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INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME 80

ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN GARY G. BORISY PIET BORST BHARAT B. CHATTOO STANLEY COHEN RENE COUTEAUX MARIE A. DIBERARDINO CHARLES J. FLICKINGER OLUF GAMBORG M. NELLY GOLARZ DE BOURNE YUKIO HIRAMOTO YUKINORI HIROTA K. KUROSUMI GIUSEPPE MILLONIG ARNOLD MITTELMAN AUDREY MUGGLETON-HARRIS

DONALD G. MURPHY ROBERT G. E. MURRAY RICHARD NOVICK ANDREAS OKSCHE MURIEL J. ORD VLADIMIR R. PANTIC W. J. PEACOCK DARRYL C. REANNEY LIONEL I. REBHUN JEAN-PAUL REVEL JOAN SMITH-SONNEBORN WILFRED STEIN HEWSON SWIFT DENNIS L. TAYLOR TADASHI UTAKOJI ROY WIDDUS ALEXANDER L. YUDIN

'

INTERNATIONAL

Review of Cytology EDITED BY

G. H. BOURNE

J. F. DANIELLI

St. George's University School of Medicine St. George's, Grenada West Indies

Worcester Polytechnic Institute Worcester, Massachusetts

ASSISTANT EDITOR K. W. JEON Department of Zoology University of Tennessee Knoxville, Tennessee

VOLUME 80 1982

ACADEMIC PRESS

A Subsidiary of Harcourt Brace Jovanovich, Publishers

New York London Paris San Diego San Francisco SBo Paulo Sydney Tokyo Toronto

'/

COPYRIGHT @ 1982, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED O R 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.

ACADEMIC PRESS,INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published b y ACADEMIC PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London NW1 7DX

LIBRARY OF CONGRESS CATALOG CARDNUMBER:5 2 - 5 2 0 3 ISBN 0-12-364480-0 PRINTED IN THE UNITED STATES OF AMERICA 82 83 84 85

9 8 76 5 4 3 2 1

Contents CONTRIBUTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

DNA Replication Fork Movement Rates in Mammalian Cells LEONN . KAPPA N D ROBERT B . PAINTER I. I1 . Ill . IV.

Introduction . . . . . . . . . . . . . . . Methodologies . . . . . . . . . . . . . . DNA Replication Fork Movement Rates . Concluding Remarks . . . . . . . . . . . References . . . . . . . . . . . . . . . .

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1

2 9 22 23

Interaction of Viruses with Cell Surface Receptors MARCTARDIEU. ROCHELLE L . EPSTEIN.A N D HOWARD L . WEINER I . Definition of Viral Receptor Sites . . . . . . . . . . . . . . . . . . .

I1 . Biological Characteristics of Viral Attachment to Cells 111. Membrane Components Which Interact with Viruses .

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

IV. Viral Components Which Recognize Cellular Receptors . . . . . . . . . V. Virus-Receptor Interactions and Pathogenicity . . . . . . . . . . . . . VI . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

27 30 37 49

52 56 57

The Molecular Basis of Crown Gall Induction W. F? ROBERTS I . Introduction

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

63 64 65

I1 . The Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111. IV. V. VI . VII . VIII . IX .

X. XI .

The Crown Gall Bacteria . . . . . . . . . . . . . . . . . . . . . . The Physiology of the Gall . . . . . . . . . . . . . . . . . . . . . . Involvement of Wounding in Gall Induction . . . . . . . . . . . . . The Opines . . . . . . . . . . . . . . . . . . . . . . . . . . . . Involvement of Plasmids in Tumor Induction . . . . . . . . . . . . Significance of Crown Gall Induction to Agrobucteriurn . . . . . . . . The Evolutionary Origin of Crown Gall . . . . . . . . . . . . . . . Agrobacteriurn and Genetic Engineering . . . . . . . . . . . . . . Future Work and Prospects . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . V

65

. .

. .

.

66 68 70 83 84 86 87 88

vi

CONTENTS

The Molecular Cytology of Wheat-Rye Hybrids R . APPELS

I. I1. I11. IV. V. VI . VII . VIII .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Genetic Relationship between Rye and Wheat Chromosomes . . . . The Molecular Structure of Rye and Wheat Chromosomes . . . . . . . Translocations in Wheat-Rye Addition or Substitution Lines . . . . . . Polymorphisms in Regions of the Chromosomes Containing Repeated Sequence DNA . . . . . . . . . . . . . . . . . . . . . . The Biological Effects of Rye Chromosomes (or Rye Chromosome Fragments) in Wheat-Rye Hybrids: Specific Effects Related to Heterochromatin . . . . . . . . . . . . . . . . . . . . . . . . . . The Possible Origins of Polymorphism in Rye Heterochromatin . . . . . Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

93 94 99 109 113

121 123 127 127

Bioenergetic and Ultrastructural Changes Associated with Chloroplast Development A . R . WELLBURN I. I1 . 111. IV. V. VI . VII. VIII .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plastid Development in Different Systems . . . . . . . . . . . . . . . Semicrystalline Structures . . . . . . . . . . . . . . . . . . . . . . Storage Reserves and Mobilization during Plastid Development . . . . . Mitochondria and Respiration during Plastid Development . . . . . . . Transfer between Cell Compartments during Photomorphogenesis . . . . Biogenesis of Photochemical Activities . . . . . . . . . . . . . . . . Influence of Light and Hormones . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

134 134 139 145 149 156 169 174 179

The Biosynthesis of Microbodies (Peroxisomes. Glyoxysomes) H . KINDL I. I1. I11. IV V. VI .

.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Concepts of Organelle Biosynthesis . . . . . . . . . . . . . . Survey Obtained by in Vivo Studies . . . . . . . . . . . . . . . . . Single Steps of Assembly Studied in Vitro . . . . . . . . . . . . . . . Special Types of Cells . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

193 194 202 210 214 224 224

vii

CONTENTS

Immunofluorescence Studies on Plant Cells C. E . JEFFREE. M . M . YEOMAN.AND D . C . KILPATRICK I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ill . Applications of Immunofluorescence Microscopy to Studies of Plant Cells and Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

246 262 263

Biological Interactions Taking Place at a Liquid-Solid Interface ALEXANDRE ROTHEN

I. I1 . I11 . IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunologic Reactions . . . . . . . . . . . . . . . . . . . . . . . Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INDEX. . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF RECENTVOLUMES A N D SUPPLEMENTS . . .

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

267 269 270 302 303 305 311

This Page Intentionally Left Blank

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

R. APPELS(93), Division of Plant Industry, CSIRO, Canberra ACT 2601, Australia ROCHELLE L. EPSTEIN (27), Department of Neuroscience, Children’s Hospital Medical Center, and Department of Medicine, Neurology and Infectious Disease Divisions, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115

C. E . JEFFREE(231), Department of Botany, University of Edinburgh, Edinburgh EH9 3JH, Scotland LEONN. KAPP( I ) , Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, California 94143

D. C . KILPATRICK (2311, Regional Blood Transfusion Service, Royal Infirmary, Edinburgh, Scotland H. KINDL(193), Biochemie (Fachbereich Chemie), Philipps- Universitat, 0-3550 Marburg, Federal Republic of Germany ROBERTB. PAINTER (l), Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, California 94143

W. P. ROBERTS (63), Department of Microbiology, La Trobe University, Bundoora, Victoria 3083, Australia ALEXANDRE ROTHEN(2671, The Rockefeller University, New York, New York 10021 MARCTARDIEU‘ (27), Department of Neuroscience, Children’s Hospital Medical Center, and Department of Medicine, Neurology and Infectious Diseuse Divisions, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115

‘Present address: lnserm U56, HBpital de Bicetre, Bicetre 94270, France. ix

X

CONTRIBUTORS

HOWARD L. WEINER (27), Department of Neuroscience, Children's Hospital Medical Center, and Department of Medicine, Neurology and Infectious Disease Divisions, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115 A. R. WELLBURN (133), Department of Biological Sciences, University of Lancaster, Lancaster, England

M. M. YEOMAN (231), Department of Botany, University of Edinburgh, Edinburgh EH9 3JH, Scotland

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 80

DNA Replication Fork Movement Rates in Mammalian Cells LEONN. KAPP AND ROBERTB. PAINTER Laboraton, of Radiobiology and Environmental Health, University of California, San Francisco, California

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

......... Rates ...................... A. Fork Rates in Human Cells,. . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Fork Rates in Nonhuman Mammalian Cells . . . . . . C. Fork Rates in Synchronized Cells.. ...................... D. Chemicals Affecting Fork Movement Rates.. . . . . . E. Fork Rates in Mutants and in Virro ...................... IV. Concluding Remarks ........................ References . . . . . . . . . ........................... 11. Methodologies.. 111. DNA Replication

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

1 2 9

9 15 15 18 21

22 23

I. Introduction The DNA replication rate is formed by two components: the number of actively operating replicating units (replicons), and the average linear rate at which the DNA replication fork moves along the parental DNA (DNA fork movement rate). The number of replicons in an average mammalian cell is about 100,000 (Painter et al., 1966; Okada, 1968), and the number of replicons active at any one time during S phase appears to be the main factor affecting changes in the DNA synthesis rate (Painter and Schaefer, 1971). DNA fork movement rates vary considerably from one cell type to another; published values range from 0.1 pdrninute in human cells to 2.5 pdminute in Chinese hamster ovary (CHO) cells. The majority of rates reported for human cells are in the range of 0.4 to 0.7 pdminute. The reasons for the reported variations are not completely known, but some factors are species, ploidy , whether normal or transformed, growth conditions (media, serum, etc.), and the experimental techniques used. Various aspects of DNA replication have been surveyed in recent reviews (Edenberg and Huberman, 1975; Painter, 1976 Sheinin and Humbert, 1978; Hand, 1978, 1979; De Pamphilis and Wassarman, 1980). In this article, we review DNA fork movement rates only in mammalian cells.

1 Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form resewed. ISBN 0-12-364480-0

2

LEON N. KAPP AND ROBERT B. PAINTER

11. Methodologies

The first report on fork movement rates utilized DNA fiber autoradiography. Cairns (1966), extending a technique he first used with bacteria (Cairns, 1963), incubated HeLa cells for 45 or 180 minutes with [3H]thymidine. The cells were then lysed and the DNA was spread out and coated with a photographic emulsion. After a sufficiently long exposure time, the emulsion was developed, and the tracks produced by the radioactively labeled DNA were measured. Cairns found that a 45-minute labeling time produced labeled DNA lengths ranging from 10 to 30 pm, whereas the 180-minute labeling time produced lengths ranging from 50 to 100 pm. Thus, human DNA appeared to have replicated at a rate of 0.5 pdminute or less. Subsequently, other workers improved the DNA fiber autoradiography technique to obtain more precise results and more information about DNA replication. Huberman and Riggs (1968) first pretreated cells with fluorodeoxyuridine (FUdR) before labeling with [3H]thymidine. The FUdR depleted the cells of DNA thymine precursors, so when [3H]thymidine was added later it became the sole source of thymine for DNA replication. This led to an abrupt start of the labeled segment of DNA as well as a higher specific activity of thymidine, thus producing heavier grain tracks in the fiber autoradiograms. Another innovation used by Huberman and Riggs was to label cells first with [3H]thymidine with a high specific activity followed by [3H]thymidine with a low specific activity. This resulted in a high grain density track followed by a low grain density track. These variable grain density tracks allowed the findings that (1) replicons are arrayed tandemly (i.e., linearly or sequentially) along the DNA fiber, (2) many replicons on one fiber are of uniform size, and (3) DNA fork movement rates can be measured within each replicon. Huberman and Riggs reported that fork movement rates ranged from 0.5 to 2.5 pdminute in CHO cells. Recently, Yurov (1980) attempted to increase the information obtained by fiber autoradiography by isolating single labeled cells on microscope slides and then lysing the cell in situ. The resulting fiber autoradiograms then represented DNA from only one cell, thus allowing analysis of DNA replication in one nucleus. Examples of fiber autoradiograms are shown in Fig. 1. The advantages of DNA fiber autoradiography in examining DNA fork movement rates are that (1) single DNA chains are resolvable, (2) information can be obtained about replicon sizes, and (3) information can be obtained about relative times of replicon initiation. It should be noted that this technique was also used to show that DNA replication occurs bidirectionally (Huberman and Riggs, 1968). Limitations of this technique are: ( 1 ) Nonrepresentative sampling. Since the average mammalian cell contains approx. lo5 replicons and most autoradiographic data represent 150-300 tracks, only a very small percentage of the replicating DNA is represented. (2) Low resolution. The limit of resolution in

FIG. I . Autoradiograms from mouse L5 l78Y cells. (a) Thirty-minute pulse-labeling. Fibers show tandem arrays of replicons. (b) Labeling with high specific activity [3H]thymidine (hot pulse) for 30 minutes followed by labeling with low specific activity [3H]thymidine (warm pulse) for 30 minutes. The two dense unbroken tracks represent the origin regions of replicons that initiated operation after the beginning of the labeling period (i.e., a postpulse figure), whereas the gap represents the origin of a replicon that initiated operation before the beginning of the labeling period (i.e., a prepulse figure). The dense areas are the result of labeling by high specific activity [3H]thymidine, whereas the lighter trailing tracks represent the DNA labeled by low specific activity [3H]thymidine. The direction of replication was from the high to low specific activity areas. (c) A comet of tangled DNA. This may represent DNA from one cell or a portion of one cell. To measure the fiber lengths, it is necessary to find a region of the slide or comet where the tracks are sufficiently clear and separated. These autoradiographs were provided by S. Sawada, Kumamoto University, Kumamoto, Japan.

4


DNA fiber autoradiographs is reported as 1.5-5 pm (Huberman and Riggs, 1968; Ockey and Saffhill, 1976). For a cell with an average fork movement rate of 0.5 pndminute incubated for 30 minutes, the error of resolution in the resulting DNA fiber autoradiogram would be 10 to 30%. Longer pulse times could reduce this error, but when pulse times become too long, adjacent replicons fuse, making fork movement rates unmeasurable. (3) Coincidence counting problems. All tracks scored may not be from separate DNA fibers. Despite these shortcomings, DNA fiber autoradiography is the most commonly used technique for examining DNA fork movement rates in mammalian cells and, when carefully performed, gives consistent and valuable information. Several ultracentrifugal methods have been used to estimate DNA fork movement rates. These methods utilize the distribution of DNA along a gradient in an ultracentrifuge tube. The gradient can measure differences in molecular weight or differences in density, and these differences are used to calculate DNA fork movement rates. Lehmann and Ormerod (1970) calculated DNA fork movement rates by examining the sedimentation of labeled DNA on alkaline sucrose gradients. They incubated mouse L5 178Y cells with [3H]thymidinefor various lengths of time. The cells were next exposed to X rays to introduce random breaks into the DNA, lysed on the top of a 5-20% alkaline sucrose gradient to release single-stranded DNA, and then centrifuged. From the resulting distributions of the labeled DNA on the gradients, the average molecular weights were computed. By labeling the cells for different lengths of time and observing the change of the average molecular weights in the resulting gradients, the DNA replication fork movement rate was calculated. Similar approaches, utilizing the increase in DNA molecular weights as observed by sucrose gradient centrifugation, have been used by several other workers (Lanotte et al., 1977; Laughlin and Taylor, 1979) to calculate fork movement rates (Fig. 2). Painter and Schaefer (1969, 1971) utilized incorporation of [3H]thymidineand unlabeled bromodeoxyuridine (BUdR) in sequential pulse-labeling and CsCl equilibrium density gradients to measure DNA fork movement rates. With this technique, cells were first incubated with [3H]thymidinefor a short time (10 to 30 minutes), followed by incubation with BUdR for 1 to 2 hours. The DNA was then isolated and sheared, and one aliquot was analyzed by velocity sedimentation to determine the average molecular weight of the sheared fragments. A second aliquot was analyzed on CsCl equilibrium density gradients to determine the fraction of DNA that was distributed on the heavy side of the normal-density DNA, i.e., that which contained molecules that had 3H at one end and BUdR at the other. From this shift, DNA fork movement rates were calculated using published equations (Painter and Schaefer, 1971; Roti-Roti and Painter, 1977) (Fig. 3). More recently, Povirk and Painter (1976) pulse-labeled cells with [3H]BUdR

5

DNA REPLICATION FORK MOVEMENT RATES

0.6

0.3

24

12

S

l

o

b

2

0

Fraction number FIG. 2. (a-e) Alkaline velocity sedimentation profiles of pulse-labeled nascent DNA segments from CHO cells blocked by hydroxyurea at the beginning of S phase until 14 hours after division. The pulse times were (a) 4 minutes, (b) 8 minutes, (c) 12 minutes, (d) 16 minutes, and (e) 20 minutes. Each pulse was terminated by submerging the cultures in SSC (0.15 M sodium chloride, 0.015 M sodium citrate) at 0°C. The cells were lysed at 0°C with standard lysing solution containing 500 p,g/ ml proteinase K. The DNA from these lysates was denatured and then sedimented in alkaline NaI velocity gradients 9 hours after lysis. The gradients were centrifuged at 20,000 rpm for an 02t of 10' I rad2/second. The vertical arrows indicate the position of the center of the T7 marker DNA band. The T7 DNA included in these samples sedimented in each case as expected for a homogeneous population of phage-sized DNA molecules. Sedimentation was from left to right. Reprinted from Laughlin and Taylor (1979) with the permission of the authors and Springer-Verlag, Inc.

for short times, and then exposed them to several fluences of 313 nm light. Under the experimental conditions used, the only breaks induced in the DNA were caused by the action of the 3 13 nm source on the BUdR-substituted residues in the DNA. The cells were then lysed on the top of an alkaline sucrose gradient and centrifuged. The resulting DNA gradient profiles showed that increasing the

6

LEON N. KAPP AND ROBERT B . PAINTER

F R A C T I O N NUMBER


7

exposure to 3 13 nm light caused an increased shift of the labeled DNA toward low molecular weights. This shift can be quantified and used to estimate the length of DNA labeled with BUdR. Dividing the length of labeled DNA by incubation time with [3H]BUdR yields the average fork displacement rate (Fig. 4). Other techniques or variations of some of the described techniques have also been used to calculate fork movement rates. For example, Planck and Mueller (1977a) labeled cells briefly with BUdR, and then with I3H]thymidine, and then exposed the labeled cells to 313 nm light. The light ruptured the DNA at the BUdR-labeled regions and released the "-labeled DNA. The [3H]thymidine incubation times were varied and the growth of labeled DNA chains was analyzed by velocity gradient sedimentation. Taylor ( 1968) used variable incubation times with [3H]BUdR and estimated the ratio of 3H in completely substituted DNA to that in partially substituted DNA as a function of incubation time. The rate of fork movement was estimated from the changes in this ratio. Gradient methods have the advantage of speed (most determinations take only 1 to 5 days) when compared to autoradiography, which often requires developing times of up to 1 year. In addition, gradient techniques also yield results that reflect an average of all the replicating DNA in the S phase cells. However, they give no information about distributions of fork movement rates within a cell. Several workers have directly compared various techniques for measuring fork movement rates. Laughlin and Taylor (1979) compared an alkaline velocity gradient method with fiber autoradiography in CHO cells. The alkaline gradient method gave an estimated fork movement rate of 0.5 to 0.6 pdminute. Fiber autoradiographic data from the same cells were in excellent agreement with the sedimentation results, indicating that two independent techniques can both result in the same findings. In another comparison of sedimentation and autoradiographic techniques, Richter and Hand (1979b) measured fork movement rates in monkey CV-1 cells. Fiber autoradiography gave a value of 0.56 pdminute, whereas the equilibrium density method described by Painter and Schaefer (1 971) gave a value of 0.36 to FIG. 3. CsCl equilibrium density gradient profiles of HeLa S3 DNA labeled for 30 minutes with [3H]thymidine and then for 2 hours with BUdR and sheared (upper panel) to produce number-average molecular weight DNA (E) of 1.3 X 107 and fraction of ['HIDNA at densities greater than normal ( F ) of 0.225 or sheared (lower panel) with ultrasound to produce B of 0.26 X lo7 and F of 0.026. A, 3H radioactivity; 0, I4Cradioactivity (adjusted). The ultrasound F value is considered the minimum possible and is primarily due to thymidine pool mixing of [3H]thymidine and BUdR. This is subtracted from the F value for the 12,000 rpm shearing to give F,,,. From B and F,,,, L , the average molecular weight of DNA labeled during the pulse with [3H]thymidine, can be estimated. Since about one-twelfth of the total DNA must be synthesized in 30 minutes (S period = 360 minutes), the total number of sites replicating DNA DNA molecular weight per ceW12 X L. Reprinted from Painter and Schaefer (1969) with permission of the authors and Academic Press, Inc.

8


BOTTOM

TOP

FRACTION

FIG. 4. Alkaline sucrose gradient profile of DNA from pulse-labeled E-11 human diploid cells that were exposed to various fluences of 313 nm light, lysed on a 5-20% alkaline sucrose gradient, and centrifuged. Cells were exposed to 0 (O), 60 (O), or 180 (0) seconds of 313 nm light. Such gradients allow calculations of parameters that can be used to estimate fork movement rates. Reprinted from Kapp and Paintep (1978) with permission of the Biophysical Society.

0.38 pdminute. Richter and Hand concluded that the equilibrium density method provided an objective measurement of fork movement rates, although it underestimated the actual rate. However, if these authors had used the equilibrium density gradient method exactly as described by Painter and Schaefer (1969, 1971) (one necessary step was omitted), the results from the two methods would have been in closer agreement. Kapp and Painter (1979) compared two sedimentation techniques: the equilibrium density method of Painter and Schaefer (1971) and the BUdR-313 nm photolysis method described by Povirk and Painter (1976). It was found that for asynchronous CHO cells, both techniques gave the same average value and range (about 1 pdminute). The same methods were used with synchronous CHO populations and resulted in the same conclusions: a constant rate of about 1 pm/ minute throughout S phase. Rates in synchronous HeLa cells were also measured using the BUdR-313 nm photolysis method and a 2- to 3-fold increase in rate from early to late S was found. This confirmed the earlier report of Painter and Schaefer (1971) for HeLa cells using the equilibrium density method. From this work, it appears that different sedimentation techniques using different approaches can be in excellent agreement. The work discussed above indicates that most of the techniques currently in use give comparable results. Therefore, it appears possible that the reported variations in fork movement rates actually are due to differences in the rates in


9

different cell types or in the same cell type under different culture conditions, rather than to inappropriate techniques. To examine this question in more detail, Kapp and Painter (198 1) split a single cell culture into two equal subcultures and measured fork movement in them using the BUdR-313 nm photolysis method. All measurements were made in duplicate, portions of each cell culture being run in separate centrifuge rotors. The idea was to determine, when cell cultures were as identical as possible, how much variation in fork rate measurements was inherent in the experimental techniques. There was less than 10% variation between the most extreme values in this set of measurements. However, under normal experimental conditions, rates measured in any single cell type vary by about 30%. Again, these results suggest that the differences measured with different techniques are largely due to variations in cell types or culture conditions.

