the order of lo2 to lo3 base pairs (Britten and Kohne, 1968) or even much lower (6-12) (Southern, 1970). Such sequences are collected in clusters with a molecular weight of several million, but these clusters are scattered throughout the genome (Maio and Shildkraut, 1967, 1969)

22

0. P. GEORGIEV

and concentrated mainly in centromere of chromosomes (Pardue and Gall, 1970; Jones, 1970). Owing to the very high degree of reiteration satellite renaturates very rapidly. Although being universal, the satellites even in closely related species are very different one from another. They are characterized by high species and even strain specificity (see, for example, Hennig et al., 1970). As far as is known, the satellite DNA is not transcribed (Flamm et al., 1969; Yasmineh and Yunis, 1969; Yunis and Yasmineh, 1970; Southern, 1970; Hennig et al., 1970). Besides satellite one can find another kind of reiterated base sequence-so-called “intermediate or kinetic fraction” (Britten and Kohne, 1968; Walker, 1971). These sequences are less multiple ( lo2 to lo4 repetitions per genome), and for this reason the Cotlx for them comprises 0.1 to 100 moles/liter x second 10-25% of the whole genome belongs to this fraction. The intermediate sequences are much more resistant in evolution, and closely related species, such as mouse and rat, contain many similar base sequences in this fraction, which leads to cross-renaturation and cross-hybridization. I n contrast to satellite, the intermediate fraction is effectively transcribed and the hybridizable dRNA mainly belongs to RNA synthesized on these repetitive DNA sequences. The significance of intermediate sequences is obscure. This fraction is probably nonhomogeneous. For example, some of it may correspond to repetitive operons such as operons for ribosonlal RNA synthesis, which are represented by about lo3 copies per genome. These are rather long, identical repetitive sequences, but they may explain the existence of only a small part of intermediate repetitions. The poor melting profiles indicate that most of the intermediate fraction renaturates imprecisely. This may be because the repetitions are rather short and the renatured molecules have long single-stranded ends. Another explanation is the imprecise character of pairing along the whole strand. Using the DNase SI which selectively destroys single-stranded parts of DNA, Sutton (1971) showed that the first suggestion is more likely. A significant part of the DNA was destroyed by enzyme from the renatured material, and the remaining “core fraction” possessed much more precise double helices. The length of such multiple sequences containing “core fraction” is not very high (probably 100400 nucleotides) . These sequences are also scattered throughout the whole genome. The in situ hybridization data indicate that they are not concentrated in centromere regions but localized in euchromatic parts of chromosomes also (Pardue and Gall, 1970; Jones and Robertson, 1970; Hennig et al., 1970). The possible role of intermediate repetitive sequences will be extensively discussed.

1.

TRANSCRIPTIONAL UNITS I N EUKARYOTIC CELLS

23

H. THESELECTIVE INHIBITION OF TRANSCRIPTION FROM REPETITIVE BASESEQUENCES IN THE CELL-FREE SYSTEM It is well known now t h a t chromosomal deoxyribonucleoprotein or chromatin is much less active as template than free D N A and that the histones, basic chromatin proteins are responsible for the inhibition of template activity (Huang and Bonner, 1962). The nature of this inhibition has been analyzed in the following way. RNA’s were synthesized with the aid of E . coli RNA polymerase on the D N A or D N P o (original, complete chromatin deoxyribonucleoprotein) templates from animal cells and then they were compared in hybridization and competitive hybridization experiments. It was found that in the presence of the excess of DNA the percentage of RNA hybridized is several times higher in the case of R N A synthesized on D N A (RNADNA) than of RNA synthesized on DNP, (RNADNPo)(Georgiev et al., 1966). As only those RNA’s which have been synthesized on repetitive D N A base sequences may hybridize with DNA in usual conditions, one can conclude that in D N P the synthesis of RNA on highly reiterated DNA base sequences is specifically inhibited (Ananieva et at., 1968). Limborska and Georgiev (1972) analyzed the hybridization Cot curves according to the Melli et al. (1971) technique and found that the difference between RNADNPo and RNADNA depends mainly on highly reiterated sequences. I t was also shown that RNA,,, saturates more DNA sequences than RNADNPo(Paul and Gilmour, 1966a; 1968; Bekhor et at., 1969). Moreover, in competition experiments the specificity of inhibition was demonstrated : nuclear R N A inhibited the hybridization of RNA synthesized on the DNP, from the same tissue more than of RNA transcribed from heterological DNP, or from free DNA (Paul and Gilmour, 1966b; Georgiev et al., 1966). I n other words, the restriction by the proteins of D N P complex possesses some specificity and reflects to some extent the in vivo restriction. Thus the proteins of D N P complex inhibit the RNA synthesis on certain DNA sequences, particularly on the reiterated sequences. The next question was which chromosomal proteins are responsible for the specific restriction of RNA synthesis. It was found that the removal of f l histone and some nonhistone proteins by 0.6 M NaCl extraction leads to the loss of the restriction (Georgiev et al., 1966; Smith, 1970). I n reconstruction experiments with D N P , treated with 0.6 M NaCl (DNP, G ) and proteins extracted from DNP, by 0.6 M NaC1, only histone f l possessed the inhibitory action of transcription (Juhasz et al., 1971). It was concluded that histone f l is probably responsible for the inhibition of the transcription from the repetitive base sequences.

24

G . P. GEORGIEV

However, the specificity of histone action on RNA synthesis depends probably on the interaction of nonhistone proteins with DNA. Histones themselves could hardly recognize specific base sequences. On the other hand, nonhistone proteins may interact with DNA much more specifically. Reconstruction experiments on D N P formed from DNA and proteins, and analysis of RNA transcribed from such D N P showed that the specific character of inhibition of transcription is completely lost after the removal of nonhistone proteins (Paul and Gilmour, 1968). They were thought to play a role of activators preventing inhibitory action of histones on certain sites of genome (Frenster, 1965; Paul and Gilmour, 1968). Some nonhistone proteins may also inhibit transcription directly, such as a recently isolated inhibitor of ribosomal RNA synthesis (Crippa, 1970). This question will be discussed. In general, one can suggest that the proteins of the D N P complex are engaged in regulation of transcription and that they specifically inhibit the transcription from the highly reiterated DNA base sequences. 111. Models of the Transcriptional Unit in Eukaryotic Cells To explain the above findings, different models have been suggested. The main question to be explained concerns the nature of dRNA, or dRNA that is degraded inside the cell nucleus.

A. CASCADE REGULATION HYPOTHESIS One of the first general models was described by Scherrer and Marcaud (1968) (Fig. 9 ) , who suggested that the transcriptional units are probably polycistronic (see also Samarina, 1964; Samarina et al., 1965b) and may contain genes that, are not connected functionally. Also these units may contain some nonfunctional sequences. As a result, the giant dRNA is formed during transcription. Only very crude regulation takes place a t this step, and newly formed RNA chains contain much more information than the cell needs. The second step of regulation of gene expression is the reduction of the information stream a t the level of dRNA transport, namely during the processing of dRNA. It was postulated that special recognizing proteins are engaged in the protection or destruction of certain dRNA sequences. The third step of restriction is the regulation a t the translational level. Thus this model postulated a complex transcriptional unit although it does not detail its structural organization. This model explains the presence of dRNA, and dRNA, sequences in the same giant precursor molecules. On the other hand, it predicts that certain RNA sequences degraded inside cell nucleus correspond to real mRNA’s and thus they may in some conditions reach the polysomes.

1.

25

TRANSCRIPTIONAL UNITS IN EUKARYOTIC CELLS

In favor of this prediction are results (Church and McCarthy, 1967; Church et al., 1969) indicating that in certain conditions the amount of RNA transferred from the nucleus to cytoplasm may be changed. For example, in regenerating liver and hepatomas the RNA sequences which belong to the class of dRNA2 in normal liver may be found in the cytoplasm. These results, however, are not yet quite conclusive as the leakage of RNA during the isolation of nuclei could not be excluded

A. Monocistronic

Level Transcription Intermediary or posttranscriptional regulation during metabolism and

Translation

8. Polycistronic

lNo sense;

--

Rejected; decays I*--

Regulatory fmtion; decoys

+

+++++t+++

Rejected;

+

1

Expressed +++++++Expressed+++++++

a

8

FIG.9. The model of the transcriptional unit according to Scherrer and Marcaud (1968). (A) Monocistronic transcriptional unit. The molecule contains only one cistron (a) which will be translated. The excess RNA contains no information but may have a structural or regulatory function prior to decay. (B) Polycistronic transcriptional unit. Several cistrons ((I, p, y, 6) linked together in the genome are transcribed into a single molecule. I t may correspond to an operon or to several independent cistrons, which become separated during metabolism. Some of these individual cistrons reach the polysomes and are translated independently ; some are immediately rejected and destroyed; and others are rejected and stored a t the intermediary level. The decay of the immediately rejected cistrons accounts for the observed nuclear turnover of dRNA. The polycistronic molecule may contain, in addition, sequences without structural information, such as those proposed for the monocistronic unit.

in the different kinds of cells, especially those with a high mitotic level. The main difficulty with this model is the necessity to postulate the presence of a number of recognizing proteins in the nucleus which determine whether a particular RNA molecule has to move to the cytoplasm or be destroyed in the nucleus. However, a t present only one kind of protein subunit has been found in purified nuclear d R N P complexes (see above). The presence of minor protein components (less than 5% of total protein) could not yet be excluded. In any case before this cascade hypothesis can be accepted, more positive data must be obtained.

2i3

O . P. OEORGIEV

B. THEACTIVAT~R RNA MODEL Another idea is that the dRNA, regulates transcription directly by combining with DNA or indirectly by participation in the synthesis of nuclear proteins. This suggestion has been made by many authors (for review, see Georgiev, 1967), but was presented in detail only reS ! j [

-

Integrator gene

Activator

UL-

RNA-

I : i --

Receptor T

- _ I genes

P roducer gene

FIO. 10. Examples of organization of transcriptional units according to Britten and Davidson (1969). (A) Integrative system depending on redundancy among the regulator genes. (B) Integrative system depending on redundancy among the integrator genes. These diagrams schematize the events that occur after the three sensor genes have initiated transcription of their integrator genes. Activator RNA's diffuse (symbolized by dashed line) from their sites of synthesis-the integrator genes-to receptor genes. The formation of a complex between them leads to active transcription of the producer PA,Pn,and PP.

cently by Britten and Davidson (1969) (Fig. 10). The latter postulated the presence of two types of transcriptional units, or operons. The operons of the first type consist of sensor genes, recognized by repressor or derepressor proteins and integrator genes, producing activator RNA. The operons of the second type contain receptor genes interacting with

1.

TRANSCRIPTIONAL UNI T S I N EUKARTOTIC CELLS

27

activator RNA (or protein synthesized on the template of activator RNA) and producer genes which encode different proteins. Many of the integrator and receptor genes are multiple, and this explains why the same signal can produce a massive effect on the transcription. Thus Britten’s model explains the role of repetitive DNA base sequences scattered throughout the genome. However, no attempts to check cxperimentally the predictions from the model have been made. The main difficulty this hypothesis met is the absence of data showing the existence of activator RNA. The authors identified dRNA, with the latter. As was shown above, dRNA, is not translated before degradation. On the other hand, it does not combine with DNA as may be suggested by a direct involvement of dRNA, in the regulation of transcription. Practically all dRNA, is complexed with informofers during its whole life. Thus the data on dRNA transport do not agree with the model, although some modification may allow one to fit the theoretical scheme to the experimental results.

C. THEAUTHOR’SMODELOF OPERONSTRUCTURE IN EUKARTOTES At the same time as Britten and Davidson’s (1969) model, another model was described (Georgiev, 1969). It will be discussed in more detail, since it allows one to test predictions experimentally and some results have been obtained recently in this direction. 1. Description of the Hypothesis

The following organization of the operon in cells of higher organisms is postulated (Fig. 11). a. Each operon consists of two main zones: acceptor or noninformative zone and structural or informative zone. The first is larger than the second. The structural zone contains cistrons carrying the information for the synthesis of proteins. These may be structural proteins, enzymes (structural genes), or regulatory proteins (regulatory genes). The acceptor zone contains loci (sites) which do not carry any information for protein synthesis but may be recognized by different specific proteins : enzymes, repressors or derepressors, and structural proteins of the D N P complex. Thus the acceptor sites correspond to the promoter (recognized by RNA polymerase) , to operators (recognized by repressors and derepressors), and probably to some parts of the DNA involved in the specific interaction with structural proteins of chromatin. b. Acceptor loci (all or most of them) are localized in the initial

28

G. P. QEORGIEV

or promoter-proximal part; the structural of informative loci being located in the tail (or distal) part of the operon. c. Most of the acceptor loci correspond to operators, ie., sites of the genome interacting with regulatory proteins. I n bacteria the acceptor noninformative zone is very small and usually consists of one promoter and one operator, both occupying only about 100 base pairs. I n higher organisms, the acceptor zone is very large and contains a number of different operators, interacting with different regulatory proteins. On the other hand, many acceptor loci from different operons are identical or very similar; i.e., there is a multiplicity of acceptor loci. Acceptor

Structural m e

ZOne

._ 6 c

-

i

I-

Ribosomes ------ _________ 4’

‘\.--_-_-- 5’p +

mRNA

3‘4H

FIG.l l . Model of transcriptional unit according to Georgiev (1969). p , promoter; a, . . . am,acceptor sites; ar, at, other service sequences, 8, . . . S, structural genes, m > 1 < n .

The most reiterated of these are localized closer to the promoter (Fig. 11). As a result of such organization of the operon each act of dRNA synthesis begins with the transcription of the acceptor zone. Only after completion may the structural cistrons be transcribed. Each newly formed dRNA contains sites complementary to the acceptor sites grouped near 5’ end of the molecule and sites complementary to the structural cistrons grouped near the 3’ end of the molecule. Then, the noninformative part of dRNA (the 5’ part of molecule) is degraded, the informative (3’ part) being conserved and transferred into the cytoplasm. The information for the destruction of the noninformative part of dRNA should also be coded in RNA. For example, some specific base sequences are sensitive or nonsensitive to the action of corresponding exo- and endo-

1.

TRANSCRIPTIONAL UNITS I N EUKARYOTIC CELLS

29

nucleases, participating in the degradation and cleavage of dRNA molecules. Such is the transcription in the absence of inhibiting factors. However, as was mentioned above, acceptor loci may interact with inhibitory proteins of chromosomes. This interacting prevents the movement of RNA polymerase along the DNA strand and thus either switches off the distal parts of the operon from transcription or decreases the rate of their transcription. It is obvious that the transcription of the whole informative part of the operon will then be blocked or inhibited. Since there are several different acceptor loci in the operon, different regulatory proteins may influence the rate of transcription of the same operon. In other words, the activity of every structural gene is under the control of many different regulatory agents reacting with different acceptor sites. On the other hand, several of the acceptor loci are localized a t the proximal ends of more than one operon; they are thus multiple and are scattered over various parts of the genome. Therefore the same regulatory protein may influence the transcription of a number of different operons. I n the discussion of the role of acceptor loci, one can consider two regulatory schemes: negative or positive. According t o the negative concept, the regulatory protein which can recognize a DNA base sequence is the repressor. According to the positive concept, the proteins recognizing a DNA base sequence play the role of derepressors which remove the inhibitory action of histones in RNA synthesis. I n higher organisms there are no conclusive facts proving either of these two possibilities, but in either case the main statements of the hypothesis remain unchanged. 2. Explanation of the Above-Mentioned Facts in the Light

of the Hypothesis

The suggested hypothesis makes it possible to give a satisfactory explanation of all the facts mentioned in the first part of this paper. The large size of newly formed dRNA is the result of the transcription of the whole operon including its noninformative acceptor part. The presence of two functional classes of dRNA may be explained by the assumption that dRNA, or dRNA restricted to the cell nucleus is the copy of the acceptor sites. On the other hand, dRNA, or the real messenger RNA, is the copy of the informative part of the operon. As the cleavage of giant precursor molecules and the degradation of dRNA, proceeds in the nucleus, it is clear that relatively short chains of dRNA accumulated after long-term labeling should contain a higher percentage

30

G. P. GEORGIEV

of dRNA, than the original giant molecules. This was actually observed in experiments of Arion and Georgiev (1967). The presence of both kinds of RNA in the same molecule is similarly found in the case of a newly formed rRNA which contains the cytoplasmic ribosomal RNA sequences as well as unstable nuclear rRNA. Thus the ribosomal operon is not an exception to the general rule. The hypothesis explains the appearance of dRNA, only in the nuclear complexes with informofers, but not with ribosomes. It is clear that the noninformative part of dRNA should not be translated a t all. According to recent data, informofers contain specific RNase (Niessing and Sekeris, 1970) which may participate in the cleavage and degradation of dRNA,. After the degradation of dRNA,, dRNA, is transferred from informofers to ribosomes. The hypothesis also explains the existence of repeated DNA base sequences (“intermediate fraction”) scattered throughout the genome. Certain parts of them may he regarded as multiple acceptor loci, located in the initial parts of different operons. It is impossible to ascribe this function to all of the repeated DNA. It was mentioned above that the hybridization reaction in the case of nucleic acids from higher organisms recovers only those RNA species which are synthesized on reiterated DNA base sequences. Therefore the hybridizability of RNA indicates that part of a given RNA which is transcribed from these sequences. It was demonstrated (see above) that the hybridizability of dRNA, is greater than the hybridizability of dRNA,. This is in line with the suggestion that many reiterated base sequences predominantly correspond to noninformative acceptor loci. I n conclusion the hypothesis explains the specific inhibition of transcription from reiterated DNA sequences by proteins of the D N P complex. If inhibitory proteins combine with acceptor sites, a significant part of which are represented by multiple base sequences, then the latter will be switched off. On the other hand, owing to the nonspecific binding of RNA polymerase in vitro, this blockage of acceptor sites may not prevent the transcription of structural genes in vitro. 3. Conclusions and Predictions That Follow from the Hypothesis and Approaches for Checking T h e m

The main statements of the hypothesis-( 1) the proximal localization of noninformative (acceptor) sites and distal localization of the structural genes; and (2) the identity of many acceptor sites, localized in different operons-may be checked experimentally. It is evident that the base sequence of the whole operon is represented in the newly formed dRNA before the degradation of its noninformative part.

1.

TRANSCRIPTIONAL UNITS I N EUKARYOTIC CELLS

31

Therefore such dRNA may be used for the analysis of the operon structure. For this purpose one must have techniques for the study of different parts of nuclear dRNA precursor molecules, namely its initial part (5’ end) and end part (3’ end). Different approaches may be used. a. Ultraviolet Radiation Technique. One can suggest that by producing obstacles on the way of RNA polymerase along the DNA strand, uncompleted RNA chains may be formed. These chains should be shorter and they should not contain the 3’ end part of the normal molecules. The condition necessary for the realization of this situation is the absence of interference of the inhibitory agent with the normal initiation process.

400

E, 2; c ._ > ._ t

a

200

g c?

Fraction No.

FIG. 12. The influence of UV preirradiation on the properties of newly formed nuclear dRNA (sedimentation and hybridizatdity) (Mmtieva and Arion, 1969). I, Control; 11, UV radiation. Base composition ( G C/A U) : I. 0.89; 11, 0.79. RNA hybridized to DNA excess at low Cot vnlue (7% of input) : 1-3.274; II--12.1%.

+

+

Abnormal initiation as well as reinitiation after the obstacle should be excluded. These requirements limit to some extent the validity of inhibitory experiments, making them indirect, although some preliminary information may be obtained from them. Different agents inhibiting RNA synthesis may be used. Mantieva and Arion (1969) studied the influence of UV preirradiation on the properties of rapidly labeled RNA of Elirlich ascites carcinoma cells. At the dose of UV radiation used, the 30-60 minute incorporation of precursor into nuclear dRNA and rRNA decreased about 3-fold. The average molecular weight of RNA synthesized after UV radiation was also about three times less than that of RNA synthesized in normal conditions (Fig. 12). Thus as a result of damage to DNA (thymine

32

G. P. GEORGIEV

dimers?) produced by UV radiation, shorter, presumably unfinished, RNA chains were formed. It is interesting that similar results have been obtained with bacterial cells in vitro as well as in vivo (Starlinger and Kolsch, 1964; Michalke and Bremer, 1969). Thus the above-mentioned interpretation seems to be very probable. Then the hybridization properties of RNA’s were studied. dRNA synthesized after irradiation hybridized with an excess of DNA two to three times more effectively than dRNA from untreated cells (Fig. 12). This means that after UV-radiation RNA containing more replica from the reiterated base sequences is synthesized. Ifl after irradiation, chains lacking the 3’ end accumulated, one can conclude that the main part of replica from reiterated DNA is localized in the 5’ end of dRNA. This is one of the main postulates of the model. I n principle, many different inhibitors of RNA synthesis can be used for this kind of analysis. One interesting inhibitor is cordycepin or 3‘-deoxyadenosine, which may incorporate into the growing RNA chain and abrupt it. Penman et al. (1970) observed that cordycepin inhibits the incorporation in mRNA much more effectively than in nuclear dRNA, suggesting the independent synthesis of these two RNA classes. However, this result may be easily explained if one assumes the localiaation of mRNA near 3’ end of giant dRNA. Moreover, recently Darneli et al. (1971) found that low doses of cordycepin selectively inhibit the formation of poly (A), which is engaged in the transport of mRNA from the nucleus to the cytoplasm (see below). This fact restricts the employment of cordycepin in the analysis of dRNA structure. b. Exonuclease Digestion, Theoretically exonuclease digestion of dRNA is an ideal technique for the analysis of the properties of 5’ and 3’ end sequences. There are exonucleases attacking RNA from the 3’ end [for example, snake venome enzyme and actinomyces exonuclease, obtained by Tatarskaya et al. (1970) ] and the exonucleases digesting RNA from 5‘ end [the best one is the spleen exonuclease described and purified by Bernardi and Bernardi (1966) 1, Unfortunately, all the exonucleases (even if highly purified by all available methods) are contaminated with traces of endonuclease activity. Only rare preparations obtained occasionally from R. I. Tatarskaya produced less than 1 endonuclease break per 1000 exonuclease breaks. These samples were used to digest giant dRNA from Ehrlich ascites carcinoma cells. The digestion of one-half to three-fourths of the dRNA resulted in 2- to 3-fold increase of hybridizability of dRNA, supporting the idea that the 5’-termini of dRNA are enriched with copies of reiterated DNA.

1.

33

TRANSCRIPTIONAL UNITS I N EUKARYOTIC CELLS

If exonuclease is more contaminated this result could not be reproduced. For this reason, the exonuclease approach has not as yet provided conclusive results. c. 5’ End Analysis. The most direct results have been obtained with the aid of end-group labeling techniques. As mentioned above, newly formed giant dRNA contains triphosphate groups a t the 5’ end, which may be detected as tetraphosphates after the alkaline hydrolysis of RNA. Triphosphorylated 5’ ends may be considered as markers of true starting points of operons as only the first nucleotide conserves its 7 and P-phosphates (Maitra and Hurwitz, 1965). For this reason, the hybridization analysis of the RNA sequences neighboring to triphosphate 5’ ends allows one to make conclusions about the properties of the initial parts of operons. TABLE I1 HYBRIDIZATION PROPERTIES OF 5’ END SEQUENCES O F HEAVY NUCLEAR dRNA“ ~

~

Percent of total s*P

Hybridization percent

Expt. no.

RNA fraction

In pppxp ppXp

In pXp

Total RNA

1

Hybridized Nonhybridized Hybridized Nonhybridized

0.118 0.027 0.062 0.018

0.027 0.021 0.03.5 0.07

11 7 -

2

+

PPPXP 36 -

20.5 -

From Ryskov et al. (1971).

Giant nuclear dRNA was hybridized with excess of DNA, and after alkaline hydrolysis the content of tetraphosphates in hybrid and in nonhybridized material was determined according to a modified Roblin (1968) technique (Table 11). In the hybrid it was 4 to 10 times higher, depending on the conditions of hybridization. I n some experiments the binding of 50% of pppX ends was achieved. Thus the sequences localized near the original 5’ ends of nascent dRNA are reiterated and probably similar in different operons. On the other hand, the concentration of nionophosphorylated 5’ ends in hybridized and nonhybridized RNA’s are roughly the same. This means that a t least B significant part of them are not formed from the triphosphorylated ends by dephosphorylation but are produced as a result of exo- or endonuclease attack during the processing of dRNA (Ryskov et at., 1971; Georgiev et al., 1972a1.

34

G. P. GEORGIEV

Thus the results of the hybridization of 5’ end sequences are in good agreement with the UV-block experiments and give a direct evidence of the high reiteration of initial sequences in different operons. The important question now under investigation is whether polysomal dRNA does or does not compete with 5‘ end sequences, which would indicate whether these sequences contain real mRNA. The preliminary data indicate the absence of such competition in agreement with predictions from the model. d. 3‘ End Analysis. More results have been obtained with 3’ end analysis, as the 3’ end may be easily labeled chemically, for example, by the periodate oxidati~n-borohydride-~Hreduction technique (Leppla TABLE 111

BASECOMPOSITION OF 3’ ENDSI N NUCLEAR dRNAa Nuclear dRNA Material Rat liver

Ehrlich ascites carcinoma

a

fractions Total Heavy Intermediate Light Heavy, hybridized Heavy, nonhyhridized Light, hybridized Light, nonhybridized

End nucleoside (% of total 3’-ends) A

U

G+C

70

20 13 14 19 19 15 17

4

71 77 68 65 62 75 80

12

+

6 18 9 13 16* 23b 8 8

From Coutelle et al. (1971).

* These figures may be overestimated. et al., 1968). As the reaction is not quite specific, it is necessary before counting to hydrolyze RNA with alkali, to separate the nucleosides and nucleotides by Dowex 1 chromatography, and to isolate individual nucleosides by paper chromatography (Fink and Adams, 1966). At first it was found that in both rat liver and Ehrlich carcinoma cells dRNA, the 3’ end nucleoside is predominantly (about 75%) represented by adenosine. About 20% was uridine and ~510% guanosine+ cytidine (Coutelle et al., 1970; see also Tamaoki and Lane, 1967). The activity of two latter nucleosides may depend on some contamination as the figures are low. It is important to note that such a distribution is typical of all fractions of nuclear dRNA: heavy (235S), intermediate (20-30 S))and light (12-20 S) (see Fig. 4, Table 111). Recently, it was found that about a quarter of dRNA chains have a short polyadenylic sequence a t its 3’ end. Most of them consist of

1.

35

TRANSCRIPTIONAL U N I T S I N E U K A R T O T I C CELLS

six adenylate residues. Again such ends are present in all kinds of dRNA, either heavy or light (Ryskov et al., 1972b). Similarly Barr and Lingrel (1971) described short poly(A) of the same size a t the 3’ end of hemoglobin mRNA. These facts indicate the conservation of 3’ end sequence during the processing of giant dRNA in the cell nucleus and probably during the transfer into the cytoplasm. Then the hybridization properties of 3’ end sequences (3H labeled in the end nucleoside) were compared with those of the whole molecule (randomly labeled with ‘T or 3?1))(Table I V ) . It was observed that the hybridizability of 3’ end sequences is two to three times higher than that of total dRNA both in the giant and TABLE IV COMPETITION OF POLYSOMAL mRNA WITH NUCLEAR dRNA’s LABELED W I T H 14C RANDOMLY A N D 3H I N 3‘ E N D ”

Source of RNA Ehrlich Carcinoma Rat

RNA fraction Heavy ( > 3 5 S ) Light (-18 S) Heavy ( 2 3 5 s )

Liver

Light (-18 S)

Polysoma1 RNA added -

+ + + +

111 hybrid 0.44 1.25

In nonhYbridixed RNA 1.8 -

0.08 0.56 0.14 21.0 52.0 32.6 2.8 6.6 3.1 -

Hybridization

(%I “C

3H

5 . 2 18.3 4.7 Tj.7 3 . 2 19.2 2.5 8.8 12.7 26.5 12. 8 17. 2 11.3 2 2 . 9 7 . 2 13.3

Competition (%) 14C

3H

-

-

-10

-69

-

-

-22

--54

0 -37

-3.5 -42

From Coutelle et a / . (1971).

light dRNA. Thus not only 5’ end, but also 3’ ends, of nuclear dRNA molecules contain copies of repetitive D N A and probably are similar in different dRNA’s. I n competition experiments between polysomal RNA (which contains true mRNA) and giant nuclear dRNA, the former inhibited the binding of 3’ end sequences much more than the binding of total RNA. Thus 3’ ends contain the sequences transferred into the cytoplasm to polysomes and probably corresponding to mRNA. The competitive inhibition of binding of the 3’ end sequences of giant dRNA led to only slight inhibition of overall hybridization. One can conclude that the readily hybridizable 3‘ end sequences correspond

36

G.

P. GEORGIEV

to only a small part of all hybridizible sequences in the molecule and are probably rather short. On the other hand, in low molecular weight dRNA the inhibition of 3’ end hybridization by polysomal RNA is of the same order as that of total RNA. This may indicate that the 3’ end sequence comprises a significant part of hybridizable material in this kind of dRNA. In other words, 5‘ end reiterated sequences probably disappear during the formation of low molecular weight RNA molecules (Coutelle et al., 1970, 1971; Georgiev et al., 1972a). What is the nature of reiterated sequences localized a t the 3’ ends of different operons? The question is open now. One can speculate that they are termination sequences of transcription and/or translation or some other kind of ancillary sequence. It is interesting that cytoplasmic mRNA probably contains some sequences that are not engaged directly in the translation. For example, the length of hemoglobin mRNA is somewhat higher than that calculated on the basis of a number of amino acids in the polypeptide chain (Williamson et al., 1971). The last sequence in this mRNA consists of 5-6 adenylic acids (Barr and Lingrel, 1971). At the same time hemoglobin does not contain amino acids encoded by AAA (lysine) a t the carboxyl end. It is also shown that purified mRNA can to some extent be hybridized with homologous DNA in conditions where only copies of reiterated DNA sequences may hybridize. For example, hemoglobin mRNA saturates about 0.5% of the total genome (R. Williamson et al., 1970) although the number of hemoglobin genes in genome is less than 10 (Bishop et al., 1972). On the other hand, these hybrids are not very exact, as shown by the melting curves. If the true structural genes were reiterated, the hybrids should be perfect. All these data may be easily explained if similar short sequences are present a t the ends of different genes, if they are transcribed in newly formed giant dRNA, and then conserved during the processing and transfer with mRNA to polysomes. At which step of the gene expression such sequences play a functional role is not yet known. One can conclude that the 5’ end and 3’ end studies gave support to at least two main statements of the model: the presence of reiterated DNA base sequences in promoter-proximal parts of operons, and the localization of structural genes near the ends of the operons. They also discovered the presence of short ancillary sequences a t the end of the operon. It is clear that many other questions may be studied with the aid of end-group labeling techniques combined with hybridization and structural analysis.

1.

TRANSCRIPTIONAL UNITS IN EUKARYOTIC CELLS

37

e. T h e Analysis of Certain Specific Base Sequences in Different dRNA’s and m R N A . It follows from my model as well as from Britten and Davidson’s that certain base sequences may occur in many different dRNA’s, and this allows one to detect them in the very heterogeneous population of dRNA’s. These sequences may serve as chemical markers in the analysis of dRNA structure. One such specific sequence is the long polyadenylate discovered in both the mRNA (Lim and Canellakis, 1970) and the nuclear dRNA (Edmonds and Caramela, 1969). These poly (A) sequences 100-200 nucleotides long are covalently bound to mRNA and nuclear dRNA. Almost every mRNA chain contains poly (A) sequence. The poly (A) content in mRNA comprises about 5-870, in nuclear dRNA only about 0.5% (Edmonds et al., 1971; Lee et al., 1971; Darnel1 et al., 1971). Several authors using different experimental approaches localized long poly(A) stretches a t the 3’ ends of mRNA and of nuclear dRNA. One of the evidence is the isolation of adenosine from the alkaline hydrolyzate of poly(A). Another is the greater susceptibility of poly(A) to exonucleases attacking 3’ end of KNA (hfendecki et al., 1972; R . P. Perry, personal communication). Ryskov et al. (1972a) looked a t the nature of the ends in poly(A) isolated from light nuclear dRNA. It was found that probably every poly(A) contains pAp groups a t 5’ end, and about a quarter of light nuclear dRNA from Ehrlich ascites carcinoma cells are started with a long poly(A) sequence. On the other hand, no 3‘ end nucleoside [labeled with 3H before poly(A) isolation from RNA]) were found in poly(A). Thus one can conclude that poly(A) sequences are localized a t the 5’ end of short dRNA’s, and possibly of mRNA which is originated from the former. However, this observation is in controversion with other evidences in the same field. Philipson et al. (1971) obtained strong evidence that the poly(A) sequences are added to nuclear dRNA after the completion of its synthesis. In particular they showed that adenovirus-specific RNA produced in the infected cells contain poly(A) although the genome of virus does not contain the corresponding template. Thus poly(A) sequences synthesized on the host template are added to the virus-specific RNA. This addition is probably very important for the mRNA transfer into the cytoplasm as the inhibition of poly(A) synthesis by cordycepin inhibits the transport of virus-specific RNA to polysomes (Philipson et al., 1971). The independent synthesis of poly(A) from the rest of nuclear dRNA was also demonstrated by V. L. Rlantieva (in preparation), who found the insensitivity of poly ( A ) synthesis to UV irradiation, The doses of UV

38

G. P. GEORGIEV

producing 2- to 3-fold inhibition of dRNA synthesis do not influence or even slightly increase the incorporation into poly(A). Also the data on distribution of poly(A) among nuclear dRNA’s of different sizes demonstrate the very low content of poly (A) in dRNA sedimenting a t 2 3 5 S. Thus the addition proba‘bly takes place after the endonuclease break of dRNA. Another approach is the study of the base composition of hybridizable sequences of dRNA. It was found that the hybrids isolated from cytoplasmic mRNA or light nuclear dRNA contain a high percentage of adenylic acid (Schemer, 1971; Besson et al., 1972). This may depend on the presence of above-mentioned poly(A) in these RNA’s. On the other hand, hybrids from giant dRNA are rich in both adenylic and guanylic acids (Besson et al., 1972). Thus G-rich hybridizable sequences disappear from dRNA in the course of processing, and they may be used as markers of nucleus-restricted dRNA sequences. Finally, Ryskov et al. (1972~)and Jelinek & Darnell (1972) found the special RNAse stable sequences which are present exclusively in giant nuclear dRNA. These sequences have roughly the same size, as long poly(A) sequences, but they are different from the latter in many respects. They are not combined with poly U a t 5OoC, do not stick to the nitrocellulose filters, have the symmetrical base composition, and give irregular oligonucleotide patterns after complete RNAse hydrolysis at low ionic strength. These sequences probably contain double-helical hairpinlike structures. In the course of dRNA processing they are destroyed. It is interesting that after the RNAse treatment and following melting, these sequences hybridize very effectively to DNA. This indicates their origin from highly reiterative DNA sequences. In general, this line of study is only beginning now, but it may give valuable information in the future. f. On the Structure of Ribosomal Operon. It is rather difficult to analyze the structure of transcriptional units for dRNA synthesis as the dRNA is a heterogeneous population of molecules. On the other hand, the ribosomal 45 S precursor is a very convenient material for analysis. The only problem is that in these two cases the principles of organization may be different. It is known that the rRNA is synthesized by RNA polymerase I which is different from that participating in dRNA formation (RNA polymerase 11) (Chambon et al., 1970). On the other hand, in rRNA processing the methylation reaction is involved whereas the dRNA is not methylated. The careful study of molecular weight and base composition of intermediates in rRNA processing allowed Weinberg and Penman (1970)

1.

TRANSCRIPTIONAL UNITS I N EUICARTOTIC CELLS

39

to localize the 28 S, 18 S, and nucleus-restricted sequences of 45 S rRNA precursor in the following way: X

188

X

28s

Then it was observed (Penman e t al., 1970) that the formation of 28 S rRNA is much more sensitive to cordycepin than the synthesis of 18 S rRNA, indicating that 28 S rRNA sequence is localized more distal to 18 S rRNA sequence. Reder and Brown (1970) made the same conclusion on the basis of studies of R operon transcription by RNA polymerase in cell-free system. The synthesis of 18 S rRNA preceded 28 S rRNA formation. Mantieva and Arion (1969) examined the hybridization properties of rRNA synthesized after UV irradiation of the cells. It was found that the unfinished chains of rRNA formed in these conditions hybridized with DNA more efficiently than usual 45 S rRNA. As the hybridization was performed in the presence of the nonlabeled 28 S and 18 S rRNA, only nucleus-restricted labeled sequences could hybridize. Therefore one can conclude that the nucleus-restricted sequences are mainly localized near the 5’ end of the precursor. This result is in a good agreement with the above-mentioned scheme (Weinberg and Penman, 1970). The latter is compatible with the model, as the noninformative part of RNA in mainly localized in the 5’ end part of the molecule. Quite the opposite conclusion was drawn by Choi and Busch (1970) who found that 45 S rRNA, 32 S rRNA, and 28 S rRNA have the same 5’ end base sequence. They isolated from the alkaline hydrolyzates of all these rRNA’s alkaline stable pCpUp. The conclusion has been drawn that 28 S rRNA is localized a t 5‘ end of 45 S precursor. New experiments are necessary to explain this difference. They will provide us with the more precise structure of one kind of operoii in mammalian cells.

D. TANDEM REPETITIONS IN

THE

GENOME A N D THEIR POSSIBLE ROLE

There are also some Observations that could not be easily fitted to the model. These observations indicate the possibility that some structural genes in eukaryotic cells are reiterated. On the basis of the autoradiographic experiments with lampbrush chromosomes, Callan (1967) suggested that the genes in eukaryotic cells are multiplied several times, being arranged linearly and covalently bound. Each loop of the lampbrush chromosome represents a battery of identical genes. It was postulated (Callan, 1967; Whitehouse, 1967)

40

G . P. GEORGIEV

that a family of identical genes contains one master gene and a number of slave genes. Only the master gene is engaged in recombination during meiosis. After this a special correction process takes place which consists of changing of sequences in slaves to make them identical to master sequences. The main difficulty with this model is that the correction process is rather speculative. It is interesting that the loops correspond to giant transcriptional units, as was shown recently by electron microscopic analysis (Miller and Beatty, 1969). For this reason the analysis of the loop structure is very important for understanding the operon structure. Callan’s data on the identity of genes in the loops are indirect. However, recently they received some support from experiments by Thomas et al. (1970),who demonstrated the tandem organization of the eukaryotic genome with the aid of electron microscopy. The authors digested the opposite ends of sheared DNA with the aid of 3’- or 5’-exonuclease and then annealed the samples containing the single-stranded ends. I n a very high percentage (up to 35%) of strands, circular or more complex closed structures were formed. Thus the ends of DNA strands contain complementary sequences. As the shearing is random, one could conclude that DNA strands consist of several identical pieces arranged one after another. The length of DNA in rings varies in the range of 1-10 p that corresponds to molecular weight of (2-20) x lo6 daltons. The authors calculated that about 70% of Necturus genome are represented by such tandems. As the repetitive sequences (satellites intermediate fraction) correspond to a smaller amount of genetic material, one can conclude that so-called unique DNA sequences also represented by families of rather limited number of members, as postulated by the “master and slaves” hypothesis. It is not yet clear whether the rings found by Thomas correspond to transcribed DNA of the genome. Even if this is so, it remains unclear whether the repeated sequences correspond to independent transcriptional units or to structural genes of the same transcriptional unit (as suggested by master-slaves models) . The classical example of tandemly repetitive genetic elements is the multiple genes for ribosomal RNA synthesis collected together and arranged linearly one after another (with the spacer sequences which separate them) in the nuclear organizer region of chromosomes (Birnstiel et al., 1968; Miller and Beatty, 1969). In this case, one deals with repetition of transcriptional units, each of which contains only one pair of ribosomal genes. It is interesting that according to the hybridization test different ribosomal operons have very similar, perhaps identical, base sequences. The mechanism which maintains their identity in evolution is completely unclear.

+

1.

TRANSCRIPTIONAL UNITS I N EUKARTOTIC CELLS

41

Another example of repeated true genes is the cistrons responsible for histone synthesis which are repeated about 100 times per genome (in the case of the frog) (Kedes and Birnstiel, 1971). I n this case it is yet unclear whether a number of identical structural genes for histones are combined in the same transcriptional units or the whole operon is a repetitive unit, as in the case of ribosomal operons. Recently Karavanov and Jordansky (1971 ) found that Cot values for half-renaturation of the “unique DNA sequences” isolated from Viba faba and V . sativa are the same. However, the size of genome of V . faba is 6 times higher than that of V . sativa. The fraction of unique sequences in both cases comprise about 65% of total DNA. This means that a t least in the case of V . faba most of so-called “unique sequences” are actually repeated several times (on average 6 times more than in the case of V . sativa). These species are closely related, and the result obtained may be explained by the assumption that in V . faba the tandems contain 6 times more repetitive units than in V . sativa. Again in this case, it is not clear which kind of genetic elements is repeated. The only indication of the possible presence of several identical genes in the same transcriptional unit derives from the data of Daneholt et al. (1969a), who studied the properties of RNA isolated from different parts of salivary gland cells of Drosophila and Chironomus larvae after fixation and dissection. It is known that the DNA of Balbiani rings encodes the formation of salivary proteins (Beermann, 1965). Each Balbiani ring is engaged probably in the formation of messengers for one or two salivary proteins (Grossbach, 1969). It was observed that the newly formed dRNA isolated from the individual Balbiani ring is heterogeneous. In contrast to data obtained with other cells, the cytoplasm of salivary glands contained dRNA also of rather high molecular weight with sedimentation properties similar to those of nuclear dRNA (Daneholt et al., 1969a). Further, it was found that dRNA isolated from individual Balbiani ring may have different sedimentation patterns in different experiments. The higher the level of RNA synthesis, the heavier the product of transcription. Thus the same locus may produce RNA’s of different size (Daneholt et al., 1969b). The authors suggested that the structural genes for salivary proteins are repetitive, and, depending on some regulatory factors, a different number of these genes may be transcribed as a single polycistronic mRNA. This RNA would be transferred into the cytoplasm without breaking. However, some other explanations are not excluded. I n support of the presence of some repetitive sequences in the same transcriptional unit is also the finding of rather short circles in Thomas’

42

G. P. GEORGIEV

experiments. These conclusions, if they are true, a t first seem to be in contradiction with the model, but some modifications can take account of these new experimental facts (see the schemes in Fig. 13). One possibility is that some of the acceptor sites or groups of acceptor sites are tandemly repeated inside the acceptor zone of the operon. This may explain most of the above-mentioned data. On the other hand, the repetition of the same acceptor site in the same operon may be very important for the reliability of the regulation process; otherwise, because of the large size of the genome in eukaryotes, the finding of the correct sequence by a regulatory protein may be difficult.

, 01

om

s,

at

SI O? SI

----------__-_ dLd*--------O / # # 0 /

t

01

s,

at

/

B

(terminator factor)

FIG.13. Modified models of the transcriptional units to explain the existence of tandem repetitions. (A) With repetitive acceptor sites. (B) With repetitive structural genes.

Another possibility is that operons may contain a number of identical structural genes each of which contains the terminator sequence at the end. I n this case, depending on the position of binding of the terminator factor, a larger or smaller dRNA may be produced (as was shown in Drosophila cells). This may be an additional way for regulation of the production of mRNA’s in the cell. Finally, C. L. Markert and F. Crick (personal communications) suggested that the possibility of the existence of several promoters in the same transcriptional unit should be considered. This also may explain the difference in the size of giant dRNA’s produced in the same site.

E. GENETICDATAON

THE

STRUCTURE OF OPERONS IN EUKARYOTES

This is a special question that I.will not discuss in detail. As pointed out above, success in understanding the transcriptional unit organization

1.

TRANSCRIPTIONAL UNITS I N EUKARYOTIC CELLS

43

in eukaryotes is limited compared to that in bacteria. There are many reasons for this, in particular the absence of good models for complex biochemical and genetic experiments. Recent progress in this field is reviewed by Beermann (1972). To determine the size of genetic units in Drosophila, Judd and coworkers (Kaufman et al., 1969; Shannon et al., 1970) determined the number of complementation groups and compared i t with a number of bands in a region of the X chromosome with a well known ultrastructure. More than 100 mutants were studied, covering the entire region. About 20 complementation groups were found in the zone z-fa. At the same time, this zone contains 23 bands as resolved by high-resolution microscopy or by electron microscopy. Thus the number of functional units exactly coincides with the number of bands in chromosomes. I n other words, one band, or chromomere, probably corresponds to one functional unit of chromosome. The amount of DNA in one haploid chromoniere of Drospophila comprises from 7000 to 140,000 base pairs (2-40 p ) or about 50,000 base pairs (15 p ) on average (see Beerniann, 1972). This is much more than the size of a single gene. On the other hand, the size of newly formed dRNA in Drosophila, as well as in other animal cells, is 2 to 10 X 10G (Daneholt et al., 1969a,b), which is comparable with the amount of DNA per band ( 5 x lo7 on average). Thus it is quite probable that each band corresponds to one or a very limited number of transcriptional units or operons. I n this respect, it seems very important to analyze the structural organization of some bands by genetic methods. Such analysis is in progress now for gene ZL'localized in the 2C2 band of X chromosome of Drosophila. It was found that a rather large part of this band may be eliminated without loss of function (Green, 1967; Beermann, 1972). In other words, the structural gene a' corresponds only to a small part of the chromomere or to the interband part of the chromosome. On the other hand, Green (1969) observed that gene w consists of two functional parts. Mutations in either of them inactivate the gene. However, if the intact part inactivated by the mutation in another site is transferred to chromosome 111, the activity of the gene is reconstituted, This result may be interpreted as follows: the mutation was localized in the acceptor zone of transcriptional unit, and for this reason the transfer of structural gene to another transcriptional unit leads to the recovery of its activity. Other explanations could not be excluded. It seems clear that the detailed analysis of certain genes may be

44

G. P. GEORGIEV

one of the quickest ways to check the hypothesis describing the structure of transcriptional units in eukaryotes.