111. DNA Replication Fork Movement Rates

DNA fork movement rates have been examined in a wide variety of mammalian and human cells (Tables I and 11). The values shown in the tables are the average values reported in the papers. As mentioned above, large variations were seen around the mean values in most of the reports. Since not all means and variances were shown in the same manner, they are omitted in the tables for the sake of uniform presentation and comparison. In addition, some reports discussing fork movement rates presented comparisons but did not present data on actual fork movement rates, and thus are not listed here (Wickremasinghe and Hoffbrand, 1979; Giannelli et al., 1977; Hand, 1975a). For human cells (Table I) the population mean and standard deviation is 0.60 f .021 pdminute. For other mammalian cells (Table II), it is 0.75 f 0.33 p d minute. Considering the variety of cell types, culture techniques, and measurement techniques, this is a surprisingly narrow range. In contrast, fork rates in bacteria are reported to be up to 15 pdminute (Cairns, 1963), and workers examining amphibians have found relatively low values of 0.02 pdminute (Hyodo and Flickinger, 1973). A. FORKRATESIN HUMANCELLS

Kapp and Painter (198 1) measured fork movement rates in 20 human cell types using a single technique (BUdR-3 13 nm photolysis) and uniform culture conditions. The overall mean fork rate was 0.53 f 0.08 pdminute (population mean and population standard deviation). For individual cell types the means and standard deviations were much larger. For example, GM637, an SV40-transformed cell line, had an average fork movement rate of 0.75 ? 0.20 pdminute,

10

LEON N. KAPP AND ROBERT B. PAINTER TABLE 1 FORKMOVEMENT RATESIN HUMANCELLS ~~

Cell type

Technique

A. Diploid cells Skin fibroblasts Autoradiography Skin fibroblasts Autoradiography Skin fibroblasts Autoradiography Skin fibroblasts Au toradiography Skin fibroblasts Autoradiography Skin fibroblasts Autoradiography Skin fibroblasts Autoradiography Skin fibroblasts BUdR-3 13 nm photolysis Skin fibroblasts BUdR-3 I3 nm photolysis GM498 fibroblasts BUdR-3 13 nm photolysis Lymphocytes Autoradiography Lymphocytes Autoradiography Embryonic fibroblasts Autoradiography Embryonic fibroblasts Autoradiography Embryonic fibroblasts Autoradiography HuP, embryonic lung fibroblasts Autoradiography MRC-5 fetal lung fibroblasts, passage 16 Autoradiography MRC-5 fetal lung fibroblasts, passage 58 Autoradiography WI-38 fetal lung Equilibrium density gradient fibroblasts Fetal brain cells Autoradiography Fetal heart cells Au toradiography Fetal retinal epithelial cells Autoradiography Fetal fibroblasts Autoradiography Fetal fibroblasts Autoradiography Fetal hepatocytes Autoradiography B. Cells from donors with genetic diseasesb XP fibroblasts XP fibroblasts XP fibroblasts Bloom’s fibroblasts Bloom’s fibroblasts Bloom’s fibroblasts

Autoradiography BUdR-3 13 nm photolysis BUdR-3 13 nm photolysis Autoradiography Autoradiography Autoradiography

Rate (pdminute)

Reference

0.8 0.65 0.64 0.62 0.60 0.6 0.35O O.5la 0.5 0.42 0.6 0.41 0.70 0.6 0.6

Ockey (1979) Hand and German (1 977) Hand and German (1975) Hand ( 1977) Yurov (1980) Yurov (1977) Heenen and Galand (1980) Kapp and Painter (1981) Kapp and Painter (1978) Kapp et al. (1979a) Yurov (1977) Hand and German (1977) Yurov (1979a) Yurov and Liapunova (1977) Yurov (1979b)

0.67

Ockey and Saffhill (1976)

0.5

Petes et al. (1974)

0.38

Petes et al. (1974)

0.9P 0.6 0.6

Painter and Schaefer (1969) Yurov (1977) Yurov (1977)

0.4 0.6 0.6 0.6

Yurov (1977) Yurov (1 977) Yurov and Liapunova (1977) Yurov (1977)

0.6 0.52a 0.46 0.75 0.47 0.51

Yurov (1978) Kapp and Painter (1981) Kapp et al. (1979a) Ockey (1 979) Hand and German (1975) Hand and German (1 977) (continued)

11

DNA REPLICATION FORK MOVEMENT RATES TABLE I (Continued)

Cell type Bloom's lymphocytes Bloom's fibroblasts Fanconi's anemia fibroblasts Fanconi's anemia fibroblasts Fanconi's anemia fibroblasts A-T fibroblasts A-T fibroblasts A-T fibroblasts Trisomy-2 1 lymphocytes Trisomy-21 fibroblasts Trisomy-7 fibroblasts C. Transformed cells HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa Chang AC297 1 KDT Basal cell nevus Basal cell carcinoma GM637 GM637 XPl2RO Fanconi's anemia (SV40-transformed)

Technique

Rate (pdminute)

Reference

Autoradiography BUdR-313 nm photolysis

0.24 0.26a

Hand and German (1977) Kapp and Painter (1981)

Autoradiography

0.67

Hand (1977)

Autoradiography

0.65

Hand and German (1975)

BUdR-313 nm photolysis Autoradiography Autoradiography BUdR-3 13 nm photolysis Autoradiography BUdR-313 nm photolysis Autoradiography

0.55" 0.79 0.65 0.50" 0.6 0.43 0.8

Kapp and Painter (1981) Ockey ( I 979) Hand (1977) Kapp and Painter (1981) Yurov (1978) Kapp et al. (1979b) Yurov (1978)

Autoradiography Autoradiography Autoradiography Autoradiography BUdR/sedimentation BUdR-313 nm photolysis BUdR-3 13 nm photolysis BUdR-313 nm photolysis BUdR-3 13 nm photolysis Equilibrium density gradient Equilibrium density gradient Equilibrium density gradient Autoradiography BUdR-3 13 nm photolysis BUdR-313 nm photolysis Autoradiography Autoradiography BUdR-313 nm photolysis BUdR-3 13 nm photolysis BUdR-313 nm photolysis

0.79 0.5 0.35 0.32 0.3a 0.9 0.65 0.55 0.42 1.6 0.95 0.8" 0.86 0.53 0.57 0.87 0.24 0.83 0.75 0.53

Ockey and Saffhill ( I 976) Cairns (1966) Edenberg (1976) Stimac et al. (1977) Planck and Mueller (1977a) Povirk and Painter (1976) Kapp and Painter (1978) Kapp and Painter (1981) Painter (1980) Gautschi and Kern (1973) Gautschi et a / . , (1973) Painter and Schaefer (1969) Ockey and Saffhill (1976) Kapp and Painter (1981) Kapp and Painter (1 98 1 ) Ockey (1979) Heenen and Galand (1980) Kapp er al. (l979a) Kapp and Painter (1981) Kapp er al. (1979a)

BUdR-313 nm photolysis

0.57

Kapp and Painter ( I 98 1 )

"These papers did not present a single average fork movement rate as shown here; an average was calculated from the published data. bXP, Xeroderma pigmentosum; A-T, ataxia telangiectasia.

12

LEON N. KAPP AND ROBERT B. PAINTER TABLE I1 FORKMOVEMENT RATESI N NONHUMAN MAMMALIAN CELLS

Cell type Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster Chinese hamster

ovary ovary ovary ovary ovary ovary ovary ovary ovary ovary ovary ovary ovary ovary ovary ovary ovary V79 V79 V79 V79 V79 B 1 IFAF B 1 1FAF lung lung

Technique Autoradiography Equilibrium density gradient BUdR-313 nm photolysis BUdR-3 13 nm photolysis BUdR-313 nm photolysis Equilibrium density gradient BUdR-3 13 nm photolysis Alkaline sucrose gradient Autoradiography Autoradiography Autoradiography Autoradiography Alkaline sedimentation Alkaline sucrose gradient Autoradiography Autoradiography Autoradiography Autoradiography Au toradiography Equilibrium density gradient Autoradiography Autoradiography Autoradiography Autoradiography Autoradiography Equilibrium density gradient

Rate (pdminute) 0.5-2.5 I .4a 1.3 1.1

I .o 1 .o 1 .o

0.98 0.88 0.85a 0.8 0.6a 0.6* 0.6 0.5 0.32 0.31 1.O" 0.93° 0.9 0.61 0.36 0.8 0.930 0.58 0.3°

Reference Huberman and Riggs (1968) Taylor (1968) Povirk and Painter (1976) Kapp and Painter (1978) Kapp and Painter (1981) Kapp and Painter (1979) Kapp and Painter (1979) Walters et al. (1976) Ockey and Saffhill (1976) Hori and Lark (1973) Lark et al. (1971) Laughlin and Taylor (1979) Laughlin and Taylor ( 1979) Kurek and Taylor (1977) Taylor (1977) Taylor and Hozier (1 976) Stimac et al. (1977) Dahle et al. (1978) Yurov ( I979a) Painter and Schaefer (1969) Dahle er al. (1980) Doniger (1 978) Yurov and Liapunova ( 1977) Yurov (1979a) Martin and Oppenheim (1977) Martin and Oppenheim (1977) (continued)

or a range of 54% around the mean value. Hydroxyurea-treated HeLa cells used for comparison had fork rates of only 30% of normal, and rodent cells (CHO and S-49 mouse lymphosarcoma cells) had rates of 1 .O and 1.1 pdminute, indicating that the technique used was sensitive enough to detect differences in fork rates in the range of interest (0.3 to 1.0 pdminute) (Fig. 5). Similar variation can also be seen in autoradiographicexperiments. For example, Probst el af. (1980) reported mean fork movement rates in synchronous cells ranging from 0.75 to 0.91 pdminute. The standard deviations for these values were 0.23 and 0.44 pdminute, or 31 and 48%, respectively. From these values and from an examination of reported data, the 30% or greater variation in fork movement rates again appears regardless of the technique used or cell lines examined.

13

DNA REPLICATION FORK MOVEMENT RATES TABLE I1 (Continued) Rate Cell type Mouse L cells Mouse L929 Mouse L929 Mouse L cells Mouse L929 Mouse L cells Mouse lymphoma L5 178Y Mouse L5178Y Mouse L5178Y Mouse S49 lymphosarcoma Primary mouse thymocytes Mouse Ehrlich Mouse Friend erythroleukemia Mouse 3T3 Mouse 3T3 (SV40-transformed) ordnii) Rat (0. Rabbit CBL Mole MI5 Shrew S3 Shrew S3/4 Monkey CV-I Monkey CV-I

Technique

( pndminute)

Reference

Equilibrium density gradient Autoradiography Autoradiography Autoradiography Au toradiography Au toradiography Alkaline sucrose gradient Autoradiography Equilibrium density gradient BUdR-3 13 nm photolysis Alkaline sucrose gradient Autoradiography

1.6 0.830 0.7O 0.55 0.53 0.41 0.7 0.54 0.04 I .o 0.6

Painter and Schaefer (1969) Hand and Tamm (1972) Hand and Tamm (1973) Jasny er a / . (1980) Hand and Tamm (1977) Stimac er a / . (1977) Lehmann and Ormerod (1 970) Watanabe (1974) Okada (1968) Kapp and Painter (1981) Lanotte er al. (1977) Probst et al. (1980)

Autoradiography BUdR-313 nm photolysis

0.43 0.2

Kundahl et al. (1981) Kapp et al. (1979a)

BUdR-313 nm photolysis Autoradiography Equilibrium density gradient Equilibrium density gradient Autoradiography Autoradiography Autoradiography Equilibrium density gradient

0.33

Kapp et a!. (1 979a) Hori and Lark (1974) Painter and Schaefer (1969) Ockey and Saffhill (1976) Ockey and Saffhill (1976) Ockey (1978) Richter and Hand (1979b) Richter and Hand (1979b)

1.1

1 .o 1 .o

0.95 0.87 1.13 0.56 0.37a

aThese papers did not present a single average fork movement rate as shown here; an average was calculated from the published data.

The slowest value reported for human cells is 0.1 pdminute (Yurov, 1980), and the highest value is 1.6 pdminute (Gautschi and Kern, 1973). Yurov (1980) reported that the average rate was 0.5 to 0.6 pdminute, in excellent agreement with other investigators, but that fiber autoradiographs from a preparation originating from a single cell displayed a range of values from 0.1 to 1.2 pdminute, indicating that the cellular average is made up largely of differing individual rates. There are no clear trends in fork movement rates that can be used to subclassify the human cells. Normal diploid fibroblasts yield a mean of 0.58 ? 0.15 pdminute; transformed cells (such as HeLa) have a mean of 0.66 ? 0.30 p d minute, and these two means are not significantly different. When transformed and normal cells are measured simultaneously, the same conclusion can also be

14


1,

r

IW

z

3s:

s

B 3t

V

DIPLOID HUMAN

RODENT

CELL TYPE FIG.5 . The mean rates of displacement of DNA replication forks are shown for each of the cell lineb used. The vertical bars represent the standard deviation for each mean. Data for each cell type were obtained from a minimum of six separate determinations. The horizontal solid line and dashed lines through the points represent the population mean and its standard deviation (0.53 2 0.08 pm/ minute). Reprinted from Kapp and Painter (1981) with permission of ElseviedNorth-Holland Biomedical Press.

drawn (Painter and Schaefer, 1969; Kapp and Painter, 1978; Ockey, 1979). Only Heenen and Galand (1980) have reported that transformed cells have slower rates than normal cells. However, their reported values (0.3 to 0.4 pdminute for normal and 0.22 to 0.26 pdminute for transformed cells) are different by approximately 30%, which again is in the range of variation seen for most fork movement rates.


15

Cells from patients with the genetic disease, Bloom’s syndrome, are apparently the only human cells that have a slower than normal fork movement rate. This was first reported by Hand and German (1975), using fiber autoradiography, who found that Bloom’s syndrome fibroblasts and lymphocytes both displayed low fork movement rates when compared to normal fibroblasts and lymphocytes. (Bloom’s lymphocytes’ fork rates were 59% of normal and Bloom’s fibroblasts were 78% of normal.) Giannelli er al. (1977), using alkaline sucrose gradient analysis of replicating DNA, confirmed these results but did not measure fork movement rates directly. However, Ockey (1979), using fiber autoradiography, found that Bloom’s syndrome cells had almost normal fork movement rates except when measured within 24 hours of subculturing, when the Bloom’s syndrome cells’ rate was significantly slower. Most recently, Kapp (1982), using BUdR-313 nm photolysis, found that fork movement rates in Bloom’s syndrome lines were 55-65% of those in normal controls, and found no indication of the subculturing effect mentioned by Ockey (1979). Thus, the bulk of evidence suggests that Bloom’s syndrome cells have DNA replication fork movement rates that are slower than normal. B. FORKRATESIN NONHUMAN MAMMALIAN CELLS The individual values for nonhuman mammalian cells (Table 11) range from 0.04 pdminute for a mouse line (Okada, 1968) to 2.5 pdminute in Chinese hamster ovary cells (Huberman and Riggs, 1968). As with the reports for human cell lines, the average values are in a relatively narrow range, despite the variety of cell types examined and the range of techniques used. Average values (calculated from Table 11) for the various species measured are mouse, 0.71 0.36 pdminute; Chinese hamster, 0.79 & 0.31 pdminute; moukey, 0.47 0.13 pdminute; shrew, 1.13 pm/minute; and rabbit, 1 .O pm/ minute. These values suggest that, in culture, rodent cells have fork movement rates higher than human cells.

* *

C. FORKRATESIN SYNCHRONIZED CELLS Early observations in synchronized cells indicated that the rate of DNA synthesis was not uniform throughout S phase. This could be explained either by a variation in the number of operating replicons or by a variation in the fork movement rate in individual replicons at different times in S phase. In the reports published to date, the number of operating replicons appears to be the main factor regulating the rate of DNA synthesis. Variation in fork movement rates during S phase does not follow any universal pattern and appears to vary among the different cell lines used, regardless of the technique used (Table 111). In the earliest report, Huberman and Riggs (1968), using CHO cells partially

TABLE 111 FORKMOVEMENT RATESIN SYNCHRON~ZED CELLS Cell type

Synchrony technique

Fork movement technique

CHO

12 hour FUdR

Autoradiography

HeLa S3

Mitotic selection

Equilibrium density gradient

HeLa S3

Equilibrium density gradient

CHO

Thymidine double block Mitotic selection

Shrew

Mitotic selection

Autoradiography

Normal human fibroblasts

Mitotic selection

BUdR-3 13 nm photolysis

CHO CHO HeLa

Mitotic selection Mitotic selection Mitotic selection

BUdR-313 nm photolysis Equilibrium density gradient BUdR-313 nm photolysis

Monkey CV-I Monkey CV-1 Mouse ascites

Serum-induced S phase Sorting of normal population

Fiber autoradiography Equilibrium density gradient Autoradiography

L5 178Y

Thymidine and colcemid block

Fiber autoradiography

Autoradiography

Results No change in rates in early, middle or late S (52.5 pdminute) 2- to 3-fold increase from early to late S (O.&l. 1 pdminute) 2- to 3-fold increase from early to late S (0.4-1.1 pdminute) 2- to 3-fold increase from early to late S (0.2 or 0.3-0.6 or 0.7 pdminute) No change during S phase ( I .1-1.2 p d minute) Minimum rate in middle S (0.55 pdminute); maximum rate early and late (0.8 p d minute) Constant rate (- 1 pdminute) Constant rate (- 1 pdminute) 2- to 3-fold increase from early to late S (0.8-2.5 pdminute) Constant throughout S (0.34 pdminute) Constant throughout S (0.15 p n h i n u t e ) In vivo: constant (1.1-1.2 pdminute); in vifrot slight increase in late S (0.75-0.9 pdminute) 3-fold increase from early to late S (0.32-1 .O pdminute)

Reference Huberman and Riggs (1968) Painter and Schaefer (1971) Painter and Schaefer (1971) Housman and Huberman (1975) Ockey (1978) Kapp and Painter (1979)

Kapp and Painter (1979) Kapp and Painter (1979) Kapp and Painter (1979) Richter and Hand (1979a) Richter and Hand (1979a) Probst ef al. (1980)

S. Sawada and T. Enomoto (personal communication)


17

synchronized with FUdR, found that fork movement rates varied from 0.5 to 2.5 prrdminute at each point in S phase where this was measured. In a later study, Painter and Schaefer (197 I ) , using an equilibrium density gradient technique with HeLa cells, found a 2- to 3-fold increase from early to late S phase. This was found whether the cells were synchronized by mitotic selection or a double thymidine block. Housman and Huberman (1975) then reported a 3-fold change in fork movement rates in mitotically selected CHO cells as measured by fiber autoradiography. Lowest rates were at the beginning of S phase, with a rapid increase in rate during the first 1 or 2 hours and a fairly constant rate for the remainder of S phase. In contrast to these results, however, other workers have reported constant fork movement rates in S phase. Ockey (1978), using autoradiography with mitotically selected shrew cells, found a constant rate of fork movement. Kapp and Painter (1979), using mitotically selected CHO cells, reported a constant rate of fork movement throughout the S phase, including the first hour of S. Kapp and Painter used two techniques-the equilibrium density gradient method of Painter and Schaefer (1971) and the BUdR-313 nm photolysis method of Povirk and Painter .(1 9 7 6 t a n d obtained identical results with both methods. However, using the BUdR-3 13 nm photolysis method with HeLa cells, a 3-fold increase in fork movement rates from early to late S phase was found, thus confirming the earlier report by Painter and Schaefer (1971) for the same cells using a different technique. Constant fork rates through S phase were also reported by Richter and Hand (1979a) with serum-induced synchrony in monkey CV-1 cells. This result was obtained using both fiber autoradiography and the equilibrium density gradient method of Painter and Schaefer (1971). Probst et al. (1980), using autoradiography, also found a constant fork movement rate through S in mouse ascites cells. They examined the cell cycle by sorting cells according to DNA content (and thus position in S phase). Using this approach, they tried to avoid all artifacts that might have resulted from an induced synchrony method. They also found a constant rate regardless of whether the cell population was labeled in vivo or in vitro. Recently, however, S. Sawada and T. Enomoto (personal communication), using fiber autoradiography, found a 3-fold increase in fork movement rates as mouse L5178Y cells progressed through S phase. Fork movement rates in synchronized cells therefore seem to follow one of two patterns, a constant rate throughout S phase or an increasing rate through S phase. However, to say that there are two patterns is probably an oversimplification. For example, HeLa cells (Painter and Schaefer, 1971; Kapp and Painter, 1979) and L5178Y cells (S. Sawada and T. Enomoto, personal communication) seem to have monotonically increasing rates of fork movement in S . However, CHO cells, when they do exhibit an increasing fork movement rate (Housman and Huberman, 1975), show most of the increase in early S phase. More uniform

18


findings may result if more investigators use similar patterns of sampling. Different cell lines may behave differently depending on their history, origin, and culture conditions, and this may partially explain the different results found for CHO cells by two different groups of investigators (Housman and Huberman, 1975; Kapp and Painter, 1979), and the variation in results for different lines of mouse cells (Probst et al., 1980; S . Sawada and T. Enomoto, personal communication). Regardless of whether the fork movement rates vary by a factor of 3 during S phase or are constant throughout, the variation in overall DNA synthesis during S phase is much larger than 3-fold and therefore must be primarily caused by the different numbers of operating replicons (Painter and Schaefer, 1971; Kapp et al., 1979b).

FORKMOVEMENT RATES D. CHEMICALS AFFECTING Although there have been a number of studies of the effects of drugs and other compounds on DNA synthesis, very few have explicitly examined the effects of these agents on DNA fork movement rates; the results of these studies are presented in Table IV. Hydroxyurea inhibits DNA synthesis by inhibiting ribonucleotide reductase and reducing the production of precursors of all four DNA bases (Young and Hodas, 1964). Inhibition of DNA synthesis by hydroxyurea depends on the concentration of the drug, but in the range 10W5 to 10 - 4 M , DNA fork movement rates are about 30 to 40% of those in untreated controls (Painter, 1980; Kapp and Painter, 1981; Gautschi er al., 1973). Inhibitors of protein synthesis also depress fork movement rates. Cycloheximide (0.1 to 1.0 kg/ml) slows fork movement rates to 25-35% of normal (Gautschi et al., 1973; Gautschi and Kern, 1973), and Stimac et al. (1977) M . Puromycin at 200 kg/ml reported rates of 55% of control at 1.8 X (Hand and Tamm, 1972) also inhibits fork movement rates to about 55% of control. Gautschi and his co-workers concluded that the depression in DNA synthesis in the presence of protein synthesis inhibition is completely accounted for by the depression in fork movement rates (Gautschi et al., 1973; Gautschi and Kern, 1973). However, Hand and Tamm (1972, 1973) and Hand (1975b) found that there must also be some inhibition of initiation in the presence of protein synthesis inhibition. Gautschi (1974) examined the effects of puromycin on DNA synthesis and found that its action was very similar to that of cycloheximide and that all of the depression of DNA synthesis could be accounted for by the slowing of fork movement rates. He concluded that fork movement was probably mediated through protein synthesis. Similar conclusions were also reached by Planck and Mueller (1977a). Stimac et al. (1977) examined the effects of a number of protein synthesis inhibitors on DNA synthesis using fiber autoradiography. They found that at

TABLE IV CHEMICALS AFFECTINGFORKMOVEMENT RATES

Cell type HeLa HeLa CHO Mouse CHO HeLa L929 HeLa HeLa HeLa HeLa Mouse Mouse CHO CHO HeLa HeLa Mouse Mouse

L

L929 L929

L929 L929

Chemical Cycloheximide (0.1 pg/ml) Cycloheximide ( I .O pg/ml) Cycloheximide (10 pglml) M) Cycloheximide (1.8 X Cycloheximide (1.8 X 10 - 4 M ) Cycloheximide (1.8 X 10-4M) M) Cycloheximide (1.8 x Hydroxyurea (10 - 5 M ) Hydroxyurea M) Hydroxyurea ( 10- 5 M) Hydroxyurea (0.1 mM) Puromycin (20 pglml) Puromycin (200 pg/ml) Puromycin (20 pg/ml) Puromycin (4.2 X M) 2,4-Dinitrophenol 2,4-Dinitrophenol Dichlorobenzimidazole riboside (60 pglml) Dichlorobenzimidazole riboside (90 pg/ml)

Technique Equilibrium density gradient Equilibrium density gradient Equilibrium density gradient Autoradiography Autoradiography Autoradiography Autoradiography Equilibrium density gradient Equilibrium density gradient BUdR-313 nm photolysis BUdR-313 nm photolysis Autoradiography Autoradiography Autoradiography Autoradiography Equilibrium density gradient Autoradiography Autoradiography Autoradiography

Fork movement rate (9% of control)

36 23 25 54 55

37 25 47 38 40 30 80 55 100

50 100

70 82 75

Reference Gautschi er al. (1973) Gautschi er al. (1973) Gautschi and Kern (1973) Stimac et al. (1977) Stimac et al. (1977) Stimac et al. (1977) Hand and Tamm (1973) Gautschi er a/. (1973) Gautschi er al. (1973) Kapp and Painter (1981) Painter ( 1980) Hand (1975b) Hand (1975b) Hori and Lark (1973) Hand and Tamm ( 1 973) Gautschi er al. (1973) Stimac er al. (1977) Hand and Tamm (1977) Hand and Tamm (1977)

20


early times after inhibition of protein synthesis, the decrease in fork movement rate could account for the decrease in overall DNA synthesis, and after later times the decrease in fork movement rates may still have been sufficient to explain the decrease in overall DNA synthesis during severe inhibition. However, if DNA synthesis was only moderately inhibited, the decrease in fork movement rate was not sufficient to account for the decrease in DNA synthesis, and under these conditions it appeared that initiation of new replicons must also have been inhibited. In marked contrast to the previously mentioned reports, Hori and Lark (1973) reported that puromycin had no effect on fork movement rates and therefore must act solely by blocking initiation of replicons. However, this is the only report that failed 'to observe an effect on fork movement rates by inhibition of protein synthesis. In reviewing the work that has been done, it appears that inhibition of protein synthesis rapidly inhibits DNA chain elongation and that the effect on initiation probably depends on the concentration of inhibitor and the length of treatment. Other inhibitors of protein synthesis, emetine and pactamycin, gave results similar to those described for puromycin (Stimac et al., 1977). Stimac er al. also claimed the same effect for 2,4-dinitrophenol, a general metabolic inhibitor. However, this was in contrast to Gautschi et al. (1973), who reported no effect of dinitrophenol on fork movement rates. The reported data of Stimac et al. (1977) do not completely support their contention that the effects of 2,4-dinitrophenol on overall DNA synthesis can be explained solely by its effect on rate of fork movement. Another agent examined in detail for its effects on fork movement rates is dichlorobenzimidazole riboside, which inhibits RNA synthesis. At concentrations that inhibit most heterogeneous nuclear RNA (hnRNA) synthesis, DNA synthesis is inhibited by 20 to 25% and this can be accounted for entirely by a reduced fork movement rate (Hand and Tamm, 1977). Methyl methanesulfonate was also reported to inhibit fork movement rates to 15 to 50% of normal, depending on the concentration used (Dahle et al., 1978). It was concluded that this drug produces lesions that can inhibit fork movement rates and, since the depression in fork movement rates does not account for the total depression of DNA synthesis, may also inhibit replicon initiation. However, there are other interpretations of the effect of this drug (Painter, 1977). Ultraviolet light strongly inhibits DNA synthesis in mammalian cells. Most reports have concluded that ultraviolet light exposure results in a lesion that blocks fork movement, but fork movement rates between lesions appear to be unaffected (Povirk and Painter, 1976; Edenberg, 1976; Dahle er al., 1980). Doniger (1978), however, reported that fork movement rates were depressed by ultraviolet light at 10 J/m2 but not at 5 J/m2. X Rays also affect the rate of DNA synthesis. However, low to moderate


21

doses of X rays do not affect fork movement rates (Kapp and Painter, 1978), but primarily inhibit initiation of replicons (Watanabe, 1974; Makino and Okada, 1975; Dahle et al., 1979; Laughlin and Taylor, 1980). In summary, agents that directly or indirectly reduce the supply of DNA precursors slow the rate of fork displacement in mammalian cells. DNA-damaging agents, in contrast, probably do not affect the rate of fork displacement but inhibit DNA synthesis by blocking initiation of replicons or by completely blocking fork movement. E. FORKRATESIN MUTANTS AND in Vitro Mutants have been very important for DNA replication studies in bacteria, and many investigators have tried to obtain mutant mammalian cell lines with alterations in DNA synthesis pathways. Zannis-Hadjopoulos er al. (1980) studied DNA replication in a purine-auxotrophic mutant cell line derived from Chinese hamster V79 cells. When purines were removed from the medium, DNA synthesis slowed to approximately 35% of the rate in cells growing in purinesupplemented medium. By observing the effects of starving and refeeding the cells with purines, it was concluded that purine deprivation had a direct effect on fork movement. In addition, there appeared to be no effect on initiation of replicon operation at the level of individual replicons. A temperature-sensitive mutant, tsBN-2 of BHK-2 I cells, exhibits a depression of DNA synthesis at a nonpermissive temperature of 39.5"C. Examination of DNA synthesis in these cells (Eilen et al., 1980) indicated that the fork movement rate in the wild-type cells at 39.5"C was 0.74 to 0.86 pdminute, whereas the mutant had a fork movement rate of approximately 1 pdminute. Both wild-type and mutant cells have lower rates of fork movement at 33.5"C. Autoradiography indicated that the average distance between replication origins in the mutant increased from approximately 80 to 98 pm at the nonpermissive temperature of 39.5"C, whereas there was little effect on interorigin distance in wild-type cells. Thus, because the depression in DNA synthesis in the mutant BN-2 cells cannot be explained by slower fork movement, it appears to be due solely to fewer initiations at the nonpermissive temperature. Hyodo and Suzuki (1982) isolated a temperature-sensitivemutant from mouse FM3A cells that was found to have defective DNA replication at a nonpermissive temperature of 39.5"C. Cell cycle analysis indicated that most of the cells were arrested in S phase. DNA fiber autoradiography revealed that DNA fork movement rates were approximately 1.04 pdminute at the permissive temperature and 0.47 pdminute at the nonpermissive temperature. Thus, the reduced rate of DNA replication in these cells did result from a slow fork movement rate. Although many mutant cell lines have been isolated in the past few years, and some of them may represent mutations that affect DNA synthesis, most of them

22


have yet to be characterized or studied with respect to specific DNA synthesis parameters such as fork movement rate. Eventually, if such mutants are found and characterized, they should be of value as model systems for examining fork movement rates in mammalian cells. A number of in vitro systems have also been employed to examine DNA synthesis in mammalian cells, but very few workers have examined fork movement rates directly in these systems. Most investigators have reported that fork movement rates are retarded in such systems and range from as low as 10 to as high as 60% of the rate in vivo (Planck and Mueller, 1977b; Fraser and Huberman, 1977; Gautschi et al., 1977). In all cases, the depression in DNA synthesis rate was caused by a lowered fork movement rate (Planck and Mueller, 1977b; Fraser and Huberman, 1977; Gautschi ef a!., 1977; Hand and Gautschi, 1979; Griffiths and Carpenter, 1980) as well as premature chain termination (Hand and Gautschi, 1979) and lack of new initiations (Planck and Mueller, 1977b). These in vitru systems are incapable of maintaining DNA synthesis for more than a short time after their preparation (Hand and Gautschi, 1979; Fraser and Huberman, 1977). Such systems will become more useful when they are better characterized and standardized and are capable of supporting DNA synthesis at higher levels and for longer periods of time.

IV. Concluding Remarks DNA fork movement rates in most mammalian cells appear to fall into a range of approximately 0.2 to I pdminute. The average value for human cell lines is 0.60 k 0.21 pdminute, whereas the average value for nonhuman mammalian cells is 0.71 5 0.36 pdminute. In addition, most of the rates reported for human cell lines are in the range of approximately 0.4 to 0.7 pdminute. In contrast, bacterial fork movement rates are reported to be as high as 15 p d minute (Cairns, 1963) and amphibian rates are reported to be as low as 0.02 p d minute (Hyodo and Flickinger, 1973). There appears to be little variation among the various human cell lines examined. The rates are similar in diploid cells, cells from donors with genetic diseases, and in transformed cells. The only human cell type that appears to have a low fork movement rate is Bloom’s syndrome. Although a number of techniques have been used to measure fork movement rates in mammalian cells, most yield values in the same range and thus give comparable results. The variations reported for fork movement rates are probably due mainly to actual variations between the cells examined and, to a smaller extent, to differences caused by various experimental and cell culture conditions. Individual fork movement rates can vary by almost 10-fold within a single cell type. However, the average fork movement rate can either increase or remain


23

constant throughout S phase, depending on the cell type examined. Because even the highest variation in fork movement rates observed in synchronized cells cannot account for the variation in cellular DNA synthesis rates, the main factor affecting the overall cellular rate of DNA synthesis must be the number of replicons operating at one time rather than the replication fork movement rate. There have been few studies examining the effect of chemicals or other agents on actual fork movement rate. In addition, there are still no clearly defined mutants in which an identifiable alteration or mutation can be correlated with an altered fork movement rate. In vitro systems in mammalian cells are still incapable of supporting a high level of DNA synthesis for more than a short period of time. Future work to examine the regulation and role of fork movement rates in mammalian cells will probably depend on developing new or improved methods, e.g., defined mutants, better in v i m systems, and new probes or techniques to measure fork movement rates.

ACKNOWLEDGMENT This work was supported by the U.S.Department of Energy.

REFERENCES Cairns, J. (1963). J. Mol. Biol. 6, 208-213. Cairns, J . (1966). J. Mol. Biol. 15, 372-373. Dahle, D. B., Griffiths, T. D., and Carpenter, J. G. (1978). Mol. Pharrnacol. 14, 278-289. Dahle, D., Griffiths, T. D., and Carpenter, J. G. (1979). Radiat. Res. 78, 542-549. Dahle, D., Griffiths, T. D., and Carpenter, J. G. (1980). Photochem. Phorobiol. 32, 157-165. DePamphilis, M . L., and Wassarman, P. M. (1980). Annu. Rev. Biochem. 49, 627-666. Doniger, J. (1978). J. Mol. Biol. 120, 433-446. Edenberg, H. J . (1976). Biophys. J . 16, 849-860. Edenberg, H. J . , and Huberman, J. A. (1975). Annu. Rev. Genet. 9, 245-284. Eilen, E., Hand, R., and Basilico, C. (1980). J . Cell. Physiol. 105, 259-266. Fraser, J . M . K., and Huberman, J. A. (1977). J. Mol. Biol. 117, 249-272. Gautschi, J . R. (1974). J. Mol. Biol. 84, 223-229. Gautschi, J. R., and Kern, R . M. (1973). Exp. Cell Res. 80, 15-26. Gautschi, J. R., Kern, R. M . , and Painter, R. B. (1973). J. Mol. Biol. 80, 393-403. Gautschi, J . R., Burkhalter, M., and Reinhard, P. (1977). Biochim. Biophys. Acta 474, 512-523. Giannelli, F., Benson, P. F., Pawsey, S. A,, and Polani, P. E. (1977). Narure (London) 265, 466-469. Griffiths, T. D., and Carpenter, J. G. (1980). Exp. Cell Res. 130, 470-473. Hand, R. (1975a). J. Cell Biol. 64, 89-97. Hand, R. (1975b). J . Cell Biol. 67, 761-773. Hand, R. (1977). Hum. Genet. 37, 55-64.

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Hand, R. (1978). Cell 15, 317-325. Hand, R. (1979). In “Cell Biology; A Comprehensive Treatise” (D. M. Prescott and L. Goldstein, eds.), Vol. 2, pp. 389-437. Academic Press, New York. Hand, R., and Gautschi, I. R. (1979). J. Cell B i d . 82, 485-493. Hand, R., and German, J . (1975). Proc. Narl. Acad. Sci. U.S.A. 72, 758-762. Hand, R., and German, I. (1977). Hum. Genet. 38, 297-306. Hand, R., and Tamm, 1. (1972). Virology 47, 331-337. Hand, R., and Tamm, I . ( 1973). J . Cell B i d . 58, 4 10-4 18. Hand, R., and Tamm, I. (1977). Exp. Cell Res. 107, 343-354. Heenen, M., and Galand, P. (1980). Nature (London) 285, 265-267. Hori, T., and Lark, K. G. (1973). J . Mol. B i d . 77, 391-404. Hori, T., and Lark, K. G. (1974). J. Mol. Biol. 88, 221-232. Housman, D., and Huberman, J . A. (1975). J. Mol. Eiol. 94, 173-181. Huberman, J . A., and Riggs, A. D. (1968). J . Mol. Biol. 32, 327-341. Hyodo, M., and Flickinger, R. A. (1973). Eiochim. Biophys. Actu 299, 24-33. Hyodo, M., and Suzuki, K. (1982). Exp. Cell Res. 137, 31-38. Jasny, B. R.. Cohen, J . E., and Tamm, I. (1980). Chromosoma 79, 207-214. Kapp, L. N. (1982). Biochim. Biophys. Acta 696, 226-227. Kapp, L. N., and Painter, R. B. (1978). Biophys. J . 24, 739-748. Kapp, L. N., and Painter, R. B. (1979). Eiochim. Biophys. Acta 562, 222-230. Kapp, L. N., and Painter, R. B. (1981). Eiochim. Biophys. Acta 656, 36-39. Kapp, L. N., Park, S. D., and Cleaver, J. E. (1979a). Exp. Cell Res. 123, 375-378. Kapp, L. N., Millis, A. J. T.. and Pious, D. A. (1979b) In V i m 15, 669-672. Kundahl, E., Richman, R., and Flickinger, R. A. (1981). J. Cell. Physiol. 108, 291-298. Kurek, M. P., and Taylor, J. H. (1977). Exp. Cell Res. 104, 7-14. Lanotte, M., Moerman, C., and Panijel, J . (1977). Exp. Cell Res. 109, 191-200. Lark, K. G., Consigli, R., and Toliver, A. (1971). J. Mol. Biol. 58, 873-875. Laughlin, T. J., and Taylor, J. H. (1979). Chromosoma 75, 19-35. Laughlin, T. J., and Taylor, J. H. (1980). Radiut. Res. 83, 205-209. Lehmann, A. R., and Ormerod, M. G. (1970). Biochim. Biophys. Actu 204, 128-143. Makino, F., and Okada, S. (1975). Radiat. Res. 62, 37-51. Martin, R. G., and Oppenheim, A. (1977). Cell 11, 859-869. Ockey, C. H. (1978). Exp. Cell Res. 114, 446-45 I . Ockey, C. H. (1979). J. CellSci. 40, 125-144. Ockey, C. H., and Saffhill, R. (1976). Exp. Cell Res. 103, 361-373. Okada, S. (1968). Biophys. J . 8, 650-664. Painter, R. B. (1976). I n “Handbook of Genetics” (R. C. King, ed.), Vol. 5, pp. 169-186. Plenum, New York. Painter, R. B. (1977). M u m . Res. 42, 299-304. Painter, R. B. (1980). J . Mol. Biol. 143, 289-301. Painter, R. B., and Schaefer, A. W. (1969). J . Mol. B i d . 45, 467-479. Painter, R. B., and Schaefer, A. W. (1971). J. Mol. B i d . 58, 289-295. Painter, R. B., Jermany, D. A., and Rasmussen, R. E. (1966). J. Mol. Biol. 17, 47-56. Petes, T. D., Farber, R. A., Tarrant, G. M., and Holliday, R. (1974). Nature (London) 251, 434-436. Planck, S. R., and Mueller, G. C. (1977a). Biochemistry 16, 1808-1813. Planck, S. R., and Mueller, G. C. (1977b). Biochemistry 16, 2778-2782. Povirk, L. F., and Painter, R. B. (1976). Biophys. J. 16, 883-889. Probst, H., Blutters, R., and Fielitz, J . (1980). Exp. Cell Res. 130, 1-13. Richter, A,, and Hand, R. (1979a). Exp. Cell Res. 121, 363-371.


25

Richter, A., and Hand, R. (1979b). J . Cell. Physiol. 101, 407-418. Roti-Roti, J. L., and Painter, R. B. (1977). J. Theor. B i d . 64, 681-696. Sheinin, R., and Humbert, J. (1978). Annu. Rev. Biochem. 47, 277-316. Stimac, E., Housman, D., and Huberman, J. A. (1977). J. Mol. B i d . 115, 485-51 I Taylor, J. H. (1968). J . Mol. B i d . 31, 579-594. Taylor, J . H. (1977). Chromosoma 62, 291-300. Taylor, J. H., and Hozier, J. C. (1976). Chromosoma 57, 341-350. Waiters, R. A,, Tobey, R. A,, and Hildebrand, C. E. (1976). Biochim. Biophys. Actu 447, 36-44. Watanabe, 1. (1974). Radiat. Res. 58, 541-556. Wickremasinghe, R. G., and Hoffbrand, A. V. (1979). Biochim. Biophys. Acta 563, 46-58. Young, C. W., and Hodas, S. (1964). Science 146, 1172-1174. Yurov, Y. B. (1977). Cell Differ. 6, 95-104. Yurov, Y. B. (1978). Hum. Genet. 43, 47-52. Yurov, Y. B. (1979a). Exp. Cell Res. 123, 369-374. Yurov, Y. B. (1979b). Chromosoma 74, 347-353. Yurov, Y. B. (1980). J . Mol. Eiol. 136, 339-342. Yurov, Y. B., and Liapunova, N. A. (1977). Chromosoma 60, 253-267. Zannis-Hadjopoulos, M., Taylor, M. W., and Hand, R. (1980). J. CellEiol. 85, 777-785.


INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 80

Interaction of Viruses with Cell Surface Receptors MARCTARDIEU,~ ROCHELLE L. EPSTEIN,AND HOWARDL. WEINER Department of Neuroscience, Children's Hospital Medical Center, and Department of Medicine, Neurology and Infectious Disease Divisions, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachussets I. Definition of Viral Receptor Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Biological Characteristics of Viral Attachment to Cells . . . . . . . . . . A. Techniques Used to Study Viral Attachment B. Mathematical Analysis of Viral Binding . . . . . . . . . . . . . . . . . . 111. Membrane Components Which Interact with Viruses. . . . . . . . . . . . A. Density and Affinity of Viral Receptor . . . ....... .. omponents B. Relationship of Viral Receptors to Other S C. Definition of Cellular Receptor Units by Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Specificity of Cellular Receptor Sites for Viruses. . . E. In Vitro Manipulation of Cell Receptor Sites. . . . . . . F. Age Dependency of Viral Permissiveness . . . . . . . . . . . . . . . . . G. Genetic Control of Cell Receptor Site Expression. . . . . . . . . . . IV. Viral Components Which Recognize Cellular Receptors . . . . . . . . . A. Picomaviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Adenoviruses . . . . . . . . . . . . . . . ................ C. Reoviruses . . . . . . . . . . . . . . . . . . . . . . . . .

E. Retroviruses . . . ............... F. Coronaviruses. , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Virus-Receptor Interactions and Pathogenicity B. Role of Virus Attachment Proteins in Pathogenicity C. Induction of Cell-Specific Autoimmunity following Viral VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References

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

27 30 30 35 37 37 38 39 40 45 48 48 49 49 50 50 51 52 52 52 53 54 55 56 57

I. Definition of Viral Receptor Sites The definition of a receptor ultimately depends upon the structural and functional identification of a site that is specifically recognized by a ligand. The most rigorous characterizations of receptor-ligand interactions have been derived 'Present address: Inserm U56, HGpital de Bicetre, 94270 Bicetre, France. 27 Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-364480-0

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MARC TARDIEU ET AL.

from binding studies of radiolabeled ligands in neuropharmacology and endocrinology. It has been more difficult to study the interactions of viral particles with the cell surface in this rigorous a fashion. Viral particles are several orders of magnitude larger than conventional ligands, making it impossible to perform many of the manipulations that are routinely done with hormones, for example. In addition, viruses contain repeating subunits and many copies of the viral attachment protein are present on the virion surface. Thus a single virion may interact with many receptor sites on the cell surface. Nonetheless, most of the principles of receptor-ligand interactions established for other ligands apply to viruses as well and this establishes a framework for the definition of viral receptors. At the simplest level, a viral receptor is the structure on the membrane surface of a cell to which virus binds prior to entering the cell. Often the presence or absence of a viral receptor determines whether or not the cell can be infected by virus. It is also possible that there are structures on the cell surface to which a virus binds but which do not serve as conduits for viral entry into the cell. Viral binding to such sites could have other important biologic consequences such as affecting cell function by triggering surface structures which affect cellular metabolism or being improtant for the generation of an immune response against the virus. In the broadest sense, then, a viral receptor is a structure on the cell surface to which a virus binds, the binding of which is of biological importance and can be measured in a biologically relevant way. The definition of viral recognition sites as “receptors” involves three major criteria which are derived from models of ligand-receptor interactions (Bennet, 1978). These include saturability, specificity, and competition. SaturnbiliQ. If virus interacts with the cell surface at discrete sites along the membrane, only a finite number of sites will be available for viral binding and high concentrations of virus should be able to fully occupy or “saturate” them. It should therefore be possible to increase the concentration of particles presented to the cell surface until no further binding results. This can be shown experimentally by determining viral binding as a function of increasing viral concentration. Saturation is demonstrated if a plot of the result is a hyperbolic curve (see Fig. IA). Specificity. Specificity is first demonstrated according to “biologic parameters.’’ In other words, viral binding is observed only to cells that the virus infects or to cells where viral binding induces some other biologically measurable response. The second measure of specificity relates to the binding assay itself. Even when virus binds to a biologically relevant cell, a certain proportion of viral binding is nonspecific and unrelated to specific viral receptors. For example, electrostatic forces result in some of the nonspecific adherence of particles to the cell surface. Nonspecific binding is contained in any binding curve but the nonspecific component is usually not saturable and therefore it is a

VIRUSES AND CELL SURFACE RECEPTORS

29

12 V-ImL7

,?‘?KF

Total Blnding

BOUND isaturable CPM x fO ’J

FIG. 1 . (A) Binding of 125I-labeled reovirus type I to L-cell fibroblasts. Each point represents the arithmetic mean of three separate determinations of uptake to 250,000 cellsisample equilibrated at 25°C for 60 minutes. The linear nonspecific binding was determined by mixing each concentration of labeled virus with an equal volume of high titer unlabeled virus containing 2.0 X 1013particlesiml. The saturable binding curve results from subtraction of the linear component from the total binding curve. The maximum uptake is the uptake value at saturation. and Kd is the virus concentration at the half saturation point. (9)Scatchard plot of ‘2SI-labeled reovirus bound to L cells. After subtracting the linear component, boundifree was calculated by dividing the corrected cpm by the viral concentration expressed in particles divided by Avogadro’s number (to result in a molar expression). Linear regression analysis of the data provides the slope to estimate K d .

linear function which can be measured, and subsequently subtracted from total binding to reveal “specific” binding (Fig. 1A). Competition. It should be possible to competitively inhibit specific binding using a second ligand which is known to bind to the same receptor. In neuropharmacology, for example, specific binding of acetylcholine to the muscarinic acetylcholine receptor is defined as that component of total binding which can be blocked by atropine. In viral systems, specific blockers of this type are usually unavailable and the only certain method for competition is to block the binding of

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MARC TARDIEU ET AL.

radiolabeled virus with unlabeled virus or, if possible, the viral attachment protein. A variety of nomenclatures have been used to describe viral-receptor interactions, and depending on the context of the discussion several abbreviations will be used in this article. The protein by which the virus attaches to cells is the viral attachment protein (VAP). The “receptor” on the surface of the cell to which the virus binds is the cellular receptor site (CRS). Finally, it has been postulated that the CRS may be composed of multiple units and these units, which may bind a single VAP, are termed cellular receptor units (CRU) (Lonberg-Holm, 198 1).

11. Biological Characteristics of Viral Attachment to Cells

A. TECHNIQUES USED TO STUDYVIRALATTACHMENT A variety of approaches have been used to study the interaction of viral particles with cell surface receptors or reception sites. As described below, each approach has advantages and limitations. A rigorous study of viral-receptor interactions requires the use of more than one technique since different approaches provide complementary information about viral binding. 1. Infectivity Assays

These assays measure the number of infectious viral particles that either remain in the medium following viral binding to the cells or that have attached to cells. Attached virus may be detected by measuring infected cells or “infectious centers,” or by dissociation of receptor bound infectious virus, providing that receptor-mediated or cell-mediated modification (‘‘eclipse”) of the virus particles can be prevented or accounted for. The advantage of infectivity assays is that binding of very small amounts of virus can be detected and viral replication, a biologic function, is being measured. Infectivity assays have the following limitations: (1) Only binding of infectious viral particles can be measured. Most viral preparations have particle to plaque-forming unit (PFU) ratios of 10 to 100 and it is likely that noninfectious virus also binds to cellular receptor sites. (2) The measurement of infectious particles by plaque assay is time consuming, cumbersome, and statistically error-prone, making the generation of quantitative data difficult. (3) When infectious centers are being measured, cells which bear membrane receptors but which are unable to support viral replication cannot be detected. Attachment to such cells must be detected by measurement of virus removal from the media. (4) Factors which modify penetration and replication can affect results, since the data reflect only the end product of infectivity. In one series of experiments, investigators surmounted some of these limita-

31


tions. Infectivity assays were used to study receptor-determined host restriction for ecotropic and xenotropic murine leukemia viruses. In this approach, pseudotype virions were created which contained the envelope of one virus and the infectious genome of another. Pseudotype virions were generated by the mixed infection of VSV (for which the cells being studied were permissive) and various retroviruses (for which the cells were not permissive). Resultant virions that contained VSV envelopes were then inactivated by anti-VSV neutralizing antibodies. The remaining pseudotype virions (retrovirus envelope VSV genome) bound to the cell surface receptor for the retrovirus being studied and resulted in. VSV replication. Thus, VSV replication measured the presence of surface receptors for retroviruses on a variety of host cells, independent of their ability to replicate the virus (Besmer and Baltimore, 1977).

+

2 . Hemagglutination Hemagglutination assays depend on the ability of many viruses to agglutinate red blood cells. Although red cell receptors responsible for viral hemagglutination might not be the same as viral receptors on host cells, hemagglutination has provided important information about virus-receptor interactions. Of note is that hemagglutination depends upon lattice formation and is very dependent upon conditions (e.g., temperature). Thus, it does not measure “attachment” direct!y. Studies using hemagglutination have detected differences in neuraminidase sensitivity of erythrocyte receptors for various enveloped viruses. For example, the erythrocyte receptor for parainfluenza virus type 3 appears to be neuraminidase resistant, while receptors for influenza virus serotypes A and B (Hirst, 1950) are inactivated by neuramininidase. Adenovirus type 9 does not bind to neuraminidase-treated erythrocytes while types 2 and 7 do (Wadell, 1969; Boulanger et a l . , 1972). For some nonenveloped viruses, hemagglutination is totally resistant to neuraminidase (e.g., reovirus, Gomatos and Tamm, 1962). A modified version of hemagglutination is the rosetting technique. In this method, cells to be tested are added to aliquots of chronically infected cells. Cells which express viral receptors adhere to the surface of infected cells which express the the viral attachment protein (VAP). In order to be used, the rosetting technique requires that a sufficient number of VAPs be expressed on the surface of infected cells. Hemadsorption of erythrocytes to cultures of virus-infected cells is a variation of the rosetting technique. In this method rosettes are formed only if the erythroctyes have viral “receptors” on their surface. The technique of viral-induced agglutination has also been used for cells other than erythrocytes, specifically, lymphocytes. Woodruff and Woodruff ( 1972, 1974) have used the agglutination of T lymphocytes by viruses to define receptors for myxo- and paramyxoviruses on these cells (discussed in Section 111,D). These techniques also allow characterization of receptors by use of reagents

32

MARC TARDIEU ET AL.

which block agglutination (e.g., enzymes or antibodies) (Bankhurst et al., 1979). 3. Use of Fluorochromes Fluorescein- or rhodamine-labeled antiviral antibodies have been used to visualize the presence of virus bound to the surface of various cell types. For example, indirect immunofluorescence techniques were used to define B cells as the lymphocyte subset susceptible to EBV infection (Jondal and Klein, 1973). The major advantage of measuring virus binding by indirect immunofluorescence is that it affords a qualitative approach for the study of viral-receptor interactions. This is especially relevant for studying primary cell cultures which contain a mixture of cell types. For example, for the study of serotype-specific binding of reovirus to cells in the nervous system, we prepared cell suspensions from mouse brain which contained 75% ciliated ependymal cells; these cells are easily identified under phase microscopy by their cilia. Using indirect immunofluorescence, it was possible to demonstrate that reovirus type 1 (which has an in vivo affinity for ependymal cells) but not type 3 binds to these cells (see Fig. 2A and B, discussed in detail later). Similarly, indirect immunofluorescence was used to demonstrate that reovirus type 3 binds to a subset of murine and human lymphocytes (Fig. 2D) (Weiner et al., 1980a). “Co-capping” studies are an extension of the indirect immunofluorescence approach. These experiments take advantage of the property of physiologically active cells to modulate receptor sites bound by ligand to one pole of the cell (Fig. 2C). Using two different colored fluorochromes (one to mark bound virus and the other to label another cell surface structure) it is possible to determine if a cell surface component moves in association with the viral receptor when viral receptors are modulated to one pole of the cell. This approach demonstrated that EBV receptors on cultured cells were closely associated with complement receptors (Yefenof er al., 1976). Co-capping studies also demonstrated that reovirus receptors on murine lymphocytes are distinct from other surface antigens, such as C3 and Fc receptors (Epstein et al., 1981). Viral particles themselves may be directly fluoresceinated or rhodaminated. McGrath and colleagues have used a direct fluorescence technique to study the binding of MuLV virions to thymic lymphoma cells (McGrath et al., 1978; McGrath and Weissman, 1979). These studies allowed a quantitative as well as a qualitative measurement of binding since the amount of fluorochrome per viral particle could be compared with the total fluorescence per cell, as measured with a fluorescence activated cell sorter (FACS).