IV. General Aspects of Regulation of Transcription in Eukaryotes and the Role of Chromosomal Proteins From the above-mentioned model (Georgiev, 1969), two general consequences follow which are important for the understanding of the regulation of differentiation. First, owing to the multiplicity of some of the acceptor loci, one regulatory protein may switch many different operons on or off. These operons may not be connected functionally, i.e., may not determine the stages of the same process. These operons may be combined in groups of a higher order; for example, they may determine the proteins for a given type of cell. The high multiplicity of certain base sequences in the genome may easily produce such massive switching. One can suggest that cell differentiation is determined by switching on (off) of the most multiple acceptor loci, located a t the starting points of the operons. Their localization near the promoter completely inhibits transcription and excludes the formation of uncompleted chains. Second, due to the presence of several acceptor loci in one operon, the latter may be regulated by different factors: by some general “tissue signals,” by hormones (sites reacting with hormone receptors), by usual repressors (sites identical t o bacterial operators), etc. Thus, a system is formed that may give very precise and fine regulation and may respond to different regulatory signals. The price the cell pays for such a fine regulation is excessive RNA synthesis; i.e., the transcription of the noninformative part of the operon. According to the experimental data, half to two-thirds of newly formed RNA is degraded. In general a 2- or 3-fold excess in RNA synthesis should not be detrimental to the cellular balance of energy. It is interesting that in the ribosomal operon one can follow the increase of the nonstructural part in evolution. In Drosophila almost all newly formed rRNA is converted into mature ribosomal molecules; in plants, Amphibia, and reptiles the degraded part comprises about 25%, and in birds and mammalian cells about half of the precursor molecule (Daneholt et al., 1969a; Loening et aE., 1969; Perry et al., 1970). It is possible that the complication and improvement of regulatory apparatus in evolution requires the increase of the acceptor part of genome. The question arises what is the place of different classes of chromosomal proteins in the scheme presented above. I will briefly consider some possibilities.

1.

TRANSCRIPTIONAL UNITS IN EUKARYOTIC CELLS

45

A. HISTONES AS INHIBITORS OF TRANSCRIPTION

It has been mentioned above that the histones may be the inhibitors of RNA synthesis in vivo and that in particular histone F1 selectively inhibits the transcription of certain DNA base sequences. For the analysis of the mechanism of the histone action on the DNA template activity, experiments were developed in which different parameters of RNA polymerase reaction were determined (Kozlov and Georgiev, 1970). RNA was synthesized using RNA polymerase from E. coli and DNA or DNP templates from calf thymus or Ehrlich ascites TABLE I’

PARAMETERS OF RNA SYNTHESIS O N DIFFERENT TEMPLATES~.~ S after 33 minutes n X lo6

Template

Without rifampicin

With rifampicin added after 3 minutes

After 3 minUtes

After 33 minutes

DNA DNPo DNPo.6 DNP,,..

0.50 0.023 0.32 0.040

0.79 0.072 0.52 0.14

300 58 137 86

494 173 450 164

1

U

after for 0-8 t 33 minute minutes interval minutes 1700 400 2300 470

130 140 144 -

>30 <3 >30 5-6

From Kozlov and Georgiev (1970). S, full RNA synthesis in moles of nucleotides related to 1 mole of DNA nucleotides; n, total number of initiated chains (in moles) also related to 1 mole of DNA nucleotides; I , average chain length ( = S / n ) ;t , time of elongation; u, rate of elongation 0

b

( = Al/At).

DNP,,,. = DPNo treated by 4 Af urea, which destroys the superstructure. Before the reaction, urea was removed by dialysis.

carcinoma cells in the presence of nucleoside triphosphates randomly The incorporation of “C labeled with 14C and in y-position with 32P. gave the total template activity (S); and 32P, the number of initiated RNA chains ( n ) . It was also possible to calculate an average chain length (1 = S / n ) ; time of chain growth ( t ); relocity of chain growth (17 = A Z / A ~ ) ; velocity of initiation (1’’ = An/At). Using rifampycin block i t was possible to exclude reinitiation, allowing already initiated chains to grow freely. The main difference between DNA and DNP, as templates is that

46

G. P. GEORGIEV

on DNP, much shorter RNA chains are produced (Table V ) . The velocity of chain growth is the same in both cases. However, the time of chain growth is reduced many times with DNP, template. RNA chains on DNP, template stop growing after 3-5 minutes, whereas on DNA they grow during more than 30 minutes. This suggests that proteins of DNP, produce some kind of obstacle to RNA polymerase along the DNA chain. I n this respect, the similarity with the scheme for the mechanism of bacterial repressor action (see Section I) is obvious. If hi,stone F1 is removed from DNP, by 0.6 M NaCl extraction or more gently by incubation of DNP, solution with excess of tRNA (Ilyin et al., 1971), the above-mentioned difference in activity disappears. The chain growth becomes continuous, and RNA chains formed reach the same or greater length as with DNA template. Thus it seems probable that the histone F1 produces the stopping points for RNA polymerase movement whereas all other four histoiie fractions do not interfere with RNA polymerase reaction (Kozlov and Georgiev, 1970). It is possible that histone F1 are localized in the initial parts of operons and switch them off by preventing the RNA polymerase movement. However, it is quite clear that neither histone F1 nor any other histone can recognize specific DNA base sequences. The most they can do is combine preferentially with DNA enriched, for example, in AT or GC pairs, but the finer differences could not be recognized. The reason is a high positive charge of histones and the homogeneity of histones -only five to ten individual histones exist in the cell. Therefore some other factors should be present to determine the specificity of histone action. The following possibilities have been proposed: 1. Activator nonhistone proteins. According t o this model some nonhistone proteins combine specifically with certain DNA base sequences and protect them from the inhibitory action of histones (Frenster, 1965; Paul and Gilmour, 1968). It was shown that the content of nonhistone proteins in the activated parts of chromosomes (for example, in puffs or in diffuse regions) is higher than in the inert parts, the histone content being very similar (Frenster, 1965; Swift, 1964; Comings, 1967; Gorowski and Woodard, 1967). In in vitro experiments, the complexes of histones and nonhistone phosphoproteins possess lower inhibitory action than pure histones (Langan and Smith, 1966). Theoretically, nonhistone proteins are much better candidates for substances recognizing specific base sequences than histones, as they do not possess a strong positive charge which makes

1.

TRANSCRIPTIONAL UNITS I N EUKARTOTIC CELLS

47

interaction nonspecific. In fact, it was recently demonstrated that a significant fraction of chromatin phosphoproteins can specifically combine with certain D N A base sequences (Teng et al., 1970). Finally, in reconstruction experiments (Paul and Gilmour, 1968; Gilmour and Paul, 1969, 1970) the nonhistone proteins were found to be factors determining the specificity of RNA synthesis. The nonhistone proteins are a large and very heterogeneous group of proteins (Teng et al., 1970; Kleinsmith et al., 1970), and now it is necessary to isolate and characterize chemically and functionally individual components of this fraction. 2. Histone modification. It is known that in the cells are a number of very specific enzymes which are responsible for the modification (phosphorylation, acetylation, methylation) of certain histone fractions (Allfrey et al., 1964; Kleinsmith e t al., 1966; Stocken, 1966). Some histone phosphokinases and acetylases were isolated and shown to modify the histone in only one definite position (Langan, 1968, 1969, 1972; Allfrey, 1968). Furthermore, only a part of total histone molecules in the nucleus are modified and the extent of modification depends on the physiological conditions (Langan, 1972). Usually, the stimulation of the cell activity (for example, after the hormone treatment, during the regeneration, etc.) stimulates acetylation and phosphorylation of histones (Allfrey, 1968; Pogo et al., 1966, 1968; Langan, 1968). Finally, in the model systems the chemical acetylation of histones led to the decrease of histone inhibitory activity (Allfrey et al., 1964). These data give evidence in favor of the idea presented, but all of them are indirect. 3. Low molecular weight RNA recognition model. The idea is t h a t special low-molecular weight RNA’s combined with nuclear proteins recognize certain D N A base sequences by complementarity (Huang and Bonner, 1965). There are some data on the presence of low-molecular weight RNA with unusual properties in chromatin preparations (Huang and Bonner, 1965) although its functional role remains obscure. I n any case, all the possibilities mentioned remain hypothetical, and new experimental data are required for any final conclusion.

PROTEINS AS POSSIBLE REPRESSORS B. NONHISTONE I n the preceding section the possible activator role of nonhistone proteins was considered. However, it is equally possible that they play a role as repressors interacting with some of the acceptor sites of the genome and excluding corresponding operons from transcription. The clearest example was obtained by Crippa (1970) with the ribosomal operon. The author isolated from amphibian oocytes a protein fraction which binds specifically only with D N A of the nucleolar organizer. I n

48

G. P. OEORGIEV

both the cell-free system and the cells in vivo, this nonhistone protein specifically inhibits the synthesis of ribosomal RNA. It is important that in the cells from which this protein was isolated the rRNA synthesis is arrested. This is a first case of isolation of a regulatory protein of repressor type recognizing certain base sequences from eukaryotic cells. The repressors which were isolated before from bacterial cells (Gilbert and Muller-Hill, 1966; Ptashne, 1967) are also nonbasic proteins. Nonhistone proteins were also found to be targets of some of the steroid hormones (see review by Tomkins and Martin, 1970), although in this case it is not clear whether the control is positive or negative. C. THEPOSSIBLE REGULATION OF TERMINATION One additional possibility should be also considered about the tandem organization of genome in the light of data presented above. If in the end part of operon a number of identical structural genes containing the terminator sites are localized, one can assume that the hypothetical terminator protein combines with some of these sites. As a result, dRNA chains of different length may be produced depending on the place of the first terminator binding. The probability of premature chain termination would depend on the terminator concentration. It is not excluded that F1 histones play a role of such terminators of RNA synthesis. As was pointed out above, they produce obstacles for RNA polymerase movement. The length of RNA’s formed with D N P template is of about 400-600 nucleotides, that is of the same order as the size of an average cistron. From this part of the paper, one can conclude that almost nothing is known definitely about the role of different chromosomal proteins, although many facts have been obtained that allow one to hope for more rapid progress in the future. D. THREE-DIMENSIONAL ORGANIZATION OF TRANSCRIPTIONAL UNIT (CRICKMODEL) The above-mentioned data do not warrant wide generalizations concerning the role of nuclear proteins in the structure and function of chromosomes. Also, the above models of the transcriptional unit structure, being one-dimensional, could not be easily related to the known chromosomal structures. The first attempt to construct general threedimensional model of chromosome has been done by Crick (1971), who analyzed theoretically the structure of polytenic giant chromosomes. Crick suggested that other interphase chromosome (monomeric) are built

1.

TRANSCRIPTIONAL UNITS IN EUKARYOTIC CELLS

49

in the same manner. His linear presentation of chromosonie coincides mainly with my model (if one accepts n = 1 in most of the cases). Further, Crick postulated that the structural cistrons or coding DNA comprise only a small part of total genome and are localized in interband of polytenic chromosome. It is in the extended form and for this reason designated as fibrillar DNA. On the other hand, the bands contain only regulatory DNA that correspond to acceptor zone. This DNA is in the complex supercoiled state and is designated as “globular DNA.” The recognition sites in globular DNA corresponding to promoters and operators are represented by unpaired DNA sequences which are formed as a result of forces produced by histone interaction with DNA. The base sequence in unpaired regions is important for recognition by regulatory protein or RNA whereas the base sequence in paired regions of acceptor zone is not significant a t all, and for this reason may be easily changed in evolution. This idea explains well the large differences between species in respect to the content and the reiteration degree of repetitive DNA base sequences, as well as the presence of tandem repetitions. The latters may obviously be meaningless. This model considers many facts. I n particular many data mentioned in this chapter may be explained in terms of the Crick model. However, its description is outside the scope of this paper, and the reader is sent to the original paper (Crick, 1971). V. O n the Possible Mechanism of Cell Transformation by Oncogenic Viruses

The above-nientioncd hypothesis of the structure of the transcriptional unit may give an explanation for the mechanism of oncogenic action of some viruses. It is well established now that a number of tumors have a virus origin. Oncogenic viruses were found among viruses containing DNA as well as RNA. I n the cells transformed by DNA-containing oncogenic virus, a number of virus genomes were found to be incorporated into the cellular genome (Westphal and Dulbecco, 1968; Sambrook et nl., 1968). Virus DNA is hound with host DNA covalently in an uninterrupted polynucleotide chain (Sambrook et al., 1968). No free viral genomes were found in transformed cells. It was also shown that a part of virus DNA is actively transcribed in transformed cells. I n different lines of cancer cells, different parts of the virus genome are transcribed. As a rule, early genes are predominantly involved in RNA synthesis. Moreover, RNA complementary to virus DNA is incorporated into polysomes and translated (Fudjinaga and Green, 1966; Benjamin, 1966; Oda and Dulbecco, 1968; Sauer and Kidwai, 1969). Last, it should be

50

G. P. GEORGIEV

pointed out that incorporation of viral DNA produces activation in the synthesis of enzymes involved in DNA synthesis (Kara and Weil, 1967). All these data, although very important, do not explain how the integrated virus genome prevents regulation of cell growth. I n the transformed cells, some of viral genes are translated. This results in the appearance of virus-specific antigens: T-antigen, localized in the nucleus of transformed cells and transplantational antigen localized on the surface of the cell (Habel, 1961; Melnick and Rapp, 1965; Tevethia et al., 1965). However, sometimes these antigens may disappear from the cells without the loss of neoplastic properties, that is, the capability of uncontrolled growth (Hare, 1967). Thus the attempt to connect the transformation with the appearance of virus-specific proteins meets some difficulties. On the other hand, the loss of viral genome from the cell normalizes the latter (Martin and Macpherson, 1969). The mechanism of transformation could be explained in terms of the hypothesis of operon structure. Each virus genome probably possesses a t least one promoter. If the virus genome becomes incorporated in the middle part of the operon (for example, in an operon responsible for DNA synthesis or for other processes controlling cell multiplication), then it transfers an additional promoter into this operon, i.e., a new site t o which RNA polymerase can be bound. If the virus genome becomes incorporated between the acceptor and structural zones of the operon, then all regulatory signals which are realized through the interaction of proteins with acceptor loci should not affect the transcription of the structural zone. This is the case because no acceptor loci, and as a result no block, would appear between the new promoter and the structural genes of the operon. The synthesis of the corresponding mRNA would not be controlled. It is clear that newly formed dRNA should consist of a copy of the viral genome in its 5‘ part and a copy of the host genome in its 3’ part. The scheme of Fig. 14 illustrates the mechanism of the alteration of the regulation process after virus genome integration. The appearance of the new effective promoter P, makes the transcription of the genes sl, s2 , . . s,, independent of the interaction between regulatory proteins and acceptor loci. RNA synthesized on such a new operon should contain in its initial part early (el, e,,, e , ) viral mRNA’s followed by cellular mRNA’s. On the other hand, most of the late (11, Z, /,) viral genes which entered the operon containing the host promoter (PH)should not be transcribed a t all. Actually, their transcription would depend on the acceptor loci of the operon, but in conditions of uncon-

1.

TRANSCRIPTIONAL U N I T S I N E U K A R T O T I C CELLS

51

trolled multiplication and growth these loci would receive an excess of signals inhibiting operon transcription. A somewhat different situation with the viral genome incorporated inside the acceptor zone may also be considered. In this case, a partial Viral and host genomes before integration

-

Pw

a, a2

.... a, U

5 , . s2 . . . . 5, --- ----- --

-I-

Integration leading to complete loss of regulation a, a2

PH-

.. .

a,

,,Ic t2

t

3' dRNA

Integration leading to partial loss of

-

PH

a, a2

. ..

I, t2Pv e,e2e3

1,

a, s, s2

... ..

6)- - - - - - - - m'

-

' 5

3' dRNA

FIG.14. The hypothetical explanation of cnrcenogenesis under the influence of DNA-containing oncogenic viruses (Georgier, 1969). P,, Virus promoter; P H host promoter; el, 8?, ex, early genes (or their copies) ; L , L, I , . late genes (or their copies) ; RP, R N A polymerase; r,, rz . . . rn,, regulatory proteins; m, complementary sites of viral and host genomes; -+, nonregulatable transcription ; ---; regulatable or partly regulatable transcription.

regulation of the operon would survive. For example, such cells which become nonsensitive to contact inhibition of DNA synthesis would remain sensitive to hormonal control. Therefore cell strains with different rates of growth and different malignancy may be obtained. On the other hand, the point of cleavage of the viral genome during integration affects

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the size of the viral genome which is transcribed in the transformed cell. Thus the hypothesis interprets the degree of malignancy of the cell as a direct result of the incorporation of the viral genome into the host genome. It explains also why a part of the viral genome is transcribed and translated in the transformed cell. It is significant that the specific nature of the virus is not important for malignancy. The only condition is that the virus genome should be able to incorporate into the host genome operons related to cell replication or its regulation. This property may depend on the presence of small complementary sequences in the viral DNA and in the corresponding host operon. The hypothesis also explains why different tumors differ in their dependence on different regulatory signals. The present model can be tested experimentally as described before. It follows from the model that virus-specific RNA should be a part of the giant dRNA and be localized in the 5' part of it. At the 3' end of giant newly formed dRNA, the host sequences may be expected. The first prediction from the model, namely the synthesis of virus-specific RNA in the form of giant precursor, have been proved recently (Tonegawa et al., 1970; Lindberg and Darnell, 1970) with the cells transformed by SV40 virus. Moreover, it was found that this giant precursor consists of both the copies of virus genome and host copies. I n the cytoplasm of transformed cell the shorter virus-specific chains not containing attached host sequences were observed (Tonegawa et al., 1970; Wall and Darnell, 1971). Similar data were also obtained with polyoma-transformed cells (Georgiev et al., 1972b). The end-labeling analysis will allow of checking the other predictions followed from the model. The mechanism of transformation produced by RNA-containing viruses now also may be explained in similar terms. It was shown that all oncogenic RNA-containing viruses contain enzymes : RNA-dependent DNA-polymerase and DNA-dependent DNA-polymerase responsible for the synthesis of DNA on the RNA template followed by replication of the DNA. Thus a t least a part of viral RNA gives rise to complementary DNA (Baltimore, 1970; Temin and Mizutani, 1970; Spiegelman et al., 1970). It is very probable that some of this DNA integrates with the host genome. Thus one may suggest that the mechanism of transformation is very similar in both DNA- and RNA-containing viruses and related to the incorporation of viral DNA into the host genome as to lead to uncoupling of acceptor and structural parts of the operon. The presented hypothesis is not the only explanation of mechanism of transformation by virus. At this moment it seems more probable that the transformation is

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a result of activity of certain virus genes. This idea is supported by finding ts-mutants of polyoma. The cells transformed by this mutant virus lose their ‘(tumor propertics” a t nonpermissive temperature (Dulbecco and Eckhart, 1970). In this respect, one can suggest that integrated virus uses the host promoter for its own transcription. If this is so, virus-specific sequence should be localized near the 3’ end of giant precursor. Again the possibility may be checked experimentally by the technique described in Section 111. From the above material one can see that the models describing the structure of the transcriptional units in eukaryotic cells may be checked experimentally and that the first results in this direction have been already obtained. Progress in understanding the operon structure of eukaryotes may lead to the solution of many problems of differentiation and carcinogenesis.

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Southern, E. M. (1970). Nnture (London) 227, 794. Spiegelman, S., Burny, A., Das, M. R., Keydar, J., Schlorn, J., Tmvnicek, M., and Watson, K. (1970). Nature (London) 227, 1029. Spirin, A. S. (1963). Progr. Nucl. Acid Res. 1, 301. Spirin, A. S. (1969). Eur. J . Biochem. 10, 20. Spirin, A. S., Belitsina, N. V., and Aitkliozhin, M. A. (1964). J . Gen. Biol. (Moscow) 24, 321. Starlinger, P., and Kolsch, E. (1964). Biochem. Biophys. Res. Commiin. 17, 508. Stevenin, J., Mandel, P., and Jacob, M. (1969). Proc. N o t . Acad. Sci. U.S. 69, 490. Stocken, L. A. (1966). In “The Histones” (A. V. 8. DeReuck and J . Knight, eds.), p. 62. Churchill, London. Sueoka, N. (1961). J . Mol. Biol. 3, 31. Summers, D., and Maizel, L. (1968). Proc. N o t . Acnd. Sci. [J.S. 59, 466. Sutton, W. D. (1971). Biochim. Biophys. Acln 240, 522. Swift, H. (1964). I n “The Nucleohistones” (J. Bonner and P. 0. P. T’so, eds.), p. 169. Holden-Day, San Francisco, California. Takanami, M. (1966). Cold Spring Hnrbor Symp. Qrtont. Biol. 31, 611. Tamaoki, T., and Lane, B. G. (1967). Con. J . Biochem. 45, 2041. Tatarskaya, R. I., Lvova. T. N., Abrosimova-Omeljanchic, N. M., Korenjako, A. I., and Baev, A. A. (1970). Eur. J . Biochem. 15, 442. Temin, H. M., and Mizutani, S. (1970). Nnture (London) 226, 1211. Teng, C. T., Teng, C. S., and Allfrey, V. G. (1970). Biochem. Biophys. Res. Commun. 41, 690. Tevethia, S . S., Katz, M., and Rapp, F. (1965). Proc. Sac. Exp. Biol. Med. 119, 896. Thomas, C. A., Hamkalo, B. A,, Mirsa, D. N., and Lee, C. S. (1970). J. Mol. Biol. 51, 621. Tomkins, G. M., and Martin, D. N. (1970). Annu. Rev. Genet. 4, 91. Tonegawa, S., Walter, G., Bernardini. A , and Dulhecco, R. (1970). Cold Spring Harbor Symp. Quant. Biol. 35, 823. Tsanev, R. (1968). In “Biochemistry of Ribosomes and mRNA” (R.Lindigkeit, P. Langan, and J. Richter, eds.) p. 293. Akademie-Verlag, Berlin. Tyndall, R. L., Jacobson, K. B., and Teeter, E. (1965). Biochim. Biophys. Actu 108, 11. Vesco, C., and Giuditta, A. (1967). Biochim. Biopliys. Actn 142, 385. Walker, P. M. B. (1972). Progr. Biophs. Mol. Biol. 23, in press. Wall, R., and Darnell, J. E. (1971). Nntuie (London) 232, 73. Warner, J. R., Soeiro, R., Birnhoim, C., Girard, M., and Darnell, J . E. (1966). J. Mol. Biol. 19, 349. Weinherg, R. A., and Penman, S. (1970). J . Mol. Biol. 47, 169. Weinberg, R. A,, Loening, U., Willems, M., and Penman, S. (1967). Proc. Not. Acad. Sci. U.S. 58, 1088. Weinberg, R. A., Vesco, C., and Penman, S. (1969). A n n . Embryol. Morphol., Suppl. 1, 63. Weisherger, A. S., and Armrntrout, S. A. (1966). Proc. Not. Acad. Sci. U.S. 56, 1612. Westphal, H., and Dulhecco, R. (1968). Proc. N u t . Acad. Sci. U.S. 59, 1158. Whitehouse, H L. K. (1967). J . Cell Sci. 2, 17.

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Willems, M., Wagner, E., Laing, R., and Penman, S. (1968). J . Mol. Biol. 32,

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779. Yoshikawa, M., Fukada, T., and Kawada, Y. (1964). Biochem. Biophys. Res. Commun. 15, 23. Yoshikawa-Fukada, M., Fukada, T., and Kawada, Y. (1965). Biochim. Biophys. Acta 103, 383. Yunis, J. J., and Yasmineh, W. G. (1970).Science 168, 263. Zillig, W., Fuchs, F., Palm, P., Rabussay, D., and Lechel, K. (1970). Proc. 1st Znt. Lepetit Colloq. p. 151.

CHAPTER 2

REGULATION OF SPORULATION IN YEAST James E. Haber and Harlyn 0. Halvorson ROSENSTIEL BASIC MEDICAL SCIENCES RESEARCH CICNTER, BRANDEIS UNIVERSITY,

I. 11. 111. 11'. 1'. VI.

WALTHAM, MASSACHUSETTS

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

61 62 64 66

72 77 82

1. Introduction

Sporulation in both eukaryotic and prokaryotic organisms is one of the simplest developmental processes accessible to experimentation. I n both, morphological and physiological changes take place. In recent years numerous laboratories have undertaken studies in the hope that elucidation of the regulatory controls governing the transition from vegetative growth to spore formation may provide new insights into the control of differentiation or development in higher systems. The recurring question is whether there are new principles governing differentiation other than those already described for the regulation of vegetative cells. One interesting finding in the studies of sporulation in Bacillus subtilis illustrates this possibility. I n this organism the onset of sporulation is accompanied by the appearance of a serine protease which modifies a number of vegetative enzymes to an altered form (Sadoff, 1970). The most striking modification is a change in the size and template specificity of DNA dependent RNA polymerase (Leighton et al., 1972). This unexpected finding clearly illustrates that normal vegetative gene products may be expressed through sporulation but modified to carry out different functions. Thus differentiation processes, such as sporulation, may be controlled both a t the transcriptional as well as posttranscriptional level. Sporulation of bacteria has been under active investigation in the past two decades and the subject of numerous symposiums and reviews (Schaeffer, 1969; Hanson e t al., 1970). Of increasing concern is whether the principles of regulation arising from studies with bacteria are applicable to eukaryotic cells. Studies on sporulation in simple eukaryotic microorganisns provides an attractive system (s) for comparison with 61

62

J A M E S E. HABER A N D HARLYN 0. HALVORSON

bacteria. Among these there has been an increasing interest in sporulation in Saccharomyces cerevisiae. There is not only a wealth of information on the physiology and biochemistry in this organism, a strong foundation for a genetic analysis, but also many of the same technical advantages which have proved useful in studies of bacteria. More important, studies with yeast introduce some intriguing complexities not seen in sporulation in bacteria. Sporulation in yeast leads on the one hand to the production of semidormant spores, but on the other hand

/

Mitosis

/

@

ASCI

@ I

Digdstion of ascus wall

FIG.1. Life cycle of yeast.

introduces the process of meiosis (i.e., reductive division of genome). At the moment there is relatively little information on the process and control of sporulation in yeast. In this review we shall summarize the current status of knowledge and discuss a number of promising approaches to examine this phenomenon. II. From Vegetative Growth to Ascus Briefly let us review the life cycle of Saccharomyces cerevisiae. I n its stable form, this yeast is diploid (a/& mating type) in the vegetative phase (Fig. 1). Vegetative cells divide by budding with an average generation time of 2-3 hours in favorable media. When maintained as an exponential culture, spores are not observed. It has long been recognized that when yeast cultures are transferred to a nitrogen-deficient starvation medium, sporulation ensues (for review, see Fowell, 1969) and leads

2.

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normally to the production of 4 refractile haploid spores within an ascus. Thus the overall process involves in its early phases nieiosis with accompanying reductive division and random segregation of chromosomes and in its later stages (ca. 20 hours) the partitioning of the cell (ascus) into 4 discrete spores. Two of the spores are of the a mating type and two of the a mating type. Unless these spores are isolated they may fuse with their opposite mating type neighbors ( a and a) in the ascus to restore the diploid state. Alternatively spores may germinate and grow vegetatively in the haploid state. Diploids can also arise from a mating of two vegetative cells of opposite type. Much effort has been directed toward defining the physiological conditions necessary for sporulation. It is clcar that like bacteria, sporulation requires aerobic metabolism (Fowell, 1967; Croes, 1967b), is sensitive to glucose repression (hliller et al., 1955) and is inhibited by many nitrogen containing compounds (Miller, 1963). As a general procedure sporulation is initiated by growing cells vegetatively to stationary phase (exhaustion of glucose) and then transferring them a t relatively high density (2 to 5 x lo7 cells/ml) to a simple potassium acetate medium a t pH 7.0. At stationary phase, the cells are adapted to oxidize the end products of glucose fermentation. Consequently this procedure was simplified by growing cells vegetatively in potassium acetate medium containing nitrogen so that the cells from exponential cultures can be directly transferred to sporulation conditions (Roth and Halvorson, 1969). Some of the gross biochemical changes accompanying sporulation have been described. These include extensive synthesis and turnover of RNA and protein, doubling of DNA, increases in dry weight and the accumulation of carbohydrate and lipid reserves (Croes, 1967a; M . S. Esposito et al., 1969). Although these studies have demonstrated the dependence of sporulation on continued protein synthesis, and described some of the periods of active macromolecular synthesis, they have not as yet revealed much about the expression and control of sporulation specific events. I n the past several years attention has turned to providing answers to more specific questions on sporulation. I n the following sections we will examine a number of approaches which appear promising in prooiding answers to the following questions. 1. Can one define a series of morphological stages in sporulation? 2. How many sporulation specific genes can be identified? 3. What biochemical changes accompany these morphological stages? 4. Are these sporulation specific components? 5. What is the nature of the regulatory controls governing the onset and completion of sporulation?

64

JAMES E. HABER A N D HARLYN 0. HALVORSON

111. Morphological Changes during Sporulation With the light microscope one can detect only the most generalized changes in nuclear organization during meiosis. With appropriate staining it is possible to observe the segregation of chromatin into a binucleate and finally tetranucleate state (Pontefract and Miller, 1962). By these procedures one cannot detect individual chromosomes. Over the last decade several serious attempts have been made to describe the morphological changes observed during sporulation in the electron microscope. These studies have primarily been concerned with changes in the nucleus accompanying meiosis. Initially Hashimoto et al. (1960) reported that during the first meiotic division, the nucleus elongates and constricts in the center forming two nuclei which later divide producing four daughter nuclei. However, more recent investigations by thin section (Moens, 1971; Moens and Rapport, 1971a) and by freeze-etching techniques (Guth et al., 1972) indicate that all four daughter nuclei bud off from the parent nucleus after the second meiotic division. In studies with frozen dry yeast, Mundkur (1961) had previously concluded that meiotic division occurs without the dissolution of the nuclear membrane. The more detailed and informative studies of the ultrastructural changes occurring in a single nucleus during meiosis come from the reports of Moens (1971) and Guth et al. (1972). The major sequence of events can be summarized as seen in Fig. 2. During the first few hours in sporulation medium the yeast cells have small indistinct spindle plaques. I n the early stages of meiosis the plaque becomes distinct and the number of spindle tubules increases. By 8 hours one observes two plaques side by side (Fig. 2b). Subsequently the spindle plaques migrate to opposite sides of the nucleus (Fig. 2c) and microtubule interconnections between the two plaques can be observed. A t this time the nucleus elongates as the plaques move apart (Fig. 2d), representing the end of the first meiotic division. The onset of the second meiotic division is signified by the replication of each of the two plaques (Fig. 2e) and the subsequent migration of each pair to form two spindles perpendicular to the plane of the first spindle (Fig. 2 f ) . These spindles then elongate and the beginnings of a forespore wall can be seen around each nuclear lobe (Fig. 2g). Completion of forespore wall and dissolution of the central nuclear body complete the second meiotic division (Fig. 211, i) . Further details of this process can be seen by freeze etching. I n Fig. 3 the formation of the first meiotic spindle is visible. Subsequently in Fig. 4 the nucleus is elongated. A second nuclear division follows with the spindle fibers perpendicular to the original axis of the nuclear elongation. At this time also 4 discrete forespore membranes appear a t the lobes of the dividing nucleus (Fig. 5 ) . A more complete encapsula-

2.

REGULATION OF SPORULATION I N YEAST

65

tion of the nucleus can be seen in Fig. 6. Even in these high resolution studies there are no clear descriptions of chromosomes. Most recently, despite the lack of chromosomal condensation, synaptonemal complexes have been reported by Engels and Croes (1968) and by Rloens and Rapport (1971b) . This is an interesting development, and the transitions of these complexes during meiosis may provide clearer definition of the stages of meiosis in yeast. Several important questions await further structural studies. Although thickening of the spore wall has been noted (Guth e t al., 1972)

FIG.2. Sporulating yeast. Figures 2a-2i are sketches showing the development of spindles, spindle plaques, and ascospores. From Dr. P. Moens.

little attention has been given to the final stages in sporulation. hlore important sporulation is dependent on mitochondria1 function. The fate of these organelles and of the cytoplasm during sporulation is little understood. Guth et al. (1972) noted that during the spindle formation small vesicles containing endoplasmic reticulum appear and are observed until the ascospore wall is completed. These were not found in vegetative cells. The function and fate of these vesicles is as yet unknown. In bacteria, electron microscopy studies have defined a discrete set of stages in sporulation. The studies with yeast are a t present in a more preliminary stage, but have already suggested a related series of stages based on morphological changes in the nucleus. Since these are primarily meiotic events, one would hope that further such studies would detail additional stages in sporul a t'1011.

66

JAMES E. HABER AND HARLYN 0. HALVORSON

FIG.3. A sporulating Saccharomyces cerevisiae cell illustrating the nucleus undergoing the first meiotic division. ERV, endoplasmic reticulum ; N, nucleus; NM, nucleus membrane; S, spindle fibers. Data from Cuth et al. (1972), by permission of the American Society for Microbiology, Washington, D.C.

IV. Mutations Affecting Sporulation In a complex and integrated developmental sequence, one approach is to dissect the sequence by mutation. An extensive mutant search not only can reveal the number of specific loci governing this process but

2.

REGULATION OF GPORULATION I N YEAST

67

FIG.4. A subsequent stage of sporulation (meiosis I ) : spindle fibers (S) extend in the direction of nuclear elongation. N, nucleus; M, mitochondria; NP, nuclear pore; V, vacuole. Data from Guth et al. (1972), by permission of the American Society for Microbiology, Washington, D.C.

also may aid in the identification of specific biochemical events. The understanding of the genetic control of metabolism and the vegetative cell cycle has advanced considerably in recent years (Halvorson et al., 1971). On the other hand, only recently have attempts been made to obtain sporulation mutants. The major difficulty, which has discouraged efforts in this area, is the problem of generating sporulation mutants in a diploid. Since recessive mutants are the most frequent class observed, the overwhelming problem is the selection and recognition of sporulation mutants.

68

JAMES E. HABER AND HARLYN 0. HALVORSON

FIG.5. Saccharomyces cerevisiae cells apparently undergoing the second meiotic division. Spindle fibers (S) are oriented perpendicular to the direction of nuclear elongation. FS, forespore ; N, nucleus; V, vacuole; ER, endoplasmic reticulum. Data from Guth et al. (1972), by permission of the American Society for Microbiology, Washington, D.C.

There have been two approaches to overcome this difficulty. First, M. S. Esposito and Esposito (1969) took advantage of the presence of a diploidizing (D) gene in Saccharomyces described by Winge and Roberts (1949). A diploid homoaygous for the D gene produces, after sporulation, 4 haploid ascospores which each contain the D gene. After germination and a few mitotic divisions, the haploid cell diploidizes yielding an isogenic diploid. It is therefore possible to mutagenize by

2.

REGULATION O F SPORULATION I N YEAST

69

FIG.6. Snccharomyces ceTevisine cells app:lrently undergoing the second meiotic division. Note the appearance of portions of the dividing nucleus ( N ) into the forespore (FS). M, mitocl~ondrin;L. lipid grmule. D:ita from Guth et ol. (1972). by permission from the American Society for Microbiology, Washington, D.C.

UV irradiation the spores of a lioniothallic diploid and then germinate the haploid spores and allow them to diploidize. Mutations induced in a haploid carrying the D gene will then be carried in an isogenic diploid, so t h a t all recessive mutations will be observed. hl. S. Esposito and Esposito (1969) used this approach to select a large number of teniperature-sensitive mutants which are unable to sporulate but are not affected in vegetative growth. Recent analysis of the coniplenientation of these mutants suggest that there are approxi~nntely 50 k 25 separate genes which show an asporogenous phenotype (R. E. Esgosito et nl., 1972). Three of 14 complementation groups studied are dominant to the wild type.

70

J A M E S E. HABER AND HARLYN 0. HALVORSON

A very similar approach for sporulation deficient mutants in Schizosaccharomyces pombe was developed by Bresch et al. (1968). They isolated a large number of spontaneous mutants and have reported 17 complementation groups, of which 5 do not complete meiosis. A second novel approach designed to select mutants deficient in meiotic recombination has recently been reported by Roth and Fogel (1971). This method takes advantage of recent isolations of a haploid strain which is disomic for chromosome I11 (Shaffer et al., 1971) which contains the mating alleles. Although an a/a disomic is unable to complete sporulation (Kadowaki and Halvorson, 1971b), these strains are able to enter sporulation and to complete meiotic DNA replication and recombination, By means of heteroallelic pair of leu2 markers (also on chromosome 111), they could easily select mutants which failed to undergo recombination and thus are unable to grow in absence of leucine. Approximately 100 presumptive mutants have been isolated by this procedure. Some of the temperature-sensitive (ts) mutants isolated by Esposito and Esposito also appear to be defective in meiotic recombination (R. E. Esposito e t al., 1971). With deference to the principle that it is better to have a mutant, one is nevertheless faced with genes in search of a function. I n none of the mutants reported to date can the function of the mutated locus be identified. Nevertheless one can inquire ( a ) whether these are mutations in a developmental sequence, (b) a t what time in sporulation is a particular function expressed, and (c) can the function of the locus be inferred from the morphology or biochemical changes a t the point of arrest? The attempt to answer these questions can be illustrated by studies of three of the sporulation mutants (Spo 1, Spo 2, and Spo 3) isolated by M. S. Esposito and Esposito (1969). Collectively the available data suggest that these are mutations in a developmental sequence. From nuclear staining and DNA content Spo 1 a t the nonpermissive temperature is mononucleate and the sporulating cells only undergo half of the normal DNA replication, Spo 2 nearly completes DNA replication an& about 15% become trinucleatz or tetranucleate, and Spo 3 increases in DNA content by 40% but nearly 25% are tri- or tetranucleate (M. s. Esposito et al., 1970). By this criterion Spo 1 is blocked earlier than Spo 2 and Spo 3. From electron microscopy, Spo 1 is arrested a t stage a and Spo 3 a t stage c-d of Fig. 2 (Gray, 1970). A further indication of the ordering of the mutant functions can be seen from measurements of the onset and escape from the sensitivity to the nonpermissive temperature. Spo 1 becomes sensitive to high temperature a t 0.5 of the sporulation cycle and escapes a t 0.8 of the sporulation cycle.

2.

REGULATION OF SPORULATION I N YEAST

71

The corresponding sensitive periods for Spo 2 and Spo 3 are 0.2-0.6 and 0.65-0.7, respectively. Since Spo 1 and Spo 2 appear reversed by this criteria, the point of execution of the function may differ from the point of arrest. The effect on meiotic recombination of the three mutants is also under investigation. All of these Spo mutants are able to begin intragenic recombination in sporulation medium, however the levels of recombination are significantly reduced a t the restrictive temperature (R. E. Esposito e t al., 1971). Conditional sporulation mutants can also be examined for any significant biochemical changes a t the restrictive temperaure. When the three mutants discussed above were examined for any changes in protein synthesis, it was found that even a t the permissive temperature (25OC) the rate incorporation of arginine-lT into protein was substantially different from the time course measured for the wild type. I n particular, Spo 2 showed a very extended period of protein synthesis which continued nearly twice as long as that in the wild type. At the restrictive temperature the period of synthesis for Spo 2 was even longer and the apparent rate of synthesis nearly doubled. This dramatic increase was not attributable to changes in protein turnover (nil. S. Esposito et al., 1970). Subsequent experiments have shown that when radioactive lysine was used to follow protein synthesis, the pattern of synthesis of Spo 2 was identical to that found with arginine (Halvorson, 1971). On the other hand, when labeled phenylalanine was used, the dramatic increase in the apparent rate of protein synthesis was not found. These results suggest that either the transport of the basic amino acids is greatly facilitated a t the restrictive temperature or that lysine and arginine rich proteins may be selectively synthesized a t 34OC with Spo 2. The observations with the Spo mutants raise several problems in finding the function associated with mutant. First, the point of arrest determined from morphological stages and the point of execution of conditional function may not be coincident. This raises the possibility that the arrest a t a particular stage may be a secondary effect of the mutant function. It is quite likely that alterations in a developmental sequence will exert a number of pleiotropic effects, thus making it difficult to define the central function. For example, Spo 2 shows a very substantial alteration in meiotic recombination (R. E. Esposito et al., 1971) as well as in the duration and rate of protein synthesis. Spo 3 also exhibits several temperature-sensitive effects which are not readily reconciled. This mutant has a temperature-sensitive period of very limited duration, yet a loss of survival of Spo 3 a t the restrictive temperature can be detected as soon as sporulation a t 34OC is initiated

72

JAMES E. HABER A N D HARLYN 0. HALVORSON

(M. S. Esposito e t al., 1970). I n fact, an exponential loss of viability of cells returned to vegetative conditions was measured, so that 30% of the cells have lost viability by the onset of the t, period (as assayed by spore-forming capacity), These results indicate a major problem confronting all developmental systems, namely, the very tight interconnection between various parts of a developmental sequence and the consequent differences of interpretation of altered function depending on the criteria applied. V. Sporulation-Specific Biochemical Events What constitutes a sporulation-specific event? This problem, which has preoccupied microbiologists since studying bacterial sporulation, has been the subject of considerable debate and possibly diversion from the main features of the process. There have been in the last several decades attempts to identify components which are unique to the spore, and thus presumably represent new gene transcripts. Only recently has a renewed interest in understanding the supporting physiology of the sporulation process been reinitiated. Since spore formation is critically dependent upon a supporting physiology, coupled with the unique conditions of starvation, physiological pathways which are essential for sporulation may be optional for vegetative growth. Thus one can well imagine mutations in sporulation which are in fact affecting enzymes known to be present in the vegetative cell, but not essential for vegetative growth. An understanding of the supportive metabolism for sporulation may be as critical to an understanding of the overall process as identifying the unique components. This problem, which was seen dramatically in the case of acetate metabolism in bacteria, is equally demonstrable in yeast. It is well known that acetate metabolism is characteristic of the sporulating cell, and of the ability to sporulate, whereas vegetative cells can readily grow under anaerobic or aerobic conditions. Thus, aerobic metabolism is essential and necessary for sporulation but not vegetative growth. One can imagine that sporulation specific events encompass several different important categories. First, there is a possibility of qualitative changes resulting from the expression of portions of the genome completely suppressed during vegetative growth. Second, and probably more likely, the rate of production of a cell constituent may change dramatically during sporulation. This could occur, for example, by changes in controls a t the transcription or translation level, or by posttranscriptional regulation. I n these instances, both qualitative as well as quantitative changes may be found. The resulting change in concentration of a component during sporulation

2.

REGULATION OF SPORULATION I N YEAST

73

from a very low level to a critical threshold may be an essential event in the sporulation process. In situations such as sporulation in yeast which are under conditional starvation, rather than the steady growth of vegetative cells, this type of a control may be very likely. Thus, we shall consider in this section those biochemical processes which dramatically change during sporulation as potential sporulation specific events. The biochemical changes associated with the transition from the vegetative state to sporulation have been well studied (for an excellent review, see Fowell, 1969). As mentioned before, sporulation is initiated by the imposition of starvation conditions. Generally, cells which have adapted to growth on acetate or other oxidizable carbon sources a t stationary phase (Croes, 1967a,b) or by pregrowth in a rich medium containing acetate (Roth and Halvorson, 1969) are placed in a nitrogen-free medium containing acetate for sporulation. Although a carbon source is present, a nitrogen source for the de novo synthesis of amino acids or nucleotides has been removed. Consequently the turnover of both proteins and RNA to regenerate pools of constitutents for further synthesis becomes important. The importance of a protease to maintain necessary synthesis has been implicated in bacterial sporulation (Hanson et al., 1970) and in yeast as well (Ramirez and Miller, 1964). A strain lacking a protease which appears with high activity in the early stages of sporulation fails to sporulate (Chen and Aiiller, 1968). I t is not known whether there are a number of proteases necessary for sporulation or whether the one protease implicated to date acts as a generalized turnover mechanism, or as in the case of the serine protease in the sporulation of Bacillus subtilis, it produces a limited set of specific modifications or other proteins (Schaeffer, 1969; Hanson et al., 1970). Sporulation is subject to glucose repression. If cells are introduced to 2% acetate sporulation medium Containing 0.3% glucose, sporulation is nearly totally inhibited (Miller et d.,1955). Darland (1970) has extended these observations to a large number of fermentable carbon sources and hexose derivatives which do not support growth such as 2-deoxyglucose and glucosamine and found that sporulation is inhibited by all these compounds. A similar inhibition of sporulation is observed in the presence of amino acids. Miller and his co-workers have shown that a large number of amino acids, when added to acetate sporulation medium, prevents sporulation (Miller, 1963). Both of these types of repression have been taken as evidence that new sporulation functions are expressed when the levels of fermentable sugars and amino acids fall below some critical threshold (Miller and Hoffmann-Ostenhof, 1964). From the available

74

J A M E S E. HABER AND HARLYN 0. HALVORSON

data it is not clear that the initiation of sporulation is primarily dependent on the continued derepression of new functions when critical metabolites fall below threshold levels. Rather, it is also possible that the addition of glucose or amino acids to sporulation medium redirects preexisting and competing pathways so that the accumulation of certain important precursors is prevented, without shutting off the synthesis of sporulation-specific functions. Certainly during sporulation there is a redirection of metabolism and the accumulation of large amounts of certain cellular constituents not found in significant amounts during vegetative growth. Toward the end of spore formation there are a number of changes in the ascosporal cell wall (Snider and Miller, 1966) and in the formation of spore coats. One component which accumulates during sporulation and which is specifically localized in the spores is trehalose. Roth (1970) has reported that two-thirds of the dry weight increase found during sporulation was attributable to carbohydrate and that trehalose synthesis was a major fraction of the total increase. Trehalose accumulation is found only when there is either no assimilable nitrogen source or else when there was no cell division or growth (Trevelyan and Harrison, 1956). Panek (1962) proposed that trehalose could accumulate only if some intermediate which also was used in amino acid biosynthesis (such as glucose 6-phosphate) was not utilized primarily by the amino acid pathways. Trehalose accumulation is therefore another possiblc example of significant biochemical changes promoted by changes in environment (i.e., presence or absence of a nitrogen source) rather than by the induction of new enzymes during development. I n the early portion of sporulation there have been virtually no apparently sporulation-specific biochemical events identified. One phenomenon which has attracted considerable interest is the appearance of a stable 20 S RNA (Kadowaki and Halvorson, 1971a,b) ; 20 S RNA can be detected soon after sporulation begins, and this species accumulates during the first 24 hours of sporulation to a final level of nearly 4% of the total RNA in the cell. We have found that this RNA is quite different from 18 S and 26 S ribosomal RNA in base composition (Table I) and is substantially submethylated in comparison to the two ribosomal forms (Fig. 7) (Sogin et al., 1972). These observations lent support t o the possibility that a stable sporulation specific message had been detected. Recent investigations now appear, however, to suggest an alternative interpretation. When 32Plabeled 20 S RNA and 18 S RNA are compared in their RNA-DNA filter hybridization saturations 20 S RNA has a somewhat higher saturation level which can be extrapolated to a level approximately 40% higher than for 18 S (Fig.