4 . Enrichment for Receptor-Positive Cells In an extension of the qualitative approach described above, it is possible to use the virus itself to select from a heterogeneous population those cells which


33

FIG. 2. Binding of reovirus to isolated ependymal cells, lymphocytes, and neurons demonstrated by fluorescence staining. (A and B) Unstained isolated ependymal cells examined by phase microscopy and the same fields seen by fluorescence microscopy showing bright labeling of the cells after incubation with reovirus type 1. Viral binding was demonstrated by indirect immunofluorescence with rabbit antibody to reovirus and with FITC-conjugated goat-anti-rabbit Ig. (C and D) Fluorescence microscopy showing labeling of lymphocytes with reovirus type 3 after capping of the receptors (C). (D) shows the appearance prior to capping. (E and F) Phase contrast photomicrograph of cultured neuronal cells (arrow) and of neuronal cells overlayed with reovirus type 3 and then labeled with FITC-labeled antireovirus antibody. Staining can be seen on the neuronal cell body surface and neuronal processes.

34

MARC TARDIEU ET AL.

bind the virus. For example, cells that have bound virus and then have been labeled with fluoresceinated antiviral antibody can be sorted on the FACS. Another method uses plate-adherence. We have developed the plate-adherence technique for the study of reovirus-receptor interactions. In this technique, cells are first incubated with virus and then with a rabbit antiviral antibody. These cells are then plated on a Petri dish previously coated with a goat-anti-rabbit immunoglobulin and separated into adherent and nonadherent populations. Using the plate-adherence technique, the percentage of reovirus type 3-positive murine splenic T lymphocytes was enriched from 2 1 % in the initial unseparated population to 88% in the plate-adherent population (Epstein et al., 1982).

5 . Radiolabels Radiolabeled virions permit the most quantitative measurement of viral binding, either by measuring loss of radioactivity from media, or more usually, by measuring attachment of labeled virus to cells. Because of impurities in labeled preparations (i.e., labeled particles that do not bind), these two approaches are not identical (Richter, 1976), and the uptake of labeled virus to cells is preferred because it is the more direct approach. Furthermore, measurement of uptake is more accurate. For example, 2% uptake can be more accurately measured than 2% loss from the medium (100 to 98%). For radiolabeled studies, many investigators have grown virus in the presence of 3H- or 14C-labeled amino acids (Lonberg-Holm, 1964; Lonberg-Holm and Whiteley, 1976; Fries and Helenius, 1979). These preparations had specific activities in the range of 1013 viral particles/@ (Lonberg-Holm, 1981), and have been used to study the time course of viral-receptor interactions, and competition for receptors by different viruses. The physical conditions which affect viral binding, such as ionic strength, pH, temperature, and cell concentration have also been studied (Lonberg-Holm and Whiteley, 1976; Lonberg-Holm and Philipson, 1974, 1980). Lactoperoxidase-catalyzed iodination labels viral surface components which contain tyrosine residues (Marchalonis et al., 197 1). Although it is more likely to result in a preparation containing inactivated virions (K. Lonberg-Holm and B. Korant, personal communication; Epstein et al., unpublished data), iodination of virus yields labeled virus with higher specific activities. With reovirus, it is possible to obtain specific activities of approximatively 10l2 particledyci. In general, the greater the specific activity of a ligand, the more accurate the quantitative measurements which can be made. Radiolabeled purified viral attachment proteins (i.e., retrovirus spike glycoproteins or adenovirus fibers) have also been used to study binding to cellular receptors (DeLarco and Todaro, 1976; Choppin et al., 1981). Finally, 1251-labeledprotein A has been used for the indirect study of surface


35

interactions of mouse mammary tumor viruses with mouse and rat cells. Virus bound to the cell surface was detected using monoclonal-antiviral antibodies that then bound labeled protein A (Altrock et a f . , 1981). B . MATHEMATICAL ANALYSIS OF VIRALBINDING The interaction of viral particles with receptors is dependent upon a number of physical conditions, including receptor affinity and density, viral concentration, temperature, pH, and ionic strength of the bathing medium. It is useful to study binding interactions using standard values for these variables and then varying one parameter at a time. Mathematical models for receptor interactions have been derived from equations for enzyme kinetics which were developed by Michaelis and Menten (Lehninger, 1975). This approach assumes reversible bimolecular binding, as represented below by Eq. ( l ) , a condition which is not necessarily true for viral binding to receptors. One approach which we have found useful for characterizing interactions of reovirus particles as ligands (L) with receptor sites ( R ) on cells has been to study binding under equilibrium conditions where a simple reversible bimolecular reaction holds:

where R*L represents viral particles bound to receptors (receptor-ligand complex), and k , and k - represent the forward or association and backward or dissociation rate constants for virus binding to receptors. The equilibrium dissociation constant for the reaction, K d , describes the relative concentrations of these reagents at equilibrium, or the ratio of the rate constants, and is represented by the equation:

,

Kd

=

k - , / k , = [Rl [LlI[RLl,

(2)

where the square brackets represent concentrations. For most binding interactions the concentration of [R] is unknown, and the cell number rather than the number of sites can be manipulated in experiments. With cell number held constant in an experiment, and viral concentration varied, an uptake curve of virus binding to receptors may be obtained (see Fig. 1A). The saturable hyperbolic binding curve in Fig. 1A can be derived from Eq. (2) above, and from Eq. (3), indicating that the total number of receptors, Rtota,,is the sum of the free (R) plus bound (RL) receptors:

+

(3) Rtota, = R RL Using Eq. (3) to eliminate [R] in Eq. ( 2 ) , one may express the number of bound virus receptors [RL] as a function of viral concentration [L] in terms of two

36

MARC TARDIEU ET AL.

parameters, the Kd value and Rtotal,the total number of available binding sites in the preparation:

The concentration at which [ L ] = Kd is a useful point on this curve, since it is the concentration of ligand at which 50% of the total receptor sites are occupied ([RL]= '/2 [R,,,,,]). Thus, Kd, the equilibrium dissociation constant, is a measure of the affinity of a receptor for its ligand, since a small Kd value indicates a tightly binding (high-affinity) ligand which saturates its receptor at a low ligand concentration (see Fig. IA). Hence, Kd is a standard measure used to compare the binding of ligands to a variety of receptors. It is important to note, however, that these equations express Kd in terms of the concentration of free ligand when equilibrium has been reached. At this point, the value of L , the concentration of free ligand, must be very close to the value of Ltota,,since as for R , the value of Ltotal = L LR. Therefore, binding studies are usually performed in a range where only small amounts of free ligand are removed from the solution, usually not more than 10- 15%, so that L can be approximated to equal Ltotal.Otherwise the free ligand concentration must be measured directly at equilibrium. The dissociation and association rate constants for binding, k - and k,, can also be estimated directly by measurement of cell-associated virus as a function of time. Rate equations fork, and k- in Eq. (1) can be derived from the same model of a bimolecular interaction, and can also provide estimates of Kd (see Bennett, 1978, for detail). Another approach to analyze the ligand-receptor interaction shown in Fig. 1A is to transform the data into a form which can be represented as a linear equation. One such form, the Scatchard plot shown in Fig. IB, plots [boundIfree ligand] against [bound ligand]. The purpose of this analysis is to allow the binding parameters to be directly estimated from the linear plot. Rearranging Eq. (4) to this form one obtains the expression:

+

,

,

If the resulting plot is a straight line, the x intercept is the maximum binding of the ligand at saturation [R,,,,,], and the slope is the negative reciprocal of the K d . Of concern, however, is that the manipulation of data for Scatchard analysis propagates errors in uptake measurements to both the x and y axis and changes the relative weighting of various regions of the binding curve. Since measurement of viral binding already has significant uncertainty, this magnification of errors further reduces the accuracy of binding data. Although it is used extensively to analyze ligand interactions in neuropharmacology and endocrinology, the Scatchard plot has not been used in the viral receptor field (see Incardona,


37

1981). Nonetheless, quantitative viral binding data can be expressed using Scatchard analysis (see Fig. 1B).

111. Membrane Components Which Interact with Viruses

A. DENSITYAND AFFINITY OF VIRALRECEPTORS Estimates of the density of cell membrane receptor sites for certain viruses have used either growth assays or radiolabeled binding techniques. Despite the variety of different cell types and viruses used, these estimates have been remarkably consistent, with values in the range of lo4 to los sites per cell. For instance, Lonberg-Holm and Philipson (1974) estimated 1 X lo4 receptor sites for poliovirus on HeLa cells, and found a similar value for adenovirus virions and other enteroviruses on several different permissive cell lines. Similarly, Birdwell and Strauss (1974) arrived at an estimate of lo5 sites per cell for binding of Sindbis virus. Recent studies from our laboratory using 251-labeledreovirions have provided estimates in this range for reovirus type 1 and 3 receptor sites on L-cell fibroblasts, and for reovirus type 3 receptors on murine lymphocytes (Epstein et al., unpublished). McClintock et af. (1980) arrived at a slightly higher estimate (1-5 x los siteskell) for EMC virus binding to HeLa cells. Studies using purified subviral binding components, e.g., the fiber protein of adenovirus, have usually led to higher estimates of receptor density than studies using whole virions. For example, Lonberg-Holm and Philipson (1974) demonstrated a 1 log increase in receptor site density when purified adenovirus fibers were used in place of virions (from lo4 to los sites per cell). Similarly, 5 x los sites per cell were estimated when DeLarco and Todaro (1 976) studied binding of the gp7 1 binding glycoprotein from Rauscher murine leukemia virus to NIH/3T3 cells. Such studies have supported the concept that viral receptor sites consist of multiple receptor “units” which can each bind an individual viral attachment protein, and that viruses can bind to the cell surface in a multivalent fashion. Few studies have quantitatively measured the affinity of viral binding to receptors in terms of the Kd of the equilibrium binding reaction (see Section 11,B). However, it has long been assumed that the virus-cell interaction is of very high affinity, both because of the rapid time course of viral binding and the difficulty in disrupting bound virus by physical means (Lonberg-Holm and Philipson, 1976). Although it has been suggested that in certain cases viral binding is virtually irreversible, some dissociation of bound virus probably occurs for most interactions (Lonberg-Holm, 1981). Rapid penetration after binding is one reason that dissociation has been difficult to measure. Initial studies from our laboratory have estimated very low Kd values for reovirus binding to L cells and

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lymphocytes, demonstrating extremely high-affinity binding sites for reovirus (Epstein et al., unpublished data). B. RELATIONSHIPOF VIRAL RECEPTORS TO OTHERSURFACE COMPONENTS It is unlikely that the structures on the cell surface which serve as viral receptors evolved merely for the purpose of virus binding. “Viral receptors,” most probably, serve other biological functions for the cell. It is known, for example, that bacteriophage receptors are components of transport systems for low-molecular-weight sugars (Hazelbauer, 1975). Influenza virus receptors on erythrocytes have been extensively characterized (Kathan et al., 1961). These are sialoglycoproteins (called glycophorins) which have multiple functions, including M or N blood group activity (Springer et al., 1966) and lectin binding activity (Jackson et al., 1973). Extensive biochemical characterization and purification of these receptors (see Burness, 1981, for review) and analogous studies on other types of cells have confirmed that sialic acid-containing glycoproteins are important structural features of myxo- and paramyxovirus reception sites. Receptors for the gp7 1 proteins of Rausher MuLV (previously described as the VAP) appear to be lipoproteins, since binding activity to fibroblast membranes is destroyed by protein-denaturing agents or treatments with chymotrypsin or phospholipase (Kalyanaraman et al., 1978). McGrath et al. (1978) have suggested that T-lymphoma cell surface receptors for MuLV might be identical to the T cell antigen-binding receptor. However, a recently isolated 190,000 dalton dimeric protein from thymocytes which retains Maloney MuLV binding activity differs in size from previously reported values for T-cell idiotype receptors (Binz and Wigzell, 1976; Schaffar-Deshayes et al., 1981). It also differs frcm immunoglobulin and Fc receptors which have also been suggested as candidates for the MuLV receptor. In other studies, co-capping experiments have shown a relationship (though not identity) between EBV receptors and receptors for complement components C3b and C3d (Yefenof et al., 1976). Helenius et al. (1978) reported that solubilized receptors for Semliki Forest virus (SFV) were enriched in HLA antigens but subsequent studies by Oldstone et al. (1980) showed binding and growth of SFV in cells lacking HLA antigens, indicating that HLA antigens were not biological receptors for SFV binding. More recently, acetylcholine receptors (AChR) have been proposed to function as rabies virus receptors on mouse muscle cells. This conclusion was based on the similar anatomic distributions of bound virus and acetylcholine antagonists as observed by fluorescence microscopy. In addition, blocking studies showed that pretreatment of cells with AChR blockers (a-bungarotoxin or dtubocurarin) decreased viral replication in susceptible cells (Lentz et al., 1982).


39

It is possible that more than one surface structure might function as a viral receptor under special different circumstances. Daughaday et a!. (1981) studied dengue virus binding to human macrophages and found that, although viral receptors were destroyed by trypsin, addition of specific nonneutralizing antibodies allowed penetration and replication of virus in the cell. They postulated that Fc receptors served as “nonneutralized viral receptors” in this instance by allowing binding of nonneutralized viral-antiviral antibody complexes to the cell via the Fc portion of the immunoglobulin molecule that was attached to the virus.

C. DEFINITION OF CELLULAR RECEPTORUNITS BY MONOCLONAL ANTIBODIES Monoclonal antibodies directed against viral receptors represent a new approach for the characterization of viral cellular receptor sites. The fine specificity of monoclonal antibodies should permit elucidation of the precise cell surface antigenic domains involved in viral binding. Although minimal data using this approach are currently available, it is timely to review some of the technical options and difficulties associated with this approach. One avenue is to screen a panel of monoclonal antibodies raised against the entire cell membrane, in hopes of isolating one which is specific for the viral receptor. These antibodies could be screened for based upon their ability to inhibit viral binding, viral growth, or hemagglutination. We have attempted this approach with reovirus. In order to find an antibody specific for the reovirus type 3 receptor on lymphocytes, a large panel of monoclonal antibodies against murine lymphocytes produced by Springer and colleagues (Springer, 1980) were tested for their ability to bind to lymphocytes which had been enriched for those bearing reovirus type 3 receptors (see Section 11,A). Monoclonal antibodies which showed significantly more binding to viral receptor-positive lymphocytes were then tested for their ability to inhibit the binding of 12%labeled reovirus type 3 to lymphocytes. Only one antibody was found which had some effect: it minimally reduced viral binding. In an analogous approach, Campell and Cords ( 1982) generated monoclonal antibodies against HeLa cells and have identified monoclonal antibodies which block binding of coxsackievirus but not poliovirus to HeLa cells. A second approach we have used takes advantage of the natural regulation of the immune response through the idiotype-antiidiotype network. To explain how this network might function during the normal immune response to a virus, assume that the virus has one antigenic determinant. That determinant will bind to B lymphocytes with an appropriate receptor or “idiotype” on the cell surface. The antiviral antibodies produced by the B lymphocytes bear idiotypic determinants that can bind the virus. These idiotypic determinants are themselves immunogenic and serve as antigens, so that antiidiotypic antibodies are made. These

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antiidiotypic antibodies do not bind virus, but may bind to the cell surface structure that originally bound the virus. Thus, in theory, they could be used as specific antireceptor antibodies. This approach was first used in endocrinology. Antiidiotypic antibodies were raised against antibodies to retinol binding protein and to insulin. These antiidiotypic antibodies were shown to recognize the cell surface receptor for either retinol binding protein or insulin. Furthermore, it was found that antiidiotypic antibodies made against insulin antibodies mimicked the biological effects of insulin on adipocytes (Sege and Peterson, 1978). We have attempted a similar approach to obtain an antibody against the reovirus type 3 receptor. In these experiments a polyclonal xenogeneic antiidiotype antiserum was made by injecting rabbits with hemagglutinin-specific mouse antireovirus type 3 antibodies (the viral hemagglutinin is the reovirus VAP) (Nepom et af., 1982). The antiidiotype antiserum was then absorbed with normal mouse immunoglobulins, and purified by using a monoclonal antibody to the neutralization site on the hemagglutinin of reovirus type 3. These purified antiidiotypic antibodies mimicked the virus in their binding patterns to various cell lines. Moreover, they bound to primary cultures of murine neuronal cells (which bind reovirus type 3) but did not bind to freshly prepared ependymal cells (which bind reovirus type 1 but not type 3). In addition, they appear to mimic the virus in its interaction with murine T lymphocytes (see Section V,A) (Nepom et al., in preparation; Tardieu et al., 1982). Thus, it appears that these antiidiotypic antibodies might recognize the CRS for reovirus type 3 on neurons and lymphocytes. Work is in progress to further characterize the properties of these antiidiotypic antibodies in terms of blocking viral growth and determining structures on the cell surface which they recognize. Using a similar approach, McGrath and Weissman, studying a spontaneous murine B cell lymphoma (BCLl ) recently produced a monoclonal antiidiotypic antibody against the BCLl-IgM (which binds BCLl -associated retrovirus) and demonstrated that the monoclonal antiidiotypic antibody blocks the binding of the retrovirus to BCLl cells (M. S. McGrath and I. L. Weissman, personal communication). D. SPECIFICITY OF CELLRECEPTOR SITESFOR VIRUSES

In some instances, structures on the plasma membrane which serve as viral receptors have specificity for a single virus, and in other instances, different viruses may share the same receptor. The concept of “viral receptor families” was introduced by Lonberg-Holm et al. (1976). In their experiments, they were able to block the binding of one virus to the cell surface by preincubating the cells with an unrelated virus (see Boulanger and Philipson, 1981, for review). Binding was measured either by infectivity (using UV-inactivated virus for blocking), by radiolabeled virus (using unlabeled virus for blocking), or by


41

immunofluorescence (using specific antiviral antibody which recognized the blocking virus). These studies established that HeLa cells have receptors for a variety of picornaviruses, and that some viruses share common receptors. Thus, the three poliovirus serotypes compete for a common receptor, which is distinct from the receptors that bind the six groups of Coxsackie B viruses (Crowell, 1966, 1976). Coxsackie viruses A13, A15, and A18 have a distinct receptor from the other two groups and Echoviruses and human rhinoviruses have separate receptors from the other picomaviruses (Crowell and Siak, 1978). Moreover, receptors were shown to be shared between viruses from different groups. Thus, the binding of adenovirus type 2 to HeLa cells was blocked by Coxsackie B3, and binding of human rhinovirus type 14 was blocked by Coxsackie virus A21 (Lonberg-Holm ef al., 1976). Confirmation of “receptor families” as defined in these experiments will ultimately depend upon biochemical characterization of the viral receptor sites. The serotype specificity of some viruses is also associated with serotypic differences in their receptors. For example, two serotypes of reovirus, types 1 and 3, differ in their ability to bind to primary cell cultures. Reovirus type 3 binds to neurons and lymphocytes whereas reovirus type I binds to ependymal cells (see below). In addition, binding experiments using 1251-labeledvirions suggest different binding patterns of the two reovirus serotypes to L cells even though both serotypes do bind to and grow in this continuous cell line. (Epstein ef al., unpublished). Specific receptors for the two serotypes of Herpes simplex virus have also been described (Vahlne et a l . , 1979). In these experiments, Herpes simplex type 1 (HSV 1) interfered with the adsorption of HSV 1 but not of HSV2 to human, monkey, and rabbit permanent cell lines. The adsorption rate was measured by assaying infective virus remaining in the medium or by measuring cell associated [3H]thymidine-labeled HSV. Adsorption profiles demonstrated that the monkey kidney cell line and the rabbit cornea cell line had more HSVl than HSV2 receptors, while HeLa cells expressed more receptors with affinity for type 2 than for type 1. Human embryonic lung cells and a cell line derived from a human carcinoma of the larynx showed equal amounts of HSV 1 and HSV2 receptors. Our experiments demonstrate that HSV 1 binds significantly more to murine ependymal cells than HSV2 (Tardieu and Weiner, 1982). A “viral interference” assay was used to study the specificity of cell surface receptors for retroviruses. Steck and Rubin (1966) first demonstrated retroviral interference by showing that chicken fibroblasts persistently infected by an avian retrovirus were not susceptible to superinfection by the same virus (Rubin, 1960, 1961). Later studies established that the interference resulted from a blockade of viral receptors by endogenously produced viruses (reviewed in Weiss, 1981). A similar approach has been used to study another group of retroviruses, murine leukemia viruses. Murine leukemia viruses (MuLV) are classified as ecotropic, xenotropic, or amphotropic depending on their ability to infect mouse

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cells, cells of other species, or cells of both mice and other species, respectively. Using the “cross-interference” approach, it was shown that cells infected with one ecotropic virus failed to bind a second ecotropic strain (R-MuLV), whereas cells infected with a xenotropic virus bound R-MuLV as well as uninfected cells (Hartley and Rowe, 1976; Besmer and Baltimore, 1977). Thus, murine ecotropic and xenotropic viruses appear to recognize different receptors on the murine cell surface, but various strains of ecotropic MuLV utilize the same receptors, since they are subject to cross-interference (Sarma et al., 1967); DeLarco and Todaro (1976) subsequently showed that infection of murine cells with various ecotropic viruses (S2CL3, AKR, R-MuLV, M-MuLV) prevented the binding of radiolabelled R-MuLV gp71 (the VAP of R-MuLV) to the surface of the infected cells. Interference studies using purified gp7 1 demonstrated that the ecotropic viruses used the same family of receptors despite marked differences in the antigenic properties of the viruses. It was then confirmed that the murine xenotropic, as well as amphotropic viruses, use a different family of receptors from murine ecotropic viruses, since they did not interfere with viral infectivity or gp7 1 binding. Similarly, a class of mouse mammary tumor viruses (MMTV) which share an antigenically similar surface glycoprotein gp52 (the VAP of C3H MMTV and GR MMTV) recognize a common cell surface receptor which is different from the surface receptor recognized by other MMTV which have an antigenically different gp52 (CH3 MMTV and RIII MMTV) (Altrock et al., 1981; Schochetman et al., 1979)). In these experiments, viral binding was studied by measuring binding of 1251-labeledprotein A to immune complexes composed of a C3H MMTV gp52 type-specific monoclonal antibody and receptor bound MMTV, or by directly measuring radiolabeled 3H C3H MMTV binding to the cells. Viruses which share class-specific gp52 determinants also share common surface antigen receptors involved in virus adsorption. Finally, the VAPs of type-C and type-D primate retroviruses recognize the same receptors (Moldow et al., 1979), a finding that might reflect a relationship between type-C and type-D VAPs as suggested by their immunological cross-reactivity (Stephenson et al., 1976; Devare er al., 1978). Most of the above studies were performed using continuous cell lines. It is important, however, to also study viral binding to cells which may be the target of viral infection in vivo. This approach has been most easily implemented using freshly isolated lymphocytes for the study of virus-receptor interaction with lymphocyte subpopulations. For example, Epstein-Ban virus selectively binds to human B lymphocytes, mouse adapted cytomegalovirus binds to murine B lymphocytes, and measles and murine leukemia virus bind to human T lymphocytes (Greaves, 1976). Woodruff and Woodruff (1974) have done a variety of studies on the binding of myxoviruses and paramyxoviruses to murine lymphocytes. They have found that Sendai virus, Newcastle disease virus (NDV),


43

influenza B virus and influenza A (H2N2) or (H3N2) virus agglutinate T lymphocytes in v i m , and, presumably, T lymphocytes have a receptor for these viruses. The receptors on T lymphocytes for the paramyxoviruses they studied (Sendai and NDV) differ from those for the myxoviruses (influenza). This was shown by differences in binding between these group of viruses according to temperature or in vitro treatment with fetuin, N-acetyl neuraminic acid, or periodate. In addition, after elution of NDV from lymphocytes, the lymphocytes are agglutinable by influenza virus but not by NDV or Sendai virus. The nine serotypes of influenza A virus they studied also demonstrated differences in their ability to bind T and B cells: five strains agglutinated T and B cells whereas four agglutinated only B lymphocytes. Thus lymphocyte receptors can distinguish among various serotypes of influenza A virus. Using an indirect immunofluorescence technique, we have found a receptor for reovirus type 3 on murine and human lymphocytes whereas only minimal binding was visualized for reovirus type 1 (Weiner et al., 1980a). These studies have recently been extended by quantitative studies using 1251-labeledvirions which show saturable binding of reovirus type 3 to lymphocytes and only minimal binding of reovirus type 1 with no saturable component (Epstein et al., in preparation). Thus, lymphocytes have different receptors for reovirus serotypes and only the receptor for reovirus 3 has a sufficiently high affinity for the binding to be characterized. More recently, the in v i m affinity of 3H-labeled mouse hepatitis virus 3 for macrophages and lymphocytes from both naturally resistant and susceptible mice was shown to be identical (Krystyniak and Dupuy, 1981). In addition, 3Hlabeled encephalomyocarditis virus bound to resident peritoneal macrophages. In contrast, unstimulated splenic lymphocytes did not have detectable numbers of EMC virus receptors, but these receptors could be induced on both T and B lymphocytes by mitogenic stimulation (Morishima et al., 1982). In addition to lymphocytes and macrophages, other cells of biological interest can be studied. We have a particular interest in viral receptors on nervous system tissue and techniques exist to obtain freshly isolated cells from the central nervous system, such as oligodendrocytes (Snyder et d . , 1980), astrocytes (Farooq and Norton, 1978), ependymal cells (Manthorpe et al., 1977), or in some instances neurons (Farooq and Norton, 1978). Freshly isolated cells can then be used to identify viruses which have an affinity for them. We have initiated this approach to study viral receptors on freshly isolated human and murine ependyma1 cells (Tardieu and Weiner, 1982). In these experiments, viral binding to the ependymal cells was demonstrated by indirect immunofluorescenceusing specific antiviral antiserum. Reovirus type 1 (which induces hydrocephalus in mice) bound to the surface of isolated human and murine ciliated ependymal cells whereas reovirus type 3 (which does not induce hydrocephalus in vivo) did not. The binding property of reovirus type 1 to ependymal cells was then mapped to

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the viral hemagglutinin (the VAP) with the use of single-segment recombinant clones between reovirus 1 and 3 (Weiner et al., 1980~).Clone 3.HA1, which contains nine genes from reovirus type 3 and one, the S1 gene, which encodes for the viral hemagglutinin, from type 1, bound to ependymal cells, whereas the reciprocal clone 1 .HA3 did not. In addition, mumps virus, measles virus, parainfluenza type 3, and Herpes simplex virus type 1 bound to murine ependymal cells, whereas Herpes simplex virus type 2 and poliovirus type 1 did not. (Further work on CNS viral receptors by McLaren and Holland is described later in this article.) Thus, it can be demonstrated that unrelated viruses may share a common receptor on the cell surface, and that viruses with the same VAP usually bind to the same receptor even if other parts of the virus are different (retroviruses, recombinant clones of reoviruses). On the other hand, different serotypes of a virus may (polioviruses, coxsackie A viruses) or may not share (reoviruses, Herpes simplex viruses, influenza viruses) a common cell surface receptor. Does a virus bind to different cells using identical or different cell receptor sites? This issue is particularly relevant since most studies of viral binding have utilized permanent cell lines. It is not known to what extent there is homology between surface receptors on the different cell lines to which a virus binds, or, more importantly, whether results obtained from binding studies using transformed cells can be generalized to in vivo virus-surface receptor interactions. Early investigations suggested that different structures served as viral receptors on different cell lines (Kodza and Junglebut, 1958; Sabin, 1959; Holland and McLarren, 1961). One experimental approach to address this issue is to compare two different permissive cell lines for such variables as number of receptors per cell, affinity of these receptors for virus, or susceptibility of receptors to inactivation by agents such as proteolytic enzymes. For example, Sindbis virus replicates in both mammalian and mosquito cell lines. Smith and Tignor (1980) studied the attachment of two Sindbis virus strains (avirulent or neurovirulent) to these cell lines both before and after enzyme treatment of the cells. Mammalian cellular receptors for the avirulent strain were sensitive to proteolytic cleavage while mosquito cells were insensitive to protease, phospholipase, and neuraminidase. The difference was less striking but still present for the neurovirulent strain. Reovirus type 3 binds both to freshly isolated lymphocytes and to L cells, a murine fibroblast cell line. Although binding studies using 1251-labeledvirus suggested similarities between receptors on these two types of cells, xenogeneic antiidiotypic antibodies raised against hemagglutinin-specific antireovirus type 3 antibodies (see Section III,C) bind to the same lymphocyte subpopulations as reovirus type 3 but do not bind to L cells (Nepom et al., in preparation; Tardieu et al., 1982). Assuming that this antiidiotypic antibody recognizes the CRS for reovirus type 3 on lymphocytes and neurons, it would appear that the receptor sites on lymphocytes and neurons express an antigenic determinant which is absent from the receptor site on L cells.


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In an analogous approach, the attachment kinetics of radiolabeled encephalomyocarditis virus were compared on established murine and human cell lines (McClintock et a l . , 1980). The receptor for this virus on human cells had a higher affinity for virus than that on murine cells. In addition, the attachment of the virus to HeLa cells was temperature-independent over the range 0 to 40°C whereas attachment to murine cells progressively decreased with increasing temperature (associated with an increased rate of dissociation of virus). Thus, from these three models, it appears that there may be structural differences between receptor sites for a given virus on various cells. A definitive answer to this issue requires the isolation and characterization of the CRU for a particular virus and the determination of which component of the receptor the virus binds.

OF CELLRECEPTOR SITES E. In Vitro MANIPULATION

The attachment of a virus to a cellular receptor site is only the first step in a series of events (internalization, uncoating, replication, and assembly) that ultimately results in viral replication. The presence of a specific receptor on the cell surface is a necessary but not sufficient condition for viral replication. Thus, for nonpermissive cells, an important question is whether restriction occurs at the receptor or intracellular level. 1. Transfer of Epstein-Barr Virus (EBV) Receptors to Receptor-Negative

cells In vitro, EBV infection occurs only in human and some primate lymphocytes, and EBV receptors are present only on B lymphocytes (Jondal and Klein, 1973; Greaves 1976). To determine if the host-range restriction of EBV growth was receptor-mediated, EBV receptors from purified Raji cell membranes were transferred into the membranes of murine lymphocytes and cells from a human T cell line both of which were nonpermissive for the virus (Volsky et al., 1980). Transfer was accomplished using vesicles reconstituted from a mixture of purified Raji membranes and Sendai virus envelope proteins. Successful implantation of receptor-rich membranes into the membrane of the nonpermissive cells was demonstrated by monitoring the fate of radioiodinated donor membrane, and was confirmed by the detection of surface EBV receptors and complement C3 receptors (which are closely associated with EBV receptor) (Yefenof et al., 1976) on implanted cells. EBV receptors could be detected for 36 hours after implantation and radiolabeled EBV bound specifically to receptor-implanted cells. Furthermore, the implanted receptors were biologically functional, since virus penetration and replication were demonstrated in the normally resistant cells as measured by the expresssion of EBV early nuclear antigen and EBV capsid antigens.

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2. Bypassing Receptor-Mediated Barriers to Infection Replication of a virus can occur in a cell lacking appropriate viral receptors if the barrier to infection at the cell surface is circumvented. This has been achieved with poliovirus by two methods: (1) direct inoculation of viral nucleic acid into the cytoplasm of a cell lacking poliovirus receptors and (2) physical entrapment of the virus into the cell by fusion of the cellular membrane with Sendai virus (which incorporate polioviruses bound nonspecifically to the cell surface) or with virus-containing liposomes (Enders et a l . , 1967; Wilson et al., 1977). 3. Binding of Polyoma and Sendai Virus to Specific Gangliosides Many studies have demonstrated the importance of sialic acids in the binding of Polyoma or Sendai virus (and other myxo- and paramyxoviruses) to the cell surface, and sialidase treatment of host cells can prevent viral infection with these viruses (Hirst, 1942; Klenk et a l . , 1955). These cellular receptors have been identified as glycoproteins with N-acetyl neuraminic acid as the terminal sugar in the carbohydrate side chains (Gottshalk, 1957). To further elucidate the role of sialic acid in cell permissiveness, binding of virus to isolated, highly purified gangliosides of defined structure was studied (Svennerholm and Fredman, 1980). Initially, binding to polystyrene Petri dishes coated with different gangliosides was studied (Holmgren et al., 1980), then host cells were made resistant to Sendai virus by removal of endogenous viral receptor with Vibrio cholerae sialidase (Markwell and Paulson, 1980). These receptor-negative cells were then incubated with individual purified gangliosides. Incubation of cells with gangliosides containing the sequence NeuAccu2, 3 GalPl , 3GalNAc fully restored susceptibility to Sendai virus infection. Furthermore, incubation with gangliosides with a sequence ending with two sialic acids in a NeuAca2, 8NeuAc linkage, rather than a single sialic acid, was 100 times more effective (Markwell et al., 1981). In an analogous way, susceptibilityto Polyoma virus infection was restored by implantation of the sequence NeuAca2, 3GalP1, 3GalNAc but not the sequence NeuAca2, 6GalP 1, 4GlcNAc even though the latter sequence contained a comparable amount of sialic acid (Fried et al., 1981). Thus, Sendai and Polyoma virus interact with specific ganglioside sequences and cell susceptibility to infection can be modified by implantation of different gangliosides into the cell membrane. 4. Inhibition of Receptor Binding Using Antireceptor Antiserum The attachment of enteroviruses to HeLa cells can be inhibited by heterologous antiserum raised against HeLa cells, suggesting that these antibodies in some way affect cell surface viral receptors (Quersin-Thiry, 1958; Axler and Crowell, 1968; Much and Zajac, 1973). This approach is limited, however, by the lack of fine specificity of the antiserum. Monoclonal antibodies directed


47

against the cell surface offer a more specific avenue for the generation of antireceptor antibody and recent work showing the blocking of Coxsackie virus binding to HeLa cells by monoclonal antibodies demonstrates that this should be a feasible approach (Campell and Cords, 1982). A similar approach has recently been described for monoclonal antibodies made against the thyrotropin receptor. These monoclonal antibodies competitively block binding of thyroid stimulating hormone (TSH) but are unable to stimulate adenyl cyclase activity as TSH does. This result suggests the existence of a second domain on the receptor which is associated with the stimulating activity (Yavin et al., 1981). 5. Modification of the Receptor Associated with Viral Binding It has recently been shown that binding of insulin can alter the conformation of insulin receptors on fat cells (Pilch and Czech, 1980). A similar question can be raised for viral receptors: can viral binding to the cell surface modify its own receptor? There are only a few investigations related to this issue. Following the incubation of human lymphoblastoid cells with Epstein-Barr virus (Hinuma et al., 1975), or of Ehrlich ascites tumor cells with mengovirus (Geschwender and Traub, 1979), modulation of cell-surface viral receptors (capping) was observed. This occurred after binding of virus alone. In our investigations using reovirus, capping of reovirus type 3 receptors on the surface of murine lymphocytes required cross-linking by antiviral antibody (Epstein et a l . , 1981). Levanon et al. (1977) have demonstrated that adsorption of infective encephalomyocarditis virus enhances fluidity of the plasma membrane. Binding of paramyxo- and orthomyxoviruses to the cell surface can result in destruction of the receptor itself. The hemagglutinin of paramyxoviruses binds to neuraminic acid-containing cell surface receptors and has neuraminidase activity which eliminates natural neuraminic acid-containing receptors from infected cells (reviewed by Choppin and Scheid, 1980). These two functions reside on two separate proteins in orthomyxoviruses. The role of neuraminidase activity, which is paradoxically present on the same viral protein that determines viral binding, is unclear. It has recently been shown that these two opposing activities can be regulated by environmental conditions such as chloride concentration and pH: high concentrations of halide ion enhance hemagglutinating activity and decrease elution from erythrocytes, while they inhibit neuraminidase activity (Merz et al., 1981). Studies with a specific chemical inhibitor of neuraminidase (Palese et al., 1974a; Palese and Compans, 1976) and temperature-sensitive mutants (Palese et al., 1974b) suggest a role for neuraminidase activity during the release of newly synthesized virus. Because of enhancement by low chloride ion concentration and an acidic pH, the neuraminidase activity is most prominently expressed intracellularly . In contrast, the ionic environment in the extracellular fluids favors virus attachment over receptor-destroying activity.

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F. AGE DEPENDENCY OF VIRALPERMISSIVENESS The age dependency of susceptibility to viral infection is relevant to the question of the role of receptors in the nonpermissiveness of a cell. In several instances, cells from adult animals are not as permissive for viral replication as cells from newborn animals, even though they appear to have the appropriate viral receptors. For example, allantoic sac cells from young chicken embryos are more permissive for influenza or vesicular stomatitis viruses than cells from older embryos. This age-dependent difference in permissiveness is due to a late intracellular event since viral attachment and penetration are the same in cells from young and old chicken embryos (White, 1959; Morahan and Grossberg, 1970). In our investigations, we have found that both isolated newborn and adult ependymal cells have receptors for reovirus type 1 even though ependymitis is observed more prominently in newborns (Tardieu and Weiner, 1982). In contrast, in studies of Coxsackie B5 infection of fibroblasts, Kunin (1962) reported that a slightly decreased ability to absorb the virus occurs in adult as compared to newborn cells, correlating with a decreased permissiveness of adult cells for viral replication. In this instance, the age-dependent reduction in permissiveness may be related to a change in receptor affinity for virus in older cells.

G. GENETIC CONTROL OF CELLRECEPTOR SITEEXPRESSION

In a few cases, a genetic basis for the expression of viral receptors on different cell types can be demonstrated. For example, cells from different mammalian species differ in their susceptibility to poliovirus, i.e., human cells are susceptible while murine cells are not. Somatic hybrids made between permissive (human) and resistant (rodent) cells (Belehradek and Barski, 1969; Wang et al., 1970) demonstrated that hybrids could be infected by poliovirus only when human chromosome 19 was present (Miller et al., 1974). Since viral replication does not require the presence of human genes once the viral nucleic acid has entered the cell (Holland et al., 1959; Wang et al., 1970), these experiments demonstrated that chromosome 19 carries the structural gene for the poliovirus receptor. On the other hand, permissiveness of cells for echo-7 and rhino-1A viruses could not be linked to the presence of a specific human chromosome (Miller et al., 1974). The genetic basis for the specificity of retrovirus cell surface receptors has recently been reviewed (Weiss, 1981). Utilizing interspecies somatic cell hybridization techniques, the gene encoding for the CRS for ecotropic MuLV on murine cells has been assigned to chromosome 5 (Oie et al., 1978; Ruddle et af., 1978; Marshall and Rapp, 1979) and the gene encoding the CRS for endogenous feline C-type virus (RDll4) on human cells assigned to human chromosome 19 (Schnitzer et al., 1980). It should be noted, however, that, for MuLV, the gene


49

expressed on chromosome 5 does not solely regulate leukemia virus replication. Another gene (Fv-I), located on murine chromosome 4, regulates the replication of the virus at a postpenetration step and may influence integration of proviral DNA into the host chromosome (Rowe and Sato, 1973; Gazdar e t a ] . , 1977). AS noted by Ruddle et a / . ( 1 978), additional host control is exerted at the level of differentiation, since bone marrow-derived but not thymus-derived lymphocytes are able to support replication of exogenous MuLV. The genetic regulation of receptor expression is, therefore, only one part of the genetic regulation of cell permissiveness for the virus.

IV. Viral Components Which Recognize Cellular Receptors Identification of the subviral components which are responsible for binding of viruses to cell surfaces has preceded structural understanding of the cellular receptors themselves. This section briefly summarizes current data concerning the viral attachment protein (VAP) of selected viruses. A . PICORNAVIRUSES

Picomaviruses are small nonenveloped viruses with icosahedral symmetry. The 22- to 30-nm capsid contains 60 copies of a “structural unit” consisting of four separate polypetides, VPI , 2, 3, and 4. Some picornaviruses contain a few copies of a precursor protein (VPO) which contains uncleaved VP2:VP4. It has been known for many years that shortly after poliovirus binds to cells, a fraction of the attached virus elutes (Halperen et a / ., 1964). This eluted virus has lost the polypeptide VP4 and is no longer infectious (Lonberg-Holm and Philipson, 1974). These experiments suggested that VP4 was the attachment protein. Further studies, however, provided contradictory evidence: ( I ) VP4 could not be labeled by techniques which label surface proteins such as ‘2sl-labeling using lactoperoxidase (e.g., Lonberg-Holm and Butterworth, 1976). (2) Naturally occuring empty capsids (top component) of some picornaviruses demonstrated identical binding characteristics as native virus, but were shown to lack VP4. (3) Antibodies to VP4 do not recognize the surface of native virions (Talbot et al., 1973). Other evidence suggests that VPl is the binding protein. VP1 is expressed on the surface of the capsid, and trypsin treatment of virions (which renders them incapable of binding to cells) appears to primarily cleave VPI , although some studies also have shown cleavage of VP3 (Boulanger, 1975; Boulanger and Lonberg-Holm, 1981). For Coxsackie virus B3 there is also evidence that VP2 is present at the capsid surface (Philipson el ul., 1973). Some investigations have suggested that no single protein functions as the viral attachment site, but that cooperative interactions among the viral proteins result in a unique conforma-

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tional state which allows binding. This view is supported by the demonstration of differences in antigenicity between native virions ( “D antigenic”) and inactive subviral particles ( ‘‘C antigenic”) for several enteroviruses and rhinoviruses. Native particles carry few of the antigenic determinants of inactive particles (Lonberg-Holm and Yin, 1973). Since the ability to attach to cells is irreversibly lost during the conversion from D- to C-antigenicity (Lonberg-Holm and Yin, 1973), this suggests that the conformation of capsid polypeptides may play a key role in the ability of the virion to attach to host cells, perhaps by regulating exposure of a polypeptide sequence carrying the determinants required for binding activity.

B. ADENOVIRUSES The adenoviruses (mammalian and avian) are larger and more complex than picornaviruses, and (for mammalian viruses) are classified according to speciesspecific hemagglutination properties. Adenovirus capsids are icosahedral and contain 252 capsomers. Most of these are called hexons because each has 6 neighbors. The 12 apical capsomers are surrounded by only 5, and are therefore called pentons. Each of these consists of a penton base and a 10- to 30-nm projection called a fiber. The fiber consists of three polypeptide chains. It has been shown that adenovirus binds to cells via determinants located in the terminal knob of the fiber. The fiber is also the hemagglutinin (Norrby et al., 1969). The ability to solubilize and purify the adenovirus fiber protein has led to the demonstration of serotypic differences in hemagglutinin among subgroups of adenoviruses. C. REOVIRUSES Reoviruses are nonenveloped viruses consisting of two concentric icosahedral capsid shells that surround a segmented double-stranded RNA genome. The outer capsid is composed of three polypeptides ( p l C , a3, and al) which are individually coded by three different viral genes. The a 1 polypeptide makes up I-2% of the outer capsid (24 copies per virion) and is located at the vertices of the icosahedral structure. The a1 polypeptide is the major determinant of reovirus interactions with cells. It is the viral hemagglutinin, elicits the formation of neutralizing antibody, and is responsible for development of delayed type hypersensitivity, generation of suppressor T cells, and generation of cytolytic T lymphocytes (Weiner and Fields, 1977; Weiner et al., 1980b; Greene and Weiner, 1980; Fontana and Weiner, 1980; Finberg et al., 1979). As described previously, it also determines the serotype specificity of viral tropism for different cells in the nervous system and the ultimate pattern of CNS virulence (Weiner et al., 1977, 1980~;Tardieu and Weiner, 1982). Tryptic peptide analysis of the


51

a1 polypeptide from reovirus serotypes 1 , 2 , and 3 has demonstrated both unique and common methionine- and tyrosine-containing peptides. This suggests that certain regions of the hemagglutinin have been conserved, while others have “drifted” with resultant serotypic changes (Gentsch and Fields, 1981). Monoclonal antibodies prepared against the viral hemagglutinin of reovirus type 3 have defined at least four antigenically different domains. One class of antibodies had neutralizing activity; a second class only hemagglutination inhibition (HI) activity. One monoclonal antibody had neutralizing and HI activity and a fourth class of monoclonal antibodies had no detectable neutralization or HI activity. These results suggest that marked functional specialization exists within regions of the reovirus type 3 hemagglutinin (Burstin et al., 1982). This separation of regions for hemagglutination and neutralization raises the possibility that, for other viruses as well, there might be separate viral determinants which bind to either red cells (hemagglutination) or host cells (infection). This suggests that RBC receptors and receptors on other cell types may not be homologous.

D. MYXOVIRUSES AND PARAMYXOVIRUSES For paramyxoviruses, the two glycoproteins which project from the viral surface have been isolated and purified (see Scheid, 1981, for review). The HN glycoprotein, which possesses both hemagglutinating and enzyme (neuraminidase) activity, is the viral receptor-binding protein and exists on the surface in a dimer configuration. It has been suggested that a single active site serves both functions, but this remains to be clarified (Scheid et al., 1972). The F glycoprotein is responsible for fusion activity (and thus hemolysis) and is involved in virus penetration into the cell (to be discussed in the next section). Morbilii viruses (measles, canine distemper virus) lack neuraminidase and their binding glycoproteins are designated H, rather then HN. The three serotypes of influenza virus comprise the myxovirus group and, unlike paramyxoviruses, influenza virions contain separate spikes for the hemagglutinin (HA) (present as a trimer) and the neuraminidase (NA) (present as a tetramer) (Schild, 1979). Influenza C virions differ as they have no neuraminidase. HA is the glycoprotein responsible for hemagglutination or adsorption to host cells and antigenic variations in this protein are largely responsible for periodic epidemics of influenza. Monoclonal antibodies raised against the HA of influenza A have identified three or four nonoverlapping antigenic domains on the protein (Wiley et al., 1981; Wilson et al., 1981). Direct correlation of these domains with functional differences have yet to be defined, but the structural definition of the HA has provided initial answers. The host-receptor binding site and antigenic determinants are located on a globular region which lies on top of a long fibrous coiled coil; the fusion activation peptide is located

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near the virus membrane end of the molecule (Laver and Valentine, 1969; Gerhard et al., 1981; Wiley et al., 1981; Wilson et al., 1981). E. RETROVIRUSES The viral attachment proteins of several retroviruses have been isolated and used in two types of experiments: (1) cross-interference experiments as discussed in Section III,D (reviewed by Weiss, 1981) and (2) experiments to define antigenic domains on this molecule with monoclonal antibodies. These latter experiments were performed using the major external glycoprotein (gp52) of mouse mammary tumor virus (MMTV). Two topographically distinct sites have been identified on gp.52. One site functions as a target for neutralization antibody and was defined by the observation that all monoclonal antibodies (MAb) which neutralized virus infectivity also competed for binding of a neutralizing MAb (used as a standard). The second site bound antibody but this binding had no effect on neutralization. This site was topographically distinct and its MAb could not compete for binding of a second neutralizing MAb (Massey and Schochetman, 1981a). It was further shown that the neutralizing site described above was not the receptor binding site but was adjacent to it as monoclonal antibodies were found which competed for the binding of the first neutralizing MAb but did not neutralize the virus. These antibodies functioned as blocking antibodies and protected virus particles from neutralization (Massey and Schochetman, 1981b).

F. CORONAVIRUSES Two glycoproteins are associated with the envelope of the A59 strain of mouse hepatitis virus (MHV): the E2 glycoprotein which makes up the peplomers of the virus and the El glycoprotein which is deeply embedded in the viral membrane (the portion of the El glycoprotein which protrudes from the viral membrane contains a small glycosilated portion) (Holmes et al., 1981). Monospecific antibodies directed against E2 glycoprotein prevent viral attachment. Virions lacking E2 (either because of growth in the presence of tunicamycin or treatment with bromelain) do not attach to the cell membrane. In addition, isolated E2 binds to the same receptor as intact virus since pretreatment of cells with unlabeled, concentrated MHV blocks the binding of raidolabeled E2. Thus, the E2 glycoprotein appears to be the virus attachment protein for the A59 strain of MHV (K. Holmes, personal communication; Holmes et al., 1981).

V. Virus-Receptor Interactions and Pathogenicity A major feature of certain viral infections is selective damage to specific tissues, and in some instances to specific cells within a tissue. The classic


53

example is poliovirus infection of anterior horn cells in the spinal cord. Other examples include selective infection of the limbic system by rabies virus, infection of ependymal cells by mumps virus (reviewed in Johnson, 1980), and infection of pancreatic beta cells by encephalomyocarditis virus or Coxsackie B4 virus (Craighead and McLane 1968; Boucher and Notkins, 1973; Yoon et al., 1980). It has long been postulated that the in vivo tropism of viruses is mediated in part by the presence or absence of specific receptor sites for viruses on the surface of the target cells (Holland, 1961).