2.

75

REGULATION OF SPORULATION I N YEAST

TABLE I

BASECOMPOSITIONS OF 26 S, 20 S, A N D 18 S RNA ISOLATED FROM SPORULATING Saccharomyces cerevisiaea C

A

G

U

% GC

20.3 19. 5 19.6 19.8

26.8 26.5 26.6 26.6

27.9 28. 5 28. 7 28.4

25.0 2.5.5 25.3 25.3

48.2

28.6 28.6 27.5 28.6 28.6

18.3 18.5 18.7 18.6 18.6

29.4 29.1 29.7 29.1 29.1

23.7 23.8 23. I 23.7 23.7

57.7

19.5 19.8 19.4 19.6 19.6

27. 0 26.5 25. 7 26.4 26.4

25.4 25. 6 25.5 25.9 25.6

28.1 28.2 29.3 28.1 28.4

45.2

26 S 1 2 3 AVG. 20 s 1

2 3 4

AVG. 18 S 1 2 3 4 AVG.

a RNA was extracted from strain D 649 after 16 hours in sporulation medium containing 3pP.The labeled RNA was separated by polyacrylamide gel electrophoresis and the three species were eluted from gel slices and subjected to alkaline hydrolysis. The individual bases were then resolved by paper electrophoresis and the separate spots were then identified and the amount of radioactivity determined.

8A). When both labeled species are competed against unlabeled 18 S ribosomal RNA in a filter hybridization competition series, it becomes evident that approximately 70% of the 20 S RNA which hybridizes to DNA is homologous to 18 S RNA (Fig. 8 B ) . Such experiments lead to the conclusion that the 20 S RNA which accumulates during sporulation is a precursor to 18 S RNA, similar but not necessarily identical to the transient 20 S RNA precursor found in vegetative cells (Udem and Warner, 1972). Unlike the processing of ribosomal RNA in vegetative cells, the 20 S RNA does not become totally converted to 18 S RNA. Pulse experiments with 3zP-labeled phosphoric acid reveals that the precursor to 26 S ribosomal RNA, 27 S RNA is either totally processed or degraded so that no significant amount of 27 S RNA accumulates during sporulation. What role the accumulation of 20 S RNA may play in sporulation is not a t all clear. Most likely the accumulation is a secondary

76

J A M E S E. HABER AND HARLYN 0. HALVORSON

I500

-

It

1000

0

500 400

a

2

N

n

I n

5

300

!,

E

500 200

; u

9

100

1

2

3

4

5

6

7

8

5

9

cm

FIG. 7. Methylation of 20 S RNA in sporulating Saccharomyces cereuisiae. Stationary phase cells were transferred to 1% potassium acetate containing 5 pCi/ml methionine-’H and 0.1 .uCi/ml 3zP-labeled phosphoric acid. RNA was extracted from cells after 18 hours and separated by polyacrylamide gel electrophoresis.

effect reflecting an alteration in the physiology of the sporulating cell. Udem and Warner (1972) have shown that both 27 S and 20 S precursor RNA’s accumulate for a short time if cycloheximide is added to a vegetative spheroplast. The implication of this finding is that processing of ribosomal RNA requires the synthesis of some of the ribosomal proteins which must bind to the RNA before processing occurs. I n the sporulating cell, the rate of protein synthesis is restricted and certain classes of proteins may be further selectively restricted. Such stringency might then account for the lack of processing of some of the 20 S RNA. Whether the 20 S RNA which’is stable is chemically distinct from the 20 S which was processed during sporulation to 18 S RNA is not known. Certainly the very low level of methylation of the stable 20 S RNA is different from the 20 S RNA observed by Warner and Udem. In this regard, it is also of interest to note the studies of Mundkur (1961f , who concluded from electron microscopic studies that the number of ribosomes in sporulated cells was significantly lower than in vegetative cells. These observations have not, however, been corroborated by more

2.

REGULATION OF SPORULATION I N TEAST

77

A 0

u

075

p!

/s-

I

0 n

2

050

a

2 0 L

0.25

C

0.5

10

15 pg/ml

20

25

15

20

30

RNA

B 100

c

c

al

? a

5

10

18 S (Unlabeled)/ ( 3 2 P ) R N A

FIG.8. RNA-DNA filter hybridization of 18 S and 20 S RNA. ( A ) Saturation of 18 S and 20 S RNA. RNA labeled with 32Pwas isolated from sporulated cells on polyacrylamide gels, and the level of saturation was measured for 18 S ( - - - - ) and 20 S (-) RNA. (B) Competitive curves of 32P-labeled 18 S and 20 S RNA (during sporulation) versus 18 S ribosomal RNA isolated from vegetative cells. The competition of 32P-labeled 18 S versus unlabeled vegetative 18 S extrapolates to zero, while the same competition of labeled 20 S RNA versus 18 S extrapolated to approximately 30%. From the data of Sogin et al. (1972).

direct physical and biochemical tests. Nevertheless these observations may indicate a very direct mechanism for the reduction of protein synthesis in the sporulating cell. VI. Cell Cycle Dependency of Sporulation

One of the main problems in the study of specific morphological

or biochemical changes during sporulation is lack of synchrony, as reflected by both the kinetics and extent of sporulation. The broad time course of the completion of sporulation in a population and similarly

78

JAMES

E.

HABER AND HARLYN 0. HALVORSON

broad periods of DNA replication (Croes, 1967a) may reflect asynchronous sporulation. I n studies of the vegetative cell cycle of S. cerevisiue, the use of synchronous cultures has permitted a precise determination of the temporal order of synthesis of particular enzymes in discrete steps or of the point of execution of various cell cycle functions (Hartwell et aZ., 1970). Both in examining the time and stage of arrest in mutants during the sporulation cycle and especially in characterizing the associated sensitive chemical events during the sporulation cycle, synchronously sporulating cultures are desired. The study of sporulation is further complicated by the fact that in general only 5&70% of the population completes sporulation, as evidenced by the formation of refractile spores within an ascus. Thus the measurement of the appearance of particular enzymatic activities may be further obscured or distorted by the presence of cells which fail to complete sporulation, since these cells may still attempt to maintain growth or sporulation processes. The need for more homogeneous, synchronously sporulating cultures prompted an investigation of the degree of asynchrony in commonly applied methods of sporulation. By using cell separation techniques which have proved most successful in understanding cell cycle events during vegetative growth, it has been possible to determine the basis of asynchrony in sporulating cultures and to develop new methods for the study of more homogeneous synchronously sporulating cells (Haber and Halvorson. 1972). Asynchrony in the sporulation of a random population of cells was tested by comparing the kinetics and extent of sporulation of homogeneous subpopulations ohtained by zonal rotor centrifugation (Sebastian et al., 1971). Cells growing exponentially in an acetate growth medium can be sporulated, without first being grown to stationary phase (Roth and Halvorson, 1969). I n Fig. 9 the kinetics and extent of sporulatioii of the two extreme size classes, the smallest single cells and the large double cells about to divide, are compared with the unfractionated random population. The random population does not sporulate in a synchronous fashion, but in fact represents a summation of widely different rates and extents of sporulation of cells a t different points in the cell cycle. I n the small, single cell fraction not only are the asci produced a t a later time, but the asci produced also differ morphologically from those in the fraction containing large, budded cells. More than 65% of the asci formed in the small cell fraction contained only 2 spores, whereas more than 80% of the asci formed in the large cells contained 3 or 4 spores. The extent of sporulation in the small cell fraction did not increase beyond 30%. This percentage is strain dependent and in some cases is less than 10%.

2.

79

REGULATION OF SPORULATION I N YEAST

p’

LARGE BUDDED CELLS,

SMALL SINGLE CELLS-

0

8

10 12 14 16 18 20 HOURS IN SPORULATION MEDIUM

22

24

FIG.9. Effect of cell size on the kinetics of sporulation of Saccharomyces cerevisine (Y-55).A culture was harvested in logarithmic growth on KAc synthetic medium and fractionated according to size on a sucrose gradient in the zonal rotor. Small single cells (ca. 0.1-0.2 of cell cycle) and large budded cells (cn. 0.8-0.9 of cell cycle) were collected from the gradient. The cells were suspended in potassium acetate sporulation medium, aerated a t 30°C and all asci containing 2 or more spores were counted. Data from Haber and Halvorson (19721,by permission of the American Society for Microbiology, Washington, D.C.

B y the use of synchronized populations the capacity of a cell t o complete sporulation is highly dependent on its stage in the vegetative cell cycle (Haber and Halvorson, 1972). I n the early part of the cell cycle, up t o the time of bud initiation and DNA replication, the intrinsic capacity of a cell to complete sporulation is low (Fig. 10). Later in the cell cycle, however, the ability to complete sporulation rises significantly. The control of such periodic fluctuation of sporulation capacity is not known. It is possible that sporulation is simply limited by the availability of one or more essential compounds which either fluctuate periodically throughout a cell cycle or are simply present in greater amounts in cells of greater volume (i.e., later in the cell cycle). These results might also be explained by the existence of a control mechanism for the expression of sporulation-specific functions which would be inducible only during a limited period of the vegetative cell cycle. Certainly a substantial portion of the decrease in sporulation capacity after cell scission can be attributed to a sharp difference in the sporulation capacity of newly formed daughter cells from that of mother cells. The observation that sporulation occurs in only one of the halves of a double cell about to divide 85% of the time is in close agreement with the conclusions reached by Yanagita ef al. (1970) in studying sporulation of mother and daughter cells from late stationary phase. These investigators found that cells which contained bud scars (mother cells)

80

JAMES E . HABER AND HARLYN 0. HALVORSON

20

w-2

120

240

360

650

Minutes

FIO. 10. Cell cycle dependency of the ability of Saccharomyces cerevisiae to complete sporulation. Cells of D649 were grown vegetatively in an acetate growth medium for 36 hours and harvested in logarithmic growth. Single cells were isolated by zonal rotor centrifugation and returned to acetate growth medium to begin synchronous growth. At intervals samples were taken to assess the ability of cells at each stage of the cell cycle. A t the time of cell scission the sporulation capacity drops.

sporulated well, whereas newly formed daughter cells without bud scars sporulated poorly. The difference between mother and daughter cells should therefore lead to a fluctuation of 50%. However, as seen in Fig. 10, the variation in sporulation exceeds a factor of 2 attributable to the sporogenic activity of mother and daughter. I n the second cell cycle shown, the drop in sporulation is closer to 50% and may be explained by the difficulty in maintaining synchrony in the acetate vegetative growth medium. Thus newly formed daughter cells as well as mother cells require some period of growth after cell scission, a t least up to the point of bud initiation and the onset of DNA synthesis, before total sporulation is regained. Some indication of the nature of the control of sporogenic activity can be found in the studies of the fate of cells which do or do not

2.

REGULATION OF SPORULATION I N YEAST

81

sporulate throughout the cell cycle. If the capacity of cells to complete sporulation, as measured by the appearance of refractile spores within an ascus, were limited primarily by the total concentration of biosynthetic reserves, then it is likely that cells which did not complete sporula-

FIG.11. Vegetative growth of sporulated and nonsporulated cells. Saccharomyces cerevisiae (Y-55)growing exponentially in potassium acetate growth medium was separated according to size by centrifugation. The small cells corresponding to the beginning of a cell cycle were returned to acetate growth medium to begin synchronous growth. At intervals samples were removed, washed, and introduced into sporulation medium. After 48 hours in sporulation medium each sample was examined to determine the percentage of cells which were full budded. Separate determinations were made for cells which had produced asci containing 3 or 4 spores and those which had failed to sporulate. Cells which had sporulated (0) exhibited the same distribution of bud incidence as the samples had contained just a t the time of introduction into sporulation medium 48 hours previously ( 0 ) .Cells which had failed to complete sporulntion, however, continued vegetative growth, as evidenced by an increase in bud incidence in the early part of the cell cycle and a decrease in budded cells toward the end of the cell cycle (X). Data from Haber and Halvorson (19721, by permission of the American Society for Microbiology, Washington, D.C.

tion would be arrested a t some intermediate point. An examination of cells which did not sporulate revealed that these cells were apparently not arrested a t an intermediate step of sporulation, but rather had not entered sporulation. (Fig. 11). Cells which did not sporulate exhibited

82

JAMES E. HABER A N D HARLYN 0. HALVORSON

substantial vegetative growth, as measured by bud initiation and enlargement. On the other hand, cells which did complete sporulation apparently ceased all vegetative growth very soon after transfer to sporulation medium. The differences between sporulated and nonsporulated cells indicate that the control of sporulation is determined by mechanisms more specific than the concentrations of biosynthetic reserves. The very early shift of the cell along a pathway to sporulation precedes the irreversible point of commitment to sporulation. As defined by Sherman and Roman (1963), commitment is reached when cells are unable to resume vegetative growth without first completing sporulation. The interval just after transfer to sporulation medium appears to be a critical period which merits further study. The ability of Saccharomyces cerevisiae to undergo sporulation is predetermined by the stage in the vegetative cell cycle from which the cells were derived. I n many respects this phenomenon is similar to the observation that Bacillw subtilis sporulation appears to be initiated only during the period of the cell cycle during which DNA replication occurs (Dawes et al., 1971). ACKNOWLEDGMENTS This work was supported by a Public Health Service grant AI-1459, a National Science Foundation grant, B-1750, and by a National Science Foundation Postdoctoral Fellowship held by one of us (J. E. H.) REFERENCES Bresch, C., Muller, G., and Egel, R. (1968). Mol. Gen. Genet. 102, 301. Chen, A. W.-C., and Miller, J. J. (1968). Can. J . Microbiol. 14, 957. Croes, A. F. (1967a). Planta 76,209. Croes, A. F. (1967b). Planta 76, 227. Darland, G. K. (1970) Ph.D. Thesis, University of Washington, Seattle. Dawes, I. W., Kay, D., and Mandelstam, J. (1971). Nature (London) 230, 567. Engels, F. M., and Croes, A. F. 11968). Chromosoma 25, 104. Esposito, M. S., and Esposito, R. E. (1969). Genetics 61, 79. Esposito, M. S., Esposito, R. E., Amaud, M., and Halvorson, H. 0. (1969). J. Bacteriol. 100, 180. Esposito, M. S., Esposito, R. E., Arnaud, M., and Halvorson, H. 0. (1970). .1. Bacteriol. 140, 202. Esposito, R. E., Frink, N., and Esposito, M. S. (1971). Genetics 68, 517. Esposito, R. E., Frink, N., Bernstein, P., and Esposito, M. S., (1972). Mol. Gen. Genet. 114,241. Fowell, R. R. (1967). J . A p p l . Bacc‘eriol. 30, 450. Fowell, R. R. (1969). In “The Yeasts” (A. H. Rose and J. S. Harrison, eds.), Vol. 1, p, 303. Academic Press, New York. Gray, R. (1970). Personal communication. Guth, E., Hashimoto, J., and Conti, S. F. (1972). J. Bacteriol. 109, 869.

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Haber, J. E., and Halvorson, H. 0. (1972). J . Bncteriol. 109, 879. Halvorson, H. 0. (1971). Unpublished results. Halvorson, H. O., Carter, B. L. A., and Tauro, P. (1971). Advnn. Microbiol. Physiol. 6, 47.

Hanson, R. S., Peterson, J. A., and Yousten, A. A. (1970). Annu. R e v . Microbiol. 24, 53.

Hartwell, L. H., Culot,ti, J., and Reid, B. (1970). Proc. N o t . Acad. Sci. I’.S. 66, 352. Hashimoto, T., Gerhardt, P., Conti, S. F., and Nnylor, H. B. (1960). 1. Biophys. Biochem. Cylol. 7, 305. Kadowaki, K., and Halvorson, H. 0. (1971a). J . Bncteriol. 105, 826. Kadowaki, K., and Halvorson, H. 0. (1971b). J . Bacterial. 105, 831. Leighton, T. J., Freese, P. K., Doi, R. H., Warren, R. A. J., and Kelln, R. A. (1972). Spores 5, 238. Miller, J. J. (1963). Can. J . Microbiol. 9, 259. Miller, J. J., and Hoffmann-Ostenhof, 0. (1964). 2. Allq. Mikrobiol. 4, 273. Miller, J. J., Calvin, J., and Tremaine, J. H. (1955). Can. J . Microbiol. 1, 560. Moens, P. B. (1971). Can. J . hlicrobiol. 17, 507. Moens, P. B., and Rapport, E. (1971%).J. Cell Biol. SO, 344. Moens, P. B., and Rapport, E. (1971b). J . Cell Sci. 9, 665. Mundkur, B. (1961). E x p . Cell Res. 25, 24. Panek, A . (1962). Arch. Biochem. Biophys. 98, 349. Pontefract, R. D., and Miller, J. J. (1962). Can. J . Microbiol. 8, 573. Ramirez, C., and Miller, J. J. (1964). Cair. J . Microbiol. LO, 623. Roth, R. (1970). J . Bacteriol. 101, 53. Roth, R., and Fogel, S. (1971). Mol. Gen. Genet. 112, 295. Roth, R., and Halvorson, H. 0. (1969). J . Bacteriol. 98, 831. Sadoff, H. L. (1970). J . A p p l . Bacteriol. 33, 130. Sehaeffer, P. (1969). Bncteriol. Rev. 33, 48. Sebastian, J., Carter, B. L. A., and Halvorson, H. 0. (1971). J . Bacteriol. 108, 1045.

Shaffer, B., Brearley, I., Littlewood, R., and Fink, G. R. (1971). Genetics 47, 483. Sherman, F., and Roman, H. (1963). Genefics 48, 255. Snider, I. J., and Miller, J. J. (1966). Cm2.J . Microhiol. 12, 485. Sogin, S. J., Haber, J. E., and Halvorson. H. 0. (1972). J . Bacleriol. (in press). Trevelyan, W. E., and Harrison, J. S. (1956). Biochem. J . 62, 177. Udeni, S. A., and Warner, J. R. (1972). J . Mol. Biol. 65, 227. Winge, O., and Roberts, C. (1949). C. R . Trav. Lab. Cnrlsberg, Ser. Physiol. 24, 342.

Yanagita, T., Yagisawa, M., Oishi, S., Sando, N., and Suto, T. (1970). J . Gen. A p p l . Microbiol. 16, 347.

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CHAPTER3

SPORULATION OF BACILLI, A MODEL OF CELLULAR DIFFERENTIATION Ernst Freese LABORATORY O F M O L E C U L A R B I O L O G Y , N I N D S - N I H , * B ISTHESDA, MARY L A N D

I. General Remarks about Differentiation.. . . . . . . . . . . . . . . . . . . . . . 11. Morpliology and Genetics of Sporulation in Bacilli., . . . . . . . . . . . 111. Necessary Conditions for the Onset of Sporulation, . . . . . .

85 88

D. Energy Production.. . . . . . . . . .

I!. Later Spore Development., . . . . . .

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

1. General Remarks about Differentiation The recent rapid advances in genetics, biochcmistry, and molecular biology have depended to a large extent on the use of microorganisms as experimental systems. This approach was justified by the assertion that the basic biochemical structures and reaction mechanisms needed for cell replication have evolved a t the single cell stage. Presumably, only the most successful cell type survived so that all present uni- and multicellular organisms evolved from this cell prototype. In fact, the coding mechanisms of transcription and translation and the biochemical pathways of different organisms are remarkably similar, although alternate pathways and multiple enzymes allow a certain variation. With respect to differentiation, such a unifying concept is usually not expressed, perhaps because differentiated tissues of different organs look so different. Undoubtedly, the ultimate differentiation into highly specialized tissues requires very specific biochemical reactions, which can be found only in particular cell types. Nevertheless, the essential

* National Institute of Neurological Diseases and Stroke, National Institutes of Health, Public Health Service, US. Department of Health, Education, and Welfare. 85

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ERNST FREESE

features of the initial reactions converting growing into differentiating cells may be quite similar in micro- and macroorganisms. Regarding animal differentiation, two inaj or types of development are essential, which may be called determination and conrmit)nent (Fig. 1). After fertilization, the egg cell subdivides until a hlastocyst has been formed in mliich all cells are usually onmipotent. Each cell can still develop into a n y direction. Upon implantation, the anlagen for different organs develop in certain cells by the process of determination, which occurs for different cells at different times. Determined cells do

Preimplantation Period

Organogenesis in Embryonic Period Critical Period depends on organ to be developed

Fetal Period and for certain cells differentiation and regeneration throughout life

1

A (z;Determination h ;yd :& (,)

determined cells still multiply

retained

germinal

/ c mafter m i one t m \last round of DNA synthesis cells stop dividing and undergo final differentiation

tisrue

\

germcells

\\

FIG.1. Stages of animnl differentiation. Initidly omnipotent cells become sequentially more and more determined (genetic differentiation) to develop eventually only into certain functional cell types. They multiply in the determined state until t h y are coniniitted to undergo the funr.tion:iI differenti;ition.

not exhibit any apparent structural or functional changes, and they continue to multiply. Nevertheless, they retain their determined state indefinitely, as Hadorn (1965) has beautifully demonstrated for the imaginal discs of Drosophila. Each determined cell has a limited potential restricting its ultimate structural differentiation; in the extreme, it can develop only into one type of tissue. For example, myohlasts (see Holtzer, this volume, Chapter 6) are determined cells which develop only into muscle fibers. At the time when the enzymatic and structural differentiation is about to take place, the determined cells undergo one last division and they become committed to differentiate into specialized tissue. Aftcr the coni-

3.

SPORULATION O F BACILLI

s7

mitment, which may be regarded as the onset of functional differentiation, the cells are obliged to continue their predetermined dcveloprnent. Whereas the process of determination is usually not observed in plants or microorganisms, that of commitment is found for all differentiating systems. Although different microorganisms differentiate into many different forms, which are usually dormant and protected to last through adverse environmental conditions, the stimulus for their diffcrentiation is similar, i.e., effected by the deficiency of rapidly metabolizable nutrients (Cochrane, 1958; Hawker, 1950; Klebs, 1900; Lilly and Barnett, 1951; Scliaeffer, 1969). The commitment of cells in higher organisms resulting in the formation of specialized tissues may also be initiated by conditions which, in effect, change the nutritional state of the target cell (e.g., by enclosure into other cells, hormone effects). One can therefore hope that observations in any one microorganism or tissue culture which elucidate the cellular reactions leading to differentiation will help to unravel these reactions also in other organisins, although the detailed biochemical mechanisms differ. The study of differentiation in microorganisms has the advantage that many developmental mutants can Be isolated that are blocked a t different stages of development. One can then attempt to analyze their biochemical deficiencies and correlate them to the morpho1ogic:d alterations seen in the electron microscope. In addition, the mutations can be genetically mapped, and it can thereby he determined how many genes are required for each developmental stage and which, if any of them, are grouped in an operon. In tissue cultures, the isolation of mutants and the phenomenon of cell fusion makes a similar analysis feasible so that cells in tissue cultures may he considered as microorganisms. In order to avoid confusion, it seenis worthwhile to introduce a new nomenclature for developmental mutations. According to previous agreement, a mutant strain (and the genetic marker) is called auxolrophir, when a mutation has rendered it dependent on an additional nutrient (amino acid, vitamin, etc.). A strain which has the same nutritional requirements as the standard strain, from which all mutant strains are isolated, is called piototrophic. A similar notation will lie useful to identify developmental mutations distinct from auxotrophic mutations, because most developmental mutations do not affect the vegetative growth properties of the cell. We propose, thercfore, to calI the genome, which allows normal differentiation, protogenic and any developmental mutation which causes abnormal development, cncogeiiic (cacos = bad, abnormal 1 . A multiple mutant may then have both auxotrophic and cacogenic markers. It will become apparent from the work herein reported

85

ERNST FREEBE

that normal differentiation requires a much more precise control of the different biochemical reactions than does vegetative cell growth. Cacogenic mutants can therefore be altered in two basically different ways. On the one hand, they can be mutated in genes that are positively required for the performance of a certain developmental function (e.g., the coding for an enzyme needed to produce a new morphological component). If the mutation is not leaky, differentiation will be blocked in all cells; e.g., if such a cacogenic mutation affects a normally sporulating organism no spores will be obtained (asporogenic mutants), except for protogenic revertants, On the other hand, most cacogenic mutations merely cause the over- or underproduction of certain compounds or the accumulation of inhibitors, or they prevent the reutilization of previously accumulated metabolites, or they may affect developmental control mechanisms. The metabolism of cells carrying such mutations is sufficiently imbalanced that they can differentiate normally only a t a low frequency, whereas the probability is high that their development is blocked a t an early stage. The value of the differentiation probability depends on the mutated gene and on the medium. For example, if a sporulating organism suffers such a mutation, the resulting (oligosporogenic) mutant can still sporulate a t a low frequency during exponential growth, but does not undergo the massive sporulation ordinarily observed a t the end of growth. These cacogenic mutations do not affect growth in normal growth media (e.g., growth on glucose), although they may exhibit their presence when specially designed growth media are used (e.g., growth on glutamate). Since these mutants have properties similar to many human hereditary diseases, their biochemical analysis will undoubtedly improve the understanding of surh diseases. II. Morphology and Genetics of Sporulation in Bacilli Sporulation and germination of bacilli are typical developmental processes tvhich are particularly accessible to biochemical and genetic studies. The structural and physiological changes during development have recently been reviewed by Fitz-James and Young (1969), Halvorson (1965), Hanson et at. (19701, Holt and Leadbetter (1969), A. Kornberg e t al. (1968), Mandelstam (1969), hlurrell (1967), and Schaeffer (1969). At the end of exponential growth in a suitable sporulation medium or after replacement of a rich by a poor medium, developmental processes start which lead, 6 or more hours later, to the production of a spore in almost every cell (Fig. 2 ) . At a low frcquency, spores are produced and later germinate also during exponential growth, presumably because

3.

SPORULATION OF BACILLI

s9

a few cells occasionally escape the suppression of sporulation effective during that time. The titer of spores can be easily measured either by colony counting, owing to the resistance of spores to high temperatures (e.g., 20 minutes a t 75OC) or to organic solvents (e.g., octanol, chloroform), or by enumerating the refractilc spore particles under the phase contrast microscope. Since tlie onset of development is reasonably synchronized, one can observe the sequential appearance of new morphologiI

O

~

~

,,

,I

, ,

I

,

I

,

-1

HOURS

FIG.2. Growth and sporulation of Bncillits subtilis. The total titer ( = vegetative of cells inoculated a t time zero into nutrient sporulation medium titer V , 0-0) increases until all rapidly metabolizable carbon sources have been used u p (3 hours). A t the time a t which the log-growth (followed by the logarithm of the absorbancy nt 600 nw) deviates from the linear increme, the dc~elopmental reactions start which lead to sporulation. The later increase in the spore titer (S) is measured by heating the culture for 15 minutes a t 75°C nnd plating ( a t different dilutions). The ratio S/V ( O - - - O ) gives tlie fraction of colony-forming partirles that are heat resistant, i.e., spores (which may he inside or outside :L bacterial cell at the time of plating).

cal structures by electron microscopy and ineasurc the sequential production of new enzymes and other products. Since different bacilli differentiate similarly, their development can be subdivided into the stages defined by Ryter et al. (1966). Figure 3 shows the essential stages of development: the asymmetric prespore septation, followed by the membrane engulfment of tlie prespore cell, then the formation of the cell wall primordium adjacent to the inner forespore nieml)rane, the deposition of the mucopeptide-containing cortex inside the forespore double membrane, and the formation of the proteinaceous coat layers outside the outer forespore membrane. After this development the spore becomes

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ERNST FREESE

denser (the stain used for electron microscopy no longer penetrates or adheres to sections of the inner spore areas). Eventually, the mother cell lyses, liberating the finished spore. If the cell lysate contains a high concentration of spores, their germination is inhibited. After washing and heat activation, spores can be initiated to germinate (measured by the loss of refractility) by single gerniinants (e.g., L-alanine) or by combinations of other compounds (Gould, 1969; Gould and Dring,

\

Prespore Septation

Prespore Membrane Engulfment

Finished Forespore

Cell Wall

m

Primordium

Cortex

Coat

FIG.3. Asymmetric membrane invagination and further development. The finished spores are liberated by cell lysis. They are resistant to organic solvents, detergents, heat, and lytic enzymes.

1972) ; only in a more complex medium does the spore core (with its cell wall primordium) grow out of its shell, splitting it like a nutshell. Cacogenic mutants, defective in sporulation development but able to grow normally, have been isolated from several bacillus species. Bacillus subtilis is particularly easy to use because protogenic strains. usually produce brown colonies, owing to melanin that is found late in sporulation as a byproduct, whereas cacogenic mutants produce pale or white colonies. Schaeffer et al. (1965a) have shown that different cacogenic (sporulation) mutants are blocked a t different stages of development.

3.

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When a particular stage does not develop, the structural or enzymatic development of all following stages is usually also blocked, showing that each stage of development controls the next one. The synthesis of messenger RNA (niRNA) needed for the production of different enzymes during developrncnt, is also sequentially controlled, as has been shown by inhibition with actinomycin D (hlandelstain and Sterlini, 1969). However, some exceptions to tliis rule have been found in certain sporulation mutants (Balassa and Yan1anioto, 1970) and in a glucosamine-requiring mutant (E. B. Freese et nl., 1970) ; each can produce a normal coat without having innde a normal cortex. Apparently it is not necessary to form the whole morphological structure of a given stage but sufficient to produce only certain molecules in order to initiate the next stage of development. Cacogenic genes that have lieen nia])ped, mainly by transduction with phage PBS1, are widely tlistrihuted over the B. subfilis genome, even for different inutaiits blocked a t the snnie stage of development. Nevertheless, cacogcnic mutations seem to recur particularly frequently a t certain genetic locations (Schneffer, 1969 ; Hoch and Spizizen, 1969; Takahashi, 1969; Ionesco et al., 1970; Hoch, 1971; Hoch and Rlatliews, 1972). The large number of genes whose mutation causes abnormal or no development shows that normal development requires many biochemical functions that are not essential for vegetative growth (although they niay be useful or even necessary for growth in certain nutrients). 111. Necessary Conditions for the Onset of Sporulation After exponential growth has ceased, or after the cells have been transferred to a poor nitdiuni, innny Itiochemical changes occur (e.g., synthesis, alteration, or destruction of protein) which may or may not be required for sporulation. In priiiciplc, one can distinguish t h e e possibilities : 1. Necessary for sporulation under any environinental condition. For example, the synthesis of dipicolinate and the cnzyme catalyzing it are necessary for the production of heat-resistant spores (Bacli and Gilvarg, 1966; Halvorson and Swanson, 1969; Chnsin and Szulmajster, 1969). 2. Pleiotrophic for sporulation, i.e., necessary only in certain media while not required in others. For example, alaninc dehydrogenase is not required for sporulation in synthetic media free of almine, hut it is necessary in media containing alanine (E. Freese and Cashel, 1965). 3. Irrelevant for sporulation. For example, the observed effect may result from the media change a t the end of exponential growth or after transfer to a poor medium (e.g., production of amylase; Schaeffer, 1969) , or it may be a by-product of development but not needed for it [e.g.,

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the production of a brown melanin pigment is not needed, since there are strains (e.g., of B. sublilis W23) that can sporulate without producing the pigment]. Some of the sporulation by-products are useful or necessary for germination [e.g., glucose dehydrogenase (Prasad et al., 1972), ribosidase (Powell and Strange, 1956), and spore lytic enzyme (Gould, 1969, 1972) 1. For most of the morphological changes occurring during the developmental period, the required biochemical reactions are still not known, and vice versa for most of the biochemical reactions; it is unclear whether or not they are required for sporulation. I n the following, the reactions known t o occur a t the end of growth will be examined and an attempt will be made to prove that a t least four of them are necessary for the initiation of normal sporulation. The four reactions can be summarized as follows: A. The expansion of the cytoplasm, i.e., the net increase of nucleic acids and protein, has to be greatly reduced, but mRNA and proteins have to turn over. B. Cell wall (mucopeptide) synthesis has to be greatly reduced but not completely stopped. C. Membrane synthesis has to continue, and septation has to be initiated early enough in the cell cycle a t an asymmetric position. D. Energy (ATP, electron transport) for continued synthesis has to be maintained. Since conditions A and B can be satisfied (in the presence of ammonia) only if all rapidly metabolizable carbon sources have been exhausted, it is necessary that the citric acid cycle enzymes ( a t least between a-ketoglutarate and malate) and the cytochromelinked electron transport system function normally. The reasons for these conditions and the detailed biochemical reactions beginning sporulation will now be considered.

POLYMERS A. SYNTHESISOF CYTOPLASMIC Since the conditions under which the sporulation development starts are essentially shift-down conditions to a poor or incomplete growth medium, similar changes of the cytoplasmic components can he expected as the ones that have been examined in Escherichia coli for the last ten years (Maalde and Kjeldgaard, 1966; Lazzarini and Winslow, 1970). I n the electron microscope one can see that the expansion of the cytoplasm is greatly reduced but not completely stopped and that DNA, which a t first appears stretched out as an axial filament, finished a last round of duplication after which one chromosome is incorporated into the prespore cell compartment (Aubert et al., 1969 ; Fitz-James and Young, 1969). The increase of DNA stops [earlier in B. cereus

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(Young and Fitz-James, 1959) than in L'. subtilis (Szulniajster, 1964)], so that there is no more significant increase a t the time of prespore septation and no DNA turnover a t any time. The net increase of R N A and protein stops almost completely a t the end of growth, but both polymer types turn over throughout sporulation (Balassa, 1964 ; Foster, 1956; Rlonro, 1961 ; Szulmajster, 1964). The cytoplasmic expansion has to be arrested during the time of prespore septation to avoid lysis of the cells, which a t that time synthesize cell wall a t a greatly reduced rate (see Section 111,B). Before that time the slowing down of cytoplasinic expansion is probably necessary to allow septation a t an asymmetric position rather than a t the rniddlc of the cell. The synthesis of RNA, protein, and DNA will be exnmined below in more detail. 1. R N A Synthesis

The incorporation of uracil-' 'C into ribosomnl RNA (rRNA) declines rapidly after the cnd of esponential growth (Hussey et al., 19711, while the turnover of mRNA continues and both vegetative and sporulationspecific mRNA are produced (Doi, 1965; Yamakawa and Doi, 1971). The reduction in rRNA synthesis may be caused hy the presence of a guanosine tetraphosphate produced under strpdown conditions (Lazzarini et nl., 1971) ; it may result from the alteration of thc core (p-subunit) of RNA polymerase toward the end of growth (Losick and Sonenshein, 1969; Losick e t nl., 1970; Kerjan and Szulmajster, 1969; A d a et al., 1970), or i t may be due to the production of some other factor. The changes are drastic enough that certain phages can no longer develop when they infect cells after the end of esponential growth; the phage genome is incorporated into spores and gives rise to phagcs during spore germination (E'ellle and Doi, 1967; Losick and Sonenshein, 1969). But it is not clear which if any of tlicse changes are csscritial for sporulation and which merely reflect cellular alterations that occur a t the end of exponential growth. The need for the change of RNA polymerase (which also makes it impossible for certain phnges to multiply during the stationary period) is suggested by the finding that a rifampiciii-resistant mutant (in which phages can always grow) is oligosporogenic (Sonenshein and Losick, 1970). But niany other metabolically imbalanced mutants are oligosporogenic (see under Section II1,D). If the change of R N A polymerase were necessary for any sporulation, one would expect mutants that cannot perform this change to be asporogenic. In any case, the mutant properties show that an RNA polymerase which behaves normally during exponential growth is required for massive sporulation. The turnovcr of RNA depends on intratellular RNases, which are present already during vegetative growth. Toward the end of growth

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an extracellular RNase is produced which may be useful to degrade extracellular RNA. This enzyme may not be needed for sporulation, since mutants have been found which have only little of this activity but can sporulate normally (Lanyi and Lederberg, 1966). I n addition to the breakdown of existing RNA, RNA nucleotides apparently have to be synthesized de novo, for when a uracil-requiring mutant runs out of uracil (in NSM 0.1% glucose) just before the usual maximal turbidity of the culture is reached, no sporulation can be observed, while the presence of uracil allows normal sporulation (E. Freese, 1968).

+

2. Protein Synthesis

The turnover of proteins during the developmental period is so extensive (Foster, 1956; Murrell, 1967) that, e.g., 75-90% of the soluble proteins of B. subtilis spores are synthesized during sporulation (A. Kornberg e t al., 1968). Spores contain most of the enzyme activities found in vegetative cells and most of the enzymes apparently are coded by the same genes as in vegetative cells, as has been shown by the properties of purified enzymes (Spudich and Kornberg, 1968; A. Kornberg et al., 1968) and by the fact that mutants lacking an enzyme activity in vegetative cells also lack it in spores (E. Freese and Cashel, 1965; Gardner and Kornberg, 1967; Prasad et nl., 1972). Some of these enzymes are modified after their synthesis or have altered properties owing to their conformation in spores (Sadoff, 1969; Sadoff and Celikkol, 1969; McCormick and Halvorson, 1964). Only few vegetative cell components are missing in spores, but several new proteins have been identified. With the exception of some ribosomal deficiency, spores have the machinery to produce RNA and proteins (Chambon et al., 1968; Idriss and Halvorson, 1969; Kobayashi and Halvorson, 1968), whereas the synthesis of DNA starts only a long time after germination (Woese and Forro, 1960) . The disappearance, alteration, or new production of specific proteins has been followed throughout the developmental period. Enzymes whose activity decreases during the developmental period are, e.g., a lysine repressible and inhibitable aspartokinase (Bernlohr and Gray, 1969; Hampton et al., 1971), the amidotransferase that produces glucosamine P, a precursor of cell wall components (E. B. Freese et al., 1970), and phosphoenolpyruvate transferase activity (E. Freese et al., 1970). RNA polymerase (Losick and Sonenshein, 1969) and aldolase (Sadoff and Celikkol, 1969) are altered during the early developmental period. FDPaldolase is changed from a heat-sensitive to tt heat-stable enzyme by the removal of a peptide from the vegetative enzyme. This effect is the most direct evidence of a protease 011 a specific enzyme. The heat

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stability of the altered aldolase is presumably not required for sporulation but is advantageous for the germination of spores. A large number of rrizymes are repressed during exponential growth in the rich medium usually employed to grow cells rapidly before they enter massive sporulation. I n addition, the very compounds that suppress sporulation also regress the synthesis of many enzymes (“catabolite repression”). These conditions change drastically when cells enter shiftdown conditions, i.e., after growth declines from the exponential rate or stops completely, or after cells are resuspended in a poor medium. Consequently, the synthesis of many enzymes is derepressed during this time or later, after the cells have undergone some differentiation. The time a t which a given enzyme activity incrcases depends on both the growth and the shift-down medium, and also on other growth conditions. Therefore, it seems to us futile to argue whether the enzyme is really a vegetative or a sporulation-specific enzyme. The issuc is rather whether the enzyme is necessary, useful, or of no value for sporulation or for germination. For example, aconitase and other citric acid cycle enzymes usually show a very low activity during early exponential growth of B. subtilis, but their activity increases toward the end of growth in nutrient sporulation medium (NSM) (Szulmajster and Hanson, 1965 ; Fortnagel and Freese, 1968) or a few hours after transfcr to a rcplacement sporulation medium (Sterlini and iUandclstam, 1969) :I n either case these enzyme activities are necessary to allow massive sporulation in the usual media, because a functioning citric acid cycle is needed both to maintain A T P during thc developincntal period and to avoid the accumulation of a sporulation suppressor (E. Frecse et al., 1969). I n contrast, glucose cleliytlrogenase, which appears a t some timc during the developmental period (the time depending on the strain and the medium used) (Mandelstnm, 1969; Sadoff, 1966, 1969), is probably not needed for sporulation but is useful for germination, in which glucose (or deoxyglucose) can be utilized to reduce NAD via this enzyme (Prased et al., 1972). Ribosidase (Powell and Strange, 1956) and a spore lytic enzyme (Gould, 1969) are other examples of enzymes that are found during sporulation (Powell and Hunter, 1956) and may be useful or nceded for germination. Whereas the NADH (and NADPH) oxidation activity is exclusively located on the mcmbrane during yegetative growth, much of this activity is found in particulate form in the cytoplasm during the developmcntnl period (Szulmnjster and Schaeffer, 1961). Also an alkaline phosphatase activity increases during the developmental period (Warren, I968), hut apparently the same enzyme (Glenn and hlandelstam, 1971) is much less susceptible to repression by phosphate than it is during exponential growth (Cashel and

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Freese, 1964). The significance, if any, of these changes for sporulation is not known. New proteins obviously needed for normal sporulation are dipicolinate synthase (Bach and Gilvarg, 1966; Chasin and Szulmajster, 1969; Fukuda et al., 1969) and coat proteins that comprise u p to 80% of the spore protein (Aronson and Fitz-James, 1968; Aronson and Horn, 1969; Spudich and Kornberg, 1968). Very early in the developmental period, extracellular proteases are formed. I n B. megaterizcna only one metal-dependent neutral protease has been found (Aubert and Millet, 1965). I n B. subtilis in addition to the neutral protease, a diisopropyl fluorophosphate (DEP)-sensitive alkaline serine protease and an esterase have been identified ( R h l i e l and Millet, 1970). B. niegaterium contains in addition an intracellular arninopeptidase (Aubert and Millet, 1965), while in B. subtitis separate intracellular protease activities have not yet been reported. Any or all of these proteases may be used to allow the rapid protein turnover that has been observed during the developmental period. However, the extracellular, nietal-requiring, neutral protcase of B. subtilis is not required for sporulation, because a mutant lacking this activity sporulates normally (Michel and Millet, 1970). There are many mutants that lack extracellular protease activity and cannot sporulate (Schaeffer et al., 1965a; E. Freese and Fortnagel, 1967; Mandelstam, 1969; Balassa, 1969). But it is not clear whether their sporulation deficiency results from the lack of protease activity or whether they are blocked so early in sporulation that the protease production remains repressed. A promising approach has recently been made by Doi’s laboratory (Santo et al., 1971 ; Leighton et nl., 1972), which has obtained a temperature-sensitive cacogenic mutant in which the DFP-sensitive serine protease itself apparently is temperature sensitive; the result suggests that this protcase activity is required for sporulation; it could, for example, cause the alteration of RNA polymerase. Most sporulating bacilli are known to produce one or more (peptide or other) antibiotics toward the end of growth. The antibiotics are detected by their inhibition or killing effects on other bacteria or fungi. These effects may be useful for sporulation by preventing the growth of other organisms during the time during which the sporulating cells do not increase in number, and also by making the components of such organisms available for the sporulating strain. At least some of these antibiotics affect the producing organism itself (Schmitt and Freese, 1968; Sadoff, 1972) or generally inhibit polymer synthesis in v i m and in, vitro DNA (hiach and Tatum, 1964; Hierowski and KuryloBorowska, 1965; Kurylo-Borowska, 1964) ; or affect the cell wall (Siewert

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and Strominger, 1967), or cause selective membrane transport (hlueller and Rudin, 1968). It is therefore possible that some of these molecules are needed for the control of sporulation. This possibility is strengthened by the fact that all mutants unable to produce antibiotic activity are blocked very early in the sporulation clcvelopnient, i t . , before prespore septation (Jayaraman et nl., 1969; Schaeffer, 1967; Schniitt atid Freese, 1968). But some mutants have very weak antibiotic activity and yet sporulate well (Balassa, 1969; Schaeffer, 1969; Schmitt and Freese, 1968). The possibility has still not been excluded that all mutants lacking antibiotic activity are blocked very early in sporulation developnient, so that all subsequent events, including antibiotic synthesis, remain repressed. Since the antibiotic synthesis is turned on early in development, it is not surprising that all mutants blocked later in development can produce the antibiotics (Schaeffer, 1969). 3. D N A Synthesis

When auxotroplrs of E . roli are starved for an amino acid, D N A synthesis continues until the chroniosoine has been cotnpletely duplicated. This is not the case i n B. subfilis, where D N A replication can stop also a t other preferred stopping sites (Copeland, 1971). Therefore, the shift-down conditions initiating sporulation must not lie too stringent but allow D N A to finish a round of replication (Oishi e t nl., 1964), so that one chromosonie can enter the prespore cell while another one remains in the mother cell ispornngium) (Auhert et nE., 1969). But sporulation also does not take place if the shift-down conditions are effective only after D N A has finished its duplication. I n a tetnperaturesensitive mutant of B. sirbfilis which cannot start a new round of DNA replication a t 45OC, Dawes et nl. (1971) have shown that cells do not sporulate when they are transferred to a poor nicdium after DN44 has finished a round of replication ; they sporulate iioririally when they are transferred early enough during D N A replication. The authors have proposed that the cell is sensitive to the initiation of sporulation only during a certain period of chromosome replication. The correlation to chromosome replication may, however, be fortuitous. Possibly, the asymmetric septation process may have to be started before the cell has grown to the size a t which the nornial division process has been started or proceeded too far ; otherwise, e.g., the mechanisms causing the transfer of the chromosome attachment site to the prespore cell compartment might not function. In any case, both division and prespore septation can start in the same cell, as can he seen occasionally in B. subtilis (Fig. 4Ei and rather frequently for B. t))egateriiou grown on arabinose (Kretschmer, 1971). This unusual cell contains both the heginning of

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a normal division septum and a completed prespore septum; the prespore cell can later develop further, giving rise to a spore. I t is not clear why the division septum has not been completed ; the necessary mucopeptide might be missing during the stepdown conditions, or the developing prespore septum might somehow interfere with the further cell division (e.g., by pulling the niesosome away from the division septum). Whereas the amount of DNA doubles during the first 3 hours after B. subtilis has entered shift-down conditions, a 40% loss has been observed in the subsequent 2 hours (Aubert et al., 1969). Thc loss has been traced by radioautography to the 45% of cells that do not enter sporulation (Ryter and Aubert, 1969). In the sporulating cells the chromosome(s) seem to finish merely one last round of replication, whereupon one chromosome condenses in the prespore cell and becomes surrounded by the prespore membrane. I n B. szrbiilis most spores seem to contain one chromosome and perhaps 25% contain two (Aubert et al., 1969). Spores of some other species apparently have more than one chromosome, since they contain several times the amount of D N A found in B. subtilis (Doi, 1965) and because their X-ray inactivation follows multihit curves (Woese, 1959).