A. ROLEOF CELLSURFACE RECEPTORS IN PATHOGENICITY The role of specific cellular receptors as determinants of cell tropism has been extensively studied (recently reviewed by Crowell and Landau, 1979; and Crowell et al., 1981). Initial studies with picornaviruses using organ minces and homogenates demonstrated a correlation between the presence of receptors on cells and the known in vivo tropism of poliovirus. Human and monkey CNS tissue and intestine were able to adsorb polioviruses whereas tissues from human lung, heart, and skin were not. The correlation was not absolute, however, since receptors were also detected on human liver, monkey heart, and skeletal muscles. Furthermore, poliovirus vaccine strains which did not induce cell damage were shown to bind to brain tissues (McLaren et al., 1959; Holland, 1961; Kunin and Jordan, 1961; LaPlaca, 1963; Harter and Choppin, 1965). A second line of evidence demonstrating a relationship between cell surface receptors and pathogenicity was the presence of a correlation between the grouping of viruses by receptor specificities and their classification according to subgroups which were derived from patterns of pathogenesis (see Section III,C and Lonberg-Holm et al., 1976). Finally, specific organ cultures have been used to show different growth specificities for picornaviruses. For example, some rhinoviruses multiply only in differentiated organ cultures of trachea (Hoorn and Tyrell, 1966); Coxsackie viruses A1 and A5 grow in differentiating primary fetal mouse muscles cultures but do not grow in nondifferentiating mouse cultures (Came and Crowell, 1964; Landau et al., 1972). In contrast, receptors for human enteroviruses exist on tissues which are not involved in their pathogenesis and in species other than their natural hosts (Holland, 1961; Kunin and Jordan, 1961; LaPlaca, 1963; Campbell, 1965). A genetic approach has been used to define the molecular basis for the different patterns of virulence and central nervous system cell tropism exhibited by reovirus serotypes 1 and 3. Using recombinant clones derived from crosses between reovirus types 1 and 3, it has been shown that the hemagglutinin of reovirus (encoded by the S1 gene) determines the central nervous system cell tropism of the reovirus serotypes (Weiner et al., 1977, 1980~).Reovirus type 3

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and clone 1.HA3 (containing nine genes from type I and the gene encoding the hemagglutinin from type 3) cause a fatal encephalitis in newborn mice with neuronal destruction but no ependymal cell damage whereas reovirus type 1 and clone 3.HAl (the reciprocal clone to clone 1.HA3) cause ependymal infection without neuronal damage. The affinity of the two serotypes for two different cell types in the nervous system appears to be due to the specific interaction of the viral hemagglutinin with the receptors on the surface of either ependymal cells or neuronal cells. These results have been confirmed in vitro by demonstrating that reovirus type 1 and clone 3.HA1 (but not reovirus type 3 and clone 1.HA3) bound to isolated human and murine ependymal cells (Fig. 2A and B) (Tardieu and Weiner, 1982). The reciprocal results have been shown on neural cells in culture (Dichter and Weiner, unpublished data; see Fig. 2E and F). The M variant of encephalomyocarditis virus (EMC) produces a diabetes-like syndrome in certain strains of mice by infecting and destroying pancreatic beta cells. Cultured pancreatic beta cells from mice resistant to EMC-induced diabetes are less able to absorb infectious EMC virus than beta cells from susceptible strains, suggesting that genetically determined differences in surface viral receptors on these cells may be one of the factors controlling susceptibility to the disease (Chairez et al., 1978). The presence of virus receptors on lymphocytes may correlate with the specific effect that some viruses may have on the immune response. T lymphocytes have a receptor for measles virus and measles infection is associated with a depression of tuberculin skin hypersensitivity, and a suppression of helper cell activity (McFarland, 1974). Reovirus type 3 binds primarily to the Ly2,3 subset of murine T lymphocytes (the suppressorkytotoxic subset) as well as to the human counterpart (T8+ cells). This binding is a property of the viral hemagglutinin (Epstein et al., 1982). Furthermore, in vitro, reovirus type 3 induces suppressor T cells capable of suppressing Con A proliferation (Fontana and Weiner, 1980). This, the generation of functionally active suppressor T cells in vitro by reovirus type 3 appears to be secondary to the interaction of the viral hemagglutinin with a specific receptor on the Ly2,3 subset of murine lymphocytes. B. ROLEOF VIRUS ATTACHMENT PROTEINS IN PATHOGENICITY The specificity of myxo- and paramyxoviruses for particular cell types depends both on the structural and functional activity of the viral surface glycoproteins and on the ability of the cells to cleave these proteins (reviewed by Choppin and Scheid, 1980). The interaction between a paramyxovirus and the cell surface is mediated by two glycoproteins projecting from the external surface of the virion: the hemagglutinin-neuraminidase (HN) and the fusion (F) glycoprotein. Binding to cellular neuraminic acid-containing receptors is a property of the HN


55

glycoprotein. Although this activity differs considerably according to the amount and position of neuraminic acid in the molecule (Markwell et al., 1981), the abundance of sialic acid in many biologic membranes limits the importance of the binding step as a determinant of cell specificity and tissue tropism. An active fusion (F) glycoprotein, however, is a requisite for infectivity and cell-to-cell spread of infection. The fusion glycoprotein acts during viral penetration, a step beyond adsorption. Virus infectivity requires cleavage of a precursor FO glycoprotein into two subunits (Fl, F2) and the host must provide the enzyme responsible for this cleavage (reviewed in Choppin and Scheid, 1980). Thus, host-dependent cleavage of FO is required for infectivity and therefore host range and tissue tropism of virus is determined by availability of the appropriate protease (Scheid and Choppin, 1975, 1976). It has recently been shown that neuraminidase (NANase) activity of the HN glycoprotein of mumps virus contributes to cytopathology . Although the HN glycoproteins of the six studied strains of mumps virus are similar in size and antigenic composition, each strain possesses a neuraminidase with distinct enzymatic properties. Strains with active NANase cause little cytopathology and no cell fusion on African green kidney cell lines, whereas infection with strains having less active NANase cause extensive cell fusion. Thus, viral NANase appears to contribute to full expression of the activity of the F protein and ultimately to cytopathology (Merz and Wolinsky, 1981). This extends the previous observation that influenza virions with less active NANase cause more cytopathology in tissue culture and were more pathogenic in viva than virions containing active NANase (Smith and Cohen, 1956; Choppin, 1963; Choppin and Tamm, 1964). Moreover, only strains of mumps virus with less active NANase were both neuroinvasive and neurovirulent (Wolinsky and Stroop, 1978; McCarthy et af., 1980). Thus, the pathogenicity of myxo- and paramyxovirus depends upon an interaction of viral glycoproteins and the cell surface at a step beyond viral adsorption. Cell specificity is determined by the availability of a protease on the surface of the cell to cleave one of the viral glycoproteins and allow viral penetration into the cell. There are few data concerning the role of viral receptor interactions in the pathogenicity of other enveloped viruses. C. INDUCTION OF CELL-SPECIFIC AUTOIMMUNITY FOLLOWING VIRAL INFECTION

Autiommune reactions against host tissue have been reported after certain viral infections. These include the production of autoantibodies against a variety of host antigens in experimental animals and man (DNA, lymphocytes, myelin) (reviewed in Onodera et af., 1981), and in man, the well-documented immunemediated damage to peripheral nerve myelin in infectious polyneuritis (Guillain-

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Barre Syndrome) which can occur 2-3 weeks after viral infections or following swine flu immunization (Schonberger et al., 1981). The mechanisms by which a viral infection can lead to an autoimmune response are not well understood, however, two possible mechanisms are relevant to the present review: (1) autoantibodies which appear following viral infection may recognize shared antigens between a viral protein and a determinant on the surface of the target cell; and (2) through the idiotypic-antiidiotypic network (described in Section II,C) antiidiotypic antibodies could be produced which recognize the viral receptor on the cell surface. Thus, the affinity of these autoantibodies for a particular cell would be identical to the tropism of the virus itself for the cell. To test these two hypotheses, we recently performed the following experiment: splenic lymphocytes from adult mice infected with purified reovirus (type 1 or 3) particles were fused with NS-1 myeloma cells. The resultant clones were then screened by radioimmunoassay for their ability to bind virus, T lymphocytes, brain, liver, and lung tissues. We found that (1) during the course of the normal immune response to reovirus, autoantibodies were generated which reacted with normal tissue, (2) monoclonal antibodies were generated which identified shared antigenic structures between viral determinants and normal tissue, and (3) some monoclonal antibodies appeared to have the same affinity for cells as the virus (putative antiidiotypic antibodies which recognize viral receptors) (Tardieu et al., 1982). In another group of experiments, performed by Onodera et u1. (1981), it was shown that mice infected with reovirus type 1 developed transient diabetes and a runting syndrome. Sera of infected mice contained autoantibodiesthat, by immunofluorescence, reacted with cytoplasmic antigens of the islets of Langerhans and anterior pituitary of uninfected mice, both target structures of the virus. The autoantibodies appeared to be directed against insulin or growth hormone. Since reovirus type 3 did not induce autoantibodies to growth hormone, using recombinant clones, it was possible to show that the ability to induce autoantibodies to growth hormone was a property of the viral hemagglutinin (Onodera et al., 1981).

VI. Conclusion Further progress in the study of virus-receptor interactions should occur in the following three areas: ( I ) the development of more sophisticated approaches for both quantitative (e.g., rigorous binding studies using radiolabeled virus with high specific activity) and qualitative (e.g., cell sorting techniques) measurements of viral interactions with the cell surface; (2) the production of monoclonal antibodies against cell receptors and against viral components. These reagents will lead to the isolation and biologic characterization of both the CRU and the


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functional domains on the virus attachment protein; and (3) techniques for the isolation of single cell suspensions from organs such as brain. This approach will allow direct study of viral interactions with biologically relevant cells. The comparison of viral-receptor studies on permanent cell lines with studies utilizing freshly isolated cells is important since there is increasing evidence that different structures might serve as receptor sites for a virus on cells of different origins. The role of receptors in determining the in vivo affinity of certain nonenveloped viruses for specific cell types and thus determining viral pathogenicity is well established. It must be emphasized, however, that a cell is not permissive for a virus merely because it has a cell surface receptor to which the virus binds. Finally, although additional studies are needed, receptors may play a less important role in the pathogenicity of enveloped viruses than for nonenveloped viruses.

ACKNOWLEDGMENTS We want to thank Dr. K. Lonberg-Holm for critically reviewing the manuscript. MT is the recipient of a Lilly International Fellowship. This work was supported by NIH grant No. NSAI- 16998.

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The Molecular Basis of Crown Gall Induction W. P. ROBERTS Department of Microbiology, La Trobe University, Bundoora, Victoria, Australia

1. Introduction . . . . ................................ 11. The Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111. IV. V. VI . Vll.

VIII. IX.

X. XI.

The Crown Gall Bacteria ............................. The Physiology of the Gall.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Involvement of Wounding in Gall Induction . . The Opines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Involvement of Plasmids in Tumor Induction A. The Presence and Structure of Ti-Plasmi Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Products of the T-DNA . . . ............. C. Ongocenic-Related Functions Not on T-DNA . . . . . . . . . . . . . . D. Nononcogenic Functions on the Ti-Plasmid. . . . . . . . . . E. Ti-DNA Transfer and Integration into the Plant Cell . , F. Reversion to the Normal State . . . . . . . . . . . . . . . . . . . . , . . . . , Significance of Crown Gall Induction to Agrobacteriurn . . . . . . . . . The Evolutionary Origin of Crown Gall . . . . . . . . . . . . . . . . . . . . . . Agrobacteriurn and Genetic Engineering . . . . . . . . . . . . . . . . . . . . . . Future Work and Prospects . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. References . . . . . . . . . . . . . . . . . . . . . .

63 64 65 65 66 68 70 71 74

76 78 79 82 83 84 86 87 88

I. Introduction Although Smith and Townsend demonstrated in 1907 that the neoplastic plant disease, crown gall, was caused by a bacterium, there was little progress elucidating the mechanism of gall induction until White and Braun (1941, 1942) conclusively demonstrated that the bacteria initiated a stable transformation of the plant cell. Once the disease had been induced then the bacteria were no longer required for continued growth of the gall. These reports stimulated a great deal of research but little further progress was made in the understanding of this transformation until Van Larebeke et al. (1973, 1974) reported that crown gall bacteria carried a large plasmid that was essential for pathogenicity. Since this discovery, there has been many reports revealing the role of this plasmid in crown gall induction and the molecular basis of crown gall. 63 Copyright Q 1982 by Academic Press. Inc All rights of reproduction in any form reserved ISBN 0-12-364480-0

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11. The Disease

Crown gall disease is characterized by the production of galls on plant roots. These galls often appear at the crown of the plant but may be anywhere on the root system. Occasionally galls appear on the aerial parts of the plant possibly due to bacteria being transferred from the soil. There are also reports that agrobacteria can move internally in the plant and give rise to tumors remote from the original infection point (see review by Lippincott and Lippincott, 1975). Experimentally, galls can be produced on all parts of a suitable host plant including the roots, stems, and leaves. The gall itself consists of undifferentiated cells which divide indefinitely to form a tumor. A gall may make up a very significant proportion of the dry weight of the plant and values up to 50 kg (Walker, 1969) gall weight have been recorded. As well as being a very significant drain on the photosynthetic capacity of the plant, a gall may also disrupt the phloem and xylem. This is particularly significant with galls that occur at the crown of the plant where the entire flow of nutrients up and down the plant may be affected. The overall result is that plants with crown gall grow poorly, give low yield, and may be very sensitive to stress. DeCleene and DeLey (1976) surveyed the available literature and carried out experiments to determine the host range of crown gall. They report that 643 species representing 331 genera of 91 families were susceptible to crown gall. This included gymnosperms and dictyledonous and monocotyledonous angiosperms. Only 7 out of 75 species of monocotyledonous plants tested have been reported as susceptible to crown gall and their own experiments gave only one possible positive response. Although only a few bacterial strains have been used to carry out these tests, it is clear that monocotyledonous plants are not generally susceptible to crown gall. The host list includes many economically important plants such as apricots, almonds, peaches, apples, pears, grape vines, and roses and the disease is very widely distributed (Hayward and Waterston, 1965). It is difficult to find data on the occurrence and economic loss caused by this disease but it is not uncommon to find complete orchards with every, or almost every, tree infected. The two common modes of infection are by contaminated material from the nursery or by planting into areas already infected. Many countries have legislation preventing the sale of material infected with crown gall. However, this only prevents the distribution of obviously diseased material not of material contaminated by Agrobacterium but lacking a visible gall. New (1972), for example, found that up to 40% of bundles of almond plants ready for distribution were contaminated with Agrobacterium. Pathogenic agrobacteria could not be isolated from soil not previosly used for stone fruit cultivation indicating that at least in the areas studied that it was an introduced disease. Until the introduction of a biological control method (New and Kerr, 1972; Kerr, 1972) there was no satisfactory control system for the disease. The biolog-

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ical control method depends on the production of a bacteriocin by one Agrobacterium strain (strain K84) that inhibits a wide range of pathogenic agrobacteria. However, not all pathogenic strains are controllable (see, for example, Kerr and Panagopoulos, 1977). The biological control of crown gall has been reviewed by Moore and Warren ( 1979). 111. The Crown Gall Bacteria

Agrobacterium is a gram-negative organism with peritrichous flagella. Strains can be divided into a number of groups on the basis of a range of physiological tests (Keane et al., 1970; White, 1972; Kersters et a l . , 1973; Panagopoulos and Psallidas, 1973; Kerr and Panagopoulos, 1977; Sule, 1978). The organisms causing crown gall disease have been classified as Agrobacterium tumefaciens and nonpathogens as A. radiobacter. As nonpathogenic isolates can be converted to pathogenic forms by simple genetic manipulation, pathogenicity cannot be considered a valid species differentiation characteristic and it has been suggested that pathogenicity be reduced to a lower status (Keane et a l . , 1970). These authors also reported that the organism causing galls on Rubus spp., A . rubi, and the hairy root disease organism, A. rhizogenes were indistinguishable from A . tumefaciens by a range of biochemical tests. Hairy root disease (Riker et a / ., 1930) is characterized by the proliferation of roots at the site of infection and the molecular basis of the disease is similar to crown gall (White and Nester, 1980a,b; Petit e t a / . , 1981; Chilton et a l . , 1982). Holmes and Roberts ( 1981 ) carried out a taxonomic study of Agrobacterium strains. These strains could be divided into four clusters, three of which broadly corresponded to the physiological groups already described. The fourth group contained yellow-pigmented isolates which had not previously been recognized as related to Agrobacterium. These authors concluded that the clusters corresponded to species and without regard to pathogenicity assigned the names Agrobacterium tumefaciens, A . rhizogenes, and A . rubi to three of the groups. They suggested that within these species individual isolates be designated as saprophytic, tumorogenic, or rhizogenic. Determination of the taxonomic position of the yellow-pigmented isolates was not attempted as few strains had been included in the study. Agrobacterium is quite closely related to Rhizobium and there have been suggestions that the two genera be merged (Graham, 1964; Herberlein et a / . , 1967; DeLey, 1968; Moffett and Colwell, 1968; White, 1972).

IV. The Physiology of the Gall Much of the early work concentrated on the hypothesis that the gall was induced by the production of plant hormones by agrobacteria. Indeed agrobac-

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teria are able to synthesize plant hormones, at least in culture (Clark, 1974; Chapman et al., 1967; Lui and Kado, 1979). However, with the demonstration by White and Braun (1941, 1942) that gall tissue could be cultured free of bacteria on media containing no plant hormones or growth factors, it became clear that crown gall was a transformed plant tissue. Crown gall tissue can be cultured on standard plant tissue culture media without any hormones and will continue to grow in a stable manner indefinitely. Kado ( 1976) summarizes the physiological differences between normal plant tissue and crown gall tissue. Since this review, other reports have shown that crown gall cells may have qualitative and/or quantitative differences in cytokinins (Scott et al., 1980; Stuchbury, 1979; Peterston and Miller, 1977), adenyl cyclase, and cyclic AMP phosphodiesterase activity (Rutherford et al., 1977), cellular membranes (Cockerham and Lundeen, 1979; Phillips and Butcher, 1979), cation concentrations (Lentz et al., 1979; Radiosevich and Galsky, 1979), Feulgen DNA content (De Cleene et al., 1980), opines (Firmin and Fenwick, 1977, 1978; Kemp, 1977, 1978), putrescine biosynthesis (Speranza and Bagni, 1977), and ubiquinone formation (Ikeda et al., 1976). Although these studies clearly demonstrate that crown gall cells are physiologically different from normal plant cells, none of them indicates the molecular basis of cell transformation. However, the high levels of both cytokinins and auxins suggest that the biochemical basis of the gall is that the cells become independent of the normal hormonal control of the cell. The agrobacteria are only required during the initial gall induction phase, so they must somehow transform the plant cell at this time. Braun (1947) suggested that some principle which he called the Tumor Inducing Principle or T.I.P. was produced by the Agrobacterium and was responsible for causing plant cell transformation. Much of the reported work from this time has concentrated on the possible nature of the T.I.P. and its role in tumorogenesis. A range of substances including RNA, DNA, bateriophages, proteins, and lipopolysaccharides have been suggested as the T.I.P. (see review by Kado, 1976).

V. Involvement of Wounding in Gall Induction Although Riker and Berge (1935) demonstrated that wounding was an absolute requirement for gall induction the precise role of wounding has still not been determined. There are a number of possibilities that include (1) wounding exposes plant cell-Agrobacrerium recognition sites; (2) wounding alters the biochemical state of the plant cell to make it susceptible to gall induction; and (3) wounding creates a suitable environment for Agrobacterium to synthesize and transfer the T.I.P. The involvement of a plant cell-Agrobacterium recognition system in crown


67

gall induction was suggested by Lippincott and Lippincott (1 969a). They reported that certain avirulent strains of Agrobacteriurn when mixed with a virulent strain prior to inoculation could reduce the number of galls induced on Pinto bean leaves in a quantitative crown gall assay. Heat-inactivated virulent bacteria also gave a reduction in the number of galls induced. The kinetics of the system suggested that one avirulent bacterium could prevent gall induction by one virulent bacterium and they concluded that crown gall inducation only required one bacterium binding to a specific site. However, in their system approximately lo6 bacteria were needed to induce one gall but they concluded that this represented the probability of one bacterium binding to the correct site. Similar results were reported by Beiderbeck (1973), Glogowski and Galsky (1978), and Ohyama et al. (1979). Beiderbeck (1973) reported that cell wall extracts from virulent bacteria mixed with viable pathogens gave a reduction in gall formation. Whatley et al. (1976) and Matthysse et al. (1978) extended these findings when they reported that a lipopolysaccharide extract (LPS) from some Agrobacteriurn strains could reduce gall formation. LPS from strains that were found to be site-binding by Lippincott and Lippincott ( 1969a) inhibited gall induction while LPS from non-site-binding strains did not and they suggested that the LPS component of the bacterial cell wall was the bacterial site of the specific plant-bacteria binding system essential for gall induction. This LPS fraction was found to have a similar action on gametophore induction by Agrobacterium in moss (Whatley and Spiess, 1977). Both chromosomal and Ti-plasmid genes seem to be involved in this attachment process (Whately et al., 1978; Matthysse et al., 1978). The complementary experiment was reported by Lippincott et al. (1977). They found that cell wall extracts from plant cells when mixed with virulent agrobacteria could reduce the number of galls formed. Much of the inhibitory effect of the plant cell wall extracts was eliminated by pretreatment with LPS or dead virulent bacteria and they concluded that the site for attachment of agrobacteria was in the plant cell wall. Pueppke and Benny (1981) were able to isolate a soluble uronic acid-containing fraction from potato that inhibited gall induction and which they believed contained the bacterial attachment sites. The concept that there is a period of cell conditioning before the plant cell is sensitive to gall induction comes from the studies of Braun (1947, 1952) and Braun and Mandle (1948). This cell conditioning could take place at 25 and 32°C without the bacteria being present although gall induction occurred only below 28°C. Braun (1952, 1954) reported that plant cells were most susceptible to transformation just before the onset of the first cell division after wounding and suggested that the conditioning process may involve alterations in the permeability of the plant cell and so allow entry of the T.I.P. Lipetz (1965) extended this finding and reported that the time taken to the onset of the first cell division decreased with temperature as did the time to reach maximum sensitivity to gall

68

W. P. ROBERTS

induction. At all temperatures greatest sensitivity to induction was found just before the first cell division after wounding. These results suggested that the process of gall induction is closely related to cell division. However, cell division alone is not a satisfactory condition for gall induction as bacteria added to the surface of actively dividing callus tissue do not produce galls unless wounding occurs (Riker, 1923; Riker and Banfield, 1932). There are also reports of gall induction on Pinto bean leaves (Lippincott and Herberlein, 1965) and decapitated pea seedling (Kurkdjian et al., 1969) where gall induction occurs without wounding inducing cell division. Klein (1954) suggested that wounds were essential to release substances that were necessary for gall induction. He reported that washing the wound with sterile water prevented gall induction. In another report (Klein 1955), he showed that carrot discs of a variety which normally respond only weakly to pathogenic bacteria gave a much stronger response when treated with an extract from carrots which normally respond strongly. The possible importance of wound sap composition is emphasized by the studies of Lippincott and Lippincott (1966, 1969b) who showed that bacterial nutrition was a significant factor in the expression of virulence particularly when only small wounds were involved. However, there is one report (Therman and Kulipa-Ahvenniemi, 1971) which indicates that wound washing enhances tumorogenesis. These workers attributed this to a promotion of the first cell division after wounding. A similar promotion of the first cell division after wounding by washing has been reported by Lipetz (1967). Ohyama et al. (1979) report that agrobacteria in plant wounds are associated with fibrils. Matthysse et al. (1981) extended this work and concluded that these fibrils were composed of cellulose and were synthesized by the bacteria. The fibrils anchored bacteria to the plant cells and formed a matrix in which the bacteria became trapped. Fibrils were synthesized only in the presence of plant cells or when the media was supplemented with a soya bean extract and they concluded that the plant cells may release a substance that stimulates production of these fibrils.

VI. The Opines Considerable support for the idea that the T.I.P. was bacterial DNA which was incorporated into the plant cell was due to the discovery of a range of gall specific compounds (Table I, Fig. 1). These compounds which have been found only in crown gall tissue (Scott et al., 1979; Yang et al., 1980c) appear to be related to the Agrobacterium strain inducing the gall rather than to the plant species and Goldmann et al. (1968) suggested that these compounds were coded for by bacterial DNA incorporated into the plant cell during gall induction. One very significant feature was that the strain inducing a particular set of these

69

CROWN GALL INDUCTION TABLE I THE OPINES N2-( I -Carboxyethyl)-amino acid derivatives Octopine Menage and Morel (1964) Octopinic acid Menage and Morel (1965) Histopine Kemp (1977) Lysopine Biernann et a / . (1960) N2-( 1-3-Dicarboxypropyl)-aminoacid derivatives Nopaline Goldmann et ul. (1969) Nopalinic acid Firmin and Fenwick (1977) Other Agropine Coxon et a / . ( 1980); Tate et a / . (1982) Agrocinopines Ellis and Murphy (1981)

HN~C-NH-(CH2)3-CH-COOH

H 2 N-(CH2 13-

I I

H2 N

NH

I

H3C-CH-COOH

Hg C-CH-COOH

octopine

H2 N-(CH~ )

7-

H COOH NH

octopinic acid

4

' '

-

~ HC-C-CH2~ ~

I NH I H3C-CttCOOH

~ H-COOH ~ ~ H

I

H3C-CH-COOH

lysopine

histopine

H N > ~ - N ~ - t) ~ ~3 2 H2N NH

~

I

~

- H~N-(CH ~ ~ ~

CH-COOH ~

* 3 -YH~

I

I

HOOC-(CH2)2-CH-COOH

HOOC-(CH2)2-CH-COOH

nopaline

nopalinic acid

0

agropine

FIG. I .