B. CELLWALLSYNTHESIS One can see in Fig. 4,B-E (p. 100) that the prespore septum is much thinner than the normal division septum shown in Fig. 4A (or E). Apparently, the prespore septum contains little or no cell wall material (murein). This agrees with the biochemical evidence that only little niucopeptide is formed during the first fcw hours after shift-down conditions, while mucopeptide is later synthesizcd again and incorporated into the developing spore cortex (Vinter, 1963; Pitel and Gilvarg, 1970). The reduction in murein synthesis is necessary for sporulation, becausc otherwise wall material could be depositcd between the double membrane of the prespore septum and the stiff septum could no longer grow around the prespore cell and engulf it. This can be seen in certain sporulation mutants which form asymmetric septa containing thick material but cannot develop further (Ryter et al., 1966; Yamamoto and Balassa, 1969b; FitzJames and Young, 1969). A particular mutant of this kind, producing two pseudoprespore cells is shown in Fig. 4F. The blockage of development can also be seen when cells are transferred to fresh NSM at the time a t which they have formed prespore septa or even when the-double membrane has started to engulf the prespore. Murein grows into the area between the membranes and completely separates the prespore cell from the mother cell. I n B. cereus both cells thus produced can grow and divide again, the larger mother cell expanding faster than the

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former prespore cell (Fitz-James and Young, 1969). Thus, irrespective of what else has happened in the cytoplasm of either mother or prespore cell, neither has lost the capacity to grow. I n B. subtilis only the mother cell grows after transfer, whereas the prespore cell, which is much smaller than that of B . cereus, remains stagnant without continuing the sporulation process (Frehel and Ryter, 1969). It is apparent that sporulation remains suppressed as long as the synthesis of cell wall (and its precursors) continues a t a ratc high enough to allow normal cell wall synthesis into any septum that might form. This suppression of sporulation, owing to steric restraint, is quite different from the repression of enzyme synthesis, which occurs a t the level of transcription of DNA into mRNA. One might assume from the foregoing that the formation of the prespore septum would not require any murein. However, if that were the case, it would be difficult to understand how the prespore septum can grow in a relatively well defined direction across the cell (unless one assumes a special protein to stiffen the septa1 membrane). Without any stiffening material the meinbrane would rather be expected to grow in a more or less random fashion, like the mesosomal membrane that is frequently seen in bacilli and is present in exaggerated amount in some cytochrome mutants, as shown in Fig. 4G. I n fact, if one grows a glucosamine-requiring mutant of B. subtilis (strain 60984) in NSM medium plus 1 mg/ml D-glucosamine, which is just enough to allow growth to the absorbance a t which it usually terminates, no prespore septa can be observed even several hours later, a t which time some of the cells have lysed. But when one adds glucosamine (100 pg/ml) whenever a small amount of lysis is observed, presporc scpta are formed normally. [Several hours later octanol-resistant, but not heat-resistant, spore particles are produced which have little or no cortex but a normal coat (E. B. Freese et al., 1970).] Apparently, a small amount of murein is necessary to form a prespore cortex. One can see an indication of murein a t the sites a t which the septum joins the cell wall (Fig. 4, B-E). The requirement of some cross wall synthesis has also been proposed by Hitchins and Slepecky (1969a,b), who showed that concentrations of penicillin or P-phenethyl alcohol which inhibit cell division of B. inegateriuin inhibit also the formation of prespore septa.

C. MEMBRANE SYNTHESIS A N D PRESPORE SEPTATION The fact that membrane synthesis continues during prespore septation is apparent from the increase in the amount of membrane seen in the electron microscope and has been measured by the incorporation of acetate-I4C into phospholipids (Pitel and Gilvarg, 1971). The reason for the asymmetric membrane invagination leading to

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FIQ.4. (A-G and I-M) Cells of Bacillus sublilis. (H) Cells of B. megnterium. Bar = 1 pm. ( A ) Normal cell division. (B) Prespore septation. (C) Beginning prespore septation. (D) Prespore septa in several attached cells. (E) Preapore and division septa in one cell. (F) Double prespore septation. (G) Unusual membrane development in a cytochrome mutant. (H) Sporulation in a chain of B. megaterium cells. There are septa between the adjacent spores, although they are barely visible

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prespore septation is unknown. The problem looks similar to that of minicell formation in E . coli (Adler et al., 1967) ; but whereas rninicells usually do not contain DNA, spores do. Prespore septa apparently are usually formed next to the location of either the last or, more likely, the last but one cell division. When three or more cells containing prespore septa can be seen in the electron microscope attached to one another, two septa are always found adjacent (Fig. 4D) (Kretschmer, 1971). Certain bacillus strains form chains in which most cells can sporulate (Fitz-James and Young, 1969; Yamarnoto and Balassa, 1969a). The refractile spores are usually seen at any place of the cell, probably because the spores can swim in the cell cytoplasm as one can observe under the phase contrast microscope. But when the sporulation process is followed in a chain producing B. cereus strain T (Whhren et al., 1967) and the spores are inspected under phase contrast as soon as they can be seen, there are almost always two spores found adjacent to each other (Fig. 4G) (Kretschmer, 1971). Many features of prespore septation are similar to cell division, in particular the apparent need for some murein synthesis mentioned before. Many of these similarities have been summarized by Hitchins and Slepecky (1969b), who proposed that prespore septation arises by a modified cell division. Before this proposition can be examined in detail, it is first necessary to describe the different models of cell division that are now being considered. 1. Normal Cell Division

In spite of many experiments, recently summarized by Higgins and Shockman (1971), the area of the cell in which rod-shaped bacteria expand their length is not established. As these authors pointed out, only for the coccus Streptococcus faecalis is the growth and division mechanism sufficiently understood. Its growth zone is located in the center of the growing coccus and contains a membrane invagination ending in a bag-shaped mesosome. Potential new growth (and division) in the photograph. (I) Spike of beginning septation on the cell pole opposite to the one a t which the successful prespore septum develops. (J) Multiple septation. (K) Multiple septation indicating the splitting of septation sites. (L) Membrane engulfment. On one side murein can still be seen at the site of prespore attachment to the cytoplasmic membrane. ( M ) Further membrane engulfment indicating the frequent asymmetry of the process. ( F ) , (J), and (K) were made from the cacogenic (sporulation) mutant 61421, ( G ) from the cytochrome adeficient mutant 61484 (Taber and Freese, 19721, and (H) from a particular strain of B. cereus T (Wlihren et cal., 1967). All other figures were made from the standard sporulating strain 60015.

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zones can be recognized by ridges that grow out, just after cell division, from the present growth zone. The two daughter components of the duplicating chromosome could be attached either to the underlying menibrane of either the growth zone or the two ridges. Cell division occurs in the present growth zone while the coccus grows to twice its rninimum size. I n rod-shaped bacteria the segregation of chromosomes (DNA) into progeny cells can be achieved because each chrotnosome apparently is attached to the cellular membrane in some organisms (Ryter and Jacob, 1964; Ryter, 1969) but not necessarily in all (Ryter and Landman, 1964) via a meinbrane invagination (mesosome). When a new round of chromosome replication is started, both daughter chromosomes attach to the niembrane a t different positions in a random manner (Ryter et al., 1968). The membrane between the attachment sites expands (grows), perhaps because the movement of the clironiosornes pulls their attachment sites in different directions and thereby separates the sites into different cell compartments. A certain time after the initiation of a chromosome replication the cell divides by the invagination of the membrane in the middle of the cell, between the chromosome attachment sites, accompanied by the growth of cell wall material into this invagination; the finished daughter chromosomes are thereby segregated into two cells. The cell wall material between the still attached cells is then partially dissolved by a lytic enzyme until the cells separate completely (Higgins and Shockman, 1971). [The overall time between the initiation of replication and cell separation was measured in a strain of B. szibtilis as 138 minute a t 3OoC (Paulton, 1970, 1971) 1. The membrane septation may be initiated by some protein when that has reached a critical concentr at'ion (Inouye and Pardee, 1970). In a poor growth medium, the minimal cell (just after division) contains one chron~osome leg., for E. coli (Helmstetter, 1969) or the 168 strain of B. subtilis (Donachie et al., 1971) 1, while another strain of B. subtilis apparently contains a minimum of two chromosomes (Paulton, 1971) ; in such a medium, cell division occurs after the chromosome has been duplicated. However, during exponential growth in a rich medium new rounds of chromosome replication start before the old one has finished aad before the cell has divided. Cell division takes place after one round of replication has been finished, while each of the two daughter chromosomes is already engaged in its further replication. A freshly divided cell has then two (or more) chromosome attachment sites (Helmstetter et nl., 1968; Helmstetter, 1969 ; Clark, 1968). I n a rich sporulation medium, cells have a t least two replication sites during exponential growth but toward the end of growth (at least) bacilli apparently divide to end up with one duplicating chromoson~e(Aubert et al., 1969, Kretschnier, 1971).

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I n both poor and rich media, cells divide relatively accurately in the middle. If one does not want to assume that cells can inysteriously measure the middle of a cell, one has to grant the presence of some marker on the cell envelope which determines the position of cell division. As will become apparent below, this marker would have to be located on the cell membrane. T o assure the proper segregation of such a division site, the membrane should grow only a t one site (or zone) in a cell having one duplicating chromosome (or a t two sites if the cell has two or three replication sites). [Possible models will be discussed later (Fig. 5).] Such a membrane growth site could be most easily provided by the location of a mesosome, which could deliver new membrane components accumulated from the cytoplasm by transport into the mesosoma1 vesicle. In fact, the cell division septum is often found associated with a mesosome (Ryter and Jacob, 1964; Ryter, 1968). I n seeming conflict with the idea of a single menibrane growth site are data showing the diffuse distribution of radioactively or otherwise labeled lipid components into the progeny (Mindich, 1970; Green and Schaechter, 1971 ; Tsukagoshi et nl., 1971). But recent nuclear resonance observations with spin-labeled phosphotidylcholine have shown that phospholipids can move (by exchange with neighbors) in an artificial meml)rane bilayer faster than 3 p m per niinute, which woulcl allow rapid diffusion of any lipid label (R. D. Kornberg and hlcConncll, 1971). I n a. biological nicnibrane, the lateral diffusion is slower hut still fast enough to allow randomization of label in E. coli in 15 minutes (Overath et d..1971). Therefore, the diffuse distribution of labeled lipid components observed in the next cell generations does not prove B diffuse growth of the membrane. I n fact, the temperature profile of 8-galactoside transport, induced after a shift from oleate to palmitate in a fatty acid-requiring mutant, shows that the transport protein is incorporated a t the same sitc(s) a t which new membrane is formed (Orerath et al., 1971). The existence of one membrane growth zone (in a mononucleate cell) can also be shown by progeny studies if membrane components are labeled which cannot rapidly move along the membrane, either because they are too large or because their relative positions in the membrane are fixed. For example, the proteins for p-galactoside transport ( Autissier and Kepes, 1971 ) or flagella (Ryter, 1970) are nonrandomly distributed over progeny cells, indicating the presence of only one membrane growth zone per mononucleate cell. With respect to such large membrane-associated components as a topological matrix, sites concerning DNA attachment, nienibrane growth, and cell division can he located. One could assume that cell wall expaiids also a t tlie same mesosoma1 site a t which the membrane grows by the extrusion of mucopeptide

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and its attachment to existing wall. But evidence is mounting that cell wall can expand a t many sites along the cylindrical surface of the cell. Autolysin activity apparently opens the wall a t many places because an extensive turnover of murein has been observed in some bacillus strains (Mauck and Glaser, 1970; Mauck et al., 1971), although not in others (Pitel and Gilvarg, 1970). The opening of existing murein bonds could allow not only turnover but also wall expansion, although one could argue that expansion really is a separate phenomenon (Higgins and Shockman, 1971). The almost random synthesis of cell wall is indicated by the thickening of murein during amino acid starvation or in chloramphenicol (see Higgins and Shockman, 1971), and by the random incorporation (van Tubergen and Setlow, 1961) or dissimilation (hlauck et al., 1972) of diaminopimelic acid. I t is also indicated by the observation that thick walls, formed during amino acid starvation, become fragmented over the whole cylindrical portion of the cell after transfer to a n amino acid-containing medium (Frehel et al., 1972). It appears, therefore, that murein can expand a t many sites along the rod and that the mesosome involved in membrane synthesis becomes important for murein synthesis only during cell division (or other septation-containing murein). This picture entails the surprising conclusion that the membrane cannot be fixed to the murein, except perhaps a t a few places, but must be able to move under the expanding murein layer. There are essentially two models which assume the expansion of the membrane a t one growing point (or zone) per nucleus and explain regular cell division in the middle of the rod. They are illustrated in Fig. 5 by the upper part of models A and B. Model A is the equivalent of the model proposed by Jacob et al. (1963; Ryter et al., 1968). I n the middle of the newly divided cell a chromosome is attached to the membrane. When the chromosome starts replication, the membrane develops a growth zone (dashed vertical lines) in the center from which the two sites for chromosome attachment and for future growth and division grow out on both sides (indicated by dots). During exponential growth the cell expands until it has reached the normal size a t which the formation of a division septum is triggered by some component; then it divides in the center. If the cell grows in a rich medium new rounds of chromosome replication can be started before the cell has divided (Cooper and Helmstetter, 1968; Donachie and Begg, 1970; Ryter e t al., 1968; Paulton, 1970). In this way, cells can have several zones of wall expansion and multiple chromosome attachment sites but still divide in the middle. (We shall call a model in which the freshly divided cell has two chromosome attachment sites “A?,” etc.)

3.

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-'zEi? m

'1' -

Model C

CTX-).'

._

'.

.'L"A

. -

FIG.5. Models of cell division (upper three parts of each model) and sporulation (lower parts). Growth zones are mnrked hy horizontal dashes dividing the cell, while dots indirate chromosome attachment sites. In model A, the prespore septa, indicated by i i vertical line, would be formed by the precocious septation at one of the present DNA attachment (and future division) sites. I n model B. they would appear a t the present growth zone' and \ \ o d d therefore be automatically adjacent to the pole nt. which the re11 had divided laat (new pole). In niodel C, prespore septa would invnginatc at n new growth zone that hnd been formed a t the cell end. I n both models A and C the prespore septa could appear a t either pole, the :ictual location being influrnced hy some gradirnt dependent on the position of the last, cell division. If the observation on cell chains has been correctly interpreted, this septation ~ . o u l dpreferentinlly occur :it the old pole ; it has nevertheless been shown adjacent to the new pole in order to allow a comparison to model B.

I n model B, whose cell division is patterned after Donachic and Begg (1970), the growth zone of a mononucleate cell starts from (or close to) the cell end at which the last cell division occurred. T h e new cell envelope (membrane) expands in one direction until the growth zone has ended in the middle of the cell; a t that time the growth zone

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becomes a division site. IThe model proposed by Higgins and Shockman (1971) assumes in addition that cell wall also expands only iii one dircction.] The present growth zone (mesosome) presumably serves as an attachment site for both chromosomes, whose segregation into the two daughter cell compartments must be somehow guaranteed a t the time of cell division. When cells are growing in a rich medium, the new round of chromosome duplication, coupled with a division of the growth zone, starts before the cell has divided. Growth occurs a t two (or more) zones, which in this case move from the middle of the cell by pushing cell wall out toward the middle. (We shall call the corresponding models “B2,” etc.) Such a cell (model B,) behaves similarly to that shown in model A, if the dots represent the growth zones and the dashes in the middle, the division site. 2. Asymmetric Septation

One can now attempt to explain asymmetric prespore septation within the above models of cell division (see lower parts of models A and B in Fig. 5 ) . Since examinations in B. subtilis have shown that the spore contains one-half as much DNA as the whole cell at the time of septation (Aubert et al., 1969), it is likely that the normal sporulating cell has a t this time two chromosomes, one of which enters the prespore cell. But the possibility will also be considered that the cell approaching prespore septation has two replicating chromosomes (models A2 and B 2 ) , so that of the four resulting chromosomes one is separated into a prespore. In any model one would assume that shift-down conditions result in the slowing of cytoplasmic expansion and the asymmetric production of a prespore septum. In model A, the prespore septum would be formed at one of the future septation sites a t a distance of one-half the minimal cell length (or one-fourth to one-half of the present cell length) away from one cell end. However, in electron micrographs the sites a t which the prespore septum is attached to the cell envelope usually are much closer than that to one cell end (Fig. 4, B-D).[Although the oblique cut through a single cell could create the impression of a narrow prespore cell which is actually wider, this parallax is only minimal if the next cell adjacent to the prespore side can still be seen attached (Fig. 4, C, D).] This problem is less severe in model A,, in which septation could occur as close as one-fourth of the minimal cell length (measured right after division) away from the end. As an interesting possibility explaining the strong asymmetry, one could assume that the mesosome (or membrane site) to which one arm of the replicating chromosome is attached

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(dot in Fig. 5A) would start to increase its membranous surface and simultaneously move its juncture with the cytoplasmic membranc further to one side of the cell; the mesosome in the middle of the cell would have to shrink correspondingly. I n model B the absence of cytoplasmic expansion could start septation while the growth zone is still close to the cell end, except that a round of DNA replication would have to be finislied. The prespore septum could therefore appear a t any distance from a cell end. This model would predict that sister cells contain prespore septa adjacent to the last cell division. [The earlier observations on cell chains may indicate the opposite, i.e., the production of prespore septa adjacent to the last but one cell division.] Rlodel B cannot explain three cell types containing several septa, which are observed as exceptions and will now be analyzed. a. Extra Division Site. One type of cell, mentioned before, contains both a prespore septum and a beginning division site (Fig. 4E). Kretschmer (1971) has observed this cell type in 10% of arabinose-grown B . megaterium. The cells apparently stop their division and then produce one spore per cell having such an incomplete division septum. This cell type could be explained in model A (Fig. 51, but not in model B. b. Double Prespore Septation. Occasionally, two presporc septa can be observed a t the two ends of the same cell (Fig. 4 F ) . There are also sporulation mutants in which this double prespore septation occurs regularly (Young, 1964; Ryter et al., 1966; Yamamoto and Balassa, 1969b, Waites et nl., 1970). These cells cannot develop further, but the layer between the double membrane is later filled out with murein. [Young (1964) has found that 40% of her B . cereus mutant can form one finished spore per cell if the medium contains dipicolinic acid (as metal chelator?). The asymmetry of septation apparently is needed for further spore development.] When the dou1)le prespore cells are transferred to fresh medium, they elongate and then divide in the middle, each new cell being able to grow while carrying one pseudo-prespore at the end. Both pseudo-prespores contain DNA clearly visible in electron micrographs, and the middle cell must contain DNA, since it can grow again. Therefore, this cell type apparently contains four (at least three) chromosomes a t the time of prespore septation and would have to be explained by models A, or B,. The septation of model A, would be similar to that described before. But the prespore septation of model B, would no longer have the advantage t h a t it could occur while the growth zone is still adjacent to the last cell division, because the last division, so to speak, did not occur. As stated above, model B, is very similar to model A and could also not produce prespore septa close

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enough to either cell end. Apparently, it is impossible to explain double prespore cells with either model B or B,. A careful examination of electron micrographs shows that even the cells having normal prespore septa often contain on the other cell end a spike that indicates a beginning asymmetric septum (Rytcr, 1965) (Fig. 41). Normal sporulating cells may start septation proximal to both cell ends but continue this development only a t that end to which a chromosome has been attached, while the development at the other end is aborted. Again, this phenomenon could not be explained by model B. c. Multiple Septation. 'In some exceptional cells (Remsen and Lundgren, 1965) and very frequently for certain sporulation mutants (Ryter, 1965; Balassa and Yamanioto, 1970), one can see two or more septa in series a t one end of the cell, For example, in the electron micrograph of Fig. 45, nuclear bodies can be seen in two of the three small cell compartments. Such cell types would not be expected by either model A or B. I n some cases the distribution of the multiple septa looks as though they have developed sequentially from the cell end. I n other Cases (e.g., Fig. 4K) it appears likely that a septation site has been split, producing two separate septa. It is apparent that model B alone cannot explain several of the above observations. Since models A, A,, or B, also have to be stretched to explain the strong asymmetry of prespore septation and since they cannot explain the multiple septation, it is possible that prespore septation is not simply related to normal cell division but requires a new septation mechanism that can be initiated during stepdown conditions. The possibility of such extra septation that is not necessarily coupled to DNA replication or normal cell division is indicated not only by the multiple septation, but also by the observation that transfer of B. subtilis cells to low temperature can trigger the formation of extra, often anucleate, cell compartments (Neale and Chapman, 1970). The asymmetric prespore septa may therefore be produced in growth zones that have developed from (or close to) the cell end. This type of septation could be regarded as an extension of model B ; one could even assume that the old cell end has retained from the previous cell division the ability to start a growth zone (Kretschmer, 1971). But this would raise the problem of how a chromosome attachment site, being located in model B on one side of the cell, should move all the way to the cell end on the other side. Furthermore, normal cell division occurs rather precisely in the middle of the cell and double prespore cells can be formed which can still divide in the middle. Therefore, an extension of model A (or A? or B,) appears more promising. Such a possibility is illustrated by model C in Fig. 5. I n that model, cells would normally

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grow froni the niiddle (either a t the dashed horizontal lines or anywlierc in the area between the dots) and divide in the middle. As a result of shift-down conditions, a new growth zone (mesosome) would develop a t (or close to) the end(sj) of the cell. This zone would expand the envelope somen.hat and then producc a septum [perhaps when triggered by some cornpound (Inouye and Pardee, 1970) or by the termination of a round of DNA replication]. Having separate sites for cell division and prespore septation, model C could explain all the above observations (see Fig. 5 C ) . Double prespore cells would derive from cells in which two chromosomes undergo duplication. Multiple septa, appearing a t one end of the cell, could arisc either by the repeated development of several growth zones, each producing a scparate septuin, or by the splitting of existing growth zones or septation sites. Instead of the development of new growth zones from the cell end, one could have proposed the immediate production of new septation sites. However, one would then have to explain why thin septa are observed preferentially near to one cell end and not randomly anywhere in the cell. The development of aclditional growth zones (or septation sites) from either cell end, might tie triggered, e.g., a t a fracture in the murein structure. [Such a fracture could arise each time a cell divides. For example, Higgins and Shocknian (1971) have proposed a model in which the cylindrical rod murein is sutured to the division murein.] Once they have been initiated, the asymmetric septation sites behave like division sites, producing thin (prespore) septa if niurein synthesis has been reduced and thick (division type) septa if not. Most difficult to explain in model C is the problem of how a chromosonie is included in the prespore cell. Since that inclusion occurs regularly, it seems clear that the chromosome must be nttached to the y e spore membrane, e.g., to the mesosome that is found a t the asyminetric septation site and part of which is included in the prespore (Ryter and Jacob, 1964; Ryter, 1968; Holt and Leadbetter, 1969). As one possibility, the new chromosomal attachment site might not be able to attach anywhere to the membrane in the mother cell and therefore eventually attach to the mesosome in the prespore cell. Another possibility is that the chromosome has (or develops) a second attachment site with which it attaches to the prespore membrane; such a site might be the one a t which chromosome replication can stop during leucine starvation (Copeland, 1971). A third possibility is that the production of a mesosomal membrane a t the prespore growth zone pulls the membrane over so that a t least one chromosome attachment site enters the prespore cell compartment; this mechanism is similar to the one already mentioned under model A.

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D. ENERGY PRODUCTION

In the usual sporulation media, rapidly metabolizable carbon sources are used up at the end of growth or left out in a replacement medium. Since polymer synthesis must continue throughout the devclopmental period, A T P must be regenerated by means of some carbon source, such as acetate, to allow the activation of polymer precursors. In addition, organisms (like B . subtilis) that depend on the uptake of extracellular compounds must retain an active transport mechanism, which depends for many compounds (e.g., all aniino acids) (Konings and Freese, 1972) on a n intact electron transport system. Other strains ( B . megaterium) accumulate during exponential growth poly-p-hydroxybutyrate (as fat droplets), which they can reutilize as energy and carbon source during the developmental period; they can sporulate in water (endotrophic sporulation) (Kominek and Halvorson, 1965 ; Foster, 1956; Lundgren et al., 1969; Slepecky and Law, 1961). The transition in the utilization of carbon sources can be indirectly observed by the change of the pH in a n (unbuffered) medium. Up to the end of growth (e.g., of B. cereus or B. subtilis) the pH drops ( i f the medium is unbuffered) until all glucose or other rapidly metabolizable carbon sources are used up. Subsequently, the pH rises l~ecauscpyruvate and aretate, which were accumulated in the medium are reutilized as energy supply and in part they are used to produce 1)oly-p-hydroxybutyrate (Nakata and Halvorson, 19601, except in B. subtitis (E. Freese et al., 1969). Simultaneously a high demand for oxygen is observed, reflecting the reoxidation of NADH, which has been produced by the oxidation of pyruvate (Halvorson, 1957, Taber and Freese, 1972). A large amount of evidcnce has bccn accumulated which shows that the citric acid cycle and the clectron transport system function very actively during the developmental period to oxidize poly-p-hydroxybutyrate or acetate (via acetyl-CoA) or other carbon sources (e.g., glutamate) (Murrell, 1967; E. Freese e f a/., 1969; Hanson et al., 1970). The concentration of ATP per cell is remarkably constant throughout the developmental period (Fig. 6A) (Klofat et al., 1969); the humps may indicate the change to a new carbon source. The need of the citric acid cycle during sporulation is shown by the fact that mutants lacking the activity of one of the cycle enzymes cannot maintain A T P or uracil incorporation into RNA, and they do not sporulate (Szulmajster and Hanson, 1965; Fortnagel and Freese, 1968; E. Freese et al., 1969; Hanson et al., 1970). The decrease in the concentration of ATP in an aconitase-deficient mutant is shown in Fig. 6B. Both A T P synthesis and uracil incorporation can be restored by the addition of any carbon

*

0

t

5

a

FIG.6. Change of ATP concentration. The absorhancy of the culture a t 600 mp (OD, 0-0) and the amount of ATP ( O - - - O ) per milliliter were followed during growth in nutrient sporulation medium. All ATP was inside the bacteria. (A) Standard sporulating strain of B. subtilis. (B) An nconitasedeficient mutant. When 2 mM L-glutamate was added at the time indicated by the arrow, the decline of the ATP concentration was avoided (m-.). Data of Klofat et al. (1969), by permission of the American Society for Biological Chemists, Inc., Bethesda, Maryland.

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compound which enters the cycle after the step blocked in the mutant, e.g., glutamate in the above mutant (Fig. 6B). But since this addition does not restore sporulation, the mutants apparently accumulate or are unable to destroy some inhibitor of sporulation (E. Freese et al., 1969; Yousten and Hanson, 1972). The effect of a mutation in aconitase can also be produced by a-picolinic acid; at low concentrations (2 X M ) it inhibits sporulation without affecting growth (Hanson et al., 1963). a-Picolinate, as well as other compounds that chelate transition metals, cause the specific inhibition of aconitase, by removing its ferrous ions (Fortnagel and Freese, 1968) ; inhibition can be reversed by Fe ions. The citric acid cycle can function only when the reduced coenzymes can be reoxidized by the electron transport system; consequently, mutants lacking either cytochrome a or both a and c are also unable to sporulate normally, although they can still grow vegetatively (Taber and Freese, 1972). Under the electron microscope one can see that they accumulate large amounts of membrane vesicles (Fig. 4G) (E. B. Freese, 1972). Other cacogenic mutants unable to maintain ATP during development are blocked in other so far unidentified pathways (E. Freese et al., 1969). I n most cacogenic mutants sporulation could not be restored by the addition or subtraction of any compound in the medium. But a few sporulation mutants are exceptional in this respect,. One mutant is blocked in pyruvate dehydrogenase and runs out of acetate and NADH (rather than ATP) after some time of growth in NSM (E. Freese and Fortnagel, 1969). While the addition of glutamate or other carbon sources restores the reduction of NAD (and a limited additional growth), sporulation can be restored only by acetate (Fig. 7 ) . The optimal acetate concentration is 70 mM, indicating that this compound is taken up by facilitated diffusion. Higher acetate concentrations inhibit growth and amino acid transport (Sheu and Freese, 1972; Sheu et al., 1972). For another cacogenic mutant the metabolic block has not yet been identified, but sporulation can be restored by acetate, palmitate, or several ot,her carbon compounds (Schmitt and Freese, 1968). Yousten and Hanson (1972) have recently shown that the sporulation of aconitase and isocitrate dehydrogenase-deficient mutants of B. subtilis can be restored by transfer of cells a t the end of growth into a spent medium, in which a sporulating strain had grown, supplemented with a carbon source such as gluconate. Growth of the sporulating strain apparently had removed a suppressor of sporulation from the medium. When cells approach the end of growth in a rich sporulation medium they undergo a number of changes, some of which apparently are necessary for the subsequent massive sporulation. They can undergo these changes also when they are transferred to a poor medium (having, e.g.,

3.

Id

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SPORULATION OF BACILLI

L 0.02

0.04

0.06

0.08

ACETATE / M I

FIa. 7. Curing of sporulation by acetate in a mutant lacking pyruvate dehydrogenase activity. The mutant cells of Bacillus subtilis were inoculated into nutrient sporulation medium containing the stated concentrations of potassium acetate. The titers of nll viable cells (0-0) and of heat-resistant particles (spores, O---O) were determined 27 hours later. Data of E. Freese and Fortnagel (1969), by permission of the American Society for Biological Chemists, Inc., Bethesda, Maryland.

glutamate or lactate as sole carbon source) a t any time of growth (Mandelstam and Waites, 1968) Ramaley and Burden, 1970). The poor carbon source is not used only for the production of energy, because transfer of B. subtilis to an acetate-containing medium does not allow sporulation although ATP is regenerated. To obtain sporulation it is necessary to add also glycine or L-serine, which alone do not support sporulation. While the reason for the need of acetate glycine is not known, it is interesting that in this combination the cells can sporulate only if they have been grown beyond the end of the exponential growth period (Sugae and Freese, 1970). Apparently some reaction (production of an enzyme, initiation of asymmetric septation, replication of DNA?) cannot occur after the cells have been transferred to the acetate glycine medium which does not support any growth.

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+

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IV. Suppression of Sporulation As we have seen before, sporulation occurs not only a t the end of growth but, a t a small frequency, also during exponential growth. Schaeffer et al. (1965b) have utilized this fact t o determine the frequency of sporulation in repeatedly diluted cultures growing in different media. They found that the frequency of sporulation is low when both nitrogen and carbon sources are metabolically rapidly available, as in casein

HOURS

FIQ.8. Suppression of sporulation in the presence of glucosamine. Standard-type cells of Bacillus subtilis were inoculated into a nutrient sporulation medium containing 10 mg/ml n-glucosamine. The increase in the titer of spores shown in Fig. 2 was prevented. Similar results can be obtained in the presence of n-glucose, n-fructose, n-mannose, glycerol, or L-malate (whose concentration has to be maintained because malate is metabolized much more rapidly than the other carbon sources). From E. Freese et al. (1972).

hydrolyzate, but that it is high when either nitrogen (e.g., nitrate) or carbon (e.g., lactate) is growth limiting. Apparently, sporulation can be suppressed by (at least) one nitrogen-containing organic compound. The massive sporulation, in a nutrient sporulation medium (NSM) or in a poor medium (containing excess ammonia) to which the cells have been transferred is suppressed by an excess of rapidly metabolizable carbon sources. This is shown for the case of glucosamine in Fig. 8. Sporulation of B. subtilis in NSM can be similarly suppressed by excess of glucose, fructose, glycerol, and malate, but not by slowly metabolizable compounds such as glutamate, lactate, or succinate. The attempt to determine the biochemical nature of the sporulation suppressor is

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hampered by the fact that the rapidly metabolizable carbon (or nitrogen) sources can efficiently produce all other cellular compounds. To avoid this dissimilation, mutants have been used which isolate certain regions of the biochemical pathways. For example, a mutant that lacks phosphoglycerate kinase activity (in the Embden-Meyerhof path) cuts the metabolic pathways into two disconnected parts which have been called upper and lower metabolic subdivisions (E. Freese et al., 1972). The mutant cannot grow on any carbon sources entering only one subdivision, while it grows normally when both subdivisions are supplied (e.g., by glucose malate). In nutrient sporulation medium, the mutant sporulates almost normally, but sporulation is suppressed, before prespore septation, by rapidly metabolizable carbon sources entering either the upper (e.g., glucose, fructose, glycerol) or the lower (malate) subdivision. Consequently, a t least one compound in the upper and one in the lower metabolic subdivision can suppress sporulation. I n the lower subdivision a compound suppressing sporulation apparently accumulates in citric acid cycle mutants, because for all of them development is arrested before prespore septation, and the addition of glutamate or malate does not enable sporulation. I n the upper metabolic subdivision several metabolically separated compounds can each suppress sporulation. For example, glycerol-P, whose catabolism is blocked in a mutant, and glucose-6-P, whose metabolism has been prevented in a triple mutant, each prevent sporulation (E. Freese et al., 1972). The reason for the suppressing effect of these different compounds is not known. One possibility is that they inhibit growth before it can proceed to the stage a t which another compound (s) suppressing sporulation is used up. Alternatively, the compounds might more directly inhibit or repress some reaction needed early in sporulation. In the course of these studies, a mutant of B. subtilis was isolated which lacks the activity of phosphofructokinase. I n some organisms this enzyme is inhibited by ATP and activated by ADP, while in other organisms (e.g., the sporulating B. lichenifomis) (Tuominen and Bernlohr, 1971) it is inhibited by PEP. These inhibitor mechanisms undoubtedly are important to control the rate of glycolysis. They have also been implied as controlling differentiation. However, the phosphofructokinase mutant can sporulate well, and its sporulation is still suppressed by all the above-mentioned carbon sources (E. Freese et al., 1972). It is therefore doubtful that the control mechanisms observed for this enzyme in other organisms are essential for differentiation. Recent experiments by Elmerich and Aubert (1972) have narrowed down the possible precursor of the amino group of a suppressor to

+

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glutamine rather than glutamate. When a glutamine-requiring mutant of B. megateriuin was transferred to a medium containing glucose and ammonia, sporulation was suppressed by excess glutamine, but not glutamate. One of the compounds, whose amino groups are derived from glutamine, is glucosamine, a precursor of mucopeptide, which suppresses sporulation a t least during prespore septation by preventing prespore engulfment.

V. later Spore Development After prespore septation, the double membrane grows around the prespore cell (Fig. 4L) until it fuses on the other side and thereby separates the forespore from the connection with the extracellular mileu (see Fig. 3 ) . Since the double membrane of the prespore septum is relatively stiff, it may require the action of an autolysin (Brown and Young, 1970) to soften the intramembranal material and make it flexible enough that it can engulf the prespore. One can see in Fig. 4L that the murein has dissolved a t one end of the septum, whereas some of it is still present on the other end. This may be the reason why closure of the septum often occurs on one side of the cell axis (Fig. 4M). After septation, the total membrane surface of the prespore cell increases more slowly than that of the mother cell, which probably contains more membrane growth zones. The more the prespore cell becomes surrounded by the growing membrane, the smaller should be the influx of extracellular molecules and the larger therefore the discrepancy between growth of the membrane in the mother cell and that surrounding the prespore. This consideration alone shows why it is important that prespore septation occurs a t an asymmetric position of the cell. It is not known why the membrane grows around the prespore and not randomly in any direction. One possible reason is that the pressure of the mother cell may push the growing membrane, which is flexible and mobile, in all directions, causing the membrane to expand into the area of least resistance, i.e., around the prespore cell. But the directed membrane growth may also be caused by an affinity of the membrane to some prespore component or by the molecular properties of some stiffening material between the double membrane. I n any case, prespore engulfment requires the presence of the stiff cell wall, because it cannot be observed in protoplasts produced by lysozyme treatment (Fitz-James, 1964). The rigid cell wall could be needed to maintain a pressure difference or as a guiding structure directing membrane growth around the prespore. However, after the complete forespore double membrane has been finished and the forespore swims in the mother cytoplasm, further spore development can continue even after protoplasting.

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It is clear from the mode of membrane growth shown in Fig. 3 that the layer between the two membranes of a forespore is the equivalent of extracytoplasmic space. It is therefore likely that the core of the forespore extrudes into this layer the mucopeptides which form a cell wall primordium (Murrell, 1967). During spore gerriiination the primordium becomes the wall of the emerging cell. Although forespores containing wall priinordia have been isolated from B. cereus, it has not yet been possible to stimulate their further development outside the mother cytoplasm (Lundgren et al., 1969). After the completion of the double membrane, the outer forespore membrane of B. cerem produces vesicles that eventually detach and form an exosporium which completely envelops the forespore. This structure may be used to concentrate spore coat precursors, which in B. cereus are synthesized from the beginning of the developmental period (Aronson and Fitz-James, 1968), whereas they are produced shortly before their deposition in B. subtilis (A. Kornberg et al., 1968; Spudich and Kornberg, 1968). An exosporium-like structure can also be seen in B. subtilis at the time of cortex formation (E. B. Freese et al., 1970). The finished spores of the larger bacilli (not B . subtilis) carry the exosporium as a bag around them; but the material apparently is not needed for germination (Aronson and Horn, 1969; Gerhardt and Ribi, 1964; Matz et al., 1970; Murrell et al., 1969). The deposition of the wall primordium is followed by appearance of the cortex consisting of mucopeptides which are probably excreted from the mother cytoplasm and which are assembled slightly differently than in the normal cell wall (Warth, 1968; Warth and Strominger, 1969; Murrell et al., 1969; Tipper and Pratt, 1970). While the intramembranal space widens, owing to the cortex formation, CaZ+and dipicolinate are incorporated, probably mostly into the core (Lean2 and Gilvarg, 1972). When the cortex has been finished, several layers of spore coat are formed outside the outer forespore membrane; they contain most of the spore’s protein and are rich in cysteine, which is probably used to form disulfide bridges (Vinter, 1959; Aronson and Horn, 1969, 1972). VI. Commitment to Sporulation The concept “commitment” has usually been imprecisely defined to describe the general observation that an organism becomes “committed” to continue its differentiation even when it is transferred to environmental conditions that would have prevented the development a t an earlier time. In the future it will be necessary to specify by which measurable property one defines ‘lfurther development’’ and which en-

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vironmental change is used to stop this development. If an organism can form stable mRNA containing the information for a certain protein, it may become “committed,” with respect to addition of actinomycin D or rifamycin, as soon as that mRNA has been formed. But the reverse is not necessarily true. If an organism becomes committed with respect to actinomycin to develp a protein, it does not necessarily contain a stable mRNA but may have become impermeable to actinomycin. This may be the reason why Aronson and del Valle (1964) have found that protein synthesis and sporulation of B. cereus become resistant to actinomycin D quite early in the developmental period. No such commitment was observed in B. subtilis in which most mRNA has the same half-life (1.5) during sporulation as during vegetative growth. However, Sterlini and Mandelstam (1969) have reported that in B. subtilis small concentrations (1 pg/ml) of actinomycin D inhibit the overall spore development but allow the expression of those functional properties that would be usually expressed 1 hour later, suggesting the formation of mRNA which is stable for 1 hour. This result disagrees with the observation by Chasin and Szulmajster (1969) , who found that higher actinomycin concentrations (10 pg/ml) almost immediately inhibited the further increase of dihydrodipicolinate synthase. Since a small amount of mRNA synthesis may continue a t low actinomycin concentrations, whereas high actinomycin concentrations can affect other cellular reactions (e.g., cause cell lysis during the developmental period), the existence of stable mRNA remains uncertain. Regarding overall differentiation to heat-stable spores, two phenomena of commitment are particularly interesting, because they involve membranes and are not necessarily directly related to control of mRNA synthesis. We shall call these phenomena single and double membrane commitment, respectively.

A. SINGLE MEMBRANE COMMITMENT Whereas D-glucose, added t o NSM during the exponential growth period, suppresses massive sporulation, it can no longer do so when the cells have entered the developmental process. This commitment, with respect to glucose addition, to produce heat-resistant spores, takes place long before the prespore septum has been formed. It results from the inability of the developing cell to transport glucose. The transport of glucose in bacteria is accomplished by a phosphoenolpyruvate (PEP)-dependent transferase system (Roseman, 1969) by which the incoming glucose is simultaneously phosphorylated (to glucose-6-P) . The membrane-associated component (enzyme 11) of this transport system is inducible in B. subtilis during exponential growth, but the inducibility is absent during the developmental period (E. Freese et al.,

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1970). This lack of enzyme inducibility is sufficient to explain the commitment to sporulation, because in a mutant in which glucose-PEPtransferase cannot be induced and which lacks the active transport of glucose, sporulation cannot be suppressed by glucose even during exponential growth. Glucose-PEP-transferase activity is not only not inducible during the developmental period, but it also declines rapidly during this time if it had been preinduced (E. Freese et al., 1970). This enzyme system may therefore not be functional during development either because one of its components is destroyed or inactivated or because the properties of the membrane have changed. The ability of developing cells to turn off the transport of certain molecules represents a very simple control mechanism by which the cell protects itself against changes in the environment that might prevent the continuation of differentiation. We call this effect single membrane commitment because i t occurs at a time at which the developing cell has but one membrane.

B. DOUBLE MEMBRANE COMMITMENT As was shown before, the sporulation development can be suppressed and growth can be resumed when developing cells are resuspended in fresh growth medium or when casein hydrolyzate ( 5 mg/ml) is added to the medium. This suppression is effective even while the prespore is being engulfed by a double membrane. But the cells are committed, with respect to this treatment, to develop heat-resistant spores, as soon as the forespore double membrane has fused and the forespore is completely enclosed in the cytoplasm of the mother cell. If the cells are suspended in fresh medium after this time, the forespores develop into heat-resistant spores; the mother cell cannot resume growth in B. subtilis (Frehel and Ryter, 1969), whereas it can grow and divide again in B. cereus (Fitz-James and Young, 1969). This commitment is very likely caused by the closure of the forespore double membrane, which drastically changes the transport of molecules into and out of the forespore cell. The development of the double membrane indicates that the layer between the two membranes is extracytoplasmic space (see Fig. 3 ) . If each of the membranes contained the ordinary mechanisms for active transport they would operate in opposite directions. Material could be actively transported from both the mother and the prespore cell compartment into or out of the intramembranal space, but no material would be actively transported through both membranes. Molecules could therefore pass from the cytoplasm of the mother cell into the core of the forespore only by passive or facilitated transport. If the cell is exposed to new medium, compounds (e.g., amino acids)

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can rapidly enter the mother cytoplasm by active transport, which is coupled to the electron transport system (Konings and Freese, 1972), whereas these compounds can enter the forespore cell only very slowly. A competition can now set in between the rate a t which the forespore cytoplasm expands due to incoming molecules and that a t which the spore cortex gets filled by rnucopeptide that is probably actively transported into it from the mother cytoplasm. Owing to the seclusion from the outside, certain enzymes can be induced or derepressed, greatly influencing the further development. For example, a dipicolinate synthase is now made, an enzyme whose synthesis has not been achieved in vegetative cells by the addition to or removal from the medium of any compound. The double membrane commitment may therefore be regarded as the most decisive stage in the differentiation of bacilli, after which the highly specialized compounds such as dipicolinic acid and the components of the cortex and the coat are produced. ACKNOWLEDGMENTS

I thank Dr. E. B. Freese for the electron micrographs and both her and Dr. R. Henneberry for valuable discussions. REFERENCES Adler, H. I., Fisher, W. D., Cohen, A,, and Hardigree, A. A. (1967). Proc. N a t . Acad. Sci. U S . 57, 321. Aronson, A. I., and del Valle, M. R. (1964). Biochim. Biophys. Acta 87, 267. Aronson, A . I., and FitzJames, P. C. (1968). J . Mol. Biol. 33, 199. Aronson, A. I., and Horn, D. (1969). Spores 4, 72-81. Aronson, A. I., and Horn, D. (1972). Spores 5, 19-27. AuberC, J.-P., and Millet, J . (1965). C. R . Acnd. Sci. 261, 4274. Auhert, J.-P., Ryter, A., and Schaeffer, P. (1969). Spores 4, 14S158. Autissier, F., and Kepes, A. (1971). Biochim. Biophys. Acta 249, 611. Avila, J., Hermoso, J. M., Vinuela, E., and Salas, M. (1970). Nature (London) 6, 1244. Bach, M. L., and Gilvarg, C. (1966). J. Biol. Chem. 241, 4563. Balassa, G. (1964). Biochem. Biophys. Res. Commun. 15, 236. Balassa, G. (1969). Mol. Gen. Genet. 104, 73. Balassa, G., and Yamamoto, T. (1970). Mol. Gen. Genet. 108, 1. Bernlohr, R. W., and Gray, B. H. (1969). Spores 4, 186-195. Brown, W. C., and Young, F. E. (1970). Biochem. Biophys. Res. Commun. 38, 564. Cashel, M., and Freese, E. (1964). Biochem. Biophys. Res. Commun. IS,541. Chambon, P., Deutscher, M. P., and Kornberg, A . (1968). J . Biol. Chem. 243, 5110. Chasin, L. A., and Szulmajster, J. (1969). Spores 4, 133-147. Clark, D. J. (1968). Cold Spring Harbor Symp. Qztant. Biol. 33, 823. Cochrane, V. W. (1958). “Physiology of Fungi.” Wiley, New York. Cooper, S.,and Helmstetter, C. E. (1968). J. M o l . Biol. 31, 519. Copeland, J. C. (1971). Science 172, 159.

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CHAPTER 4

THE COCOONASE ZYMOGEN CELLS OF SILK MOTHS: A MODEL OF TERMINAL CELL DIFFERENTIATION FOR SPECIFIC PROTEIN SYNTHESIS Fotis C . Kafatos T H E BIOLOQICAL LABORATORIES, HARVARD UNIVERSITY, CAMBRIDGE,

MASSACHUSETTS

I. Introduction. . . . . . . . .. A. A Definition of D ........................... B. A Class of Phenomena: Terminal Differentiation for LargeScale Protein Synthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Cocoonase Production: Morphological Studies.. . . . . . A. The Escape of Moths from the Cocoon.. . . . . . . . . . . . . . . . . . B. Morphological Differentiation in the Galea. . . . . . C. Autoradiographic Studies on Zymogen Synthesis ................. port, . . . . . . . 111. The Differentiat Galea: Biochemical and Enzymological Characterization. . . . . . . . . . . . . . . . . . . . . . . . . A. The Enzymology of Cocoonase.. . . . . . . . . . . . . . . . . . . . . . . . . . ........................... IV. Quantitation of

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

.......................... D. Differentiation as a Changi thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1’. Transition Points in Zymogen Synthesis during Development. . . VI. Progressive Increase in Zymogen Synthesis during Phase 11.. . . . A. Stability of the Differentiation-Specific Message. . . . . . . . . . . . ..... B. Implications of Messenger Stability. . . . . . pecific C. Can Highly Differentiated Cells Produce Protein As They Do with a Single Gene Copy per Genome?. VII. Concluding Remarks ................................ References. . . . . . . . . . . . . . . . . . . . ..................

125 12.5 127 129 129 130 139 142 142 144 145 145 147 149

153 159 161 161 173 178

18.5

187

I. Introduction

A. A DEFINITION OF DIFFERENTIATION It may be bad style to begin an article with a definition, but since the field of development has long been plagued by arguments about semantics such a beginning may be salutary. I consider cellular differentiation to be any change or sequence of changes which is internally 125

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programmed, or becomes independent of an external stimulus a t some point in time; such a change may involve either the form or the function of EL cell or both. This definition may not be as broad or as narrow as some might desire. For example, despite its obvious importance for any understanding of cellular regulation I do not consider the induction of p-galactosidase in Escherichia coli as differentiation, because it is utterly dependent on continued presence of the inducer. By contrast, I do consider the internally coordinated synthesis and assembly of phage in the phage-bacterium system as a valid case of differentiation. Similarly, bacterial sporulation, which a t some point in time becomes independent of the inducing stimulus, is cellular differentiation just as much as the maturation of the human erythrocytes. The ovalbumin-producing cells of the chick oviduct are differentiated because their characteristic morphology, and their capacity to function again immediately upon restimulation, persist even though ovalbumin synthesis decreases when estrogen is withdrawn (Oka and Schimke, 1969b). Admittedly, the criterion of independence from an external stimulus is somewhat arbitrary. It is adopted partly for reasons of convenience, in order to make cellular differentiation less than coterminous with the vast field of cellular regulation. I n addition, this criterion serves to highlight an important aspect of advanced forms of differentiation, commonly found in higher animals: the stability of the differentiated state, which often persists through many cell generations and which, in vivo or in tissue culture, may survive long periods without expression. On the other hand, I want to emphasize that my relatively broad definition of cellular differentiation does not necessarily imply a universality of mechanism. I firmly believe that the pursuit of “the” mechanism of differentiation is illusory. Cellular structure and function can be controlled a t many levels, and the relative importance of various controls will prove to differ among systems. As our understanding advances, we will undoubtedly find generalizations that will make comprehensible entire classes of phenomena. Some of these generalizations will be limited to only prokaryotes or onIy eukaryotes, others will span this phylogenetic gap ; still others may be limited to unidirectional differentiation, such as that which culminates in cellular death (erythrocytes, xylem elements), others will be relevant to differentiation involving a succession of distinct stages (such as the sequential polymorphism of insect cells undergoing metamorphosis) , In all humility, we must recognize that our understanding of developmental changes is still primitive. Under those circumstances, it seems a needlessly parochial folly to restrict the exchange of ideas by overcircumscribing the range of phenomena we are willing to consider as differentiation. Lest I, in turn, be accused of sectarianism, I hasten to say that

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I do not consider cellular differentiation coterminous with development. Equally important is morphogenesis, the emergence and maintenance of form in an organism (or part thereof) through the temporal and spatial coordination and delimitation of differentiative events (e.g., see MacWilliams and Kafatos, 1968; MacWilliams et al., 1970). M y preoccupation with cellular differentiation is due partly to temperament and partly to a belief that a t the present time it is the easier of the two developmental processes to study. According to our definition, particular cases of differentiation may involve primarily a change in form, rather than a major reorganization of metabolism and biosynthesis. In fact some classical systems, such as the asymmetrically developing Fucus egg or insect cells producing specific types of bristles and scales, show major changes in form with only subtle metabolic alterations. The importance of such phenomena is obvious to any developmental biologist of broad outlook. However, because contemporary tools for a mechanistic analysis are primarily (and fashionably) biochemical, most of the recent studies on differentiation emphasize metabolic changes. Among the systems used for biochemical studies of differentiation, several favorite categories can be distinguished : developing early embryos ; prokaryotes and unicellular eukaryotes ; primary or permanent cell cultures derived from multicellular organisms; and virus-transformed cells. A final category are cells terminally specialized for production of one or only a few characteristic proteins. B. A CLASSOF PHENOMENA: TERMINAL DIFFERENTIATION FOR LARGESCALE PROTEIN SYNTHESIS Cell-specific proteins are particularly convenient in biochemical studies of cellular differentiation. Consider, for example, the contractile proteins produced by muscle cells, hemoglobin produced by erythroblasts, the digestive enzymes and zymogens produced by the pancreas, collagen produced by fibroblasts, keratins produced by skin, crystallins produced by the eye lens, immunoglobulins produced by plasma cells, ovalbumin produced by the chick oviduct, silk produced by moth silk glands, the saliva proteins produced by the giant salivary cells of Drosophila, Chironornus, and other flies, or the hatching enzyme of silk moths, cocoonase, produced by modified mouthparts. All these cases have certain characteristics in common. 1. The proteins are either secretory, i.e., destined for use outside the cell, or they are part of a specialized cellular machinery. I n the former case, they are topologically outside the cell from the very time

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of synthesis, being released by membrane-bound polysomes directly into the cisternae of the endoplasmic reticulum (Redman and Sabatini, 1966). I n either case, the proteins do not function in intermediary metabolism, intriguing but sometimes exasperating in its complexity. I n a certain sense, they are “luxury molecules,” as Holtzer has called them (Holtzer and Abbott, 1968), although they are the very reason for existence of the differentiated cells. It may be a reasonable hope that their synthesis will be under relatively simple controls, since it need not be finely tuned to all the complexities of day-to-day metabolism. 2. These proteins are made in large amounts. I n most cases the fully differentiated cells devote the majority of their protein synthetic capacity to elaboration of these products. Because of their abundance the proteins are easy to purify. Therefore they can be conveniently quantified by chemical or immunochemical procedures, and their rate of synthesis can be evaluated reasonably directly through radioactive amino acid incorporation experiments. Direct assay methods are distinctly superior to estimation of minor constituents through functional criteria such as enzymatic activity ; functional activity in a complex cellular homogenate is rarely a simple reflection of enzyme concentration. 3. Large-scale synthesis of a particular protein is tantamount to large-scale expression of a particular gene, Gene expression can be controlled a t many levels of “information flow.” But whether one is interested in control a t the template level (e.g., amplification or redundancy), in transcription, in posttranscriptional steps (e.g., mRNA selection, transport, stabilization), in translation or in posttranslational events (e.g., protein cleavage, assembly, turnover), a system involving large-scale expression of a particular gene seems attractive in its simplicity, and may have methodological advantages. One example that comes t o mind is. specific mRNA isolation, which in eukaryotes has been accomplished primarily in highly differentiated cells (hemoglobin, myosin, immunoglobulin, and silk mRNA). 4. I n each case, synthesis of specific proteins occurs during a defined period, as the culmination of a precise program of sequentiaI development. Some common steps in the program are formation of the specialized cells from “stem” precursors, cell growth or multiplication, and preparation for rapid protein synthesis through the accumulation of the appropriate cellular machinery (e.g., free ribosomes or, in the case of secretory proteins, membrane-bound ribosomes and well-developed Golgi zones). In many cases, one or more of these steps are under the control of identified chemical stimuli (e.g., hormones), or of less well characterized but recognizable molecules (e.g., serum growth factors). Differentiation of highly specialized cells can thus be studied apart from the rest

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of the life cycle, as a distinct, predictable but often manipulable sequence of events. The preceding comments may have given some indication of why I have chosen to study a particular type of cellular differentiation, that involving large-scale synthesis of specific cell proteins by terminally differentiated cell types.

II. Cocoonase Production: Morphological Studies A. THEESCAPE O F MOTHSFROM

THE

COCOON

The galeae of butterflies and moths are a pair of mouthparts, normally developed into the proboscis used in sucking food from flowers. In the silk moths, which neither feed nor drink during their entire adult life, the galeae are small and have long been considered vestigial. However, they in fact play an important role in the “hatching” of the moths from the cocoon (Kafatos and Williams, 1964). The first postembryonic stage in the life cycle of a silk moth is the caterpillar. After the caterpillar grows to its mature size, it spins around itself a cocoon of silk. Metamorphosis, a most remarkable series of transformations, then ensues. First the caterpillar turns into an immobile creature, the pupa. A period of quiescence or diapause may follow, depending on the species and on environmental conditions. At the end of diapause, the pupa transforms into an adult moth over 2-3 weeks, a period known as adult development. At the end of this final metamorphosis, the moth is ready to fly away and procreate. But, of course, it first has to escape from the cocoon. I n certain species a trap door is available, built by the caterpillar from the outset. In others, the texture is loose and the moth can cut or push its way out. In still others, the cocoon is so tough that mechanical means alone do not suffice, and the moth resorts to better living through chemistry. Specialized cells develop in the galea to produce a large amount of a proteolytic hatching enzyme, cocoonme. The enzyme is extruded to the surface late in adult development and, just before hatching, it can be found as a dry semicrystalline material encrusting the galeae. I n moths of the genus Antheraea, 0.1 mg or more can be collected with forceps from each animal. The material proves to be virtually pure enzyme. At the time of hatching, it is redissolved near its pH optimum, 8.3 (in copious isotonic KHCO, secreted by another gland; Kafatos, 1968), it is brought into contact with the cocoon and is allowed to digest the proteinaceous matrix binding the silk fibers together. The structure of the cocoon is thus loosened locally and the moth can push its way to the outside.

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B. MORPHOLOGICAL DIFFERENTIATION IN THE GALEA 1.H o m o m l Control

Cocoonase is synthesized as an inactive precursor by a group of approximately 20,000 large polyploid cells in each galea, the zymogen cells. Their morphological development has been described a t the light microscope level (Kafatos and Feder, 1968), and a detailed study with the electron microscope is forthcoming (Selman, 1972; Selman and Kafatos, 1972). Differentiation of the galea cells is under the control of the developmental insect hormones, ecdysone and juvenile hormone. Ecdysone acting alone sets in motion the program of epidermal cell differentiation described below. But when juvenile hormone is also present, differentiation is inhibited and the cells remain in the state characteristic of the pupa. It is important to realize that the hormones act merely as triggers, signaling to the cells which of two alternative paths to take, pupal or adult development. That decision is made very early, as shown by the fact that juvenile hormone is ineffective in preventing zymogen cell differentiation if given after day 3 or 4 of adult development (Williams, 1956, 1968 ; Kafatos, 1972) , long before morphological differentiation begins. Once the decision is made, it is implemented in an orderly fashion over nearly 3 weeks, irrespective of the presence or absence of juvenile hormone. As will be discussed below, many important steps in zymogen cell differentiation occur after day 7, when ecdysone is no longer needed for normal development (Williams, 1947; also “hyperecdysonism” cannot be induced by injections after day 3; Williams, 1968). Thus, as is true in general (Ashburner, 1970; Williams and Kafatos, 1971; Wyatt, 1972) the insect hormones seem to serve as triggers for alternative internally coded programs, rather than as continuous guides of differentiation. 9.Formation of the Cells The metamorphosis of pupa into moth (adult development) occurs in slightly less than 3 weeks, 18 days in Antheraea polyphemus or 21 days in A. pernyi. From now on, descriptions will be made according to the polyphemus timetable; for greater accuracy, each day can be subdivided into three parts, I, I1 and 111, respectively. At the outset of adult development, the galea walls consist of only squamous epithelial cells arranged in a single layer and, like their counterparts elsewhere in the body, specialized for production of the overlying cuticle. The zymogen cells and their accessory duct cells develop from this homoge-

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neous population of “undifferentiated” cells (undifferentiated with respect to subsequent specialization). First the epithelial cells retract from the overlying pupal cuticle (Fig. 1A) , leaving behind a proteinaceous “molting gel” (with inactive proteases subsequently activated to digest the cuticle; Katzenellenbogen and Kafatos, 1970, 1971b). It is important to note that a t this stage all cells are normal epidermal cells, secreting molting gel just like their counterparts elsewhere in the body. Then a period of DNA synthesis occurs (days 1 to 2 ) , followed by a wave of mitosis (days 3 to 4 ) . The orientation of these mitotic divisions is significant. Since the epidermis in insects is composed of one cell layer, proliferative mitoses are always horizontal (with the metaphase plate and hence the division plane perpendicular to the surface). By contrast, differential divisions giving rise to epidermal derivatives (e.g., scales, glands, bristles) include a t least one that is vertical or partly inclined with respect to the surface (Kuhn, 1971; Lawrence, 1966). I n the galea, we observe metaphase plates parallel to the surface (Fig. 1B). Following the mitotic wave, the specialized duct and zymogen cells appear in a regular pattern (Fig. lC, D). I n recent electron microscopic studies, Kelly Selman in my laboratory has shown that a single duct cell is associated with two zymogen cells; the resulting three-cell “organule” (Lawrence, 1966) alternates with ordinary epidermal cells. No more cell divisions occur during development.

3.Construction of the Duct Apparatus I n subsequent days, while the epidermal cells prepare to secrete the new cuticle delimiting the galea, the organules undergo an intricate morphogenesis, resulting in a precise alignment of cell processes (Selman, 1972). Cell processes serve as a scaffolding for deposition of an elaborate cuticular duct apparatus, which will eventually permit conduction of cocoonase to the surface. Thus, the organules still function as epidermal cells, albeit of a specialized type, depositing a characteristic extracellular cuticle just like the bristle-and-socket or scale-and-socket organules of other body regions. The innermost of the two zymogen cells acquire two long, microtubule-filled pseudoflagella which extend as far as the molting gel. Around the outermost part of the pseudoflagella (nearer the surface), the duct cell secretes a single duct as a final outlet for cocoonase. Around the innermost part of the pseudoflagella (nearer their attachment to the cell body), the two zymogen cells collaborate (mostly on days 6 to 9) to form a complex cuticular apparatus, continuous with the duct. This apparatus apparently serves as a valve, regulating the outward flow of cocoonase.

FIG.1. Morphological development of the galea cells. Photographs taken from 1 to 1.5 pm glycol methacrylate sections stained with 0.05% toluidine blue with or without acid fuchsin as a counterstain. Except for (C)and (G),a part of the lateral wall of the galea is shown, with the external surface on the left and the blood on the right. (A) Pupal galea. The thin squamous epithelial layer is not specialized except for production of the overlying thick cuticle (unstained thick endocuticle and pigmented external exocutile). x 1050. (B) Mitotic period (day 4). The epidermis has retracted from the cuticle, leaving behind the molting gel 132

(left). Cell divisions have already occurred in this region, giving a pseudostratified appearance to the epithelium. Near the center of the picture a metaphase is evident, with the plate oriented parallel to the surface. A prophase is adjacent. ~ 7 7 0 . (C) Low power view of a complete cross section through the hollow galea. The tissue is embedded in the molting gel, most evident on the left and top. The lateral side of the galea (left) contains the zymogen organules, whereas the medial side (.right) has only normal epidermal cells. The cavity of the galea [continuedl 133

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FOTIS C. KAFATOS

4. Preparation for Zymogen Synthesis Of the three cells, the duct cell apparently has no function beyond cuticle production. It remains small, probably diploid, and by the end of cuticle deposition its cytoplasm shrinks substantially, just as in ordinary epidermal cells (Fig. 1F). By contrast, the two zymogen cells are also preparing for a further developmental career. Between days 5 and 12, their cytoplasm increases tremendously in size (Fig. lE, F) and, in parallel, their nuclei become polyploid through continued cycles of DNA replication. Polyploidization or polytenization are widespread in insect secretory cells: scale-forming cells in Lepidoptera and giant salivary gland cells of Diptera are just two examples. It should be considered an alternative to the cell multiplication commonly observed in vertebrates, similarly permitting an increase in the total secretory tissue. The final nuclear size, staining properties and number of nucleoli (Fig. lE,) suggest the occurrence of 6 to 7 replication cycles, resulting in a ploidy of approximately 100-200. Preliminary cytophotometric measurements of DNA using Auramine 0 confirm this estimate (Nardi, 1972). [Fig. 1 legend continued1 includes blood with blood cells, fat body cells, myoblmts, and tracheoblasts. ~ 1 1 2 .(D) Day 7. The normal epidermal cells (outermost layer of nuclei, on the left) are preparing for cuticle synthesis; their apical region is baaophilic. The larger zymogen cell nuclei are evident on the right, surrounded by basophilic cytoplasm. The duct cell nuclei are at an intermediate location (D). An intensely basophilic sleeve of duct cell cytoplasm (arrowhead) is secreting the duct; the pseudoflagella are seen protruding through the duct into the molting gel. x770. ( E ) Day 10 galea. Each organule has a single storage vacuole ( V ) filled with zymogen; a cuticular valve (white arrowhead) occludes the duct (black arrowhead). The zymogen cell nuclei are large and filled with granular chromatin and numerous prominent nucleoli. The cytoplasm is intensely basophilic except for unstained scattered Golgi zones. X448. (F) Day 15 galea, shortly before zymogen extrusion. The storage vacuole has grown tremendously through zymogen accumulation. Compare the size of a normd epidermal cell nucleus (black arrowhead) and its cytoplasm to the nucleus (white arrowhead) and cytoplasm of a zymogen cell. ~ 1 3 3 . ( G ) Low power view of a cross section through a galea a t the beginning of zymogen extrusion. Compare with Fig. 1C. The large zymogen cells are evident in the lateral side (left), and the very thin layer of normal squamous epithelial cells (black arrowhead) on the medial side (right). The blood space contains muscles (white arrowhead) aa well as tracheae, including a very large tracheal sac (upper right third of the galea; the diffuse curved lines in the sac are folds of the embedding plastic). x70. (H) Galea just before emergence of the adult moth. Except for one at the bottom of the figure, the vacuoles have emptied of zymogen; moreover, the cell cytoplasm has regressed. The ducts penetrating the cuticle are evident (black arrowhead), as are the cuticular valves (white arrowhead) within the empty vacuoles.

x 308.

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While the zymogen cells increase in size, their cytoplasm also changes in appearance. Ribosomes accumulate to a high density. At the early stages of growth, they are found primarily in the ground substance of the cytoplasm, many of them in free polysomes (Fig. 2A). As time passes, however, the endoplasmic reticulum proliferates extensively, so that after day 9 a large fraction of the ribosomes are membrane-bound (Fig. 2B). In surface views of the ER cisternae, spirals of 9-12 ribosomes (Fig. 2B) can often be seen; such a polysome size would correspond to a protein ,of 27,00036,000 daltons (Staehelin et al., 1964), in agreement with the known molecular weight of cocoonase zymogen (Section II1,B). Other changes occurring in the zymogen cells during the growth phase are the development of many Golgi zones and the depletion of stored reserves, such as glycogen deposits. As a result, the cytoplasm on day 9 and later consists mostly of a branched network of endoplasmic reticulum, interspersed with numerous Golgi zones and a few mitochondria (Fig. 2C). No marked polarity in distribution of these organelles exists. However, the secretory polarity becomes evident with the development of an extracellular cavity in the apical region of the cells. This cavity, a “storage vacuole” for zymogen, is formed jointly by the two zymogen cells of an organule (Selman, 1972), as an extracellular cavity around the valve apparatus which they had secreted earlier. One cell forms the bottom of the vacuole, and the other, being partly wrapped around the first, forms most of the side walls. The apical surface of both cells, facing the cavity, is lined with microvilli. Thus, during their growth, the zymogen cells prepare themselves for rapid synthesis of a secretory protein, cocoonase zymogen, through the large-scale accumulation of membrane-bound ribosomes and Golgi zones. Moreover, they make provision for storage by forming a vacuole in their apical region, as well as a cuticular valve regulating extrusion. 5. Zymogen Synthesis and Extrusion The morphological evidence indicates that no extensive secretion takes place until day 9 (when the main parts of the valve apparatus are formed). At that time, the endoplasmic reticulum becomes distended with granular material, dense mcmbrane-bound granules appear in the Golgi regions, and material of similar staining properties fills the storage vacuole (Fig. 2 D ) . At the light microscope level, the storage vacuole is first seen a t about day 9,, to fill with a material staining with acid dyes, such as acid fuchsin. The zymogen-secreting phase lasts from day 9 to day 16. During that time so much secretory material accumulates (Fig. 1G) that the

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storage vacuole reaches a prodigious length (110-200 pm) and volume (approximately 11 to 20 x 103 pm3). Since a single galea (of a 4-gm polyphemus) contains 45 pg of secretory protein (Section IV,A), in approximately 10,000 vacuoles, the concentration in the vacuole is apparently 2040% (weight per volume). On days 16 and 17, this concentrated protein solution is slowly extruded, past the cuticular valve, into the common duct, and finally out on the surface of the galea. It is not clear yet what the motive force for extrusion is, or how the valve operates to prevent premature extrusion. The triggering signal seems to be drying of the galea surface, which normally occurs on day 16 when the molting fluid is resorbed. Premature extrusion would lead to dissolution of the enzyme in the molting fluid and hence to dispersal throughout the body. I n preliminary experiments in vitro, day 14 and 15 galeae were made to extrude cocoonase prematurely by floating on tissue culture medium (the hydrophobic surface is not wetted) ; by contrast, submerged galeae extruded much less enzyme (Berger and Kafatos, 1971b; Mazur, 1971). I n any case, once extrusion occurs, the zymogen cells regress so that only small collapsed cells are evident after emergence of the moth (Fig. 1H). The zymogen cells persist in this degenerate condition, and never function again until the death of the animal, approximately 1 week later. FIO.2. Electron micrographs of zymogen cells fixed in glutaraldehyde and OsO, and stained with uranyl acetate and lead citrate. (A) Growing zymogen cells on day 7, prior to the turning-on of rapid zymogen synthesis. Golgi zones and endoplasmic reticulum have not developed extensively ; E R vesicles are relatively sparse in ribosomes. By contrast, ribosomes are abundant in the ground substance of the cytoplasm, mostly as free polyribosomes (insets), many of them helical. ~ 1 6 , 9 4 0 . (B) Day 13 zymogen cell. The cytoplasm consists mostly of hypertrophied endoplasmic reticulum, swollen with granular secretion, and densely studded with ribosomes. Scattered throughout the reticulum are Golgi zones (G) and mitochondria. In grazing sections of the endoplasmic reticulum sacs, membrane-bound polysomes are often seen to consist of spirals, with predominantly 9-12 ribosomes (insets). At high magnification (center inset), there is some indication that the small ribosomal subunit is directed toward the interior of the spiral; a thread which seems to connect ribosomes within the polysome (arrowhead) may represent mRNAprotein complex. Similar polysomes are also found on the outer nuclear membrane (right inset) among the nuclear annuli. ~ 1 4 , 6 3 0 .(C) [see p. 1381 Low power micrograph comparable to Fig. 20, showing the distribution of abundant E R , Golgi zones, and mitochondria in the basal zone of a zymogen cell. The basement membrane and the blood space are seen on the lower right corner. Many mitochondria are cupshaped (arrowhead) and appear as doughnuts when transected. x9240. (D) The cortex of a day 11 zymogen cell. The densely stained vacuole is seen at the top of the picture, penetrated by microvilli. Its zymogen content stains similarly to material present in Golgi vesicles (lower right corner) and in granules (arrowhead) presumably on their way to discharge at the surface. ~ 1 7 , 7 1 0 .

FIG.2(A) and (B). See legend on facing page.

FIQ.2(C) . a d (D). See legend on p. 136.

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I n sum, on the morphological level differentiation in the galea begins with formation, through differential mitoses, of zymogen and duct cells from the ordinary, cuticle and molting fluid producing epidermal cells. The one duct and two zymogen cells in each organule function a8 epidermal cells in constructing together an intricate cuticular duct and valve for the eventual extrusion of cocoonase to the surface. The zymogen cells also differentiate further for rapid protein synthesis. I n an initial growth phase, these cells becotne polyploid and their cytoplasm gradually accumulates large numbers of memhrane-bound ribosomes and Golgi zones-the characteristic organelles of cells synthesizing protein for export. By day 9, when growth has progressed (and the valve is essentially formed), the cells rather abruptly begin rapid synthesis of cocoonase zymogen. Synthesis and accumulation of zymogen then continue for approximately 7 days. Therefore, extrusion of the accumulated cocoonase to the surface occurs, and is followed by regression of the zymogen cells to an inactive state.

C. AUTORADIOGRAPHIC STUDIES ON ZYMOGENSYNTHESIS AND TRANSPORT Galeae can be excised from animals a t various developmental stages and maintained in a simple organ culture for 1-2 days (Kafatos and Reich, 1968). Under these conditions, the glands continue protein synthesis and, since even in situ they are dependent on blood for their supply of amino acids, they incorporate rapidly radioactive amino acids added to the medium. As a result, autoradiographic as well as biochemical experiments are easy to perform. 1. Kinetics of Zymogen Transport

The transport of secretory proteins from membrane-bound polysomes

to extracellular space is an important aspect of cell function in highly differentiated secretory cells. The kinetics of transport are particularly easy to study in the eymogen cells of the galea, since transport culminates in sequestration of the protein in a single large vacuole, permitting easy autoradiographic localization. We hope that this system might prove useful in a mechanistic analysis of the secretory process (Jamieson and Palade, 1967, 1971). Galea segments were pulse-labeled for 15 minutes with I e ~ c i n e - ~ H and were then maintained in nonradioactive medium for various periods of time, under effective “chase” conditions (Kafatos and Kiortsis, 1971). Autoradiography revealed that throughout the chase the nuclear label remained constant; by contrast, there was a redistribution of radioactivity from cytoplasm to vacuole, so that radioactivity in the vacuole increased from zero to a final plateau level, while radioactivity in the

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cytoplasm decreased correspondingly (Fig. 3). The translocated radioactivity corresponds t o zymogen, which is synthesized in the cytoplasm and then transported to the vacuole for storage. By filtering out of our calculations radioactivity which fails to be translocated, zymogen transport can be studied kinetically as a precursor-product conversion : zymogen which is initially 100% cytoplasmic is progressively converted into 100% vacuolar zymogen (Fig. 4). The kinetics show an initial lag phase, corresponding to the minimum time needed for local “processing” of zymogen within the ER and Golgi in the vicinity of the vacuole. A phase of rapid, almost linear transport then ensues, during which 100

0 20 4 0 60

93

125

93 125 I55 0 20 40 6080 100 120140 TIME(MIN)

I55 0 20 4 0 60

FIG.3. Zymogen transport from cytoplasm to vacuole at various developmental stages. After a 15-minute pulse with leucine-IH and a variable period of chase, gales segments were processed for autoradiography. The radioactivity in the nucleus (A-A), the cytoplasm (O-O), and the vacuole (0-0) is shown as a function of chase duration. From Kafatos and Kiortsis (1971).

the rest of the zymogen from throughout the cytoplasm is secreted. The half-transport time, t,, (the time required for an average zymogen molecule to move from the site of synthesis to the site of storage) increases with developmental age, from 38 minutes on day 10 to 74 minutes on day 15 (at 25OC). Regardless of the stage, transport is complete within 2 hours from the end of the pulse. The kinetics are surprisingly similar to those of vertebrate secretory cells operating a t 37OC: pig pancreas (Jamieson and Palade, 1967), chick embryo tendon (Dehm and Prockop, 1971), rat duodenal epithelium (Bennet, 1970)) rat liver (Glaurnann and Ericsson, 1970), mouse plasma cells (Zagury et al., 1970). 2. Relative Rates of Zymogen Synthesis during Development

Knowledge of transport kinetics permitted estimation of rates of zymogen synthesis during development, relative to total protein synthesis

4.

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T H E COCOONASE ZYMOGEN CELLS OF SILK MOTHS

in the zymogen cells. Staged galeae in culture were labeled for 1 hour and then chased for 4 hours. The synthesis of zymogen and of nonzymogen proteins was determined from the autoradiographically observed radioactivity over vacuole and cytoplasm, respectively, a t the end of chase (Kafatos, 1970). The results clearIy showed that during development a progressively larger fraction of total protein synthesis in the zymogen

I A

100.

75

-

z W u

P> 50-

0

W

w m

4

C 25-

0-

l o t

Letr3H

CHASE

I

I

I

38

60

74

TIME

0

I

163

(MIN)

FIG.4. The kinetics of zymogen transport from cytoplasm to vacuole, based on the data of Fig. 3. The figure describes the conversion of a precursor (cytoplasmic zymogen) to a product (vacuolar zymogen). For vacuole, the value of “100% labeled zymogen” corresponds to the final plateau of vacuole label in Fig. 3. For the cytoplasm, “10070 labeled zymogen” is defined as the difference in label between the time zero cytoplasmic label (total cellular label minus the earliest value of nuclear label) and the final plateau level of cytoplasmic label. The half transport time, too,is the intersect of the two curves. 0, ., day 10; A , A,day 12; 0, 0 ,day 15. From Kafatos and Kiortsis (1971).

cells is devoted to prococoonase. Zymogen synthesis is about 20% of total protein synthesis on day 10 and more than 7Q% on day 14.” This progessive specialization is also evident in Fig. 3.

* The values originally reported (in Fig. 12 of Kafatos, 1970) need some adjustment, since we now know that there are two zymogen cells per vacuole. The synthetic rates on day 14 are correct, because of the method used for estimating relative sizes of vacuole and cytoplasm a t that stage; the rates at earlier stages are as much as 50% lower than those reported.

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111. The DifferentiationJpecific Product of the Galea: Biochemical and Enzymological Characterization

A. THEENZYMOLOGY OF COCOONASE Because of its availability (approximately 0.1 mg per animal) and its ease of purification, cocoonase has been well characterized enzymologically (Kafatos et al., 1967a,b; Hruska et aE., 1969; Hixson and Laskowski, 1970; Hruska and Law, 1970, 1972; Berger et al., 1971; Felsted et al., 1972a,b; Felsted and Law, 1972; Kramer and Law, 1972). The dry material collected from the face of the moth is approximately 70% pure, the impurities being a mixture of activation peptides and degradation products. Purification to homogeneity can be accomplished by a single passage through a Sephadex G-100 or CM-cellulose column; Sephadex is preferable since some enzyme inactivation occurs upon prolonged exposure to CM-cellulose. Cocoonase shows remarkable chemical similarity to mammalian trypsin and other serine proteases. Like trypsin, cocoonase of the genus Antheraea has a molecular weight of ca. 24,000, although Bombyx cocoonase is slightly smaller (approximately 20,000; Hruska and Law, 1970, 1972). Like trypsin, cocoonase is a basic protein, with an isoelectric point higher than pH 9.5. Its amino acid composition is very similar to that of bovine trypsin (and indeed to that of all pancreatic proteases of vertebrates, but not of unrelated proteases such as papain). Recently, Kramer and Law have established a partial sequence homology with trypsin. The first four N-terminal amino acids of the A . polyphemus enzyme are identical to those in several vertebrate trypsin, and a 16 amino acid peptide which contains the active site serine residues is 70% homologous with the corresponding peptide in bovine trypsin (Kramer and Law, 1972). A further indication of similarity between cocoonase, trypsin and chymotrypsin is their immunological cross-reactivity in liquid precipitin and immunodiffusion tests (Berger and Kafatos, 1971a). The cross-reactivity is probably due to similarities in the active center, since it is also observed with subtilisin, a bacterial serine protease completely unrelated to trypsin in amino acid sequence except in the active center (Smith et al., 1966). Cocoonase resembles trypsin in its enzymatic properties as well. It reacts with the trypsin titrant, p-nitrophenyl-p‘-guanidinobenzoate,in a typical “burst” fashion, without subsequent deacylation (Hruska et al., 1969). With amino acid ester substrates, its catalytic specificity is limited to esters of arginine and lysine. With these artificial substrates, the cocoonase kinetic constants, K M and k,,,, are comparable to those

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of mammalian serine proteases, including trypsin. The strict specificity of cocoonase toward arginine and lysine was also observed with polypeptide substrates. However, cocoonase is less reactive toward certain of the protein substrates than is trypsin (Felsted and Law, 1972; Felsted et al., 1972b). For example, the rate of hydrolysis of citraconylated lysozyme by bovine trypsin is 12 times faster than with the same concentration of A . polyphemus cocoonase, and activation of chymotrypsinogen is 5000 times faster. On the other hand, peptide maps made by digesting lysozyme with these two enzymes are quite similar. Further similarity to trypsin is indicated by similar susceptibility to inhibitors. Effective inhibitors are diisopropylphosphorofluoridate (DFP, which reacts with the active center serine) , l-chloro-3-tosylamido-7-amino-2-heptanone (TLCK, a substrate analog which inactivates the enzyme by alkylating basic groups a t the active site), and soybean trypsin inhibitor (a protein inhibitor with which polyphemus cocoonase interacts a t the same reactive site as does bovine trypsin; Hixson and Laskowski, 1970). Finally, the pH for optimal activity (ca. pH 8) is similar for cocoonase and for the other serine proteases. Despite substantial similarities to trypsin, cocoonase has several peculiar properties. One is the ease of its denaturation by moderately acid pH, by heat, and by denaturing agents such as urea; this instability ,may be related to the small number of disulfide bonds in the molecule. Another peculiarity is its resistance to autodigestion. The enzyme is almost completely stable, in highly concentrated solutions and near the pH optimum for enzymatic activity, for more than a day. In nature, the active enzyme finds itself under such conditions on the surface of the galea for many hours prior to use; if it were as susceptible to autodigestion as is bovine trypsin, little active enzyme would remain for use in escaping from the cocoon. I n sum, cocoonase is a protease with substantial similarities to mammalian trypsin. I t is also similar in some respects to other invertebrate trypsinlike enzymes (for a review, see Katzenellenbogen and Kafatos, 1971a). Of particular interest is the comparison of cocoonase with the proteolytic enzymes of molting fluid (Katzenellenbogen and Kafatos, 1970, 1971a). This fluid is apparently secreted by the epidermal cells throughout the body of the insect (initially as a gel), for the purpose of digesting the old cuticle during the molt. I n developing adult A . polyphemus, approximately 0.15 ml of molting fluid contain approximately 12 pg of a nearly equimolar mixture of two trypsinlike proteases. Enzymological characterization has revealed quite unequivocally that the molting fluid enzymes are distinct from cocoonase. Thus, these enzymes are

144

FOTIS C. KAFATOS

weakly anionic, whereas co,coonase is a basic protein; they are larger in molecular weight (34,000 and 30,000 as compared with 24,000 for cocoonase) ; they are not inhibited by TLCK; and they are much more specific toward arginine as compared to lysine esters, whereas cocoonase is much more indiscriminate (the rate of hydrolysis of N-a-benzoylL-lysine methyl ester, relative to N-a-benzoyl-L-arginine ethyl ester, BAE, is 0.08 for molting fluid proteases and 0.29 for cocoonase). Moreover, the molting fluid proteases and cocoonase act differently in zymogen activation (Section 111,B). Thus, it seems that the gene whose activity characterizes zymogen cell differentiation in the galea is different from the gene (s) used by unspecialized epidermal cells, including the zymogen cell precursors, for molting fluid production. It is an interesting speculation that this difference is imposed by different requirements for control of gene activity in the two cases. B. COCOONASE ZYMOGEN Cocoonase is synthesized and accumulates in the form of an inactive zymogen (Berger et at., 1971). Conversion to the active enzyme occurs either during extrusion through the duct apparatus or shortly thereafter, since even the earliest material which can be collected from the surface of the galea is fully active enzymatically. It is quite possible that activation begins with limited proteolysis by traces of molting fluid proteases, either within the duct or left behind on the surface when the molting fluid itself is resorbed (Berger, personal communication). Like trypsin and subtilisin (but not chymotrypsin), molting fluid proteases are very effective as activators of prococoonase (Fig. 5 ) . By contrast, cocoonase itself is much less active in zymogen activation. The stored zymogen behaves as a protein of approximately 28,00033,000 MW. It has recently been ascertained that it contains a small amount of carbohydrate (Kramer, unpublished observation). Activation involves the removal of an N-terminal peptide, as in the case of trypsin. No immunological cross-reactivity was detected between prococoonase and trypsinogen (Berger and Kafatos, 1971a). Because of its high concentration in the tissues (approximately half of the total protein in day 14 or 15 galeae), cocoonase zymogen can be purified with relative ease. Very high purity can be attained by careful fractional precipitation with ammonium sulfate. At pH 6.6, at 4OC and a t a concentration of 12 galeae per milliliter, most of the nonzymogen proteins are precipitated by 2.5 M ammonium sulfate. A cut of essentially pure zymogen can be obtained between 3.1 M and 3.7 M salt (Fig. 6 ) . If desired, further purification can be obtained by

4.

THE COCOONASE ZYMOGEN CELLS OF SILK MOTHS

145

passage through a G-100 Sephadex column. The zymogen has been used in detailed studies of the mechanism of activation. I n analogy to the activation of trypsinogen, purified cocoonase zymogen exhibits an autocatalytic mechanism (Felsted et al., 1972a). Most important for present purposes, specific antizymogen sera have been prepared (Berger and

-

N

'sx u)

t

I /

T 1:300

-5 4 0 W

V

3 0 0

K

a AI

w 2 u)

a z 0

s 0

0

0.5

1

L5

10 40

80

420

HOURS OF INCUBATION

FIG.5. Activation of zymogen by serine proteases. Four polyphemus galeae (day 14 to 15) were homogenized in 1.2 ml of 0.1 M Tris.HC1, pH 7.6,lo-' M NaCN. and clarified at 28,000 g for 15 minutes. Aliquots, 200 pl, of the supernatant were mixed with 10 pl buffer containing the activator enzyme (0-0 or 0 4 , trypsin; A-A, molting fluid proteases; A-A, cocoonase). At the indicated times of incubation, a 10-pl aliquot of each activation mixture was withdrawn and assayed for cocoonase activity under standard conditions (Berger et at., 1971). Results were corrected for the amount of activator contained in the aliquot assayed. The ratios indicate the approximate proportion of activator to potentiaI cocoonase (6 x lo-* unit). Courtesy Dr. E. Berger.

Kafatos, 1971a). These antisera can precipitate cocoonase from a complex mixture of proteins and thus can be used as analytical tools for estimating zymogen content or zymogen synthesis during development.

IV. Quantitation of Zymogen Synthesis and Accumulation during Development

A. ZYMOGENCONTENT Zymogen content can be estimated by two independent procedures (Berger and Kafatos, 1971b): conversion to cocoonase by a 30- to

146

FOTIS C. KAFATOS

60-minute incubation with 1 to 5 X equivalents of trypsin, followed by spectrophotometric assay of cocoonase with BAE as the substrate; and precipitation with antizymogen serum followed by comparison of the protein content in the resulting pellet to a standard precipitin curve.

FIG.6. SDS-acrylamide gels showing the total proteins extracted from polyphemus galea ( A ) and zymogen purified from the extract by (NHJSO, ( B ) . For other details, see Berger et al. (1971).

The two methods give essentially identical results (Fig. 7). Very little aymogen is present in the galea until day 9 ; thereafter, zymogen accumulates until the end of day 15 when release to the surface (extrusion) begins. The maximal content of zymogen per galea in a 4-gm animal is approximately 45 pg, an amount consistent with the amount of cocoonase which can be harvested after extrusion. Assuming 20,000 zymogen cells per galea, and the molecular weight of zymogen as 33,000, this corresponds to 4 x 1O'O molecules per cell.

4.

THE COCOONASE ZYMOGEN CELLS OF SILK MOTHS

147

I

I

STAGE OF A D

FIQ.7. Accumulation of zymogen in the galea during development. The galeae of staged Antherea polyphemus were excised and homogenized, and the proteins were extracted first with 0.3 ml of buffer (0.1 M Tris-HC1, p H 7.0, lo-’ M NaCN) and then with 0.1 ml of buffer containing 1% sodium deoxycholate. The amount of zymogen in the protein extract was determined either by immunoprecipitation of serial dilutions with antizymogen serum (0) or by activation with trypsin followed by spectrophotometric assay of the generated cocoonase using N-a-benzoyl-Larginine ethyl ester as the substrate ( 0 ) .Results were normalized to a typical animal weight of 4 gm. The arrow indicates the time when extrusion of the zymogen to the surface and conversion to cocoonase begins. From Berger and Kafatos (1971b).

B. ZYMOGENSYNTHESIS Zymogen accumulation accelerates continuously between “turning on” (day 9) and day 14. By measuring the tangent (i.e., the derivative) of the accumulation curve (Fig. 7) we can determine (Fig. 8A) that on day 11 accumulation is a t the rate of approximately 3 pg per day per galea, but on day 14 reaches approximately 15 pg per day per galea. However, a t stage 14,,, to 15,, slightly before extrusion begins, the rate of accumulation decreases. The derivative, of course, measures the net rate of synthesis, i.e., the actual rate of synthesis minus any breakdown (Schimke, 1969). If breakdown is negligible, these results indicate

148

FOTIS C . KAFATOS

that the actual synthetic rate increases continuously between day 9 and day 14,,,, to a maximum of 1.5 X 1Olo molecules per cell per day, and then decreases again. The assumption has been shown to be valid (Section VI,A, 1). Shortly after its synthesis zymogen is sequestered in an extracellular storage cavity, apparently isolated from intracellular protein turnover.

, , , , , , , , , , , 7n

9=

Iln

13n

S T A G E OF A D

1511

,I

17n

!

I

O 7a

U Pn

,

,

,

11,

13,

STAGE

OF A D

,

, 15,

,

, 17,

FIG. 8. Zymogen synthesis during development. (A) Rate of net zymogen synthesis, determined from the derivative of the accumulation curve (Fig. 7) ; assuming that no turnover of zymogen occurs, this procedure measures the absolute rates of synthesis. In the calculations, the molecular weight of the zymogen was taken as 33,000 and the number of zymogen cells per galea of 4-gm animal as 20,OOO. (B) Zymogen synthesis relative to total protein synthesis in the entire galea. After pulse labeling staged galeae in culture with leucine-’H, relative rates of synthesis were determined from either the fraction of incorporated (TCA precipitable) radioactivity which was also precipitated by antizymogen serum (immunoprecipitation alone, 01, from the fraction of incorporated radioactivity which migrated as zymogen upon SDS acrylamide electrophoresis (electrophoresis alone, .), or from the fraction of incorporated radioactivity which was precipitated by antiserum and which subsequently migrated with zymogen upon SDS acrylamide electrophoresis (immuno-precipitation-electrophoresis, W ) , Arrows indicate the beginning of extrusion.

Another method of estimating ..rates of zymogen synthesis during development (without a significant error even if turnover occurs) consists of putting staged galeae in short-term culture, pulse-labeling them with radioactive amino acids, and determining the incorporation of label in eymogen and in other proteins (Berger and Kafatos, 1971b). Such direct estimates (Fig. 8B) affirm our analysis of the accumulation curve

4.

THE COCOONASE ZYMOGEN CELLS OF SILK MOTHS

149

(Fig. SA). Synthesis of zymogen prior to turning on is a t a low (but finite) level. Depending on the exact stage, typical values on day 8 are 0.1 to 0.5% of total protein synthesis. Between the end of day 8 and the end of day 14, the relative rate of zymogen synthesis progressively increases by about two orders of magnitude, from less than 1% to about 60% of total protein synthesis in the galea. As was suggested by the accumulation curve, zymogen synthesis decreases again a t early day 15, prior to the beginning of extrusion.