Structures of the opines

compounds could utilize the same compounds for growth in culture (Petit et al., 1970; Bomhoff et al., 1976; Montoya et al., 1977; Firmin and Fenwick, 1978; Guyon et al., 1980; Ellis and Murphy, 1981). For example, strain T37 could induce nopaline and nopalinic acid in galls and utilize these compounds in

70

W. P. ROBERTS

TABLE I1 OPINESINDUCEDI N GALLSAND CATABOLIZED I N CULTURE BY Agrobacterium Agrocinopines Octopinic Nopalinic Octopine acid Histopine Lysopine Nopaline acid Agropine A B C D

culture. It does not utilize octopine or agropine while strain B6 induces these compounds in galls and utilizes them in culture. Using these criteria, pathogenic agrobacteria can be divided up into groups: the octopine group, the nopaline group, and the agropine group (Table 11). In addition, there are a number of “defective” nopaline strains that utilize nopaline in culture but induce galls containing no known opine (Petit et al., 1970; Sciaky et al., 1978). Most nopaline strains can utilize octopine when a transport system is induced by nopaline (Petit and Tempi, 1975; Klapwijk et al., 1977b). Kemp (1978) suggested that one enzyme coded for production of all the P-( 1carboxyethyl) derivatives in the gall and the properties of the purified enzyme support this conclusion (Hack and Kemp, 1977, 1980). Kemp (1978) also suggested that one enzyme synthesized nopaline and nopalinic acid. No data are available on the properties of these enzymes or the enzyme involved in agropine and agrocinopine synthesis in the gall. Gall specific amino acids were called opines (Tempi et al., 1977) but this definition was later extended to include all crown gall-specific compounds that are utilized by agrobacteria. Opines in gall tissue may reach high levels. Scott et al. (1979) report that nopaline may constitute 19 mg/g dry weight and agropine 35 mg/g dry weight. Octopine levels tend to be much lower at up to approximately 0.5 mg/g dry weight of gall.

VII. Involvement of Plasmids in Tumor Induction Despite the evidence of the opines, none of the early work attempting to demonstrate the presence of Agrobacterium DNA in crown gall cells could be reliably repeated (Kado, 1976). Van Larebeke et al. (1973, 1974) and Zaenen et al. (1974) first demonstrated

71


that plasmids were present in pathogenic agrobacteria. They found a good correlation between the presence of a plasmid and pathogenicity. Bacterial strains lacking a plasmid were nonpathogenic. They showed that the technique of producing avirulent agrobacteria by growth at 37°C (Hamilton and Fall, 1971) cured the bacteria of this plasmid. The reciprocal experiment was reported by Watson et al. (1975) and Van Larebeke (1975) who were able to transfer the plasmid back into cured strains using the in vivo transfer method of Kerr (1971). Nonpathogenic strains that received the plasmid became pathogenic. These experiments unequivocally established that this plasmid (that was named the Ti or tumor-inducing plasmid) was required for crown gall induction. The Ti-plasmid determines both the catabolism of opines in culture and the synthesis of opines in galls (Watson et al., 1975; Bomhoff et al., 1974; Montoya et al., 1977; Van Larebeke et al., 1975) and Ti-plasmids corresponding to all opine groups exist. These results suggested but by no means proved that the Ti-plasmid was the T.I.P. and that all or part of it was transferred to the plant cell during tumor induction where it coded for tumorogenicity and opine production. The simplest interpretation, that synthesis and breakdown were due to the activities of the same enzyme working in opposite directions in gall and bacteria, was eliminated by the reports that the enzyme systems from bacteria and gall were biochemically and genetically distinct (Bomhoff, 1974, cited in Montoya et al., 1977; Montoya et al., 1977; Petit et al., 1970; Sciaky et al., 1978). A. THE PRESENCE AND STRUCTURE OF TI-PLASMID DNA

IN THE

GALLCELL

The first convincing report of the presence of Ti-plasmid DNA in crown gall cells is that of Chilton et al. (1977). They used isolated restriction endonuclease DNA fragments of an octopine type Ti-plasmid to perform liquid-liquid hybridizations with DNA isolated from the crown gall cells and demonstrated that part of the Ti-plasmid was in the gall cells. This DNA became known as transfer or TDNA. Chilton et al. (1978) presented a restriction endonuclease map of an octopine Ti-plasmid that showed that the restriction fragments present in the gall cell were contiguous on the Ti-plasmid. This suggested that only one discrete region of the Ti-plasmid was stably incorporated into the plant cell. Merlo et al. (1980) and Thomashow (1980a,b) extended this study to a number of crown gall tumor lines induced by different octopine strains. They concluded that each tumor contains a core of T-DNA that is colinear with the Ti-plasmid, a given Tiplasmid does not always give rise to the same T-DNA complement, and that most, if not all the T-DNA, is integrated into plant DNA. In addition, these studies showed that the copy number of parts of the T-DNA varied between tumor lines and suggested that in some lines the T-DNA may be present as tandem repeats and integrated into repeated sequence plant DNA. Up to approx-

72

W . P. ROBERTS

imately 13 X lo6 daltons of Ti-piasmid DNA may be present in octopine type gall cells, depending on the tumor line (Thomashow et al., 1980a; Fig. 2 ) . The structure of the T-DNA in nopaline tumors has also been looked at in detail (Yang el al., 1980a; Zambryski et al., 1980; Lemmers er a!. , 1980; Yadav er al., 1980; Fig. 3). The T-DNA in nopaline tumor cells is larger, with approximately 18 X lo6 daltons of Ti-plasmid DNA (Chilton et al., 1980; Lemmers er al., 1980; Yang et al., 1980a) being present in tumor cells. Like the octopine TDNA it represents one continuous fragment from the Ti-plasmid and internal rearrangement does not take place after insertion into the cell. Each tumor line contains only a few T-DNA plant DNA junction fragments suggesting that the TDNA is inserted only once or a few times in the cell and does not “jump about.” Although there appears to be certain preferred ends of the T-DNA, these are not an absolute requirement as transposon mutagenesis of the ends will still allow tumorogenesis (Lemmers et al., 1980). Like the octopine T-DNA the nopaline T-DNA also seems to be inserted in repetitive sequence plant DNA. Unlike the octopine T-DNA, the nopaline T-DNA does not appear to vary in copy number along the T-DNA region. 1200

60

FIG. 2. A map of the B653 octopine plasmid. The map is calibrated in 10 megadalton segments with the zero site at an endonuclease Smal site common to both octopine and nopaline plasmids (Depicker e t a / . . 1978; Chilton et al., 1978). The extent of the T-DNA is indicated as well as areas known to be essential for pathogenicity (line within map) and areas homologous with the C58 nopaline plasmid (solid blocks). Also indicated are areas involved in determining gall morphology (Gm), octopine synthesis (Ocs), arginine catabolism (Arc), phage API exclusion (Ape), plasmid transfer (Tra), octopine catabolism (Occ), and agropine catabolism (Agc).


73

FIG. 3 . A map of the C58 nopaline plasmid. The map is calibrated in 10 megadalton segments with the zero site at an endonuclease Smal site common to both octopine and nopaline Ti plasmids (Depicker et a / . , 1978; Chilton e t a / . . 1978). The extent of the T-DNA is indicated as well as areas known to be essential for pathogenicity (line within map) and areas homologous with octopine plasmids (solid blocks). Also indicated are the areas involved in nopaline synthesis (Nos), arginine catabolism (Arc), nopaline catabolism (Noc), incompatability (Inc), indole acetic acid production (Iaa), phosphorylated sugar catabolism (Psc), Agrocin 84 sensitivity (Agrs), plasmid transfer (Tra), and phage API exclusion (Ape).

Octopine and nopaline plasmids show extensive areas of homology (Chilton et al., 1978; Depicker et al., 1978a,b; Drummond and Chilton, 1978; Hepburn and Hindley, 1979; Van Montagu eral., 1980; Figs. 2 and 3). The areas of homology include a substantial part of the T-DNA region and areas to the left of the T-DNA in both octopine and nopaline type plasmids. Mutagenesis of the T-DNA area common to both nopaline and octopine plasmids abolished pathogenecity or resulted in galls having altered morphology. Mutagenesis of common DNA regions outside the T-DNA also affects oncogenicity (Holsters et al., 1980; Garfinkel and Nester, 1980; DeGreve et al., 1981). This suggests that both octopine and nopaline tumors share the same mechanism of oncogenicity. However, it is interesting to note that White and Nester (1980b) found little homology between A. rhizogenes plasmids and the common T-DNA region of A . tumefaciens plasmids. A similar lack of homology was reported by Thomashow et al. (1980, 1981) for certain narrow host range plasmids. However, in both cases homology was detected at lower levels of stringency suggesting that common

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W. P. ROBERTS

sequences were present but were perhaps arranged in a different manner to the “normal” Ti-plasmids. Although these studies showed that there were unique fragments formed at the ends of the T-region in plant cells indicating that the T-DNA was covalently linked to plant DNA none of them differentiated between chromosomal, mitochondrial, or chloroplast DNA. Chilton e r a / . (1980), Yadav e t a / . (1980), and Willmitzer et a / . (1980) isolated DNA from subcellular fractions of tumors. TDNA probes were used to hybridize to restriction digests of the isolated plant DNA. Both groups reported that hybridization occurred only with DNA isolated from plant cell nuclei and concluded that the T-DNA was covalently linked to plant nuclear DNA. This supports the report of Nuti et a/. (1980) who showed that Ti-plasmids hybridized to the Feulgen-positive DNA of tumor cells. At this stage it is difficult to generalize about the structure of the T-DNA in the tumor cell except to conclude that all tumors studied to date include an area of common T-DNA. It is presumably this area that is involved in maintaining the tumorous state. It is wise, however, to remember that all the tumors studied have been tobacco tumors induced by only a few strains of Agrobacterium. In most cases the tumor lines had been in culture for long periods so it is possible that the structure of the T-DNA in the cell may simply reflect the general gene rearragement that takes place in tissue culture (see, for example, DeCleene et a/. , 1980). B. PRODUCTS OF THE T-DNA 1. Transcription Drummond et al. (1977) showed that transcription takes place from the TDNA in tumor cells. They isolated pulse-labeled RNA from octopine tumors and normal tobacco tumor tissue and hybridized this to digested Ti-plasmid on cellulose nitrate. They obtained hybridization between the tumor RNA and a digestion fragment from the right-hand end of the T-DNA. This is the area thought to be involved in opine synthesis (see later). No hybridization to the common TDNA region was observed. Gurley et al. (1979) extended these findings when they reported transcription of T-DNA in three sunflower tumor lines incited by octopine strains of Agrobacterium. Their results showed that an area to the righthand end of the T-DNA was always strongly transcribed and the rest of the TDNA to a varying degree. Parts of the common DNA were transcribed strongly to not at all, depending on the particular tumor line. Yang er al. (1980a,b) looked at a nopaline tumor line of tobacco. They detected transcripts from both ends of the T-DNA but the intervening sequences were not transcribed at a level that they could detect. Willmitzer (1980) pulse-labeled RNA in highly purified nuclei of octopine tumors. This RNA was found to hybridize to restriction fragments covering most of the T-DNA in this tumor line. Bevan er al. (1981) and Murai and Kemp (1982) both report that there is extensive transcription of the T-DNA in the octopine tumors they studied. These studies confirm that the T-DNA is in


75

the plant cell nucleus and indicate that transcription can take place over most, if not all of the T-DNA. Gelvin et al. (198 1) studied the transcription of the T-DNA in the bacterium as well as in tumor cells. In the tumor cell they located specific areas of high level transcription at both ends of the T-DNA. In the bacterium slight transcription of the whole T-DNA occurred. This suggests that the T-DNA has eukaryote rather than prokaryote transcription signals and does not depend on specific insertion downstream of plant cell transcription signals. The conclusion from all these data is that the T-DNA may be transcribed to varying degrees in different tumor lines. The fact that transcription of the common DNA is detected in at least some tumors suggests that tumorogenesis involves the production of some product by the T-DNA rather than the simple integration of the T-DNA into a specific site. This will be considered in more detail in a later section. 2 . Translation McPherson er al. (1980) used RNA that hybridized to T-DNA in an in vivo translation system. RNA from one tumor line hydridizing to the right-hand end of the T-DNA produced proteins of 30,000 and 16,500 molecular weight. RNA from another tumor line lacked the smaller protein. One tumor line which did not produce octopine and in which the T-DNA region was altered in the right-hand end produced no proteins from RNA that hybridized to this region. The octopine synthase protein is approximately 38,000 (Hack and Kemp, 1980) which is significantly bigger than the protein detected in the in vitro traiislation system. McPherson et al. ( I 980) suggested that perhaps the enzyme is glycosylated thus accounting for the extra size. RNA hybridizing to the common T-DNA region from all tumors produced a protein of approximately 15,000 molecular weight. Because this region and presumably this protein is present in all tumors, it was suggested that the protein may be involved in tumorogenesis. The expression of cloned Ti fragments in E. coli minicells was studied by Schweitzer et al. (1980). Approximately five proteins with molecular weights ranging from 15,000to 45,000 were detected. Determination of the significance of these results will have to await studies looking for similar proteins in tumor cells but they do indicate the possible size and some of the properties of proteins that may be involved in tumorogenesis.

3 . Phenotypic Expression of the T-DNA The known functions of the T-DNA are opine synthesis and tumorogenesis. Several groups have attempted to map these functions more precisely using various mutagenesis mechanisms. Depicker et al. (1978a,b) found that the insertion of the plasmid RP4 into a nopaline type Ti-plasmid in the common T-DNA region abolished pathogenicity. Revertants that had lost the RP4 plamid regained pathogenicity.

76

W. P. ROBERTS

Koekman et al. (1 979) reported that a number of deletion mutants of octopine type plasmids affecting the T-DNA region were still virulent. These deletions involved approximately the right half of the T-DNA as defined by Thomashow et al. (1980a) but left approximately half of the DNA common to both octopine and nopaline Ti-plasmids. These deletion mutants were weakly virulent on Helianthus, Solanum, and Kalanchoe but avirulent on Nicotiana. None of them contained octopine. Their results suggested that opine syntheses per se was not the tumorogenic principle and that both ends of the T-DNA are not required for tumorogenicity. Ooms et al. (1980, 1981) used transpositional mutagenesis with Tn904 (Klapwijk et al., 1980) to obtain a series of mutants in an octopine Tiplasmid. They obtained a number of insertions into the T-DNA region which were virulent to weakly virulent but had an altered gall morphology. In some cases, galls similar to wild type galls could be induced by using mixtures of mutants or adding auxin during gall development. Garfinkel and Nester (1980) used Tn5 to generate a series of mutants of octopine plasmids. Like the mutants produced by Ooms et al. (1980) these were virulent to weakly virulent depending on the host and the tumors formed had an altered morphology compared to tumors from wild type strains. All these mutants produced octopine. Similar results were reported by DeGreve et al. (1981). Holsters et al. (1980) report a similar study on nopaline type plasmids using Tnl and Tn7 as mutagens. This allowed them to define the area which was essential for tumorogenicity. They also found mutations that affected the morphology of the tumor produced as well as a region to the right of the T-DNA which appeared to be involved in determining host range. The morphology of tumors formed by wild type Ti-plasmids depends on the particular plasmid/plant combination (Gresshoff et al., 1979) and even on the position of inoculation up the stem. This suggests that crown gall induction is not an all or none response and that there is some interaction between the host and the inducing bacteria. Deletions or transposon insertions into the right-hand end of the T-DNA of both octopine and nopaline plasmids abolish opine synthesis in the gall (Holsters et al., 1980; DeGreve et al., 1981) indicating that these functions are distinct from tumorogenicity. No information about the location of the genes for agropine and agrocinopine synthesis in galls is available. These functions are believed to be T-DNA encoded functions but as yet no reports indicate their position. The known functions of the T-DNA in the plant and their relative location on this DNA are summarized in Figs. 2 and 3.

C. ONCOGENIC-RELATED FUNCTIONS NOT ON T-DNA Although parts of the T-DNA are essential for tumor induction on plants, it is quite possible that other essential functions are not coded for by the T-region.


77

This may include functions involved in processing the T-DNA, its transfer to and incorporation into the plant cell DNA, survival in the wound, and bacterial attachment to the plant cell. So far, only bacterial attachment has been demonstrated to be at least partly a plasmid-coded function (Whatley et a/., 1978; Matthysse et al., 1978). Ooms et al. (1980, 1981) and Garfinkel and Nester (1980) observed that a number of transposon mutants outside the T-DNA of octopine plasmids abolished tumorogenicity. Similar results were reported by Holsters et al. (1980) for nopaline plasmids. They found at least two regions outside the T-DNA on the left-hand side of the plasmid which were essential for oncogenicity (Fig. 3). Lui et al. (198 1) also report that transposon mutagenesis into this area can abolish pathogenicity. This area appeared to be involved in the production of the plant hormone, indole acetic acid (IAA), by the bacteria and they suggested that 1AA production may be important in the initial stages of crown gall induction. All these mutations are in DNA that is common to both nopaline and octopine type plasmids again emphasizing that there probably is a common tumorogenic mechanism in all tumorogenic plasmids from Agrobacrerium. Loper and Kado (1979) and Thomashow et al. (1980~)report that the host range of Agrobacterium depends on the Ti-plasmid. Plasmids transferred from a narrow host range strain to a plasmidless strain gave a pathogen with a narrow host range and a plasmid transferred from a wide host range strain to a narrow host range strain cured of its Ti-plasmid gave a wide host range pathogen. Although these reports do not indicate where the genes for host range are located on the Ti-plasmid the mutation studies suggest that certain areas of the T-DNA may determine host range but this does not rule out the possibility that there are other areas around the Ti-plasmid involved in this function. Ooms et al. (1981) report that some transposon insertions into areas outside the T-region but in areas homologous in different Ti-plasmids gave galls with altered morphology. This is surprising as this DNA is not maintained in the gall so it is difficult to imagine how it could have a stable effect on the gall morphology. Perhaps this area is involved in transfer and processing of the T-DNA so that a mutation in this area leads to an altered T-DNA insert in the gall cell. The involvement of the plasmids in determining the morphology of the galls is further emphasized by the studies of Albinger and Beiderbeck (1977), Colak and Beiderbeck (1979), Costantino et al. (1980), and White and Nester (1980a,b) on induction of hairy root disease by A . rhizogenes. Although these studies clearly indicated that this disease is plasmid encoded, White and Nester (1980b) show that there is only slight homology between this plasmid and the common DNA of Ti-plasmids. However, it is clear that the disease is induced by the incorporation of plasmid DNA into the plant cell (Chilton et al., 1982) and that hairy root contains agropine (Petit et al., 1981). So the weak homology detected probably represents only rearrangement of the tumorogenic functions rather than a completely different mechanism.

78

W . P. ROBERTS

Several reports have shown that there is a plasmid-encoded temperature-sensitive phase in gall induction (Rogler, 1980, 1981). Rogler suggested that the temperature-sensitive step in gall induction was the temperature-sensitive replication mechanism for the Ti-plasmid (Hamilton and Fall, 1971) and that this replication mechanism was involved in tumorogenesis. Schilde-Rentschler et al. (1977) report the isolation of a number of temperature-sensitive mutants with respect to gall induction. It is possible that these are mutants in the temperaturesensititive replication mechanism of the Ti-plasmid but no data are available to confirm this hypothesis. D. NONONCOCENIC FUNCTIONS ON THE TI-PLASMID The known functions of both octopine and nopaline type plasmids are summarized in Figs. 2 and 3. The best studied functions are those for the catabolism of the opines. Both these functions map to the right of the T-DNA region in both octopine and nopaline type plasmids. Holsters et al. (1980) found two regions involved in nopaline catabolism. Mutations in one of these regions affected the catabolism of argine and ornithine. These compounds are thought to be breakdown products of some opines and catabolic genes for them are located on the Tiplasmid (Ellis er al., 1979). All this region may be involved with the breakdown products. The genes for the catabolism of another opine, agropine, are further around the plasmid. Agrocinopines (Ellis and Murphy, 1981) are thought to be the true substrates for the plasmid-encoded uptake system for agrocin 84 (Engler et al., 1975; Murphy and Roberts, 1979) and so this function should map to the same region. Although Ellis and Murphy (1981) showed that Tn7 insertion into the agrocin 84 sensitivity gene prevented catabolism of one of the agrocinopines, they did not show this was due to lack of uptake and a number of agrocin 84resistant strains could catabolize certain agrocinopines. Transfer functions for both plasmid types are shown. Despite suggestions that plasmid transfer and gall induction may share common biochemical events (Temp6 et al., 1977), Holster et al. (1980) were able to obtain mutants which were transfer negative and still oncogenic. Although this does not entirely rule out the possibility that there are common mechanisms, it is clear that a complete functional transfer mechanism is not an absolute requirement for tumorogenesis. However, the transfer functions of the Ti-plasmid are involved with crown gall in a unique way. Ken et al. (1977), Genetello et al. (1977), and Petit et al. (1978) report that conjugative transfer of the Ti-plasmid in Agrobacterium was induced only when either octopine or nopaline was present. For example, the presence of octopine in the mating system increased the frequency of transfer of an octopine Ti-plasmid from an undetectable level to a frequency of l o - ' transconjugants per recipient. The transfer frequency of nopaline type plasmids

79


was much lower at approximately ]OW4 transconjugants per recipient and despite the earlier report (Ken et al., 1976) does not seem to be induced by nopaline (Petit er al., 1978; Ellis and Kerr, 1981). Petit and Temp6 (1978) and Klapwijk et al. (1977a) both studied mutants affected in opine transport and catabolism. They found mutants could be obtained which were constitutive for opine catabolism and showed a high frequency of plasmid transfer in the absence of octopine and they concluded that both processes shared a common regulatory gene but had separate operons. Klapwijk and Schilperoort (1979) extended this work and reported that there was a common repressor system for the two functions. The repressor from a quite different compatible nontumorogenic octopine plasmid was functional in a strain containing a repressor-defective octopine plasmid; this suggests a common mechanism of regulation in both these plasmids. None of these studies on opine-induced transfer and catabolism of the opines has shown any obvious relationship between transfer ability and tumorogenicity. Other functions known or thought to be located on the Ti-plasmid are bacteriophage AP 1 exclusion (Schell, 1975) incompatability between Ti-plasmids (Hooykaas et al., 1980) and periplasmic proteins (Sonoki and Kado, 1978). Despite the functions of the Ti-plasmid detailed above, there is still a very substantial portion of the Ti-plasmid with no known functions. These plasmids are large and may constitute up to approximately 5% of the total DNA of the cell and it is unlikely that they would be so stably maintained in the cell if they did not have some adaptive advantage. This argument suggests that there may well be many functions still to be discovered on the Ti-plasmid. Some of the possibilities are survival and competition in the soil and the ability to utilize other substrates.