C. SOMECOMMENTS ON METHODOLOGY I n connection with Fig. 8B, some methodological comments are in order. Rates of synthesis were expressed in relative rather than absolute terms, for the simple (but often overlooked) reason that incorporation data alone do not measure absolute rates. Even with identical tissue culture media, there is no reason to expect that the intracellular amino acid pools available for protein synthesis will be comparable in different experiments. For example, changes in cell geometry with development may affect the rate of precursor uptake from the blood; permeability changes may occur in the cell membrane; or the size of the intracellular pool may change because of changes in the relative rates of protein synthesis and degradation. Indeed, potentially misleading variations in labeling of the intracellular amino acid pool have been noted in several cases: in fertilized eggs such as sea urchin, depending on the exact time after fertilization (Mitchison and Cummins, 1966; Fry and Gross, 1970), and in several types of tissues after treatment with actinomycin D (Fry, 1970; Regier and Kafatos, 1971). Without knowledge of specific activities, incorporation data can only yield rates of zymogen synthesis as a percentage of total protein synthesis. These relative rates depend on only two assumptions: 1. Zymogen cells utilize the same amino acid pool for synthesis of zymogen and nonzymogen proteins. (This is almost certainly true, since membrane-bound and free polysomes are not spatially segregated in different parts of the cell, and since all polysomes face the ground substance of the cytoplasm). 2. I n any experiment the specific activity in all other cells of the galea is the same as in zymogen cells. (This assumption may not be valid, but the uncertainty it introduces is minor since, as shown by autoradiography, incorporation in the zymogen cells is greater than in all other tissues combined). A simple microtechnique for estimating the intracellular specific activity of an amino acid such as leucine has been devised by J. Regier in my laboratory (Regier and Kafatos, 1971). This technique depends

150

FOTIS C. KAFATOS

011 the coupling of the free amino acids extracted from a tissue sample, dinitrofluorobenzene-l*C. The resulting D N P including l e ~ c i n e - ~ Hwith , derivatives are separated by two-dimensional chromatography on TLC plates, and the radioactivity in the DNP-leucine spot is determined. The desired specific activity, "H cpm/mpmole of leucine, is proportional to the 3H:14Cratio in the spot: the 3H cpm measure the radioactive leucine molecules, and the '%cpm measure all leucine molecules. With appropriate standards, the ratio can be converted to absolute specific activity, 3H dpmimpmole leucine. A further refinement is the inclusion of radiolabeled inulin in the medium, permitting an estimate of the extracellular fluid in the tissue sample, and hence a correction for extracellular leucine. A future improvement might be to perform the coupling reaction with leucine prepared from peptidyl-tRNA, so as to eliminate the possibility that the specific activity of total extractable leucine is irrelevant because the amino acid pools are compartmentalized (Berg, 1968; Hendler, 1968). The present method requires no more than nanogram amounts of amino acid and thus should prove of general use, especially when the amount of tissue is limiting. The method is being used in an ongoing study of the absolute rate of zymogen synthesis during development, performed in my laboratory by Dr. A. Efstratiadis. He has also developed a sensitive radioimmunoassay for refined studies of zymogen accumulation. The second methodological comment relates to the procedures for apportioning the incorporation between zymogen and nonzymogen proteins. First of all, it is clearly essential to make radioactivity measurements for the two protein classes comparable, by careful attention to counting efficiencies. In addition, attention must be paid to losses inherent in the purification procedure (e.g., immunoprecipitation) and, on the other hand, to the contamination of the purified fraction with labeled nonspecific proteins. It is also necessary to take into account the amino acid composition of the proteins, The most direct method for determining relative rates of synthesis is to fractionate the total labeled proteins by a single procedure, such as electrophoresis in an SDS-107, acrylamide gel. Since essentially all proteins enter the gel and migrate no more rapidly than the tracker dye, incorporation in the specific zymogen peak can be compared directly with the total incorporation in the gel (Fig. 9 ) . As shown in Table I (p. 152), the reproducibility of such estimates is quite good. However, this method is accurate only when the specific peak has a high proportion of the total incorporation, ca. 5-10% as a minimum. A second method is to use immunoprecipitation alone (with due corrections for losses), and to consider all precipitated radioactivity as zymogen ; when labeled

4. THE

COCOONASE ZYMOGEN CELLS OF SILK MOTHS

151

zymogen is abundant, contamination of the immune pellet with other labeled proteins is minor. However, with early stages these two methods are inaccurate. The third and most sensitive method involves immunoprecipitation of “-labeled zymogen from a tissue extract, followed by dissolution of the pellet in SDS and electrophoresis on an acrylamide-SDS gel (Berger and Kafatos, 1971b). In this procedure, then, immunoprecipitation is used as a partial purification step, enriching the sample so that 236-1

SLICE NUMBER

FIG.9. Assay of relative rate of zymogen synthesis hy electrophoresis alone (see Fig. SB, 0 ) . Day 11 galeae were labeled in culture with le~cine-’~C. The labeled galeae were homogenized, the proteins were dissolved in electrophoresis sample buffer, and an aliquot was suhjected to SDS acrylamide electrophoresis. The shaded region indicates specific incorporation in the zymogen peak, over and above the background of other labeled proteins. From the relative areas of shaded and unshaded portions of the curve, the relative rate of zymogen synthesis was estimated as 18.5% (see Table I ) . In this and all subsequent gels, migration is from left to right.

zymogen can be estimated accurately in a specific electrophoretic band (as in Method 1 ) . The efficiency of the total procedure can be monitored with an internal standard of “C-labeled zymogen. It is important to realize that the specificity of the antibody does not guarantee that the radioactivity of an imniunoprecipitate is solely due to the antigen (hlethod 2 ) . For example, if even 0.17. of labeled nonspecific protein is passively trapped in the immunoprecipitate, immunoprecipitation alone cannot yield valid measurements when synthesis of the antigen is less than 1% of the total. In addition, serious error

152

FOTIS C. KAFATOS

TABLE I REPRODUCIBILITY OF ESTIMATES FOR RELATIVE RATE OF ZYMOQENSYNTHESIS USINQSDS-ELECTROPHORESIS A

B

C

D

E

Total cpm in gel

Total cpm in zymogen region4

Nonspecific cpm in zymogen region*

Net zymogen cpmC

Zymogen synthesis as % of total protein synthesisd

14,960 16,377 14,802 14,050 13,175 14,212 12,825 16,196 15, 755 15,594

4.510 4838 4226 4616 4449 4648 3541 4319 4748 4458

1644 1830 1485 1953 1876 1911 1485 1166 205 1 1.560

2866 3008 274 1 2663 2.573 2737 2056 3153 2697 2898

19.2 18.4 18..5 19.0 19.5 19.3 16.0 19.5 17.1 18.6

3,802 4,511 3,869 4,505 3,902 4,907

785

312 317 286 320 248 417

473 463 379 456 422 602

12.4 10.3 9.8 10.1 10.8 12.3

Experiment 1 Aliquot 1 2 3 4 5 6 7

8 9 10

Experiment 2 Aliquot 1 2 3

4 5 6

780 665 776 670 1019

Sum of counts in the 4 or 5 gel slices forming the radioactive zymogen peak. counts in the zymogen region, calculated from the average of the slices bordering the zymogen region on either side. D = B - C; this corresponds t o the shaded portion in Fig. 9. a

* Nonzymogen E

D

=

- X 100. A

may result if the antigen complexes with other proteins or adsorbs to particles of the tissue homogenate (ribosomes, microsomes, mitochondria, membranes, even whole cells). I n this respect, it is worth noting (Berger and Kafatos, 1971b) that contamination is considerably reduced if the tissue homogenate is treated with a suitable detergent (0.5-170 deoxycholate, or a mixture of DOC and a nonionic detergent such as Triton X-100) ; solubilization of organelles by the detergent probably reduces the ratio of antigen to nonspecific protein in adsorption complexes, and thus reduces contamination of the pellet. Even with such refinements,

4. THE

COCOONASE ZYMOGEN CELLS OF SILK MOTHS

153

overreliance on the specificity of the antigen-antibody reaction can be disastrous. I n general, immunoprecipitation from a complex tissue extract is inadequately specific (Palmiter e t al., 1971; Berger and Kafatos, 1971b). Unless the protein in question is a major fraction of the total labeled protein, imrnunoprecipitation should be combined with a second purification procedure, such as SDS acrylamide electrophoresis, if the results are to be meaningful.

D. DIFFERENTIATTON AS A CHANGING RATEOF SPECIFIC PROTEIN SYNTHESIS 1. Cocoonase Z y m o g e n

Quantitative details aside, a qualitatively similar picture is obtained with all methods available, relative (autoradiography and immunoprecipitation-electrophoresis) as well as absolute (accumulation measurements and inimunoprecipitation-electrophoresis combined with knowledge of intracellular specific activity). As diagrammed in Fig. 10, zymogen synthesis increases from zero (strictly speaking, no more than in other cells of the body) to a low but finite level no later than day 8. We do not know exactly when this transition occurs, but we speculate that it might come soon after the differential mitoses on day 4. Even though the synthetic rate then increases over time, i t remains a t a low range throughout what we call phase I. This period is analogous to the “protodifferentiated” phase described in rat pancreas (Rutter e t al., 1968). I n a second transition, zymogen synthesis exceeds 1% of the total protein synthesis late on day 8 or early on day 9, just prior to the appearance of zymogen in the storage vacuole, and during the ensuing phase I1 (or “fully differentiated” phase) it continues to increase but a t a rapid rate. After 6 days, it exceeds 70% of all protein synthesis in the zymogen cells (60% of all protein synthesis in the galea). A third transition occurs shortly before extrusion, when the rate of zymogen synthesis begins to decrease (phase 111). If we are to understand zymogen cell differentiation as expressed in specific protein synthesis, four phenomena merit special attention. Three are the transition points, identified by arrows in Fig. 10. The fourth and equally as important phenomenon is the progressive increase over two orders of magnitude, in zyrnogen synthesis during phase 11. It is important to realize that the cells are no more static during the “fully differentiated’’ phase than they are a t other times in development. This, I believe, is not an accident, but a necessary consequence of how highly differentiated cells, in general, specialize: by progressively accumulating stable differentiation-specific mRNA, which may not be syn-

154

FOTIS C. KAFATOS

thesized any faster because of limits imposed by the mechanism of transcription and by the number of gene copies present. 2. Other Systems

Unfortunately, precise data on changing rates of specific protein synthesis during development are rare in the literature. Nevertheless, sufficient information exists to indicate that the kinetics diagrammed in Fig. 10 have some generality. DAYS OF A D U L T

DEVELOPMENI

-

ln ln Y

I

+ Z

aln

f Y I-

: n.

u

-

U

u Y

n.

ln

II PREDl FFERENTIATED

‘ m

DIFFERENTIATED

P H A S E S OF

DEVELOPMEN1

FIQ.10. Diagram of the phases of development as revealed by the changing rate of specific protein synthesis. Arrows indicate the transition points between the four phases (the predifferentiated phase and the three phases of differentiation). The top abscissa indicates the time axis in the galea. For further details see the text.

In their studies of the embryonic rat pancreas, Rutter and his collaborators (Rutter et al., 1968) called attention to the existence of a “protodifferentiated” state, characterized by low levels of differentiation-specific proteins. Interestingly, the transition between the protodifferentiated and fully differentiated states, as indicated by a more or less abrupt increase in molecules per cell, differs for different secretory

4. THE

COCOONASE ZTMOGEN CELLS O F SILK MOTHS

155

products (days 15-17 of gestation). Such a protodifferentiated phase is also recognizable in the ovalbumin-producing chick oviduct ( Palmiter and Wrenn, 1971) ; in this case, the transition occurs approximately 2 days after estrogen stimulation, when the secretory cells are first found organized in tubular glands and when large secretory granules first appear. A similar transition occurs in the zymogen cells of the galea early on day 9 (Fig. 7 ) . We prefer to define the transition in terms of rate of synthesis rather than of content, since the former is an earlier step in “information flow.” I n any case, the choice of a criterion does not affect significantly the timing of the transition in any of the above systems (Figs. 8A and 1 1 ) . As discussed in Section IV,C, great care is needed in determining rates of specific protein synthesis from incorporation experiments. The most dependable data in the literature are relative rates, as percent of total incorporation. Even relative data have methodological limitations, as discussed previously, and without special precautions are probably inaccurate for rates of less than 1 or 2%. It would be indeed valuable to determine, by brief pulse labeling, the changing rates of protein synthesis in several developing systems, using appropriate methodologies so as to make accurate estimates over a range of a t least 4 orders of magnitude. Such measurements are essential for a general description and analysis of terminal cell differentiation-especially since they are irreplaceable, being uniquely insensitive to variations in protein turnover. In their absence, synthetic rates can be estimated from curves of specific protein accumulation during development. Accurate determination of the content of a specific protein in a tissue sample can often be performed over 4 or 5 orders of magnitude (see, for example, Rutter et al., 1968). Thus, the accumulation of a protein over time can be graphed accurately. As in Section IV,B, we recall that the derivative of the accumulation curve is the rate of net synthesis, i.e., the difference between rate of synthesis and rate of breakdown. If secretory proteins are assumed to be exposed to little or no turnover, the derivative can be taken as a measure of the actual rate of synthesis. The assumption is probably valid in general; it certainly holds true for cocoonase zymogen (Section IV,A, 1).From derivatives of accumulation curves we find that in the embryonic rat pancreas between days 13 and 21 (Rutter et al., 1968) the rate of synthesis per cell increases by approximately 50,000-fold for chymotrypsinogen, 20,000-fold for lipase A, and 50,000-fold for amylase. The increase takes place during both the protodifferentiated and the fully differentiated phase. Neither period should be considered an invariant state. Rather, acceleration of synthesis is a characteristic of both, and a phase transition is designated

156

FOTIS C. KAFATOS

somewhat arbitrarily when the synthetic rate increment per unit time becomes large. Data for the synthesis of fibroin by silk glands (Tashiro et al., 1968), chymotrypsinogen and lipase A by embryonic rat pancreas (Rutter e t al., 1968), and ovalbumin by estrogen-treated chick oviduct (Palmiter and Wrenn, 1971) are presented in Fig. 11. Considerable similarity with the synthesis of cocoonase zymogen (Fig. 8) is apparent. This is especially true for fibroin (Fig. l l A ) , which is produced mostly during a specific time of the life cycle, the last larval stage (instar). I n this case, the increase in rate of synthesis continues throughout the 6 days of phase 11, at the end of which time “turning off” occurs shortly before metamorphosis and the death of the secretory cells (Matsuura et aZ., 1968). In the embryonic rat pancreas (Fig. 11B) continued specialization for chymotrypsinogen and lipase A synthesis occurs throughout the first three days of phase I1 (days 16-19), and the high rates of synthesis attained remain constant a t least until birth. The absence of an immediate “turning off” is reasonable, since the pancreas is not a time-limited organ like the silk gland or the galea, but is destined for use throughout the animal’s life. Even so, a transition analogous to turning off occurs later in life, as enzyme synthesis is LLmodulated”according to the digestive needs (Reboud et al., 1966). I n the case of amylase, modulation apparently begins even before birth since, according to Rutter’s data, the rate of synthesis on days 20 and 21 is lower than the peak attained on day 19. Prior to birth, the maximum number of molecules synthesized per cell per day is 9 X los for chymotrypsinogen, 6 x IOR for amylase and 2 x lo6 for lipase A, according to my estimates. I n the oviduct, differentiation is induced by the injection of exogenous estrogen. The transition to phase I1 occurs approximately 2 days after primary hormone treatment. Thereafter, the absolute rate of ovalbumin synthesis per oviduct magnum (Fig. 11C) continues to increase for a t least 3 days (Palmiter and Wrenn, 1971). Quantitative data are not available after day 5. Nevertheless, it is known that modulation also occurs in this tissue, since estrogen withdrawal leads to a marked reduction in ovalbumin synthesis (Oka and Schimke, 1969b). I n studying rates of synthesis, it is important to know what the basis of the calculations is. If we are interested in specific protein synthesis as the expression of a specific gene, rates of synthesis should generally be calculated on the basis of DNA, optimally in terms of haploid DNA level. Normalization against parameters such as total protein content, dry or wet weight, are not informative, since these parameters bear no fixed relationship to the number of genome copies. Unfortunately, an additional complication exists: the period of specific protein

I '

DAYS

OF L A S T I N S T A R

D A Y S OF G E S T A T I O N

DAYS

1

OF

ESTROGEN

TREATMENT

FIG. 11. Changing rates of specific protein synthesis during development, determined from derivatives of accumulation curves (on the assumption t.hat no turnover of secretory proteins occbrs, i.e., that net synthesis is equal to total synthesis; cf. Fig. 8 A ) . (A) Synthesis of fibroin by the posterior silk glands in the last larval stage of Bombyz mori (Tashiro et al., 1968). (B) Synthesis of secretory proteins by the embryonic rat pancreas (Rutter et al., 1968). (C) Synthesis of ovalbumin by chick oviduct following primary stimulation with estrobut relative rates of syngen (Palmiter and Wrenn, 1971) ; the curve is drawn through the data for absolute rates of synthesis (.-a), thesis, as percent of total protein synthesis, are also shown (0).

158

FOTIS C. KAFATOS

synthesis often overlaps with the period of DNA synthesis. Even if specific mRNA accumulates, being synthesized a t a constant rate per gene c,opy (see Section VI,B), its exponential accumulation will then be paralleled by DNA accumulation. As a result, mRNA content per DNA may increase only slightly. In this case, calculations of protein synthesis per DNA unit can be misleading; relative rates, as a fraction of the total, are somewhat more meaningful. A case in point are the developing primitive erythroblasts in chick embryos (Campbell et al., 1971). The developmental career of these cells apparently involves six mitotic divisions, coincident with the period of hemoglobin synthesis. The absolute rate of hemoglobin synthesis per cell, averaged over an entire mitotic cycle, increases only very slightly during this time (and subsequently declines). Nevertheless, when the rate of synthesis by all the descendants of a single precursor cell is calculated, it is seen to increase by approximately two orders of magnitude during phase 11. Moreover, the absolute and relative rate of hemoglobin synthesis (as a percent of total protein synthesis per cell) increases during mouse fetal erythroblast development (Fantoni et nl., 1968). Similar computations can be made for the absolute rate of myosin synthesis by developing chick leg muscle (Herrmann e t al., 1970); in relative terms, it is observed that the percentage of polysomes synthesizing myosin increases progressively, from 7% on day 10 to 22% on day 17 of incubation. Fortunately, continued DNA synthesis is not a major complication in interpreting the results presented in Figs. 8 and 11; in these cases DNA synthesis ends early in phase 11. Thus, the differentiated pancreatic cells rarelv undergo division after day 16 (Rutter et al., 1968). In the zymogen cells of the galea, cell division does not occur during phase 11, and DNA synthesis ends shortly after day 11. In the silk gland, cell division does not occur and the DNA content per gland nearly doubles daily between days 1 and 3, but remains constant thereafter (Tashiro et al., 1968). In the case of the oviduct, DNA replication continues throughout the period for which quantitative data are available (Oka and Schimke, 1969a) ; this, however, is not the only explanation for the increase in rate of ovalbumin synthesis per oviduct magnum, since the rate per cell also increases (from 1 x 10" molecules per cell per day on day 3, to 6 X los on day 5 ; Oka and Schimke, 1969a; Palmiter and Wrenn, 1971). The relative rate of ovalbumin synthesis, as a percent of total protein synthesis, also increases (Fig. 11C) while the rate of nonspecific protein synthesis apparently remains nearly constant. I n sum, although continued DNA synthesis may account in part for the exponential nature of the increase in specific protein synthesis

4.

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during the early part of phase 11, the increase apparently continues even when the genome is no longer replicated. Thus, i f protein synthesis is proportional to mRNA content, it appears that in several different systems the specific mRNA produced by each genome copy progressively accumulates during the fully differentiated phase. V. Transition Points in Zymogen Synthesis during Development I n the galea, no causal analysis has been attempted with two of the three transitions (Fig. 10) : the primary transition between predifferentiated and protodifferentiated phases, and the final “turning off” from phase I1 to phase 111. Nor are these two processes understood mechanistically in other systems. At the level of phenomenology, we can say t h a t many systems undergo the primary transition in response to recognizable chemical stimuli: inducers or growth factors (e.g., see Wessells and Cohen, 1967) or, alternatively, hormones (e.g., see Oka and Schimke, 1969a,b). I n the insects, hormones do not seem to be involved directly; the epidermal cells apparently make the commitment to develop into specific organules in response to gradients (Lawrence, 1970), probably two-dimensional, which convey positional information (Wolpert, 1969). Wigglesworth has shown that during the molt cycle in Rhodnius new bristles form in places where the separation between preexisting bristles exceeds a certain threshold (Wigglesworth, 1964). Thus, formation of insect organules is controlled not by systemic hormones or by inducers operating on a large cell population, but by highly localized stimuli. As a result, organules are found in very precise distributions (e.g., alternating with normal epidermal cells in the galea). With respect to the third transition, it is surprising that not more is known. Since this transition (unlike the primary) involves easily recognizable cells, and since a t that time the cells are characterized by a rapid rate of specific protein synthesis, it should be feasible to dissect the biochemical processes involved in the slowdown of synthesis. It may just be that we are all aesthetically attracted to systems which are going up, rather than downhill. In any case, a t the phenomenological level this transition either may be controlled by systemic factors such as hormones (Oka and Schimke, 1969133 or diet (Reboud et al., 1966), or may be internally programmed in time-limited cells such as erythroblasts or lens fibers (Weintraub et nl., 1971 ; Clayton, 1970). Slightly (but only slightly) more is known about the second transition between the protodifferentiated and fully differentiated phases. In the zymogen cells it appears that the transition may be internally programmed; a t least it can occur independently of systemic hormones.

160

FOTIS C. KAFATOS

I n galeae excised from the animal a t various times on day 7 and 8 and placed in defined organ culture medium, many cells acquire a zymogen-filled vacuole approximately on schedule, as if they had been kept in vivo (Kafatos, 1970, 1972). The bulk of the zymogen appearing in the vacuole is synthesized de novo subsequent to excision from the animal (Kafatos, 1970). Moreover, recent biochemical experiments have shown that when day 8, glands are put in culture under the appropriate conditions (in medium supplemented with a macromolecular serum factor, analogous to those found essential in vertebrate tissue culture; Kambysellis and Williams, 1971), they increase their absolute rate of zymogen synthesis 2- to &fold within a day, while maintaining their total protein synthesis nearly constant. In these preparations, zymogen synthesis may reach as much as 0.6% of total protein synthesis, equivato 811rin vivo. Thus, acceleration of zymogen synthesis lent to stage can clearly occur in culture, under endogenous control, although not quite as rapidly as in v i v a The question arises whether this transition is under transcriptional or translational control. Unfortunately, a clear-cut answer is difficult to obtain by the use of actinomycin, because of the long time necessary for a substantial acceleration of synthesis even under control conditions, the toxicity of actinomycin to rapidly growing cells, and the methodological difficulties of staging animals and assessing accurately low rates of specific protein synthesis (Section IV,C), When RNA synthesis is inhibited by saturating doses of actinomycin D, day 81 galeae are sometimes able to increase in 24 hours their rate of isotope incorporation into zymogen by as much as 2-fold (two out of four experiments; Kafatos, 1972). This result would seem to favor a translational control model, especially since some cell death undoubtedly occurs as a result of actinomycin treatment, so that the stimulation of incorporation is even higher on a per cell basis. However, the specific radioactivity of the precursor free intracellular leucine proved to be higher in the presence of actinomycin than in its absence (see also Section VI,A, 1) so that the actual rate of zymogen synthesis per galea a t most remains around the initial level after 24 hours in actinomycin. A translational control model might envisage that a store of zymogen mRNA exists in the cytoplasm in an inactive (or partially active) state. The negative results of the actinomycin experiment cannot disprove this model (since activation of the hypothetical zymogen mRNA store may itself require synthesis of another RNA species, or may be sensitive to nonspecific toxic effects), but they certainly do not support it. The experiment also showed that synthesis of zymogen is much more resistant to actinomycin than is the synthesis of other proteins; non-

4.

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specific protein synthesis is reduced to roughly 10% of the initial level in the course of the experiment. Apparently, zymogen mRNA is unusually stable (Section VI,A, 1) even prior to the beginning of phase 11. Because of this differential stability, the relative rate of zymogen synthesis (as percent of total protein synthesis) increases by 2- to 13-fold and can be easily misinterpreted as “turning on” in the presence of actinomycin (Kafatos, 1970). I n fact since zymogen synthesis continues, even though a t a low rate, some cells can acquire a stainable vacuole in the presence of actinomycin. This emphasizes that great care must be exercised in interpreting an increase in enzyme specific activity after actinomycin treatment (Roth et al., 1968; hloscona et al., 1968) in terms of a translational control model. Translational control clearly operates in some developing systems, such as unfertilized eggs (Humphreys, 1971). Whether it also operates in the transition between protodifferentiated and fully differentiated phases of highly specialized cells will probably remain problematical until specific mRhTA’s can be recognized and quantitated directly. VI. Progressive Increase in Zymogen Synthesis during Phase II A.

STABILITY O F T H E

DIFFERENTIATION-SPECIFIC MESSAGE

1 . Experimental Evidence

Throughout phase I1 the messenger RNA for zymogen is very stable, much more so than the average messenger coding for other proteins in the same cells a t the same time. This is an important fact that must be considered in any model attempting to account quantitatively for changing rates of protein synthesis. The evidence is indirect, being based on the observation that zymogen synthesis is unaffected by actinomycin D even a t concentrations which inhibit more than 99% of RNA synthesis. Nevertheless the evidence is, I believe, overwhelming. For convenience, and pending exposition of the arguments in the following section, resistance to actinomycin will he interpreted in this discussion as evidence for a stable mRNA. The initial evidence (Kafatos and Reich, 1968) came from autradiographic experiments which took advantage of the known kinetics of zymogen sequestration in the storage vacuole (Section II,C, 1 ) . One galea from each animal was cultured in normal medium, and the other in medium containing saturating levels of actinomycin (60 pg/ml). After a variable period of culture, the matched galeae were labeled for 1 hour in a mixture of 3H-labeled amino Lcids, and were then chased in nonradioactive medium for 4 hours, a time more than sufficient for complete

162

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transport of the labeled zymogen into the vacuole. The tissues were then fixed and quantitative autoradiography was performed. The label which was associated with the vacuole indicated the rate of zymogen synthesis, and the label that remained in the cytoplasm the rate of synthesis of general cellular proteins. The results were clear-cut (Fig. 12). Actinomycin has almost no effect on zymogen synthesis, permitting

3'

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I

I

lo

20

30

40

HOURS

IN

ACTINOMYCIN

FIG. 12. The effect of actinomycin on synthesis of zymogen and nonspecific proteins in the zymogen cells. Matched galeae (from the same animal) were kept in 60 pg/ml actinomycin D or in control medium for the indicated period of time. They were labeled with a mixture of 'H amino acids during the last hour, and were then chased in nonradioactive medium for 4 hours to permit complete transfer of zymogen from cytoplasm to vacuole (cf. Figs. 3 and 4 ) . Through quantit.ative autoradiography, the synthesis (relative to controls) of zymogen and nonzymogen proteins by actinomycin-treated cells was estimated from the radioactivity localized over vacuole and cytoplasm, respectively. From Kafatos and Reich (1968).

it to continue a t more than 70% of the control rate even after 2 days of exposure. By contrast, the synthesis of other proteins is strongly inhibited by actinomycin, being reduced by half within approximately 2 hours. Autoradiography was the procedure of choice for these experiments. It demonstrated the differential effect of actinomycin on synthesis of the two protein classes in the very same cell, thus precluding the possibil-

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ity of a n artifact of isotope or actinomycin penetration. However, it was desirable to document biochemically that the protein whose synthesis is unaffected is zymogen. Therefore, a second type of actinomycin experiment was performed (Kafatos and Moore, 1972). Matched galeae were again cultured for variable periods of time, with and without actinomycin. They were then labeled simultaneously, but with different isotopes: the control tissues with leucine-"C, and the experimental (actinomycin-treated) with l e ~ c i n e - ~ HAt. the end of the incubation the tissues were washed, combined, and frozen. The proteins were then extracted and analyzed by SDS-acrylamide electrophoresis. Since control and experimental proteins were handled together, incorporation of the two isotopes could be compared in any gel slice without worrying about losses or about precise reproducibility of electrophoretic conditions. Since 3H-labeled proteins were synthesized in actinomycin-treated cells, a high 3H:1'Cratio in a particular gel slice indicated a protein whose synthesis was relatively unaffected by actinomycin, i.e., whose mRNA was relatively stable. Conversely, a low 3H:14Cratio indicated an unstable mRNA. This type of experiment consistently showed a very high peak in the 3H:14Cratio, coincident with the zymogen band, thus confirming the unusual stability of zymogen mRNA (Fig. 13B). As expected, no such peak was present in control experiments in which both isotopes were given to the same tissue (Fig. 13A) or to separate but identical control tissues. I n this type of experiment, it was not possible to use the I4C-DNFB method for determining absolute rates of synthesis, since leucine from two different tissues was present in a single extract. Moreover, we could not assume that the intracellular leucine pool was comparable in control and experimental tissues (see below). However, since zymogen mRNA was unusually stable, it was possible to obtain an estimate of half-life for the average nonzymogen mRNA by using zymogen synthesis as an internal standard and assuming, as a first approximation, that zymogen mRNA is infinitely stable. Essentially, we assumed that the 3H:*4C ratio throughout the gel would have been the same as that observed in the zymogen region (Fig. 13A) if no mRNA decay had occurred. The actual isotope ratio in the nonzymogen region, divided by the isotope ratio in zymogen, gave a fractional value, R t , which was a measure of how Iow nonzymogen protein synthesis had become as a result of actinomycin treatment for the time period, t. Then, assuming that mRNA decay is exponential, the half life, T , could be estimated from Eq. (1) :

n

A

9

J

J

20

YO

80

SLICE NUfl6ER

80

100

20

uo

60

SLICE NUMBER

80

100

?I

P4 20

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60

S L I C E NUHEEA

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FIG.13. Ratio counting of galea proteins labeled in culture. When different glands were labeled with the two isotopes, the glands were mixed and homogenized together prior to protein extraction. Proteins were separated by electrophoresis on SDS acrylamide gels and counted by standard procedures (Berger and Kafatos, 1971b). Electrophoretic migration is from left to right. (A) Control experiment in which galeae were labeled with a mixture of leucine-'H- and leucine-"C. As expected, the 3H:14C ratio (upper panel) is constant within counting error (solid line above and below the asterisk), indicating that counting conditions are uniform across the gel, including the zymogen (major "C peak). (B) mRNA decay experiment. Galeae previously exposed t o 60 pg/ml actinomycin for 6 hours were labeled for 3 hours with I e ~ c i n e - ~ Hwhile , matched control galeae were labeled with leucine-"C. A high 'H:"C ratio indicates a protein whose synthesis is relatively unaffected by actinomycin; a prominent ratio peak is coincident with zymogen, confirming the unusual stability of zymogen mRNA. Considerable ratio variability is observed in the nonzymogen protein region, far exceeding the counting error; we take this as evidence of variability in mRNA turnover. (C) Protein turnover experiment. Matched galeae were labeled with either leucine-"C or leucine-3H for 3 hours and were then either frozen immediately ("C) or chased for a 16-hour period to permit degradation of labeled proteins. The high 'H:"C rat,io in zymogen indicates that zymogen is an unusually stable protein.

g 5

2

4.

THE COCOONASE ZYMOGEN CELLS OF S I L K MOTHS

165

which can be derived simply from the definition of half-life. I n 11 separate experiments (in which the period t varied between 4.4 and 7.7 hours), the average mRNA half-life for all proteins larger than zymogen’ proved to be 2.5 k 0.24 hours (9570 confidence liniits)-in good agreement with the autoradiographic results. In a third experiment (Regier and Kafatos, 1971), both control and actinomycin-treated tissues were labeled with l e ~ c i n e - ~ Hand , were so as to make possible determinations of intracelluprocessed separately lar leucine specific activity and hence of absolute rates of protein synthesis. Following electrophoresis in SDS gels, incorporation in zymogen and nonzymogen proteins was determined, and control and experimental data were normalized according to the protein content of the gels, as revealed by staining and densitonietry. The effects of actinomycin on incorporation are shown in Fig. 14A. Relative to the controls, the actinomycin-treated tissues showed a progressive, approximately exponential decrease in the rate of incorporation in nonzymogen proteins, but an increase in the corresponding zymogen values. This paradoxical “superinduction” was explained by attention to the intracellular leucine pool. Under the conditions of this experiment, actinomycin progressively increased the specific activity (Fig. 14B). This did not seem to occur in the autoradiographic experiments (Fig. 12), presumably because of different labeling conditions (high total leucine in the medium and labeling with a mixture of 3H-labeled leucine, valine, serine and glycine in). the incorporation data were divided stead of only l e ~ c i n e - ~ H When by the corresponding specific activity, actual rates of protein synthesis were calculated. The results were consistent with a zymogen mRNA half-life of 99 hours, and an average half-life of 3.6 hours for all nonzymogen messengers (Fig. 14C). The latter value is probably slightly too high, because proteins smaller than zymogen, contaminated with traces of zymogen degradation products, were included ( d i k e the preceding experiment). I n sum, three independent experiments, all using actinomycin, gave a very consistent picture: zymogen mRNA has a very long half-life, probably in the neighborhood of 100 hours, whereas the average nonzymogen messenger is unstable, with a half-life of only 2 or 3 hours. It was interesting to know whether zymogen mRNA is unique in * T h e region of the gel including proteins smaller than zymogen was omitted from the calculations, because of contamination with traces of zymogen breakdown products, produced during the experimental manipulations by a process akin to activation. When zymogen breakdown is suppressed (Pringle, 1970) by the addition of the inhibitor, phenglmethylsulfonylfluoride, and brief incubation at 100°C, the proteins smaller and larger than zymogen show comparable average isotope ratios.

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0 0

7 04 -

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2

3 4 5 TIME IN ACT.D (HR)

6

0

1

2

3 4 5 TIME IN ACT.D (HR)

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2

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4

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TIME IN AC1.D (HR)

FIG.14. Effects of actinomycin D (Act. D) on the galea. 0-0, symogen; 0-0, nonzymogen. Matched galeae (days 11-15) were incubated for identical periods in the presence or absence of actinomycin (60 pg/ml) and were then labeled for 1 hour with leucine-aH in the presence of inulin-W. At the end of labeling the tissues were homogenized and diquots were electrophoresed on SDS acrylamide gels. The gels were stained with Coomassie brilliant blue, scanned at 575 nm, sliced, and counted by liquid scintillation. ( A ) Incorporat,ion into zymogen and nonzymogen proteins evaluated as in Fig. 9. Incorporation data for each type of protein are presented as the ratio of actinomycin over control, after correction for differences in the amount of protein in each gel (575 nm). (B) Change in specific activity of the intracellular leucine as a function of time in actinomycin. Correction for extracellular leucine contamination was made by use of the inulin-"C in the medium. ( C ) Rates of zymogen and nonzymogen protein synthesis in the galea after actinomycin treatment. The incorporation data of (A) were divided by the specific activity d a t a of (B) to yield estimates of protein synthetic rates; these are reported as the ratio of actinomycin over control. From Regier and Kafatos (1971).

4.

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its stability or merely a t one end of a continuous spectrum; previous experiments had merely measured the average stability of all nonzymogen mRNA's. Accordingly, in a fourth experiment we used additional protein purification, beyond that afforded by SDS electrophoresis, to follow the rate of synthesis of partially purified galeal proteins after

FIG.15. The diversity of messenger and protein stabilities in the galea. Protein and mRNA half-lives were determined from experiments analogous to those presented in Fig. 13B and 13C. To increase the resolution, the mixed 'H- and "C-labeled proteins were fractionated into 10 fractions prior to electrophoresis: extracts in 0.1 M potassium phosphate (pH 7.0), and in 1% DOC plus 1% Tween 40 were each subdivided into proteins precipitated by 2070, 3970, 55%, and 75% ammonium sulfate ; detergent-insoluble proteins and proteins soluble in 75% ammonium sulfate were the other two fractions. Precipitations were performed a t 4"C, at a concentration of 40 galeae per 1.3 ml, with pH adjustment after each salt addition. Salt concentrations are expressed as the relative saturation a t 25°C of a solution with the same weight of salt per volume of solvent. Following electrophoresis of each of the 10 fractions, components with distinct isotope peaks or unusual 'H:"C ratio values were selected (see Fig. 17). Assuming that the turnover of zymogen mRNA and protein is negligible (see Fig. 13), half-lives for nonzymogen mRNA and protein were determined by the use of Eq. (I). The asterisk indicates the average mRNA and protein half-lives in this experiment (for all proteins larger than zymogen). The arrow indicates a protein with a half-life too large to measure accurately.

actinomycin treatment, and hence to estimate the diversity of mRNA half-lives (Kafatos and Moore, 1972). A substantial variability in halflives (Fig. 15) exists among those messengers present in large enough amounts to produce labeled proteins identifiable under our conditions (approximately 0.027% of total protein synthesis as a minimum). I n our sample of 50 partially purified proteins, 49 messenger half-lives were

168

FOTIS C. KAFATOS

in the range of 1.09-4.48 hours, and one was 9.8 hours; the mean was 2.37 hours, and the standard deviation 1.26 hours. Obviously, the zymogen message is in a class by itself with respect to stability. I n this experiment, the combined proteins ( 3H-labeled for actinomycin and “C-labeled for control) were fractionated arbitrarily into 10 subclasses by ammonium sulfate precipitation, prior to electrophoresis. The rationale was as follows. When all proteins are analyzed in a single gel, the peaks and valleys in the ratio profile, although statistically significant and highly reproducible (Fig, 16), clearly underestimate the variability of messenger half-life. A single gel of 100 slices has a theoretical maximum resolution of 50 labeled proteins, whereas the number of proteins in the sample is actually in the thousands. Thus, each datum corresponds not to a single component but to a mixture of overlapping proteins. Since there is no reason to expect that overlapping proteins have similar messenger half-lives, we must conclude that overlap dampens the deviations from the mean half-life. The overlap was revealed by the scarcity of distinct peaks in the SHand 14C profiles (Figs. 13 and 16). Fractionation into 10 distinct ammonium sulfate cuts increased the theoretical resolution by an order of magnitude. Overlap of components and thus dampening of the variability undoubtedly still occurred, but 50 proteins were sufficiently purified to be evident either as sharp peaks in the isotope profiles, or as significant deviations from the mean in the ratio profile (Fig. 17, left, p. 170). Thus, although the exact values of the half-lives have a wide margin of error, i t was possible to conclude with reasonable confidence that zymogen mRNA is not only more stable than average, but also outside the range for representative nonzymogen messengers. Protein turnover studies were performed in parallel with this experiment (Kafatos and Moore, 1972). The basic experimental procedure in this case was to label identical normal tissues with the two isotopes, and then to freeze the I4C-labeled cells immediately after labeling but to culture the 3H-labeled tissues for an additional time in nonradioactive medium, allowing time for a fraction of each protein to decay. I n this case, a high 3H:14Cratio indicated a stable protein, a low ratio an unstable protein. The ratio profile showed that, once synthesized, cocoonase zymogen is very stable (Fig. 13C). This of course was expected, since zymogen is sequestered in the storage vacuole, presumably out of reach of the intracellular machinery for protein turnover. Zymogen stability enhances the reliability of calculating synthetic rates from the zymogen accumulation curve (Section IV,A) . Assuming that zymogen is completely stable, half-lives of other proteins could be determined by use of Eq. ( 1 ) . The average half-life of all proteins (larger than

4. TEE

COCOONASE ZYMOGEN CELLS OF SILK MOTHS

169

3b L I C E NUHBEF

w v 30

; L I C E NUHBEF

u 3b

; L I C E NUHBEI

FIG.16.Reproducibility of isotope ratio profiles in actinomgrin experiments. Each panel presents a separate experiment (top: 3 galeae for each isot.ope, 10 days of adult development; center: 3 galeae, 12 days; bottom: 2 galem, 13 days). Only proteins larger than zymogen are shown. I n each case preincubation in t.he presence or absence of actinomycin was for 5.5 hours, and labeling with leucine-'H (artinomycin) or leucine-"C (control) for 3 hours. Vertical guidelines were positioned by reference to photographs of the stained gels. Desiiit,e variations in gel sectioning and in the st.age of the animals used, the major peaks and valleys of the ratio profilm are reproducible. Higher ratios indicate relatively stable mHNA's (see Fig. 13B).

170

FOTIS C. KAFATOS

*

zymogen) combined was 21.0 10.4 hours (95% confidence limits; four experiments, chase duration 9.5-16 hours). The diversity of half-lives in our sample of partially purified proteins was substantial. For 49 proteins, the mean was 18.9 hours, the range 4.89-89.4 hours, and the

0.83 A

0-

A

1

n

0.

45

0 30

SLICE NUMBER

0

I C

30 SLICE NUMBER

1

45

FIG. 17. Lack of correlation between mRNA and protein stability. Portions of parallel mRNA (left) and protein turnover (right) gels from the experiment described in Fig. 15 are shown containing 3 identifiable proteins (A, B, C). Note that for component C both mRNA and protein are unstable; for component B both mRNA and protein are reasonably stable; and for component A the protein is stable but t.he mRNA is unstable.

standard deviation 14.6 hours. One protein was too stable for half-life determination. It is interesting that no correlation apparently exists between the half-life of a messenger and the half-life of the protein that it codes (depending on the method, coefficient of correlation +0.037 to $0.103, p >> 0.1; Figs. 15 and 17). This conclusion, however, must

4. THE

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be qualified, since minor components were excluded by the nature of the experiment, 2. Is the Apparent Zymogen m R N A Stability a n Artifact?

I n interpreting the experimental evidence, and given that artifacts of isotope or actinomycin penetration are excluded by the autoradiographic analysis, three main types of possible artifacts must still be considered. One is that the zymogen gene may be uniquely refractory to actinomycin inhibition, as compared with other genes in the same cell. Two parameters which are known to influence susceptibility to actinomycin are the size of the gene (a larger target being easier to hit randomly; Perry and Kelley, 1970) and its guanine content (actinomycin binding preferentially to guanine; Reich, 1966). I n terms of these parameters the zymogen gene is unlikely to escape inhibition. The molecular weight of the zymogen and therefore of the gene is not unusually low. I n our study we observed no correlation between the effectiveness of actinomycin and protein molecular weight (in the range 35,000-160,000) ; we surmise that actinomycin inhibition is so effective under our conditions that target size is unimportant. The amino acid composition of zymogen suggests that the gene does not have an unusual base composition. Susceptibility to actinomycin may well depend on additional factors. Nevertheless, direct evidence exists that the apparent stability does not result from escape of the gene from inhibition. If zymogen mRNA is really unstable, it must be synthesized rapidly in cells devoting a major fraction of their protein synthesis to zymogen production ; if this mRNA continues to be made after actinomycin treatment, when other RNA’s are not made, it should be very easy to detect. Yet, when galeae are labeled with uridine following actinomycin treatment, there is no significant peak of incorporation in RNA analyzed on sucrose gradients (Kafatos and Reich, 1968). The second type of possible artifact is that the differential stability is real, but results from actinomycin treatment-i.e., that actinomycin either destabilizes nonzymogen mRNA or stabilizes zymogen mRNA. The former possibility is unlikely, in view of the fact that the average apparent nonzymogen mRNA half-life is comparable to values obtained from numerous other eukaryotic systems, with or without actinomycin (Penman et al., 1963; Trakatellis et al., 1964; Brandhorst and Humphreys, 1971). Stabilization of zymogen mRNA by actinomycin is also unlikely, in view of the required magnitude of such an effect (40-fold stabilization) and especially in view of the apparent stabilities of other differentiation-specific mRNA’s (Marks et al., 1962; Scott and Bell, 1964; Wessells and Wilt, 1965; Wilt, 1965; Moscona et al., 1968;

172

FOTIS C. KAFATOS

Papaconstantinou, 1967; Fantoni et nl., 1968). I n reticulocytes (assuming that hemoglobin genes are exclusively nuclear), the stability of the hemoglobin message can be demonstrated without the use of actinomycin, by the persistence of hemoglobin synthesis in the absence of a nucleus. Apparent mRNA stability is among the best established characteristics of highly differentiated cells. The third possibility is that messenger stabilities are identical for zymogen and nonzymogen proteins, but actinomycin changes the relative efficiency of translation of the two messenger classes by affecting the availability of a rate-limiting factor specific for only one class. I n more general terms, we might ask whether, in general, rate of protein synthesis can be used to a first approximation as a measure of mRNA content, or whether factors other than mRNA are usually rate-limiting in protein synthesis. I n the absence of actinomycin, nonmessenger rate-limiting factors have been postulated in a variety of systems (see Palmiter, 1972, for some references). Certainly the evidence is good that many unfertilized eggs contain inactive stores of mRNA (e.g., Nemer, 1967; Crippa and Gross, 1969; Davidson and Hough, 1971 ; Humphreys, 1971). However, in many other cases no compelling evidence exists that the postulated translational control factors play a regulatory role in vivo. I n maturing Xenopus oocytes, injection of hemoglobin message reveals that mRNA can be rate-limiting (Gurdon et aE., 1971, and persona1 communication). I n the normal estrogen-treated chick oviduct (Palmiter, 1972) initiation factors and ribosomes are apparently in excess ; recent evidence indicates that the in vivo rate of ovalbumin synthesis is proportional to the amount of the messenger, which can be extracted and assayed in a cellfree system (R. Schimke, unpublished observations). I n another highly differentiated cell type of silk moths, the egg-shell producing follicular cells (Paul et al., 1972), Dr. M. Goldsmith in my laboratory has found that the rate of peptide chain elongation is about the same for differentiation-specific and nonspecific proteins ; moreover, the spacing of ribosomes in the egg-shell polysomes is ca. 1 ribosome per 37 codons, similar to those found in other eukaryotic systems, highly differentiated as well as undifferentiated (Staehelin et al., 1964; Schreiber, 1971; see also Section II,B, 4 ) . Thus the evidence, incomplete though it is to date, is compatible with the suggestion that in vim, and except for special cases (dormant cells, cells in mitosis, hormone or nutrient starved cells), protein synthesis is generally proportional to the number of polysomebound mRNA molecules, rather than being primarily modulated a t the levels of initiation, elongation, or termination (but see Hunt et al., 1969). I n the presence of actinomycin, the rate of peptide chain elongation

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THE COCOONASE ZTMOGEN CELLS O F SILK MOTHS

173

per ribosome may increase by as much as 30% in the chick oviduct (Palmiter and Schimke, 1972). This effect, if general, would affect our absolute values of mRNA half-lives. However, it appears that the elongation rate of all proteins is changed in the oviduct. Thus, this phenomenon would not invalidate our conclusion that zytnogen mRNA is differentially stable, since mRNA’s of truly equal stability would show equal apparent half-lives, relative to each other, irrespective of the peptide chain elongation rate. Of the postulated specific translational control factors, the ones for which in vivo evidence is most compelling are those thought to sequester mRNA in an inactive or “masked” form, not permitting it to be associated with polyribosomes (e.g., in unfertilized eggs). Thus, a plausible model for explaining away our results might be that zymogen mRNA is of normal stability, but is sequestered (by a mechanism requiring continued RNA synthesis) in such an excess that its decay becomes apparent only after many hours in actinomycin. Evidence against this model comes from the data (Figs. 12 and 14C) which indicate that in the presence of actinomycin zymogen synthesis decays a t a slow, consistent rate, rather than by a delayed rapid mechanism. Moreover, the absence of a true “superinduction” by actinomycin suggests that a Tomkins-type model (Tomkins et al., 1969) is inapplicable in this case. Finally, if we postulate that zymogen mRNA is of normal stability but merely overabundant, the time over which actinomycin is relatively ineffective as an inhibitor is so long (at least 13-20 nonzymogen mRNA half-lives ; Fig. 12) that an unreasonably large store of inactive message must be postulated to exist in normal cells. Clearly, the differential stability of zymogen mRNA cannot be established rigorously until the message is isolated and its metabolism is assayed directly. Nevertheless, the indirect evidence strongly supports the conclusion that zymogen rnRNA is remarkably long-lived, as compared both with the average and with the range of half-lives for other messengers in the same cell and a t the same time.

B. IMPLICATIONS OF MESSENGER STABILITY What are the consequences of differential messenger stability? The first and most obvious, although inadequately appreciated, is that differential stability reduces the transcriptional load on the differentiationspecific gene. The quantitative argument runs as follows. During any small interval of time, the net change in messenger content represents the balance between messenger synthesis (which is assumed to be independent of preexisting messenger, i.e., zero order) and messenger decay (which is assumed to be exponential, thus proportional

174

FOTIS C. KAFATOS

to the messenger present, i.e., first order) (Adesnik and Levinthal, 1970; Mosteller et al., 1970; Schwartz et al., 1970; Attardi et al., 1966; Jacquet and Kepes, 1971). dM ~- S - DM dt

where M is the amount of message, t the time, S the messenger synthetic rate, and D the messenger decay constant. Since by definition the halflife, T , is related to the decay constant,

by substituting we obtain: dnf - - = s - - V ln(2) dt T

(4)

Therefore a t a steady state, when the change of M with time is zero,

s = -Af ln(2) T

(5)

Now let us consider the zymogen cells when they are devoting 50% of their protein synthetic capacity to zymogen production. Let us assume for the moment, t.hat zymogen synthesis remains constant over time, both in absolute and in relative terms. According to our previous discussion, to a first approximation protein synthesis is proportional to messenger content; thus the cells must obey Eq. (5) as it applies to both zymogen and nonzymogen mRNA. Using z and nz as the respective subscripts in Eq. ( 5 ) and dividing, we obtain

Thus, a t steady state the synthetic rates of the two messenger classes are inversely related t o the corresponding messenger half-lives. Since a t this time zymogen and nonzymogen proteins are produced a t a n equal rate, M, = Mnz.Substituting our best estimates for half-lives, we have

S, - 2.5 s,, 100

(7)

Thus, the zymogen gene will be responsible for only 2.4% of all messenger synthesis. I n other words, a 50% translational specialization can

4.

THE COCOONASE ZTMOGEN CELLS OF SILK MOTHS

175

be maintained indefinitely with a commitment of only 2.4% of the total transcriptional activity. This analysis implies that short-term labeling experiments are futile for isolation of specific messengers, even if the cells are highly specialized. I n short (e.g., 1 hour) pulse-labeling experiments, incorporation in mRNA is proportional to S ; if the specific message is stable, it is synthesized at a relatively slow rate, and hence it is overshadowed.” By contrast, in long-term labeling experiments incorporation is proportional to M and thus the differentiation-specific mRNA should be identifiable with ease, The optimal labeling protocol is long pulse-short chase, with the chase long enough (at least 2 x T,,) to permit decay of most labeled nonspecific messengers. I n agreement with this analysis, Suzuki and Brown in their elegant isolation of fibroin mRNA failed to identify the messenger in short pulse-labeling experiments, and succeeded only when they used long labeling periods (Suzuki and Brown, 1972). The second implication, which has considerable developmental interest, is that major changes in gene expression a t the translation level can occur without parallel changes in transcriptional gene activity. I n our preceding example, if zymogen mRNA synthesis is fixed but a t somewhat higher than 2.4% of all messenger synthesis, the cells will not remain a t a translational steady state but will continue to specialize, progressively increasing the rate of zymogen synthesis, because the “excess” zymogen mRNA will accumulate. Let us assume that the rate of nonzymogen protein synthesis is constant throughout phase 11. Assuming again a proportionality between protein synthesis and mRNA content, M,, is constant. If T,, is invariable, the synthetic rate of the nonzymogen mRNA, S,,, is also constant. Suppose that a t the transition to phase I1 the “transcription thermostat” of the zymogen gene is set to a fixed point, considerably above what is needed for maintenance of the “protodifferentiated” rate of zymogen synthesis; further, suppose that this transcription rate, S,, is kept constant thereafter. I n this simple-minded model all transcription, specific and nonspecific, remains constant throughout phase 11. Nevertheless, the specific zymogen mRNA will accumulate over time, because its transcription is faster than needed to balance its unusually slow breakdown. Accumulation will continue up to a final steady-state given by *Shorter periods of labeling are worse, because of the prominence of rapidly turning-over, nonmesenger, heterogeneous nuclear (hn) RNA (Soeiro et al., 1968). In the present discussion, S refers only to the synthesis of polynucleotide sequences that will become mRNA. If hnRNA, ribosomal and transfer RNA are included, S, becomes very much less than 1% of total transcription.

176

FOTIS C. KAFATOS

Eq. (5). At any time, the amount of the message will be given by the following equation," which is obtained by integrating Eq. (4).

Thus, under this model zymogen protein synthesis (which is proportional t o M,) will increase with a precise time-course, defined by the constant values of the half-life and the synthetic rate of zymogen message, T, and S,, plus the initial state, M,. Figure 18A and B show hypothetical curves describing the change in relative and absolute rates of zymogen protein synthesis over time during phase 11. The curves were generated by an IBM 360-65 computer with associated Calcomp plotter, using a program developed by B. Molay according to the preceding discussion. In this program, given an initial state and a fixed value for the zymogen messenger half-life, a constant zymogen mRNA synthetic rate can be calculated which will lead a specified value of M , by a specified time. For Fig. 18A and B, the conditions were set so as to approximate the real conditions in the galea (Fig. 8 ) : T n z = 2.5 hours, M n z (and hence S,,,) cpnstant, T, = 125 hours, zymogen synthesis 8% of total protein synthesis on day gIrI ( t = 0) and 70% on day 14,, ( t = 120 hours). With these conditions the rate of zymogen mRNA transcription, S,, was calculated to be 10.6 times lower than the total nonzymogen mRNA transcription (Fig. 18C). According to Fig. 18, the increase in zymogen synthesis during phase I1 can be visualized as follows. During phase I the slow absolute increase in rate of zymogen synthesis suggests that zymogen mRNA transcription is relatively slow. Shortly before the beginning of phase 11,the transcription thermostat of the zymogen gene is set t o a high and fixed value (8.6% of total mRNA synthesis). As a result of the upward adjustment of the transcription thermostat, zymogen mRNA begins to accumulate rapidly. Even if it is maintained temporarily inactive under translational control, around the beginning of phase I1 it is fully activated. Thereafter, the rapidly increasing amount of the mRNA is revealed by the rapidly increasing rate of zymogen protein synthesis. Over 120 hours, between day 9,,, and 14111,the absolute amount of zymogen mRNA increases 27-fold (Fig. 18B), and zymogen production jumps from 8% to 70% *This equation is the same as that describing the approach of an enzyme to steady state, after a change in either rate of synthesis or rate of turnover (Schimke, 1969). Indeed, the analysis presented for mRNA is equally valid for proteins. By reference to Eq. ( 5 ) , it can be seen that the frrst term, ST/ln(2), is the final steady state and the second term is the difference between final and initial states, multiplied by an exponential factor.

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FIG.18. Theoretical curves of changing relative and absolute rates of zymogen synthesis, resulting from differential mRNA stability and constant mRNA synthetic rates. Curves derived by an IBM 36M1.5 computer and plotted by a Calcomp plotter. For further details, see text. Stage 9111is set a s time zero and 141x1as t = 120 hours. For further details, see the text. ( A ) Relative rate of zymogen synthesis (as percent of total protein synthesis), deduced from the calculated ratio of Mnz)x 100. See Fig. 8B. amount of zymogen mRNA to total mRNA multiplied by 100, i.e., M , / ( M , (B) Absolute rate of zymogen synthesis expressed in terms of synthesis a t time zero. Values based on the calculated ratio of amount of zgmogen mRNA a t time t over amount a t time zero, A 4 z , t / M z , oSee Fig. 8A. (C) The constant rates of zymogen and nonzymogen mRNA synthesis necessary to yield the curves shown in (A) and (B), given T,, = 25 hours and T. = 1% hours. The ordinate unit is the synthesis per hour of an amount of mRNA equal to the steadystate amount of M”:.

+

178

FOTIS C. KAFATOS

of the total protein synthesis (Fig. H A ) . If “turning off” did not intervene, mRNA accumulation would proceed asymptotically until 82.5% of all protein synthesis were devoted to zymogen production. I n rough terms, the predictions of this model show some similarity to our current estimates of the synthetic curves (Fig. 8 ) . Better approximations can be obtained by manipulating the values of the parameters. It remains to be determined how closely this, or somewhat more sophisticated models, can fit reality. A first approach is to obtain very exact data on the absolute rates of protein synthesis during phase 11, in the galea and in other systems, and to see how closely they are fitted by models based on mRNA stability. Ultimately, a more rigorous test will be possible when we can isolate and quantify specific mRNA’s directly, without depending on assumptions about proportionality between messenger content and protein synthesis. Meanwhile, this model serves to illustrate how one of the most dramatic developmental changes in differentiated cells may be caused by the stability of the differentiationspecific message, combined with a particular constant rate of transcription. It will be noted that according to Eq. (8) and Fig. 18A and B, the curve describing the increase of specific protein synthesis over time cannot be exponential, but must have a continuously decreasing slope. This is because S and T are assumed to be constant and mRNA decay first order, so that decay increases as the amount of mRNA itself increases. At most, the curve will approach linearity when far from the steady state ( T , large relative to t , and S, large relative to what would be needed for a steady state with M , a t the time zero level). By contrast, the actual data (Figs. 8 and 11) indicate that the rate of specific protein synthesis increases in an accelerating fashion during the early part of phase 11; the agreement with the theoretical curves is much better for the period llrI, to 1411,. The early discrepancy could be accounted for in several ways. The most likely possibility is that the continued replication of the genome creates new copies of the specific gene until the end of day 11, so that S, increases for the tissue as a whole even though it remains constant on a per gene copy basis (Section IV,D, 2).

C. CAN HIGHLY DIFFERENTIATED CELLSPRODUCE As MUCH SPECIFIC PROTEIN As THEYDo WITH A SINGLE GENE COPYPER GENOME? Despite its stability, zymogen mRNA must still be synthesized a t a considerable rate; 8.6% of total mRNA synthesis is a considerable transcriptional load for a single locus to bear. The question is, is the

4.

THE COCOONASE ZTMOGEN CELLS OF SILK MOTHS

179

load compatible with the existence of a single gene copy per genome, or must gene multiplicity be involved, in the form of either amplification or redundancy? The answer apparently is that a single copy of the gene per genome is sufficient. To answer the question with certainty, we would need to recognize the specific mRNA, quantitate it, and measure its rate of synthesis directly. Until then, we must resort to indirect estimates. One strategy is to start from the known rates of protein synthesis during development. With quantitative data about translational efficiencies, we can convert rate of protein synthesis to amount of active mRNA. Assuming no inactive store of mRNA, we can then interpret curves such as in Figs. 8A and 11 as curves of mRNA accumulation over time. By differentiating the mRNA accumulation curve, we obtain the net rate of mRNA synthesis, dM/dt. From that and a known value of mRNA half-life we can estimate the actual rate of mRNA synthesis, S, using Eq. ( 2 ) . Dividing by the number of genome copies we obtain the required rate of mRNA synthesis per genome. Finally, we evaluate whether that rate is possible by reference to a reasonable estimate of maximal rate of mRNA transcription per single gene copy. I n practice, an estimate can be obtained more simply by taking the highest rate of specific protein synthesis during phase 11, converting it to mRNA content and dividing by the time from the beginning of phase I1 (on the assumption that the specific mRNA is infinitely stable and is synthesized a t a constant rate throughout phase 11). Since in all these cases the mRNA half-life is of the same order of magnitude as the time over which accumulation is assumed to occur, the answers obtained are quite similar to those given by the more laborious method 1. In this analysis, a key concept is the efficiency of template utilization. As shown in Fig. 19, whenever a ternplate is copied by multiple copying units, each producing a single copy as it traverses the template, the overall efficiency of the process is determined by the packing of the units and by the speed with which they move down the template. Thus, if ribosomes are spaced 30 codons apart on the mRNA and if they move at the rate of 60 codons per minute, the efficiency of translation will be 60 _ -- 2 protein chains per mRNA per minute 30 irrespective of the length of the messenger. A single ribosome requires more time to translate a longer message, but more ribosomes can be accommodated per message so that the overall efficiency is the same.

180

FOTIS C. KAFATOS

Template

Efficiency of Copying =

velocity (v) spacing(s1

FIG.19. The efficiency of copying [velocit,y (v)/spacing (s)l any template by multiple copying units. The overall efficiency of copying depends solely on the average velocity of copying units moving along the template (template distance per time, v ) and on the average spacing of the copying units (template distance per copying unit, s). Thiw holds irrespective of what determines the spacing (frequencies of initiation and termination of copying, values of v ) . For our purposes, the template could be either DNA or mRNA, the copying unit (copying apparatus) either RNA polymerase molecules or ribosomes, and the copy either mRNA or protein, respectively. Similarly, the efficiency of gene utilization (mRNA copies per gene per minute) is a function of the average spacing of RNA polymerases on the gene and of the number of nucleotides transcribed per minute per polymerase. Table I1 presents calculations for five different systems, both insect and vertebrate, according to the simplified second method. The first column gives the translational efficiency factor and the second the maximal observed rate of protein synthesis in each system per haploid genome equivalent (amount of DNA equal to that in sperm). In the third column the data of the preceding two are used to calculate the number of active mRNA copies present per haploid genome equivalent. The constancy of this number is striking. I n the case of the silk gland, the entry in parentheses is an independent estimate, obtained from the fraction of total silk gland RNA which is fibroin mRNA (Suzuki and Brown, 19721 plus the known content of RNA per DNA unit in the silk gland (Tashiro et al., 1968). The fourth column gives the time from the beginning of phase 11; this is the time over which mRNA presumably accumulates. By dividing columns 3 and 4 we obtain column 5 , our estimate of the number of mRNA copies that must be produced per haploid genome equivalent per minute. I n Table I11 similar calculations are done by method 1. I n this case, the maximal rate of new mRNA synthesis (column 3) is estimated from the maximal acceleration in specific protein synthesis (column 2) during phase I1 (Figs. 8A and 11). The actual rate of mRNA synthesis, S, is calculated from Eq. ( 2 ) , assuming for convenience a uniform value for the half-life, 1 t = 50 hours. These values of S are in the same range

TABLE I1 CALCULATION OF TRANSCRIPTION RATES, S,

System and temperature

FOR

DIFFERENTIATION-SPECIFIC GENES

2 M a x i m i level of translational activity, and stage (polypeptides. HGE-'.min-')

1"

Translational efficiency (polypeptides. mRNA-'.rnin-')

3

4

5

Time for tranS scription (mRNA. (days) HGE-'.min-')

Jf (mRNA.HGE-')

3.8 1 . 9 X 106 (day 5 ) Ovalbumin (chick oviduct; 37°C) 4 . 9 x 104 1.6 21 Chymotrypsinogen (embryonic rat 16 3 . 0 X 10' (day 19) 1 . 9 x 104 3 4.4 pancreas; 37°C) Hemoglobin y (embryonic mouse primi16 3 . 1 X lo6 (day 10.5) 2 . 0 x 104 1.1 13 tive erythroblasts; 37°C) Cocoonase zymogen (polyphemus galea; 2.8 6 . 9 X l o 4 (day 1 4 ~ 1 ) 2 . 5 X 10' 5 3.5 25T) Silk fibroin (Bombyx mori silk glands; 2.8 4 . 5 X 105 (day 6 . 5 ) 16 x 104 .5 . 5 20 25OC) (4 x 104) (.5) Column 1: Calculated by dividing polypeptide chain elongation rates ( u ) by the spacing of ribosomes on mRNA (s) (see Fig. 19). For ovalbumin, u = 2.1 codons per second (Palmiter, 1972) and S = 33 codons per ribosome (Palmiter et al., 1971). For chymotrypsinogen and hemoglobin y, the values used are those typical of mammalian systems (e.g., Staehelin el al., 1964; Hunt et al., 1969), u = 8.5 and S = 32. For the insect systems, v = 1.6 (calculated for silk moth egg-shell proteins; Goldsmith, 1972) and S = 34 (calculated for silk moth egg-shell proteins and cocoonase zymogen; Fig. 2B; Goldsmith, 1972). Column 2: Calculated from Figs. 8A and 1 1 . For hemoglobin y, the amount of globin produced between days 10 and 1 1 of gestation was divided by the average number of HGE during that interval (Fantoni el al., 1969). HGE = haploid genome equivalents; for vertebrates, cells were assumed to be 2.1 X haploid on the average; cocoonase zymogen cells were assumed to be 150 X haploid (Nardi, 1972); and the fibroin-producing part o f the Bombyx mori silk gland was assumed to contain 2 X lo8 HGE, calculated from a rough estimate of genome size ( I pg; Shepherd and Nardi, 1972) and the amount of DNA per silk gland (Tashiro et al., 1968). Fibroin was assumed to consist of two different subunits, approximately 1.7 X 105 daltons each (Tashiro and Otsuki, 1970); the fibroin protein and HGE estimates are the least dependable in this study. Column 3: Column 2 divided by column 1. For fibroin, the value in parentheses is calculated from the direct measurement of silk fibroin by Suzuki and Brown (1972). They measured fibroin mRNA extractable from the entire cell; the reasonable agreement with the value calculated from translation (using estimates of u and s from other silkworm tissues), and especially the fact t h a t their value is not greater than ours supports the conclusion that no substantial store of unused mRNA exists in the cells during phase I1 (see Section V1,A). Column 4: Assuming that rapid synthesis of mRNA is initiated at the beginning of phase 11. For ovalbumin and hemoglobin y, the effective transcription times are given, after correction for continued DNA replication during this interval. Column 5: Column 3 divided by column 4. This method assumes that no mRNA decay occurs during this period. ~~