E. TI-DNA TRANSFER AND INTEGRATION INTO

THE

PLANTCELL

Almost nothing is known about the transfer mechanism of the Ti-plasmid to a plant cell. Possible mechanisms range from a highly specific transfer mechanism elaborated by the bacteria to a nonspecific release of DNA into the plant wound. Although the need for viable bacteria and apparent specific binding of Agrobacterium to plant cells suggest that a specific transfer mechanism is involved, Davey et al. (1980) report that Petunia protoplasts could be transformed with isolated Ti-plasmid. Transformed cells selected for hormone-independentgrowth were screened for lysopine dehydrogenase, octopine synthesis, and production of overgrowths when grafted onto normal plants. The results were consistent with the cells being transformed crown gall cells. Tests for the presence of T-DNA in the transformed tissue were not reproducibly positive but as the tissue had not been cloned it is possible that the tumors were a mixture of normal and transformed cells. Krens et al. (1982) reported reliable transformation of isolated

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tobacco protoplasts. The transformed nature of these cells was demonstrated by the detection of octopine and T-DNA in them. These experiments suggest that (1) Agrobacterium does not have to process the Ti-plasmid before it is transferred to the plant cell, (2) a highly developed transfer system involving the bacteria is not an absolute requirement, and (3) the Ti-plasmid alone is sufficient for tumorogenesis. In both the in vitro transformation experiments the efficiency of the process was very low requiring a high concentration of plasmid DNA to transform a few cells. This may be due to the lack of an Agrobacterium-mediated transfer and processing system for the T-DNA or it may simply reflect the problem of maintaining and delivering a fragile DNA molecule to the correct plant cell site in an environment that is likely to contain DNase and many other interfering compounds. It is possible that the major role of the bacteria is simply protecting the Ti-DNA and releasing it at a specific site on the plant cell to give a high level of transformation. This hypothesis can be at least partly investigated by refining the in vitro transformation techniques using methods such as liposomes to protect the DNA until it is in the plant cell. It will be interesting to see whether the efficiency of the process can be improved to the level achieved by viable agrobacteria. Very little is known about the mechanism or the precise site of integration of the T-DNA in the plant genome. Although the in vitro transformation experiments above suggest that the total Ti-plasmid enters the cell, it is not possible to eliminate the possibility that the transformation observed in these experiments was due only to a fragment of the Ti-plasmid present in the DNA samples. If this were the case then perhaps the low efficiency of transformation observed in these experiments is due to the low concentration of the required fragment in the Tiplasmid preparations. That the whole Ti-plasmid enters the plant cell is also suggested by the detection of considerable Ti-plasmid sequences in plant cells up to approximately 9 days after infection (Nuti et al., 1980). After this period no Ti-plasmid sequences were detected and it was suggested that the T-DNA was present below the level of detection by the methods used. Although this suggests that the Ti-plasmid is processed in the plant cell to give the T-DNA, it is difficult to evaluate the significance of this type of experiment. The major problem is deciding which cells are being transformed, given that crown gall induction can be a one cell process. On the available evidence then it is not possible to decide where processing of the Ti-plasmid to give the T-DNA occurs. Although it is possible that oncogenic Ti-plasmid functions not on the T-DNA region are involved in this processing, there are no experimental data supporting this suggestion. It may be significant that apparently no specific mechanism is required for the entry and integration of “foreign” DNA into cultured animal cells (Wigler et af.,1979; Hsiung et al., 1980). These reports indicate that prokaryote DNA


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including bacteriophage +x 174 and pBR322 could enter and integrate into the DNA of cultured mouse cells. Plant cell protoplasts lack a complex cell wall and so, at least superficially, are like cultured animal cells, and it is possible that the in v i m transformation experiments with isolated Ti-plasmid involve a similar nonspecific uptake and integration system as has been demonstrated with animal cells. Possibly the wound is required only to remove enough cell wall to expose the cell membrane to allow uptake of released Ti-plasmid DNA. Although the site of integration of the T-DNA into plant DNA varied from tumor line to tumor line and between different strains of agrobacteria (Merlo et al., 1980; Chilton et al., 1980; Zambryski et a!. , 1980; Lemmers et al., 1980; Thomashow et al., 1980a) in all the cases studied, it was inserted in repetitive sequence plant DNA. However, as data were available only for a few tobacco galls, it is impossible to assess the significance of this finding. In tobacco repetitive DNA of some kind makes up approximately 75% of the total genome (Zimmerman and Goldberg, 1977) so it is quite possible that random insertion of T-DNA into the plant genome could give the reported results. There are two conflicting views of the role of repetitive sequence DNA is eukaryotes. One model (Davidson and Britten, 1980) suggests that this DNA is involved in gene regulation but the other suggests that much of it is simply “selfish” DNA making no specific contribution to the phenotype of the organism (Orgel and Crick, 1980; Doolittle and Sapienza, 1980). The Davidson-Britten model would suggest that the simple integration of foreign DNA in a critical regulatory area could cause crown gall. However, the evidence reviewed above showing that the T-DNA can integrate into a number of different sites, has a strongly conserved area essential for tumorogenesis, and is transcribed and translated in the gall suggests that the simple integration of the TDNA into a specific site is not the key event leading to tumorogenesis. If this were true, it would mean that integration of DNA into the plant genome may not induce a gall if the DNA lacks the tumorogenic functions. This hypothesis, if correct, has considerable practical importance for the use of Agrobacterium for genetic engineering in plants (see later section). Assuming that the T-DNA codes for some product, two of the possible mechanisms for tumorogenesis are (1) that the T-DNA codes for the production of plant hormones that are directly responsible for the establishment and maintenance of the tumorous state or (2) the T-DNA codes for products that either directly or indirectly deregulate the normal hormonal control of the cell. Lui and Kado (1979) suggested that IAA production coded for by the Tiplasmid is necessary for gall induction but as the information for this was in an area of the Ti-plasmid that does not integrate into the plant cell, this cannot be involved in tumor maintenance. There are also many studies (see section on physiology of gall) that show that crown gall cells have qualitative and quantitative differences in hormones compared to normal cells, however, none of these

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indicates if this is due to hormone production being directly coded for by T-DNA or that the T-DNA alters the level of hormones produced by the preexisting plant genes. From the available evidence, it is not possible to decide which of the suggested mechanisms of tumorogenesis is correct. F. REVERSIONTO THE NORMALSTATE Any model of crown gall induction has to take into account that tumors can sometimes revert to an apparently normal state and in some cases then revert back to the tumorous condition. Prior to the discovery of the Ti-plasmid and the role of the T-DNA in gall induction there were many reports of reversion of crown gall tissue to apparently normal plants (see review by Lippincott and Lippincott, 1975). However, because there was no definitive test for transformed cells, it is impossible to rule out the possibility that reversion in some of these studies was due to a few normal or habituated cells in the gall, rather than a genuine reversion of transformed cells. It is necessary to clone the gall line and check for opine production and the presence of the T-DNA, both before and after reversion of the tissue. The capacity to revert to normal varies between tumor lines and with the length of time the tumor has been in culture (Turgeon et al., 1976; Sacristan and Melchers, 1977). Braun and Wood (1976) and Turgeon et al. (1976) report regeneration of crown gall tissues into apparently normal plants. In their experiments teratoma shoots were grafted onto tobacco plants and some of the grafted shoots produced flowers and set seeds. The fact that this tissue was still tumorous was demonstrated by the fact that the apparently normal tissue produced nopaline and could grow in the absence of any added growth hormone. The F, generation from the seed produced completely normal plants which no longer produced nopaline or had the ability to grow autonomously in the absence of plant hormones. Yang et al. (1980a) looked at this system at the molecular level. The “normal” plant tissue that appeared on the teratomas grafted onto tobacco plants still retained the T-DNA but at a lower concentration. They were not able to decide from their experiments if the decrease in T-DNA was due to a decrease in copy number of all or some of the T-DNA or to a specific loss of a portion of the T-DNA. They were not able to detect the presence of T-DNA in the F, generation grown from the seed set by the grafted tumor lines and concluded that they were normal because of the lack of T-DNA. Lemmers et al. (1980) looked at the same set of regenerated tumor lines and substantially confirmed the work of Yang et al. (1980a). They report that all of the T-DNA was represented in regenerated plants but there appeared to be some rearrangement of the T-DNA ends. Like Yang et al. (1980a) they found no TDNA in the F, generation from seed of the regenerated plants.


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It is not clear whether the grafting process and meoisis induce the reduction and subsequent loss of T-DNA or whether these events only selected for the cells in the tumor population that had reduced or lost the T-DNA. However, in this regard, O’Schieder (cited in Lemmers et al.. 1980) has reported that approximately 1% of the cells in one of the regenerating cell lines (BT37, Braun and Wood, 1976) is not transformed. The only other molecular-based study of regeneration into normal plants is that of Wullems et al. (1981). Shoots obtained from tumorous tissue and which contained opines were grafted onto healthy plants. Some produced male sterile aberrant flowers and were pollinated by normal pollen. Approximately 40% of the F, seedlings contained opines. They also contained T-DNA. Some of the plants did not contain opines but did contain T-DNA as shown by hybridization experiments. These results indicate that the T-DNA can survive through the process of meiosis.

VIII. Significance of Crown Gall Induction to Agrobacterium The aim of any pathogen is to obtain nutrition and/or a favorable environment from the host species and in this respect Agrobacterium is no exception. However, its method of achieving this aim is unique. The transfer and insertion of the T-DNA into the plant appears to be a specific genetic engineering system by the bacteria to induce the plant to produce opines. These opines are then used as carbon, nitrogen, and phosphorus sources by the bacteria, presumably conferring a selective advantage on those bacteria able to utilize these compounds. This process has been called “genetic colonization” by Schell et a f . ( 1979). Although there are many studies that show that Agrobacterium strains carrying Ti-plasmids can utilize opines in vitro, there are no studies that evaluate the significance of these compounds to the growth and survival of the bacteria in vivo. New and Kerr (1972) report that the ratio of pathogenic to nonpathogenic Agrobacterium was significantly greater around a gall than around a normal root: they suggested that the high number of pathogens around a gall was the cause of gall induction. However, if the opines were significant in enhancing survival and growth of Ti-carrying agrobacteria then high numbers should result from the presence of a gall although, presumably, a certain number of pathogenic bacteria is required to ensure that gall induction takes place. Opines are also involved in induction of Ti-plasmid transfer (Kerr et a f . , 1977; Genetello et a f . , 1977; Petit et al., 1978). This transfer system was first reported by Kerr (1969, 1971) who demonstrated that nonpathogenic agrobacteria acquired pathogenicity when swabbed on galls formed on tomato stems. Up to 75% of single colony isolates of the original nonpathogenic strain became pathogenic

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indicating that either the process was very efficient or that there was very strong selection for converted pathogens on the gall. The efficiency of this process under conditions approaching “natural” conditions suggests that it may be significant in the ecology of Agrobacteriurn. Perhaps the high level of pathogens around galled plants reflects both the selective advantage of opine catabolism and the conjugative spread of the Ti-plasmid to nonpathogens. Although there seems an obvious reason why agrobacteria should induce opine production, an answer to the question, why do they induce a gall is not as obvious. It is clear that opine production is not the tumorogenic principle and that strains inducing tumors containing no (known) opines have been found. It is conceivable that nontransformed plant tissues producing opines could be produced and indeed some of the revertant tumors are like this phenotypically. So it is not unreasonable to suggest that cell transformation and the resultant gall structure must have some adaptive advantage to agrobacteria other than simply opine production. One function of transformation may be to increase the number of cells producing opines and so the concentration of opine available to the bacteria. Another possibility is that the structure of the gall itself provides favorable environmental conditions for the growth of agrobacteria. The capacity for opine catabolism and agrocin 84 sensitivity is not always associated with the Ti-plasmid (Merlo and Nester, 1976; Kerr and Roberts, 1976; Hooykaas et al., 1979; Roberts, 1981) and in at least some strains that can catabolize both nopaline and octopine it is only the nopaline catabolic functions that are transferred with the Ti-plasmid suggesting that the octopine catabolism function is on the chromosome. The non-Ti-plasmid location of these functions may reflect the original genetic source of these functions but, more likely, simply reflects recombination between Ti-plasmids, other plasmids, and the Agrobacteriurn chromosome.

IX. The Evolutionary Origin of Crown Gall As crown gall induction is such a complex process involving the transfer and expression of a defined segment of bacterial DNA in a plant cell, it is difficult to think of a mechanism that would allow the slow development of the system by the mutation and selection of preexisting bacterial genes. The alternative origin is that the main elements of the process existed and, at some stage, combined to give a more or less working scheme. Perhaps the simplest hypothesis would be that the prototype crown gall organism grew around the roots of a plant which produced opines or opine-like substances. By the normal processes of selection and adaption the organism acquired the ability to utilize these compounds and possibly gained a competitive advan-


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tage in this environment. At some stage the bacteria also acquired the genes from the plant to produce these compounds and the mechanism (if there is a specific mechanism) to insert these genes into a suitable host. Of course, the major question with this scheme is how could the bacteria acquire these genes from the plant? Two possible mechanisms are (1) a plant virus picked up plant DNA and subsequently established as a plasmid in the bacteria; and (2) plant DNA perhaps from decaying plants simply transformed into and perhaps recombined with preexisting plasmids or chromosomal DNA to make the prototype Ti-plasmid. On the available evidence, it is difficult to decide which of these mechanisms is more likely or indeed whether either mechanism is likely. Of course, they are not mutually exclusive. The current methods of DNA cloning effectively duplicate the sort of process that underlies both these suggested origins of the Tiplasmid and the mechanism of gall induction. Available techniques allow DNA from any origin to be cloned into either eukaryotic or prokaryotic organisms and with the right manipulations in some cases even be expressed. The soil and indeed most natural environments are constantly being exposed to DNA from many organisms and it is quite possible that there is much more genetic exchange between organisms than is in fact realized (Reanney, 1977). These suggested possible origins of crown gall would imply that ( I ) opines were originally plant products, (2) some of the T-DNA sequences may be present in normal plant DNA, and (3) some of the T-DNA may be homologous with plant viruses. There is some evidence that compounds structurally similar to some of the opines are found in plants. Durzan and Chalapa (1976) report the occurrence of a whole range of substituted guanidines including one compound in callus tissue and seedlings of Pinus banksiana that cochromatographed with octopine. It may be worthwhile looking for the presence of opines in nonhost plants for crown gall on the basis that these plants or their ancestors may have been the origin of these genes and so may have the mechanism to control them. If so they might be resistant to gall induction. It is interesting that Thomashow et al. (1980a) reported some slight hybridization between normal plant DNA and the T-DNA. This has not been confirmed by other workers but it is quite likely that if the TDNA did come originally from plant DNA that it may have diverged in sequence considerably and so detection of any hybridization between it and plant DNA may depend on the selection of the correct plant DNA and the use of low stringency hybridization conditions. There are no reports of homology between T-DNA and plant viruses. The problem with all these predictions is that if one of the mechanisms suggested above is correct these predictions may have only held true at the time the mechanism of crown gall was evolving and evolution since this time may have obliterated any trace of original conditions. Still they are quite testable experimentally and may be worth pursuing. Certainly, it would be surprising if evolu-

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tion has not tried the sort of experiments that many molecular biologists are now attempting. Perhaps crown gall is an experiment that was successful, at least for the bacteria.

X. Agrobacterium and Genetic Engineering A good deal of the interest in the crown gall area is due to the possibility of using the Ti plasmid system for the genetic engineering of plants. The type of experiments suggested is the insertion of desirable genes into the area of the T-DNA of a Ti-plasmid which is not essential for integration of this DNA into the plant cell using available in v i m recombinant DNA technology. This would then be used to induce crown gall tissue containing the new genes. Subsequent manipulations would recover a plant carrying the desirable characteristics in a form that would be agriculturally useful. At least the first steps of this process have been shown to be feasible. Hernalsteens et al. (1980) inserted transposon Tn7 into the area of the T-DNA coding for nopaline synthesis. Tn7 was subsequently maintained in tumor tissue induced by this modified Ti-plasmid. Tn7 has a molecular weight of 9.6 X lo6 so this approach has the potential to incorporate a substantial amount of DNA into the plant genome. Prokaryotic genes such as Tn7 are not likely to be expressed in eukaryotes as they lack the necessary eukaryotic promotor. One approach around this problem is to insert the required gene in the correct phase downstream from the opine promotor in the T-DNA. This should allow the expression of genes lacking eukaryotic promotors. Although the techniques are available to allow the insertion of foreign DNA into plants using this system, how a useful plant can be produced from this is still not obvious. One of the main problems is selecting a plant that has retained the added genes but is no longer tumorous. The work on reversion reviewed earlier suggests that this may not be impossible but that the resultant plants could well be abnormal both in genotype and phenotype. It is not clear whether this is only due to the tranformation process or is at least partly due to tissue culture allowing the development of chromosomal abnormalities, but this is an area that will require much more extensive study before the system can be routinely used for genetic engineering in plants. One possibility is the separation of the transformation process from the insertion process. If these are separate processes, then a reconstructed plasmid carrying only the insertion mechanism, the desired gene, and some selectable marker may well make an efficient system for incorporating new genes into plants. However, at the present time there are few selectable markers that could be used in such an approach.


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One of the problems with using Agrobacterium for genetic engineering in plants is the host range of the disease. Although a range of economically important plants is affected some of the major food crops such as the cereals are not. This may limit utility of an Agrobacterium approach to genetic engineering unless this barrier can be overcome. If the limitation in host-range occurs at the level of survival of Agrobacterium and transfer to the plant then it may be possible to utilize in vitro transfer of the T-DNA by methods similar to those of Davey et al. (1980) and Krens et al. (1982). However, if it reflects a fundamental difference between hosts and nonhosts at the molecular level of T-DNA insertion and transformation then the limited host-range may be a fundamental barrier to the application of a genetic engineering system based on Agrobacterium.

XI. Future Work and Prospects Perhaps the area where least is known about crown gall induction is the process of transfer and integration of the T-DNA into the plant cell. This is an area which is very difficult to study as it involves the interaction of just a few bacteria with a few plant cells. Important questions in this area include: 1. Is the T-DNA involved in transfer and insertion into the plant DNA or is it only involved in tumorogenesis? 2. Does the complete Ti-plasmid enter the plant cell or only the T-DNA? 3. What is the function of the Ti-plasmid outside the T-DNA that is required for gall induction? These and other questions about crown gall induction may be answered by improvements in the in vitro transformation system (Davey et al., 1980; k e n s et al., 1982). Such a system combined with the use of in vitro modified Ti-plasmids and plasmid fragments may be crucial in determining the precise molecular mechanism of crown gall induction. Increases in the efficiency of the in vitro transformation systems may be possible using liposome incorporation of DNA and improvements in techniques for producing and handling protoplasts. There is also very little information on how the products of the T-DNA lead to cell transformation. This is a process that probably has direct bearing on gene regulation and development of plants. Crown gall offers a unique opportunity in that DNA involved in developmental changes can be isolated and manipulated conveniently and work in this area may significantly advance knowledge of developmental processes in plants. Other areas that may well bear further study are the ecology of Agrobacterium

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in relation to the opines, the significance of Ti and other Agrobacterium plasmids transfer in natural systems, and the basis of the host specificity of Agrobacterium.

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The Molecular Cytology of Wheat-Rye Hybrids* R. APPELS Division of Plant Industry, CSIRO, Canberra, Australia

1. Introduction

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

11. The Genetic Relationship between Rye and Wheat Chromosomes

.

Ill. The Molecular Structure of Rye and Wheat Chromosomes . . . . . . . IV . Translocations in Wheat-Rye Addition or Substitution Lines . . . . . V. Polymorphisms in Regions of the Chromosomes Containing Repeated Sequence DNA .................................. VI . The Biological Effects of Rye Chromosomes (or Rye Chromosome Fragments) in Wheat-Rye Hybrids: Specific Effects Related to Heterochromatin ......................................... VII. The Possible Origins of Polymorphism in Rye Heterochromatin . . . VIII. Prospects ............................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

93 94 99 109 113

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I. Introduction Biologists have, for many years, combined the genome of one species with that of another species to produce a new combination of genomes interacting in the one cytoplasm. In animal cell biology this has been achieved mainly in tissue culture by the induced fusion of cells or using microinjection techniques. This type of manipulation provided opportunities for mapping chromosomes genetically (Ringertz and Savage, 1976), defining the biological prerequisites for expression of genes characteristic of a differentiated state (Ringertz and Savage, 1976; Willing etal., 1979; Allan and Harrison, 1980; Halaban et al., 1980), and analyzing the biochemistry of nuclei undergoing extensive structural change in a short period of time (Appels and Ringertz, 1975; Linder et al., 1981). In plants, interspecies combinations of genomes have also been achieved by cell fusion (Schieder and Vasil, 1980) as well as by natural means using cross-pollination followed by chromosome doubling with colchicine (Blakeslee and Avery, 1937). The manipulation of plant genomes has attained practical levels in providing commercially grown wheat with resistance to stem rust from Elytrigia pontica (Podp.) McGuire et Dvorak (= Agropyron elongaturn) (Host.) P.B. (Sr26), from *The term hybrid is used in a broad sense to include derivatives of primary wheat-rye hybrids such as triticales and wheat-rye addition and substitution lines. 93 Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-864480-0

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Secale cereale (Sr31), from Triticum timopheevi Zhuk ( S I ~ C as ) , well as from diploid wheats (reviewed by Knott and Dvorak, 1976). The development of wheat-rye hybrids into the new crop triticale ( X Triticosecale Wittmack) highlights the potential for combining the genomes of different species (for recent reviews see Gustafson, 1976, 1982). Attempts to form hybrids between wheat and other cereals such as rye date back to 1876 (for reviews see O’Mara, 1953; Briggle, 1969). Present-day cereal cytogenetics allows alien chromosomes to be introduced into wheat in an efficient manner (Sears, 1975) and it is the aim of this article to examine in detail how such studies have allowed new features of the wheat and rye genomes to be uncovered. Emphasis is placed on the analysis of rye chromosomes because the production of wheat-rye hybrids has been particularly significant in allowing the assignment of genetic and cytogentic markers to specific chromosomes.

11. The Genetic Relationship between Rye and Wheat Chromosomes An important use of wheat-rye substitution lines has been in the investigation of the genetic relationship between wheat and rye chromosomes. The 21 chromosomes of hexaploid wheat can be partitioned into seven homoeologous groupings (each containing three chromosomes) using nullisomic-tetrasomic tests (Sears, 1966). These groups are designated 1-7 for each of the A, B, and D genomes of hexaploid wheat. Homoeology is defined by the degree to which a given chromosome can overcome nullisomy for another chromosome and is thus a measure of the genetic similarity of chromosomes. Although the sensitivity of the assay depends on the severity of the nullisomic phenotype which is being rescued, the chromosomes of Secale cereale (rye) have been placed into the homoeologous groups defined in wheat (Sears, 1968; Gupta, 1971; Koller and Zeller, 1976). The extension of homoeologous chromosome relationships beyond hexaploid wheat requires the production of wheat-alien chromosome addition lines which can then be utilized for the production of substitution lines where an alien chromosome substitutes for a wheat chromosome (Sears, 1975). This procedure can generate artifacts, as discussed later, but has clearly shown that rye chromosomes are sufficiently closely related to wheat chromosomes to allow substitution to occur. This relationship is also observed when specific molecular markers such as allozymes (Hart, 1979), seed storage proteins (Shepherd, 1973; Lawrence and Shepherd, 1980), and genomic arrangement of the DNA sequences for 5 S, 18 S, and 26 S rRNA genes (Appels et al., 1980) are examined. The comparison of 1R and 1B is particularly striking since the relative chromosomal positions of the 5 S and 18 S-26 S rDNA regions have been retained. Figure 1 summarizes a number of markers which are located in the same approximate position in the respective wheat and rye chromosomes. Figure 1 also lists the genetic markers on rye

WHEAT-RYE HYBRIDS

95

chromosomes which do not show clearly defined homoeology with wheat; the extent of this list (see also Schlegel, 1982) indicates that homoeologous relationships have not been strictly maintained during the course of evolution, a point taken up later in this section. The differences in homoeologous genetic markers such as seed storage proteins and allozymes are detectable but are not so great as to prevent their expression, and the association of heterologous subunits of enzymes into active molecules in wheat-alien chromosome combinations (Hart, 1979). The 18 S-26 S rDNA and 5 S rDNA regions of 1R and 1B are similar in the size of the basic repeating unit as estimated using restriction enzyme sites located within the conserved gene sequences (Fig. 1). Spectacular differences are, however, observed when repeated sequences such as those found in heterochromatin (discussed later) or in the spacer regions of the 18 S-26 S rDNA and 5 S rDNA regions, are examined. The 5 S rDNA regions of rye and wheat are strikingly different when a restriction enzyme such as Hue111 is used to probe the region since the enzyme has cleavage sites located in the spacer region (Appels et al., 1980; Gerlach and Dyer, 1980). Even within wheat the two basic types of repeating unit of 5 S rDNA are highly diverged with respect to their spacer sequences (Gerlach and Dyer, 1980). The 18 S-26s rDNA spacer region of wheat has been mapped in detail (Appels and Dvorak, 1982). The most significant feature of the spacer region is the presence of 11-13 130-bp repeat units analogous to the repeated structure seen in animal spacer regions (Federoff, 1979; Rae et al., 1981; Kunz et al., 1981). The two major rDNA locations on 1B and 6B can be distinguished by the length of the spacer region but sequence divergence between these two chromosomes is very small compared to their divergence from the corresponding sequence in 1R. When the 130-bp repeated sequence is used to probe the rDNA region, high levels of divergence are observed between 1R and 1B (or 6B) (Fig. 2). The melting curves in Fig. 2 are the result of hybridizing a radioactive 130-bp sequence to either wheat (cytogenetic stocks of cv. Chinese Spring) or rye (cv. Imperial) DNA immobilized on nitrocellulose filters and determining the temperature at which radioactivity is lost from the filter. The difference observed in Fig. 2 is much greater than observed in the comparison of the entire genomes of wheat and rye by analyzing isolated DNA; on average, the genomes of rye and wheat have diverged to give a T,,, difference (in the heterologous hybridization reaction) of only 4°C (Bendich and McCarthy, 1970; Flavell et al., 1977). Since this is an average of conserved gene regions and diverged spacer regions this is not inconsistent with the large T , difference observed in Fig. 2. Translocations during the course of evolution tend to diffuse the homoeologous relationships with respect to the position of a particular locus on a chromosome. Secale cereale differs from the more primitive Secale montanum (which is considered to be more closely related to wheat, Stutz, 1972) in that at least three chromosomes have been involved in translocations (Price, 1955; Riley, 1955; Heemert and Sybenga, 1972). Consistent with this observation, the

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FIG. 1. Summary of homoeologous genetic markers on rye chromosomes. The data used were taken from Appels et al. (1980). Barber et al. (1968), Gerlach and Dyer (1981), Hart (1979). Irani and Bhatia (1972), Koller and Zeller (1976), Lawrence and Shepherd (1980), May (1974), Payne and Dyer (1976), Rao and Rao (19801, Sanchez-Monge et al. (1979), Shepherd (1973), and Zeller (1976). Phosphoglucose isomerase-1 has been located to the short armof 1R (R.Koebner, unpublished data). With respect to genetic markers on rye chromosomes the reader is also referred to Schlegel (1982) and the work of J. de Vries and J. Sybenga at the Department of Genetics, Agricultural University, Wageningen (in preparation). The pachytene karyotype of rye chromosomes is from

97

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