~

~~~

~

~~

Q

m

F

F TR

182

FOTIS C. KAFATOS

TABLE I11 CALCULATION OF TRANSCRIPTION RATES,S, FOR DIFFERENTIATION-SPECIFIC GENES(METHOD 2) 15

Translational

System and temperature Ovalbumin (37°C) Chymotry psinogen (37°C) Hemoglobin y (37°C) Cocoonase zymogen (25°C) Silk fibroin (25°C)

2

3 S

Maximal rate of change in trans-

dM efficiency lational activity, (polypeptidesand stage dt (polypeptide. (mRNA. mRNA-1. HGE-l.min-1) HGE-1.min-l) min-1) 3.8

97 (day 5)

4

26

DM

=-

dt

+-MlnT(2) (mRNA. HGE-'.min-l) 37

16

117 (day 18)

7.3

10

16

Not known

-

-

18 (day 13) 97 (day 5 )

6.4 35

10 63

2.8 2.8

Column 1: See Table 11. Column 2: This is the steepest tangent to the curve of translational activity vs. time (Figs. 8A and 11). Data not available for hemoglobin y. The value for ovalbumin is a slight overestimate, since no correction was made for the continued (but slow) cell proliferation. As in Table 11, the value for fibroin is the least accurate. Column 3: Net rate of mRNA synthesis, neglecting mRNA decay. Calculated by dividing column 2 by column 1. The values are in reasonable agreement with those calculated in Table 11, column .5 (within a factor of 2). Column 4: Rate of mRNA synthesis, corrected for an assumed exponential mRNA decay with a half-life of 50 hours. 0

as those calculated in Table 11; we believe that they all are correct within a factor of two or three. But what is a reasonable maximum rate of transcription per gene? Let us start by noting that direct observations show the packing of RNA polymerases to be surprisingly uniform in several cases of highly active genes. In the case of E . coli ribosomal cistrons the packing is 75 nucleotide pairs per polymerase (Miller e t al., 1970b) ; in the amplified ribosomal RNA cistrons of Triturus it is 79, and in the lampbrush chromosomes of Triturus it is 97 (Miller et at., 1970a). The average, 82 nucleotide pairs per polymerase, is not much higher than the closest physically possible packing, 38 nucleotide pairs per polymerase (given the diameter of eukaryotic polymerase as 125 A ; Miller e t al., 1970a). Thus, these three cases probably represent the maximal reasonable polymerase packing. With respect to the rate of RNA chain elongation, good estimates exist for bacteria. The rate is remarkably constant, being

4.

183

T H E COCOONASE ZYMOGEN CELLS OF SILK MOTHS

determined almost exclusively by temperature, and being very insensitive to even drastic alterations in growth rate (Rose e t al., 1970). In eukaryotes, rates of RNA extension have been determined for poliovirus RNA and for 45 S ribosomal precursor RNA (Soeiro et al., 1968) ; they are somewhat higher than in bacteria. In Table IV the maximal reasonable transcriptional efficiencies are calculated a t 37OC (for vertebrates) and 25OC (for insects) using either the prokaryotic or the eukaryotic RNA elongation rate and RNA polymerase packing of 82 nucleotide TABLE I V MAXIMAL REASONABLE TRANSCRIPTION RATES 2 RNA elongation rate (nucleotides.min-1)

1'

Polymerase spacing (nucleotides)

Temperature 37°C

84 (minimum 38)

250c

84

}

38)}

2.7 x 4.0 x 1.0 X 1.5 X

l o 3 (bacteria) lo3 (eukaryotes) l o 3 (bacteria) lo3 (eukaryotes)

3

Transcription rate (mRNA.gene-l.min-i 32 48 12 18

(maximum 71) (maximum 105) (maximum 26) (maximum 39)

~

Column 1 : Average of polymerase spacings directly visualized in bacteria and eukaryotes; see the text. Column 2: Bacterial RNA elongation rates are averages of several determinations (e.g., see Rose el al., 1970). The eukaryotic rate a t 37°C was determined as < 5 X 10' nucleotides per minute for ribosomal precursor RNA synthesis (Greenberg and Penman, 1966) and as at Ieast 4 x lo3 for poliovirus synthesis (Darnel1 et al., 1967); for 25'C, extrapolation was made using the same Q l o as in bacteria. Column 3: Maximal transcription rates. Column 2 divided by column 1. a

pairs per polymerase. Comparison with the last column in Tables I1 and 111 reveals that a single copy of the differentiation-specific gene is indeed sufficient, if it operates a t or near maximal efficiency. The importance of mRNA stability is emphasized by Table V. According to Eq. ( 5 ) , if mRNA decay is exponential the maximum amount of mRNA that can be attained, no matter how long the time available, is determined by the ratio of mRNA half-life and synthetic rate. Using the reasonable maximum estimate for S a t the appropriate temperature, the minimum half-lives necessary to maintain the observed amount of mRNA is calculated. With a single gene copy per genome, it would not have been possible to maintain the observed amount of specific mRNA's if the mRNA's were not substantially more stable than average; for this conclusion it is immaterial how much time is available for mRNA accumulation.

184

FOTIS C. KAFATOS

TABLE V

MINIMUM mRNA HALF-LIFE,T,NEEDEDFOR ATTAINING M MAXIMUM 2

3

M maximum (mRNA.HGE-1)

T minimum

1"

-

s

System and temperature Ovalbumin (37°C) Chyrnotrypsinogen (37°C) Hemoglobin y (37°C) Cocoonase zymogen (25°C) Silk fibroin (25°C)

(hours.HGE.mRNA-') 3.6 3.6 3.6 9.6 9.6

x x x x x

10-4 10-4 lo-' 10-4 10-4

4.9 1.9 2.0 2.5 16 (4

x x

x x x x

104 104

104 104 104 104)

(hours) 18 7 7 24 154 (38)

0 Column 1: The best estimates of maximal reasonable transcription rate were used (32 mRNA.gene-1.min-1 a t 37"C, and 12 a t 25°C; Table IV). Column 2: See Column 3, Table 11. CoIumn 3: Column 1 multiplied by column 2. With half-lives shorter than this product, the observed mRNA content (column 2) could not be attained regardless of the time available, if mRNA decay is exponential and there is one gene copy per HGE.

It may be remarked that in embryonic pancreas (Fig. 11B) the kinetics of change in lipase A synthesis with time suggest that transcription is more than two orders of magnitude lower than in the case of chymotrypsinogen (if mRNA half-lives are comparable). I n a tissue such as the pancreas, which must produce several specific proteins in substantially different amounts, it may be that the protein which must be produced at the maximal rate determines the number of genome copies that must be present in the ehtire tissue. Given that number of genome copies, the transcription thermostat of other differentiation-specific genes is set during phase I1 a t various submaximal settings, according to the required rates of synthesis of the corresponding protein product. I n sum, it appears that terminal cell differentiation might proceed according to one of two strategies. One is to increase the multiplicity of the gene, by resorting either to redundancy in the germ line (Thomas, 1970) or to specific gene amplification, analogous to that observed with ribosomal RNA cistrons of amphibian oocytes (Brown and Dawid, 1968). In that case, no requirements exist for particular transcriptional efficiencies or mRNA stabilities. The alternative strategy is to depend on a single gene copy per genome, normally requiring it to transcribe a t a nearly maximal efficiency and to produce an unusually stable species of mRNA. Clearly, I believe that the second strategy is used as a rule. Evidence that has appeared since this analysis was written supports my belief. Hybridization studies with purified fibroin mRNA reveal that

4.

THE COCOONASE ZYMOGEN CELLS OF SILK MOTHS

185

the fibroin gene is not specifically amplified in the silk gland, and is present in fewer than 3 copies per genome (Gage and Suzuki, private communication). Gene multiplicity is also not encountered in cells synthesizing hemoglobin: the number of globin genes per genome i5 comparable with the number of different globin chains produced (Bishop et al., 1972), and the number of chain globin genes per genome is one (Paul, personal communication). It may be that specific gene amplification occurs only in rare cases when the gene product must be produced in high amounts during a relatively brief period, as with ribosomal RNA during oogenesis-or (for unknown reasons) with the products of “DNA puffs” in sciarid salivary gland chromosomes (Meneghini et al., 1971). (Y

VII. Concluding Remarks The study of zymogen cell differentiation leads t o certain conclusions which may have more general validity. 1. Development of terminally differentiated cells specialized for specific protein synthesis involves the sequential and coordinated occurrence of several steps. Some of these are: differential mitosis (to form the cells themselves), morphogenesis (to form the organule in the case of the galea, the tubular glands in the case of the oviduct, or the acini in the case of the pancreas), mitosis or polyploidixation (to increase the amount of secretory tissue), elaboration of an appropriate cytoplasm (rich in endoplasmic reticulum and Golgi in the case of secretory cells), synthesis of the specific cell product a t a low (phase I) and then high (phase 11) rate, and finally turning off or modulation of specific synthesis (phase 111).I n the galea, this entire program of differentiation is triggered by hormonal stimuli. Nevertheless, once set in motion the program shows a considerable degree of autonomy. For example, juvenile hormone cannot influence differentiation after the primary transition. Illoreover, the cells may turn on phase I1 without external stimulation, I n sum, terminal cell differentiation shows a high degree of internal programming. 2. The continuous increase in specific protein synthesis during phases I and I1 is quite striking. Indeed, this increase seems to be one of the most universal phenomena in terminal cell differentiation. It is particularly obvious when DNA is no longer replicated (latter part of phase I1 in the galea silk gland and pancreas), but it is also apparent a t other times if synthesis by the entire tissue is considered. 3. The mRNA for the specific cell product is unusually stable, when compared both to the range and to the mean of all nonspecific mRNA half-lives. The evidence for this conclusion, although indirect, seems overwhelming. I n the galea a t least, the differential stability of the

186

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specific mRNA is apparent even prior to phase 11. Most probably, the differential stability is an intrinsic property of the specific mRNA. Such intrinsic differential stability has recently been documented even in bacterial cells (Marrs and Yanofsky, 1971). 4. If the specific gene is very active in transcription, differential stability would lead to mRNA accumulation. Thus, stability of the mRNA can account for the marked progressive increase in translation within phase I1 without the necessity of postulating a parallel increase in transcriptional activity. The absolute increase in specific protein synthesis during phase I is much slower, suggesting that accumulation and perhaps specific gene transcription is occurring more slowly a t that time. Quantitative interpretation is complicated by the fact that DNA synthesis occurs throughout phase I . It is also possible (although not supported by the evidence) that a translational control may postpone full activation of accumulating mRNA until phase I1 begins. However, we prefer to view the transition to phase I1 as the reduction or elimination of transcriptional restraints on the specific gene (what we have called “resetting the transcription thermostat”). If this is in fact the case, it may be easier t o rationalize the apparent requirement for DNA synthesis during this transition in a number of systems (Holtzer, 1970; Rutter et al., 1968). 5. To a first approximation a t least, protein synthesis during phase I1 appears to be proportional to mRNA content. I n a related system, measurements of the translation time and of the packing of ribosomes on the mRNA suggest that the differentiation-specific mRNA attached to polysomes is translated with about the same efficiency as are other mRNA’s. When the absolute rate of protein synthesis is measured with and without actinomycin treatment, the results give no indication of a store of unused mRNA (which might be revealed by superinduction or, more generally, by nonexponential kinetics of decay), although they do not rigorously exclude it. 6. Assuming that protein synthesis is proportional to mRNA content, the increase in protein synthesis during phase I1 measures the rate a t which specific mRNA accumulates in the system. This rate of accumulation is compatible with the existence of a single copy of the differentiation-specific gene per genome, More explicitly, a single gene is adequate if the mRNA it synthesizes is unusually stable (as it is) and if transcriptional activity is near maximal. The importance of mRNA stability, in the absence of template multiplicity, can be shown quite independently of kinetics of accumulation. If mRNA decay is exponential, the high content of specific mRNA in cells late in phase I1 could never be attained with a normal mRNA half-life, no matter how long

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a time was allowed for accumulation; specific mRNA half-life must be of the order of a day or more. 7. Turning from model-building to data: a dispassionate observer would probably conclude that, for a field which aspires to be at the frontier of modern biology the field of differentiation is surprisingly poor in quantitative data. For example, most developmentalists would probably take it as self-evident that specific protein synthesis increases during differentiation. Nevertheless, a quantitative documentation of this intuition is hard to find in the literature. I n Sections IV,B and IV,D, 2, I have used a method which, with an assumption, permits calculation of synthetic rates from available data on changing protein levels during development. I n Section IV,C, I discussed the pitfalls of isotopic methods for measuring rates of protein synthesis-not in order to discourage the use of such methods, but in order to emphasize that they are so important that they must be used with care, so that they yield truly quantitative information. 8. I hope that the theoretical aspects of this chapter will be taken not as examples of loose thinking, but as attempts to make sense even before we have access to all the necessary information. Our methodologies are much superior for working with proteins than for working with nucleic acids. Moreover, we must acknowledge that developmental biology has not matured fully even as a descriptive science. Thus, it is entirely appropriate that we work at the level of protein synthesis, or at even more derivative levels of information flow. Nevertheless, as our understanding increases, as we learn what questions to ask about gene activity a t the transcriptional level, i t becomes obvious that we must learn how to study the metabolism of specific gene products directly. I n my view, major advances in developmental biology during the next decade will come from the isolation and sequencing of specific mRNA’s and specific genes, which will permit direct study of the transcriptional basis of differentiation. ACKNOWLEDGMENTS I t is a pleasure to acknowledge the contributions of my colleagues named in the text, as well as those of my research assistant, P. B. Moore, and my secretary, M. J. Randell; our collaboration has been a source of joy. The work reported has been supported by grants from the National Science Foundation (No. GB-8562), from the National Institute of Child Health and Human Development (No. HD-04701), and from the Rockefeller Foundation I thank Drs. Palmiter, Schimke, Suzuki, and Brown for communicating their manuscripts prior to publication. REFERENCES Adesnik, M., and Levinthal, C. (1970). Cold Spring Harbor S y m p . Quant. Bid. 35, 451-459.

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CHAPTER 5

CELL COUPLING IN DEVELOPING SYSTEMS: THE HEART-CELL PARADIGM Robert L. DeHaan and Howard G . Sachs* DEPARTMENT OF EMBRYOLOGY, CARNEGIE INSTITUTION OF WASHINGTON, BALTIMORE, MARYLAND

I. Introduction. . . . . . . . . . . . .................. 11. Cell Coupling in Mature Cardiac Tissue.. . . . . . . . . . . . . . . . . . . . . A. Electrotonic Coupling in Heart Tissue., . . . . . . . . . . . . . . . . . . . B. Ultrastructure of Heart Cell Contacts.. . . . . . . . . . . . . . . . . . . C. The Nexus as the Path for Cell Coupling. 111. Contacts and Junctions in the Early Embryo.. . . . . . . . . . . . . . . . . A. Coupling during Early Development. . . . . . B. Cell Coupling in the Precardiac Mesoderm an Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I V . Contacts and Coupling in Tissue Culture. . . . . . . . . . . . . . . . . . . . . A. Cell Interactions and Electrical Communication. B. Electrical Communication among Cultured Heart C. Coupling and Synchronization. ........................... V. Conclusions and Speculations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Speculations.. . . . . . ................... Keferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

193 194 194 196 203 206 205 207 209 209 212 213 222 222 223 225

1. Introduction From the time the fertilized egg undergoes its first cleavage to yield two cells, intercellular contacts and junctions come to be important to the embryo as well as to the embryologist. Contacts and junctions serve at least two vital roles-adhesion, i.e., holding the newly formed daughter cells together, and intercellular communication, allowing the passage of information and material between cells. The nature of such junctions a t the ultrastructural and electrophysiological level has been a particularly active area of investigation in recent years, with the result that we now have a growing body of evidence with which to construct models of developmental and physiological interaction of cells. Since many of our current concepts of cell coupling have arisen, historically, from considerations of the structure and electrical behavior of adult heart tissue, it is convenient to direct attention to that literature in order to define a set of terms and concepts that are required as background for a discussion of the role of cell coupling in developing systems. Moreover, we shall argue that embryonic heart cells, because of their spontaneous

* Present address : Department of Anatomy, University of Illinois, Chicago, Illinois 60680. 193

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electrical activity and contractility, provide unique advantages as a model system for further analyses of cellular communication. II. Cell Coupling in Mature Cardiac Tissue

Nineteenth century histologists recognized that heart muscle was cellular in structure ; they correctly interpreted the intercalated discs as zones of apposed junctional membranes. For example, Eberth (1866), to whom the first detailed description of the intercalated disc is usually attributed, regarded these structures as intercellular because of their strong reaction to silver nitrate (known to stain cell membranes) and because of the tendency of macerated muscle to fragment along the discs (recognized by early workers, and recently confirmed by Yokoyama et al., 1961). Ranvier (1889) also described mammalian myocardium as composed of individual rhomboidal branching cells separated by intercalated discs. Shortly after the turn of the century, however, several eminent anatomists, among them Heidenhain (1901) and Godlewski (1902) decided that the intercalated discs of heart muscle represented either contraction artifacts or were the sites of sarcomere differentiation. They concluded that cardiac tissue was syncytial in nature. Although the concept remained controversial during the first half of the present century (Jordan and Steele, 1912) as some light microscopists continued to make observations corroborating the earlier cellular view (Werner, 1910; W. H. Lewis, 1926), most investigators during this period tended to accept the syncytial hypothesis. This argument was strengthened by early physiological studies of cardiac conduction which were readily interpreted on the basis of protoplasmic continuity between heart fibers. COUPLING I N HEART TISSUE A. ELECTROTONIC Conduction of the action potential in nerve and skeletal muscle fibers had been interpreted successfully on the basis of core-conductor theory-the fiber being modeled as a long cable composed of a highresistance cylindrical membrane surrounding a low-resistance cytoplasmic core (Hodgkin, 1964). For heart muscle the law of “all-or-none” excitability and the common observation of conduction without decrement lent credence to the idea that cardiac tissue functioned in a like manner, as if it were composed of cablelike fibers, not chains of individual cells. Early application of linear cable analysis to cardiac muscle yielded reasonable first-approximation estimates for the electrical parameters of heart fibers (Weidmann, 1952). More recent analyses, in which investigators have applied more sophisticated linear (Weidmann, 1970) or various branched cable models (Spira, 1971; Tanaka and Sasaki,

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1966; Tille, 1966), have only confirmed that a heart muscle fiber indeed behaves as a functional syncytium. Nonetheless, as soon as techniques of ultrathin sectioning and electron microscopy were perfected in the early 1950’s, it was observed that fibers of mammalian heart tissue were in fact composed of linear chains of discrete cells (Sjostrand and Anderson, 1954) and that the intercalated discs represented points of specialized contact between their abutting ends (Van Breeman, 1953; Moore and Ruska, 1957; Muir, 1957). This discovery a t first raised doubts about the correct interpretation of the electrophysiological data. One group of investigators presented evidence suggesting that niyocardial cells are separated by high-resistance membranes (Sperelakis et al., 1960; Sperelakis, 1963) and proposed that the action potential in heart tissue is transmitted from cell to cell via a chemical mechanism (Tarr and Sperelakis, 1964). However, the interpretations of this group are not now widely accepted (Weidmann, 1969) since an overwhelming body of physiological evidence has emerged showing, instead, that heart cells are electrically coupled across low-resistance junctions. This evidence can be summarized as follows: 1. When current is passed through a bundle of heart fibers, the space constant (A), i.e., the distance over which the electrotonic potential falls by a factor e is of the order 0.5-1.0 mm (Kavaler, 1959; Kamiyama and Matsuda, 1966; Spira, 1971 ; Weidmann, 1970). Since the heart cell length is only about 100 p (see below), this result is possible only if the longitudinal cell-cell resistance is low. 2. Myocardial fibers from mammalian ventricle, atrium, and conduction tissue, as well as from amphibian heart have been subjected to voltage-clamp analysis (see E. A. Johnson and Lieberman, 1971, for review). With the sucrose-gap clamp commonly used, the “clamped” part of the fiber bundle has a length several times that of a single cell. Under these conditions, in most cases, the spatial distribution of the potential changes imposed on the fiber approaches reasonable uniformity, which is possible only if cells are electrically connected by low-resistance pathways. 3. When one-half of a bundle of ventricular fibers is exposed to 42K, and the other half is continuously washed with Tyrode’s solution, the radiopotassium diffuses down the length of the fibers within the myoplasm, across the low-resistance intercalated discs. A steady state with respect to tissue 42K is established within about 6 hours. Quantitative treatment of the data indicates that the permeability of the intercellular junctions to moyement of 42K from one cell to the next is about 5000 times greater than the permeability of the surface membrane to outward movement of the ion (Weidmann, 1966).

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4. When a mid-portion of a frog atrial bundle is bathed in sucrose with its two ends in Ringer’s solution, conduction of action potentials fails unless the two Ringer pools are shorted by a low ohmic resistance (Barr and Berger, 1964). This is the classical experiment that demonstrates propagation by local circuit currents, which could exist only if the linear longitudinal resistance from cell to cell were low (Osterhout and Hill, 1930). 5. When current is injected through an intracellular microelectrode into a single cell in a myocardial fiber, the degree of electrical coupling with an adjacent cell can be measured directly by comparing the change in membrane voltage produced in the two cells. With suitable controls and appropriate mathematical treatment of the results, the junctional resistance between myocardial cells is estimated a t less than 5 ohm cm2 (Woodbury and Crill, 1961, 1970; Heppner and Plonsey, 19701, as compared with a resistance of the nonjunctional surface membrane of 500-9000 ohm cmz (Spira, 1971 ; Weidmann, 1970).

B. ULTRASTRUCTURE OF HEARTCELLCONTACTS What then is the structure of the contacts that join myocardial cells? I n a recent definitive review, McNutt (1970) stated, “In considering the development of cell-to-cell contacts in cardiac muscle, it is important first to attempt to reconcile the widely differing and often confusing descriptions of the intercalated disc as interpreted by various morphologists using different preparative procedures. I n cardiac muscle, for example, the terms tight junction, close junction, gap junction, fascia occludens, and nexus are all employed by various authors to describe the same structure. The basis for this confusion in nomenclature must be explained so that the reader can appreciate how new techniques provide information essential to an understanding of this type of junction.” Relying heavily on the works of McNutt (1970; Fawcett and McNutt, 1969; McNutt and Weinstein, 1970), and Spira (1971) plus our own observations (see below), we shall attempt here to clarify this situation. The intercalated disc of all vertebrate heart muscle fibers is seen, with the light microscope, as a thick, refractile transverse band across the muscle, in register with the regular pattern of Z-lines. At low magnification in the electron microscope (Fig. 1) the intercalated disc is composed of two membranes bounded by dense granular material, and a separating interspace of 200-300 A. The membranes are continuous with the sarcolemma surrounding the fiber. The cleft between the transverse membranes is clearly continuous with the extracellular space. Within the disc the parallel membranes are thrown into folds and fingerlike projections, and rarely traverse the full width of the cell a t a single

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FIG.1. Low magnification electron micrograph of human embryonic myocardium showing a typical intercalated disc. Glutaraldehyde-osmium-methanolic uranpl acetate (block stain)-lead. Courtesy of John E. Rash.

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a.

SACHS

Z-line level. Instead, most discs are of the “step” type (Spira, 1971), with transverse segments located a t different sarcomeric levels but continuous through longitudinally oriented segments of the plasma membranes (Fig. 2 ) . Although some authors continue to use the term intercalated disc to refer only to the dense transverse portion, i t is now evident that the term should include both the transverse and longitudinal segments of the junctional complex. Mammalian heart cells are remarkably uniform in size. One study of the measured length and diameter of 96 atrial cells sampled from three hearts indicated a mean cell length of 92.2 p and a diameter (in the nuclear region) of 10.2 p (Spira, 1971). Cat ventricular cells have been estimated to be 12 X 125 p (Weidmann, 1966) , and the dimensions of rat atrial cells are given as 15 x 15 x 100 p (Woodbury and Crill, 1961). As a result, Sommer and Johnson (1970) have generalized that in all mammals (mouse through whale) cardiac muscle cells are 10-15 p in diameter. Myocardial cells of the turtle and domestic fowl are slightly smaller in diameter, averaging about 3.5-10 p for the reptile and 3-7 p for the chicken (Hirakow, 1970b). A heart muscle fiber, then, consists of chains of such cells, one or a few cells thick, joined end to end across intercalated discs. At higher magnification in the electron microscope, the intercalated disc contains three types of junctional specializations (Fig. 2B, 3 ) , which may be designated as the desmosome (macula adhaerens), the fascia adhaerens, and the nexus or gap junction. Desmosomes are disc-shaped structures, generally 0.2-0.5 p in diameter, in which the apposed plasma membranes from the two adjoining cells are separated by an intercellular space of 250-300 A, which is filled with a fibrillar qaterial. Often a thickened layer of this material forms a denser stratum midway between the adhering membranes (Karrer, 1960). I n the cytoplasm adjacent to the membranes is a region of highly condensed filamentous material into which tonofilaments 70-100 A in diameter insert (Muir, 1965 ; Fawcett, 1966, pp. 374-381). Desmosomes are usually found in longitudinal sections of the plasma membrane, i.e., those running parallel to the myofibrils and do not represent points of attachment for myofibrils. Fasciae adhaerentes, also known as cardiac adhesion plaques (Baldwin, 1970), are most often located on the transverse segments of the intercalated disc, are generally much larger in extent than desmosomes, and are the major form of junctional structure associated with the end-to-end adhesion of myocardial cells. As in the desmosome, the two apposed cell membranes in a cardiac adhesion plaque (CAP) run parallel and are separated over most of their area by an interspace of 256300 A containing a fine fibrillar material which usually

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FIG.2. ( A ) Step-type intercalated disc with nexal junrtions (arrow). Human embryo (150 mm crown-rump length) myocardium. Fixation and staining as in Fig. 1. The apposed membranes within the disc are continuous with the longitudinal plasma memhrane surrounding each cell. Courtesy of John E. Rash. (B) Nexal junction in human embryonic myocardium. Glutaraldehyde-osmium fixation, aqueous uranyl acetate hlock stain. Courtesy of John E. Rash.

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FIG.3. Desmosomes in stage 13 embryonic chick heart. Fixation and staining aa in Fig. 2R. Courtesy of Max Springer.

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lacks a central dense stratum. I n the cytoplasm adjacent to the membranes there is also a dense mat of fibrils. This has staining characteristics similar to the Z-band material (McNutt and Fawcett, 1969), into which the thin myofilaments (actin) insert (Kelly, 1967). When heart tissue is incubated in media containing the normal complement of ions, desmosomes and CAP’S appear to be regions of strong adhesion between adjacent cells (Muir, 1967). I n calcium-free medium, however, the apposed membranes in both the macula and fascia adhaerens separate, leaving the adhering cells joined only a t the more intimate contact region of the nexus (Muir, 1967). The third component of the intercalated disc, and the one of greatest interest in the present context, is the nexus or gap junction. I n a remarkably insightful paper on impulse propagation in smooth muscle, Dewey and Barr (1962) coined the term “nexus” to describe localized areas of close membrane apposition (in which the intercellular gap was reduced to less than 100 A) and discussed the role that this structure might play in transmission of electrical excitation (Barr, 1963). It is this specialized contact area to which the property of low-resistance has been attributed, and through which ions and electrical current are thought to flow freely from cell to coupled cell. The appearance of the nexus in the electron microscope is dependent to a greater extent than most structures on the procedures of fixation and staining used during tissue preparation. Although the overall thickness of the nexus-that is, the distance from one juxtacytoplasmic surface to the other-remains fairly constant, at about 180 f 10 A wide with various preparative methods, the structure can take on very different apparent configurations with these different procedures. When tissues are fixed with osmium tetroxide and stained with lead citrate, nexuses have a three-layered appearance (Muir, 1965). Fixed in potassium permanganate (Dewey and Barr, 1964) or in aldehyde-osmium with subsequent staining of the sections with uranyl acetate (Karrer, 1960) and with lead citrate (MrNutt, 1970) the nexus seems to have five layers. If tissue is fixed in either osmium or osmiumaldehyde and stained en bloc with uranyl acetate before dehydration, nexuses appear to be composed of seven distinct layers (Revel and Karnovsky, 1967; Brightman and Reese, 1969; McNutt, 1970). The middle electron-lucent layer of this seven-layered complex is generally 20-40 A thick, and a t highest resolution appears to have a granular or “scalloped” configuration (Fig. 2B). Since it is readily penetrated by extracellular marker molecules such as lanthanum hydroxide (Revel and Karnovsky, 1967) it must represent a physical “gap” between the apposed cell membranes which is continuous with the intercellular space. It was this discovery that led to the concept that the seven-layered

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complex is a gap junction (Brightman and Reese, 1969; Goodenough and Revel, 1970). Early in the analysis of the ultrastructure of cellular contacts, Farquhar and Palade (1963) described five-layered junctional contacts between columnar epithelial cells which they called “tight” junctions because the outer lamellae of the apposed membranes seemed to be completely fused. Since these structures appeared to form a beltlike seal around the apical end of each epithelial cell, connecting i t firmly with all of its adjacent neighbors they also coined the term “zonula occludens” (occluding zone), postulating that these were the structures that restricted diffusion of materials across the epithelial layer. I n contrast, the disc-like plaques of pentalaminar junctional configuration that had been seen connecting vascular endothelial cells (Karrer, 1960) and Mauthner cells (Robertson, 1963) and those seen in the intercalated discs of heart muscle, they termed “macula occludentes” (occluding spots), assuming that these, too, were “tight junctions.” We now know that there are important and reproducible differences between these two kinds of contact, which do not depend merely on preparative procedure (Brightman and Reese, 1969). Nexuses of the heart and adhesive plaques of close membrane apposition in other cell types are consistently found to be 1 8 0 k 10 A thick, as noted above, i.e., somewhat more than twice the 75 A thickness of each of the two apposed “unit” membranes. I n contrast, the zonulae occludentes of epithelial layers are equal to or less than twice as thick as the unit membrane, generally 13&150 A. While nexuses are readily penetrated by extracellular markers, lanthanum (Revel and Karnovsky, 1967), horseradish peroxidase (Brightman and Reese, 1969), and ruthenium red (Hirakow, 1970a ; Martinez-Palomo and Mendez, 1971), such compounds are always occluded by true tight junctions. Finally, the recent freezecleave method of visualizing the surfaces of unfixed cells has revealed dramatic ultrastructural differences between the surface architecture of gap junctions and true tight junctions. The former are characterized by 100 A center-to-center packed granules (McNutt and Weinstein, 1970) while the face of a tight junction is covered by a network of ridges or fibrils (Goodenough and Revel, 1970; Lorber and Raynes, 1972). The evidence, therefore, is highly convincing that nexal junctions in intercalated discs are true gap junctions, most accurately represented by a seven-layered configuration, and are not to be confused with the pentalaminar tight junctions of epithelia in which no space exists between the fused outer lamellae of the apposed surfaces (Farquhar and Palade, 1963 ; Brightman and Reese, 1969). Grazing tangential sections through a lanthanum-treated nexus show that the tracer fills a series of 20 A channels that run parallel to the

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cell surface, separating the array of electron-lucent, hexagonally packed subunits. These subunits apparently span the gap between the apposed membranes, since they exclude the lanthanum molecules (McNutt and Weinstein, 1970; Pappas et al., 1971). Freeze-etch preparations of the nexus suggest that the subunits outlined by lanthanum represent particles embedded in and protruding from the surface of the plasma membrane (McNutt and Weinstein, 1970). Thus, it should be noted that even in the nexal junction the apposed membranes appear to “fuse” at least at the crests of these protruding particles. I n the center of each particle a pit can often be visualized which is thought to represent the opening of a transmembrane 20-40 A channel which would join the cytoplasms of the apposed cells (McNutt and Weinstein, 1970; Pappas et al., 1971). It is this suggestion of the appearance of transmembrane channels, plus the consistent presence of nexuses between electrically coupled cells (Martinez-Paloma and Mendez, 1971; Revel et al., 1971) that argues for nexuses as being the anatomical site of low-resistance pathways between cells. Let us review the evidence in favor of this hypothesis.

C. THENEXUSAS

PATHFOR CELL COUPLING 1. Numerous investigators have concluded (as noted above) that current spreads from one cardiac cell to the next by electrotonic means. A partial list of these studies includes those of Weidmann (1952), Tille (1966), and Deleze (1970) on Purkinje fibers, Woodbury and Crill (1961) and Woodbury and Gordon (1965) on atrial fibers, and Van Der Kloot and Dane (1964), Tanaka and Sasaki (1966), and Weidmann (1970) on ventricular fibers. It is important to note that such investigations indicate that current not only can flow along the fiber, but also transversely to adjacent parallel fibers (Draper and Mya-Tu, 1959; Woodbury and Crill, 1961 ; Tanaka and Sasaki, 1966). Because of the steplike morphology of the disc, and the fact that cardiac adhesion plaques are not restricted to the ends of fibers, communication via intercalated discs would indeed be expected to occur not only in a linear direction but also transversely. 2. I n all cases studied thus far, cells known or inferred to be electrically coupled have revealed gap junctions at points of contact. Despite numerous early reports to the contrary, no case now exists of coupled cells lacking demonstrable regions of close* apposition. For example, THE

* Throughout this review we shall use the phrase “close apposition” or “close junction” to describe cases in which the outer leaflets of apposed membranes come to within less than 80 A of each other, when we have no further information with which to decide whether this contact represents merely a point of unusual proximity, a true nexus, or a true tight junction. “Nexus” and “gap junction” are used interchangeably.

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although it is necessary to infer that the myocardial cells of the heart of any species are electrically coupled (see above), several investigators failed to find gap junctions between heart cells in a variety of nonmammalian forms, including fish (Yamamoto, 1967), frog (Sommer and Johnson, 1970), axolotl (Gros and Schrevel, 1970)) reptile (Leak, 1967; Yamamoto, 1967), and chicken (Sommer and Johnson, 1970). However, with improved techniques, more recent studies have revealed close junctions in heart tissue of each of these species (Baldwin, 1970; Hirakow, 1970b; Martinez-Palomo and Mendez, 1971). Since, in fact, gap junctions have been demonstrated in cardiac tissue of animals representative of every class of vertebrates (hlartinez-Palomo and Rlendez, 1971 ; Scott, 1971) as well as the nonvertebrate tunicates (Kriebel, 1968; Lorber and Raynes, 1972), these junctions may be taken to represent a common structural basis for the spread of intercellular excitation in the chordate heart. Another kind of junction which has been implicated in cell coupling is the septate desmosome of the Drosophila salivary gland. Among the earliest demonstrations of electrical communication between cells was that of Loewenstein (1966) on the giant salivary gland cells of Drosophila. Since the only contact specialization between these cells appeared to be septate desmosomes, these structures were considered as likely candidates for the low-resistance pathway. More recent observations, however, have demonstrated the coexistence in similar preparations of septate desmosomes and nexal junctions (Hudspeth and Revel, 1971 ; Gilula and Satir, 1971 ; Flower, 1971 ; Rose, 1971). Thus it can be argued, both from the ubiquity of nexal junctions among electrically coupled cells, and the very low values of nexal resistance estimated by a variety of techniques (Weidmann, 1970; Spira, 1971; Woodbury and Crill, 1970) that the nexus is a t least a sufficient pathway for cell coupling. 3. There are a t least two systems in which the only membrane specializations found between electrically coupled cells are gap junctions. In a study of BHK2l fibroblasts in culture, and of interscapular brown fat cells, both of which are known to exhibit electrical coupling (Furshpan and Potter, 1968; Sheridan, 1971b), Revel e t al. (1971) have used thin sectioning and freeze-etch techniques to demonstrate small punctate nexuses between the apposed cell surfaces. But in neither case were tight junctions, desmosomes, or other surface specializations demonstrable. 4. By subjecting heart tissue to hyperosmotic solutions, it is possible to produce a gradual reduction in intimacy of contact of the cell surfaces in the intercalated discs (Barr e t al., 1965; Dreifuss et al., 1966). Conditions that separate the apposed halves of cardiac adhesion plaques and

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desmosomes but leave nexuses intact are not associated with diminished electrical communication. However, in an electrotonic synapse of the crayfish, treatments that caused physical separation of the gap junctions result in uncoupling (Asada and Bennett, 1971 ; Pappas et al., 1971). Thus, although rigorous experimental proof is still lacking, the circumstantial evidence is compelling that gap junctions are sufficient and necessary correlates of electrical communication between heart cells, and probably others as well. 111. Contacts and Junctions in the Early Embryo

A. COUPLING DURING EARLY DEVELOPMENT In some embryos (the starfish Asterias, for example), there is no electrical coupling between the first two blastomeres immediately after cleavage is completed (Ashman et al., 1964; Tupper et al., 1970). Ito and Loewenstein (see Loewenstein, 1967) have found that the sand dollar, another echinoderm, likewise lacks coupling between the first two blastomeres. In contrast, amphibian embryos all seem to be coupled from the first division: the newt Triturus (Ito and Hori, 1966), the frog R a n a pipiens (Woodward, 1968), and the South African clawed toad Xenopus Zaevk (Palmer and Slack, 1970). The possibility that invertebrate embryos lack electrical coupling a t first cleavage while vertebrates have such connections remains an intriguing but as yet untested idea. As embryos develop through the early cleavage stages, electrical coupling becomes a generally observed phenomenon. After the fifth cleavage in Asterias, electrical communication between blastomeres is found (Tupper et al., 1970). The cells of Xenopus remain coupled during this period (Palmer and Slack, 1970) as do the blastomeres of Triturus (Ito and Hori, 1966). The teleost Fundulus undergoes a series of cleavage divisions which result in incomplete sealing of cytoplasm of adjacent cells. However, in cells that have sealed completely, notably the surface cells, electrical coupling has been found (Bennett and Trinkaus, 1970). The blastula represents the first stage a t which true epithelial relationships are demonstrated in embryos. Hay (1968) has characterized an epithelium as a tissue that has a free surface and a basal surface, the latter coated by a basement lamina ; whose cells exhibit polarity of organellar localization with their nuclei disposed toward the basal side ; and in which tight or close junctions are usually seen between cells near their apical ends. By the blastula stage all embryos that have been examined exhibit electrical coupling among their cells: echinoderm (Tupper et al., 1970), amphibian (It0 and Loewenstein, 1969), fish (Bennett and Trinkaus, 1970), and chick (Sheridan, 1966; Trelstad et

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al., 1967). It seems to make no difference whether the embryo has a hollow ball form of blastula, as in the echinoderm and amphibian, or forms its blastocoel between the blastodisc and the uncleaved residue of the egg as in chick and fish embryos. I n the case of the gastrula of Fundukus, however, the outer membrane of the surface cells is of unusually high resistance (50,000-100,000 ohm cm?) permitting coupling by way of the intraembryonic extracellular space as well as via specialized junctions (Bennett and Trinkaus, 1970). It is also worthwhile considering that a t the stage when sea urchin blastomeres are not yet coupled, these blastomeres have the capacity to give rise to complete urchin embryos a t least to the pluteus stage. Yet after the first few cleavage divisions, when the blastomeres become coupled, separation of blastomeres by microsurgery reveals that they have lost their totipotency, as demonstrated clearly by Horstadius and Wolsky (1936). Blastula cells (macromeres) separated mechanically in a t least three species have been shown to couple electrically when manipulated into contact (Ito and Loewenstein, 1969; Triturus; Bennett and Trinkaus, 1970, Fundulus; Sheridan, 1971a, Xenopus) . Whether blastomeres from four- or eight-cell mammalian embryos, which can reaggregate and develop into blastocyst form after mechanical separation (Lin and Florence, 1970), undergo electrical coupling concomitant with aggregation remains to be investigated, but it seems likely that the establishment of electrical coupling is of functional importance a t these early stages of embryonic development. Hybrid embryos developing from eggs of R a n a pipiens fertilized with R a n a catesbiana sperm do not undergo gastrulation. K. E. Johnson (1972) measured the extent of contacts between presumptive mesodermal cells in such crosses and compared them with pipiens X pipiens (normal) embryos. About 30% of the junctions in the normals had gaps less than 50 A between outer membrane leaflets, while only about 10% of the hybrid gaps were of this order. It may be that electrical coupling, or a t least the establishment of close (gap ?) junctions is critical to triggering gastrulation. I n this regard i t is of interest that lithium ions, which prevent normal gastrulation and produce exogastrulae (Gustafson, 1950; Masui, 1961), have been shown to depress electrical coupling (Rose and Loewenstein, 1971). The early chick embryonic junctions have been investigated a t the ultrastructural level and provide one of the best opportunities to compare electrophysiological evidence concerning junctions with ultrastructure. Desmosomes were early implicated in many systems (e.g., Sheffield and Moscona, 1970) as having a primary role in adhesion. However, the early chick (stage 4) is noted for an absence of desmosomes (Overton,

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1962; Trelstad et al., 19671, as well as a lack of other membrane specializations such as zonula occludens and zonula adhaerens. At a stage when electrical coupling is regularly found between cells in the blastodisc, the only membrane junctions so far seen in the electron microscope have been punctate close junctions (Trelstad et al., 1966, 1967). Unfortunately, the techniques available to Trelstad et al. in these studies did not allow a distinction to be made between true tight junctions and nexuses. Only at later stages of development did desmosomes and other junctional complexes appear. The ultrastructure of the developing Fundulus has also been studied in relation to electrical coupling by Lentz and Trinkaus (1971). I n the early blastula only close membrane appositions and occasional punctate membrane fusions have been seen. As the embryo enters mid-blastula stages, desmosomes and gap junctions become increasingly extensive. Since the apical junctions of the Fundulus enveloping layer are actually composites of tight and close junctions, it is also not possible to distinguish from this study which type of contact is responsible for cell coupling.

B. CELLCOUPLING I N THE PRECARDIAC MESODERM A N D EMBRYONIC HEART During the first hours of chick embryo incubation a t 37O (stages 3 to 4 ) , the progenitors of myocardial tissue exist in bilateral regions of epiblast tissue adjacent to and roughly halfway between the anterior and posterior ends of the elongating primitive streak (Rosenquist, 1970). Precardiac mesoderm begins its invagination through the streak a t stage &along with the rest of the mesoderm. Preconus migrates into mesoderm before preventricle, and preventricle before preatrium. The precardiac mesoderm then moves anteriorly to form well-defined bilateral regions a t stage 5 centered a t the level of Hensen’s node halfway between the node and the lateral edge of the area pellucida (Rosenquist and DeHaan, 1966). While precardiac nlesoderln exists as a loose spongy layer of primary mesenchyme just after invagination, by the end of the first day (stage 6) it becomes a flat mesothelial sheet (Manasek, 1968). Near the forming head fold and shallow foregut of the stage 7 (one somite) embryo, precardiac mesoderm seems to thicken and condense, forming a layer of columnar cells that appears to move as a cohesive sheet rostrally relative to the endoderm (Stalsberg and DeHaan, 1969). Paired myocardial folds develop from these condensed regions by stage 8 (four somites) and begin to fuse a t their rostra1 ends a t stage 9 ; the heart begins to beat rhythmically a t about stage 10 (ten somites). The developing primary mesenchyme has been shown to have both

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focal tight junctions and close (20-100 A) junctions (Hay, 1968) even before it forms an epithelium. However, Manasek (1968) has noted that the cells of the splanchnic mesoderm which are destined to become cardiac mesoderm (Stalsberg and DeHaan, 1969) show desmosomes and apical junctional complexes. Although Manasek’s study was a t too low a magnification to distinguish between tight or gap junctions, such surface specializations are routinely found (Fig. 4) after staining with the

FIO.4. Contacts in the early chick heart. Glutaraldehyde-osmium fixation, uranyI acetate block stain. (A) Stage 10. Courtesy of John E. Rash. (B) Stage 13. Courtesy of Max Springer. Note more developed muscle in the later stage.

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en bloc uranyl acetate procedure (cf. Fambrough and Rash, 1971). The report of Sheridan (1966) which has been cited widely for the demonstration of electrical coupling between early chick embryonic cells is not in fact pertinent to a consideration of precardiac mesoderm, for the paraxial dorsal mesoderm that he found coupled to notochord and neural plate was too f a r posterior a t this stage (six somites) to have included the precardiac mesoderm (Stalsberg and DeHaan, 1969). So we are left with several questions concerning the early embryonic prebeating heart and its intercellular junctions, particularly as concerns electrical coupling and the occurrence of gap junctions. These questions can be summarized briefly as: 1. Are the cells of the early heart, just before the first heartbeat, electrically coupled? 2. Were they coupled when they were situated near the primitive streak (Stalsberg and DeHaan, 1969; Rosenquist and DeHaan, 1966) ? 3. If so, were they coupled at all precardiac stages-and by what means were they coupled-that is, can the presence of gap junctions or nexuses be demonstrated by the use of better staining procedures or the freeze-etch technique? 4. Until what stages does cardiac mesoderm remain coupled to neighboring tissues such as liver and lung? I n the squid embryo uncoupling has been seen to occur after organogenesis-i.e., eye was coupled to yolk cell and to other organs until well formed (Potter et at., 1966). Therefore, does the uncoupling of different organs result from their physical separation as organogenesis progresses, or does electrical uncoupling precede differentiation? Only in the latter case can hypotheses be devised that visualize uncoupling as causally related to tissue differentiation. 5 . Since precardiac mesoderm is thought to be induced by contact with the underlying endoderm, are these two tissues electrically coupled during the time the inductive interaction is taking place? IV. Contacts and Coupling in Tissue Culture The usefulness of tissue culture for probing cell interactions on a defined basis cannot be underestimated. I n a previous volume of this series Furshpan and Potter (1968) summarized the then current status of work on coupling in tissue culture. In this section we will attempt to update their summary and to give particular emphasis to the culture of embryonic cardiac myocytes. A N D ELECTRICAL COMMUNICATION A. CELL INTERACTIONS

Recent interest in electrical coupling in tissue culture has been spurred by the occurrence of such junctions in cancer models. Loewen-

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stein (1968) has long maintained that junctional uncoupling is intimately related to cancer, based primarily on studies from his laboratory and others on liver tumors (Loewenstein and Kanno, 1967), thyroid cancer (Jamakosmanovic and Loewenstein, 1968), and stomach cancer (Kanno and Matsui, 1968). Despite the apparent lack of electrical coupling in these cancerous tissues and a corresponding sparsity of gap junctions in cervical carcinoma (McNutt and Weinstein, 1970; McNutt et al., 1971), the role of uncoupling in cancer is less clear than it was perhaps several years ago. Loewenetein has attempted to clarify this situation by using tissue cultured cancer cells and transformed cells. Furshpan and Potter (1968) had demonstrated that BHK cells and their transformed counterpart-polyoma BHK-both were coupled. The lack of contact inhibition demonstrated by the polyoma virus-transformed line has been often cited as a characteristic of carcinogenicity. Here was a case of pseudocancer that showed coupling. Borek et a2. (1969) found that epithelial cells (liver) normally are coupled, but that X-ray transformation results in uncoupling. Similarly, a cloned epithelial cell line from a Morris liver tumor of the rat was found to be uncoupled in culture (Azarnia and Loewenstein, 1971). However, a cancerous fibroblastic strain from the same hepatoma, as well as transformed fibroblasts (X-ray, SV40 virus) and normal fibroblasts, are coupled. To add to present confusion is the study of R. G. Johnson and Sheridan (1971), showing that Novikoff hepatoma cells in suspension culture are coupled, and that gap junctions are demonstrable between these cells. Previously Loewenstein and Kanno had reported (1967) that solid Novikoff cancers were uncoupled. The only consistent finding extractable from such studies is that wherever coupling has been demonstrated, gap junctions can also be found, as confirmed by Gilula et al. (1972) on variants of a Chinese hamster fibroblast line. As mentioned earlier, in BHK cells the only demonstrable contacts between cells are small gap junctions (Revel et al., 1971). Perhaps more germane to our discussion are studies of tissue-cultured cells attempting to answer questions of developmental significance. It has been suggested that morphogenetic movements and differentiation may depend on communication between cells (Loewenstein, 1968; Wolpert, 1971). Organogenesis in the squid is accompanied by uncoupling of unlike (e.g., eye and liver) cells (Potter e t al., 1966), and in a recent report, Dixon (1971) noted a transient phase during which close junctions appeared in Xenopus retinal cells prior to retinal specification. Despite these suggestive bits of evidence that uncoupling may be related to differentiation, it is clear that maintenance of electrical communication be-

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tween cells is not in itself incompatible with retention of differentiated properties. I n culture, electrical coupling has been demonstrated between unlike cells by Michalke and Loewenstein (1971). Not only were rabbit lens, rat liver, and BHK coupled when cell pairs in homotypic cultures were examined, but they were all found to couple to one another in mixed cultures. It may also be concluded that coupling does not result in mitotic synchrony among cells. Fibroblasts in culture remain coupled to neighbors throughout all phases of the mitotic cycle; a dividing cell retains electrical continuity with its interphase neighbors (O’Lague e t al., 1970). The functional significance of such connections is not clear, nor is it readily apparent whether such communications form as a consequence of cultivation procedures. The electrophysiological measurements ordinarily used to assay for cell coupling show only that conjoint cells are separated by a much smaller resistance between them to the flow of current (i.e., small ions) than the resistance across the nonjunctional surface membranes. There are now, however, numerous demonstrations of coupled cells that also exhibit transfer of larger molecules, such as fluorescein (Loewenstein and Kanno, 1967; Furshpan and Potter, 1968; Sheridan, 1971a), Procion Yellow (Payton e t al., 1969; R. G. Johnson and Sheridan, 1971), sucrose (Bennett and Dunham, 1970), and even microperoxidase of molecular weight 1800 (Reese et al., 1971), none of which enter cells readily across nonj unctional surface membrane. There are several reports that dyes are transferred between coupled cells but not between uncoupled cells (Payton e t al., 1969; Sheridan, 1971b). Gilula and co-workers (1972) have recently shown that a competent strain (Don) of Chinese hamster cells can transfer “metabolically important molecules” to neighboring cells of an incompetent line, thereby correcting the metabolic deficiency of the latter cell. But this only occurs between cells that can establish electrical communication, and can form gap junctions. I n fact, 3T3 cells which have been shown to be coupled (Furshpan and Potter, 1968) can apparently transfer macromolecules up to the size of 28 S ribosomal RNA from cytoplasm to cytoplasm (Kolodny, 1971). From these data, it is tempting to conclude that the transfer of ions and the transfer of larger molecules always exist together and utilize the same route of passage. This is a logical trap; there exists no experiment in the literature in which this hypothesis has been tested critically. I n fact, Slack and Palmer (1969) have shown that electrically coupled blastomeres of Xenopus blhstulae do not pass dye, although more recently Sheridan ( 1971a) has observed the transfer of fluorescein between reaggregated Xenopus neurula cells, Furthermore, isolated Fundulus blastomeres may be manipulated together, couple, and yet not pass dye

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(Bennett and Spira, 1971). The argument of Furshpan and Potter (1968), that effective spread of dyes between BHK cells in culture is not likely to occur via the intercellular cleft because transfer was seen across small areas of contact, seems to us weak. Even a “small” appositional area (1-2 $) could allow for an intercellular compartment sealed effectively from the bulk extracellular fluid. Since pinocytotic vesicles are commonly observed in apposed junctional membranes (Porter et al., 1967; Kanaseki and Kadota, 1969; Lipton and Konigsberg, 1972) the possibility of transcellular pinocytosis-i.e., extrusion of materials by one cell into the intercellular cleft and uptake by its conjoint neighbor-cannot be ignored as a mechanism for transfer of large molecular weight components. Studies of development of neuromuscular junctions in culture have recently confirmed that functional connections can be made in vitro (James and Tressman, 1968, 1969). Shimada et al. (1969) and Veneroni and Murray (1969) provided light microscopic and histochemical evidence strongly indicative of formation of such junctions in culture, but electrophysiological evidence for functionality of these junctions has only recently come from two laboratories, those of Robbins and Yonezawa (1971) in Japan and of Fishbach (1970) in the United States. Another report of functional coupling has recently been made on cultured muscle cells that undergo fusion. The conduction of a response to iontophoretically applied acetylcholine in developing myotubes (Fambrough and Rash, 1971) has been employed to trace the events occurring prior to establishment of cytoplasmic continuity. The conduction of the acetylcholine response has been correlated with the electron microscopic appearance of nexal-like junctions (Rash and Fambrough, 1971). Such junctions have been found just before the start of membrane breakdown in fusing muscle cells, fixed within seconds after the first propagated response. At this time no other membrane specializations or cytoplasmic continuities were seen in serial sections through the total contact area between the apposed cells.

B. ELECTRICAL COMMUNICATION AMONG CULTURED HEARTCELLS The pulsation properties of cultured embryonic heart have been under study since as early as 1911. Burrows (1911) was able to maintain beating of hearts from chick embryos for from 3 to 8 days by using a plasma clot technique. For the first 3 days the rhythm appeared to remain normal, but subsequently both the force and frequency of beating periodically changed. Later studies on explants of cardiac fragments and myocytes isolated by teasing (M. R. Lewis, 1920) demonstrated that single cells could beat in isolation, and further that “the phenome-

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non of contraction exhibited by the few entirely isolated cells observed had the character of a distinct beat as though the single cell constituted in itself a minute force pump rather than an infinitesimal part in such a structure” (M. R. Lewis, 1920, p. 203). Early investigators were convinced, until this time, that the heart was not only a functional syncytium, but a morphological one as well, as documented above. A more efficient technique for isolating cells was to use trypsin to digest intercellular bonds. Cavanaugh ( 1955) cultured cells derived in this manner from chick heart and examined the reestablishment of functional connections between cells. She noted that “for the first few hours each cell pulsed with its own frequency. As contact among cells was established, the small clusters gradually began to contract synchronously” (p. 579). While the factors affecting pulsation in single isolated myocardial cells have been investigated in many laboratories, little of that work has helped our understanding of the synchronization and rate-setting processes (e.g., see Lehmkuhl and Sperelakis, 1967; Harary et al., 1967; DeHaan, 1967; Mark et al., 1967). Two major controversies have dominated the recent research on synchronization and rate-setting: (1) is the rate of a mass of heart cells in culture set by the rate of the fastest cell (Pacemaker Hypothesis), or is the rate-setting mechanism more complex; (2) are the cells electrotonically coupled to form a “functional syncytium,” or are they electrically isolated from one another. AND SYNCHRONIZATION C. COUPLING

The concept that the myocyte with the fastest intrinsic beat will set the rate in any assembly of such spontaneously beating cells has been advanced so often as to become dogma (see DeHaan, 1968, for review). Harary and Farley (1963) felt their experiments demonstrated support for the pacemaker hypothesis. They cut confluent beating sheets into two half-sheets, and noted that the rate of the intact sheet was the same as that of the faster half-sheet. But Cavanaugh (1955) noted that in the establishment of sheets of cells “subsequently pulsation became synchronous with a rate approximately equal to the average rate for individual cells in the same culture” (p. 584). More recently Goshima and Tonomura (1969) have stated that in monolayer cultures the confluent sheets beat a t a rate equal to that of the fastest isolated cells. Our experience (unpublished) has been that the rate of pulsation of the confluent sheet is not equal to the rate of the fastest cells. There might be species-specific differences involved, for Goshima and Harary

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ROBERT L. DEHAAN AND HOWARD Q. SACHS

and Farley have used mammalian heart cells, while Cavanaugh used chick cells. However, we have worked with chick and mouse and found them similar in this respect. An alternative experimental approach to the question of synchronization dominance would be to study two cells as they come into contact and synchronize. Mark and Strasser (1966) attempted to do this, although their published data show that the cells they observed were not isolated singlets. However, they concluded that the faster cell determined the rate of the synchronized group.

FIG. 5. Contact and synchronization of embryonic chick heart cells in culture. At time 0 no contact (C) or synchronization ( S ) of rate waa seen. Ninty-two minutes later contact was achieved, but not synchronization. A t 126 minutes after the first frame (38 minutes after contact), synchronization of rates waa established.

Recent studies in this laboratory have been designed to answer the following questions: (1) When cells are first isolated from embryonic hearts and then brought into contact will they achieve a synchronous pulsation? (2) Will that synchronous beating rate be related to the rate of the cell (or cells) with the fastest intrinsic rate of pulsation? This question has now been examined in isolated cell pairs (DeHaan and Hirakow, 1972; Wolf and DeHaan, 1972) and in aggregates in the hundred- to thousand-cell size range (Sachs and DeHaan, 1973), prepared by the methods developed by Moscona (1961; Fischman and Moscona, 1971).

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Strict application of the "pacemaker concept"-that is, as Aidley (1971), for example, has defined it: "the whole of the heart will then be driven by those cells which have the most rapid spontaneous frequency" (p. 275)-would predict that if two isolated myocytes in culture were to come into contact the cell with the faster rate would set the rate of the pair. A total of 37 cell pairs was studied in culture over TABLE I PULSATION RATESOF HEARTCELLPAIRSBEFORE

A N D AFTER

SYNCHRONIZATION^

Group L

Group I

Group H

RK

RL

Rs

RE

RL

Rs

RH

RL

Rs

78 78 80 90 91 100 100 109 112 120 120 128 129 142 -

70 70'

120 90 109 100 118 110 140 113 129 120 120 149 140 143

76 92 107 120 120 133 136 165

60 8' 74 7.5" 48' 80 46 120

70 67 105 100

90 95

20= 80 46" 78' 75c 93 110 90 100 98 110 109 131 120 133

20' 25 38' 75" 73" 84 108 80 100 95 110 75 103 120 130

1oc

27 78 92 10" 82 73 70 100 85 114 140 -

-

100

100 100 115 115 120 120 130 130 150 161 170 207

60"

98 54

162 -

-

~

~

~

Data from DeHaan and Hirakow (1972) and Wolf and DeHaan unpublished data. Definitions: Ra, Rate of the cell with higher spontaneous pulsation rate; RL, rate of the cell with the lower rate; Rs, rate of the pair after synchronization. Irregular, nonrhythmic beat. (I

the time course between initial contact of the cells and subsequent establishment of a synchronized rhythm (Fig. 5). This period ranged from 4 minutes to over an hour. The initial rates of each pair-member before contact and the synchronized rate of the pair are listed in Table I. The data are grouped in three categories: those pairs in which the synchronous rate was equal to, or greater, than the rate of the faster isolated cell (group H ) , those in which the synchronized rate was less than or equal to the rate of the slower isolated cell (group L) and

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ROBERT L. DEHAAN AND HOWARD G. SACHS

those which were intermediate. It is clear that only 14 out of the 37 pairs could be considered as obeying the pacemaker hypothesis. Some of the isolated cells had a regular pulsation rhythm which was maintained for periods of up to several hours. Others showed irregular rhythms, and still others changed their rate considerably over several hours. Of the 74 cells studied during synchronization 10 had irregular rates. Thus most cells appeared to be fairly regular. As a measure of the rate distribution of isolated myocytes, rates of 150 regularly rhythmical 7-day cells were determined; their distribution is shown in Fig. 6. Less than 5% of the cells beat a t greater than 200 beats per minute

250

RATE, BEATS/MINUTE

290

Fm. 6. The distribution of pulsation rates of 7-day embryonic heart cells in culture. Total N = 150. From Sachs and DeHaan (1973).

(B/M). The mean rate for this population was calculated to be 116 B/M. I n any assembly of more than a few hundred cells, one would expect to find a t least a few cells of intrinsically rapid rate. If these cells could impose their endogenous rhythm on the rest of the myocytes then the expectation would be that all assemblages of myocytes over a threshold size should beat a t fast rates. Aggregates of embryonic myocytes prepared by gyration in flasks and then plated out in Falcon tissue culture dishes remain spheroidal for several hours (Fig. 7 ) . The volume of such aggregates can be deduced from the profile seen with the inverted phasecontrast microscope, as shown in Fig. 8. If the rate of pulsation of an aggregate were set by those cells with intrinsically fast rates, all aggregates would beat rapidly. However, as

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217

FIG.7. Verification of the spheroidal nature of cardiac aggregates. Aggregates were prepared by gyration, plated in Falcon tissue culture dish, and fixed with glutaraldehyde and osmium. Plastic-embedded aggregates were photographed from top (A$) and side (B,D). Calibration in C is 200 p. From Sachs and DeHaan (1973).

FIG. 8. Calculation of aggregate volume based on the spheroidal nature of aggregates. From top view,

218

ROBERT L. DEHAAN AND HOWARD 0. SACHS #

shown in Fig. 9 the rate of pulsation was inversely related to the volume (or number of cells), This relationship was found not only in myocardial aggregates from chicks of 4, 7, and 14 days of age, but also in mouse myocardial aggregates, as seen in Fig. 10. Thus, the inverse rate-volume relationship appears to be independent of age or species.

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FIG.9. Rate-volume relations in embryonic chick myocardial aggregates. Aggregates made from 4-day (4D), 7day (7D), and 14-day (14D) chick embryo heart cells.

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.

5.

219

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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FIG. 10. Rate-voIume relations in aggregates of embryonic mouse and chick heart cells. Mouse aggregates from 16-day mouse fetal heart, chick aggregates from 14-day chick heart. Medium 818B with 1.3 mM potassium was used for both experiments. The straight lines are fitted by eye. 0 , Mouse; A , chick.

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

220

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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ROBERT L. DEHAAN AND HOWARD G. SACHS

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

224

ROBERT L. DEHAAN AND HOWARD G. SACHS

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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Lehmkuhl, D., and Sperelakis, N. (1967). In “Factors Influencing Myocardial Contractility” (R. D. Tanz, F. Kavaler, and J. Roberts, eds.), pp, 245-278. Academic Press, New York. Lentz, T. L., and Trinkaus, J. P. (1971). J . Cell Biol. 48, 455. Lewis, M. R. (1920). Contrib. Embryol. Carnegie Inst. 9, 191. Lewis, W. H. (1926). Contn’b. Embryol. Carnegie Inst. 18, 1. Lieb, W. R., and Stein, W. D. (1969). Nature (London) 224, 240. Lin, T. P., and Florence, J. (1970). Exp. Cell Res. 63,220. Lipton, B., and Konigsberg, I. R. (1972). J . Cell Bzol. 53, 348. Loewenstein, W. R. (1966). Ann. N . Y . Acad. Sci. 137, 441. Loewenstein, W. R. (1967). Develop. BioZ. 15, 503. Loewenstein, W. R. (1968). Perspect. Biol. Med. 11, 260. Loewenstein, W. R., and Kanno, Y. (1967). J. Cell Biol. 33, 225. Lorber, V., and Raynes, D. G. (1972). J . Cell Sci. 10, 211. McNutt, N. S. (1970). Amer. J . Cardiol. 25, 169. McNutt, N. S., and Fawcett, D. W. (1969). J. Cell Biol. 42, 46. McNutt, N. S., and Weinstein, R. S. (1970). J . Cell Biol. 47, 666. McNutt, N. S., Hershberg, R. A., and Weinstein, R. S. (1971). J . CeEl Biol. 51, 805. Manasek, F. J. (1968). J . Morphol. 125, 329. Mark, G. E., and Strasser, F. F. (1966). Exp. Cell Res. 44, 217. Mark, G. E., Hackney, J. D., and Strasser, F. F. (1967). In “Factors Influencing Myocardial Contractility” (R. D. Tanz, F. Kavaler, and J. Roberts, eds.), pp. 301-315. Academic Press, New York. Martinez-Palomo, A,, and Mendez, R. (1971). J . Ultrastruct. Res. 37, 592. Masui, Y. (1961). Experientia 17, 458. Michalke, W., and Loewenstein, W. R. (1971). Nature (London) 232, 121. Moore, D. H., and Ruska, H. (1957). J . Biophys. Biochem. Cytol. 3,261. Moscona, A. A. (1961). Exp. Cell Res. 22, 455. Muir, A. R. (1957). J. Biophys. Biochem. Cytol. 3, 193. Muir, A. R. (1965). J . Anat. 99, 27. Muir, A. R. (1967). J . Anat. 101, 239. O’Lague, P., Dalen, H., Rubin, H., and Tobias, C. (1970). Science 170, 464. Osterhout, W. J. V., and Hill, S. E. (1930). J. Gen. Physiol. 13, 547. Overton, J. (1962). Develop. Biol. 4, 532. Palmer, J. F., and Slack, C. (1970). J . Embryol. Exp. Morphol. 24, 535. Pappas, G. D., Asada, Y . , and Bennett, M. V. L. (1971). J . Cell BioE. 49, 173. Payton, B. W., Bennett, M. V. L., and Pappas, G. D. (1969). Science 166, 1641. Pinto de Silva, P., and Gilula, N. B. (1972). Exp. Cell Res. 71, 393. Porter, K. R., Kenyon, K., and Badenhausen, S. (1967). Protoplasma 63, 262. Potter, D. D., Furshpan, E. J., and Lennox, E. S. (1966). Proc. Nut. Acad. Sci. U.S. 55, 328. Ranvier, L. (1889). “Trait6 Technique d’Histologie,” 2nd ed. F. Savy, Paris. Rash, J. E., and Fambrough, D. (1971). Abstr. 11th Annu. Meet. Amer. SOC.Cell Biol. Abstract No. 471. Reese, T. S., Bennett, M. V. L., and Feder, N. (1971). Anat. Rec. 169,409. Revel, J. P., and Ka aovsky, M. J. (1967). J. Cell Biol. 33, C7. Revel, J. P., Yee, A. G., and Hudspeth, A. J. (1971). Proc. Nut. Acad. Sci. U.S. 68, 2924. Robbins, N., and Yonezawa, T. (1971). Science 172, 395. Robertson, J. D. (1963). J . Cell Biol. 19, 201.

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Rose, B. (1971). J . Membrane Biol. 5, 1. Rose, B., and Loewenstein, W. R. (1971).J. Membrane Biol. 5, 20. Rosenquist, G. C. (1970). Develop. Biol. 22, 461. Rosenquist, G. C., and DeHaan, R. L. (1966). Contrib. Embryol. Carnegie Inst. 38, 71. Sachs, H. G., and DeHaan, R. L. (1973). Develop. Biol. (in press). Scott, T . M. (1971).J. Anat. 110, 259. Shefield, J. B.,and Moscona, A. A. (1970).Develop. Biol. 23, 36. Sheridan, J. D. (1966).J . Cell Biol. 31, C1. Sheridan, J. D. (1971a).Develop. Biol. 26, 627. Sheridan, J. D. (1971b).J. Cell Biol. 50, 795. Shimada, Y., Fischman, D. A., and Moscona, A. A. (1969).Proc. N u t . Acad. Sci. U.S. 62, 715. Sjostrand, F. S., and Andersson, E. (1954).Ezperientia 10, 369. Slack, C., and Palmer, J. F. (1969).Exp. Cell Res. 55, 416. Sommer, J. R., and Johnson, E. A. (1970). Amer. J. Cardiol. 25, 184. Spanswick, R. M.,and Costerton, J . W. F. (1967). J . Cell Sci. 2, 451. Sperelakis, N. (1963).Circ. Res. 12, 676. Sperelakis, N.,Hoshiko, T., and Berne, R. M. (1960).Amer. J. Physiol. 198, 531. Spira, A. W. (1971).J. Ultrastruct. Res. 34, 409. Spitzer, N.C. (1970).J . Cell Biol. 45, 565. Stalsberg, H.,and DeHaan, R. L. (1969).Develop. Biol. 19, 128. Tanaka, I., and Sasaki, Y. (1966).J. Gen. Physiol. 49, 1089. Tarr, M., and Sperelakis, N. (1964).Amer. J. Physiol. 207, 691. Tille, J. (1966).J. Gen. Physiol. 50, 189. Trelstad, R. L.,Revel, J. P., and Hay, E. D. (1966). J. Cell Biol. 31, C6. Trelstad, R. L., Hay, E. D., and Revel, J . P. (1967).Develop. Biol. 16, 78. Tupper, J., Saunden, J. W., and Edwards, C. (1970). J. Cell Biol. 46, 187. Van Breeman, V. L. (1953). Anat. Rec. 117, 49. Van Der Kloot, W. G., and Dane, B. (1964).Science 146, 74. Veneroni, G.,and Murray, M. R. (1969). J. Embryol. Ezp. Morphol. 21, 369. Weidmann, S. (1952).J . Physiol. (London) 118, 348. Weidmann, S. (1966).J . Physiol. (London) 187, 323. Weidmann, S. (1969).Progr. Brain Res. 31, 275. Weidmann, S. (1970).J. Physiol. (London) 210, 1041. Werner, M. (1910).Arch. Mikrosk. Anat. 75, 101. Wolf, H. H., and DeHaan, R. L. (1972).In preparation. Wolpert, L. (1971). Curr. Top. Develop. Biol. 6, 183. Woodbury, J. W., and Crill, W. E. (1961). I n “Nervous Inhibition” (E. Florey, ed.), pp. 124-135. Pergamon, Oxford. Woodbury, J. W., and Crill, W. E. (1970).Biophys. J . 10, 1076. Woodbury, J. W., and Gordon, A. M. (1965).J . Cell. Comp. Physiol. 66, Suppl. 2, 35. Woodward, D. J. (1968). J. Gen. Physiol. 52, 509. Yamamoto, T. J. (1967). I n “Electrophysiology and Ultrastructure of the Heart” (T.Sano, V. Mizuhira, and K. Matsuda, eds.), pp. 1-13. Grune C Stratton, New York. Yokoyama, H. O., Jennings, R. B., and Wartman, W. B. (1961). Exp. Cell Res. 23, 29.

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

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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.

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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.

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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-

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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).

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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).

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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.

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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

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245

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-

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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

NS

7

' 6 m

Lo

m& 5 c

m 7 4 m c

c

0 3 C

0

.-

+ 2 L

0

ai

"

0

NS

5

S

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).

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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

t

4-6 days

BrdUrd

(0-3)

9-11 days

2 0

-2

0

2

4

6

B

10

12

1 4 - 2 0

2

4

6

8

10

12

14

CENTIMETERS

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

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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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Fischman, D. (1970). C.urr. Top. Develop. Biol. 5, 235. Fulton, C. (1970). Methods Cell Physiol. 4. Furmnnski, P., Schuman, D., and Lubin, M. (1971). Nntirre (London) 233, 413. Gurdon, J. B. (1970). Develop. Biol., SnppZ. 3, 59. Gurdon, J. B., Lane, C., Woodland, H., and Marbaix, G. (1971). Nature (London) 234, 521.

Hagopian, H., and Ingram. V. (1971). J. Cell Biol. 51, 452. Holtzer, H. (1963). Colloq. Ges. Physiol. (?hem. 13, 64. Holtzer, H. (1964). Biophys. J. 4, 239. Holtzer, H. (1968). In “Epithelial-Mesenchymal Interactions” (R. Fleisclimajrr and R. E. Billingham, eds.), pp. 152-164. Williams & Wilkins, Baltimore, Maryland. Holtzer, H. ( 1 9 7 0 ~ ) .In “Cell Differentiation” (0. Schjeide and J. de Villis, eds.), p. 476. Van Nostrand-Reinhold, Princeton, New Jersey. Holtzer, H. (1970b). Symp. Int. SOC.Cell Biol. 9, 69. Holtzer, H., and Abbott, J. (1968). In “Results and Problems in Cell Differentiation” (H. Ursprung, ed.), Vol. 1, pp. 1-6. Springer-Verlag, Berlin and New York. Holtzer, H., and Detwiler, S. R. (1953). J . Exp. ZOOZ.123, 335. Holtzer, H., and Matheson, D. W. (1970). In “Chemistry and Molecular Biology of the Intercellular Matrix” (E. A. Balazs, ed.), Vol. 3, pp. 1753-1769. Academic Press, New York. Holtzer, H., and Mayne, R. (1973). Holtzer, H., and Sanger, J. (1972). In “Research in Muscle Development and Muscle Spindles” (B. Banker and R. Pryzbalski, eds.), p. 122. Exerpta Med. Found., Amsterdam. Holtzer, H., Marshall, J., and Finck. H. (1957). J . Biophys. Biochem. Cgtol. 3, 705.

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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280

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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