Chromatin Structure and Function Molecular and Cellular Biophysical Methods

Part A

NATO ADVANCED STUDY INSTITUTES SERIES

A series of edited volumes comprising multifaceted studies of contemporary scientific issues by some of the best scientific minds in the world, as­sembled in cooperation with NATO Scientific Mfairs Division.

Series A: Life Sciences

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Volume 20 - Antiviral Mechanisms in the Control of Neoplasia edited by P. Chandra

Volume 210 - Chromatin Structure and Function: Molecular and Cellular Biophysical Methods edited by Claudio A. Nicolini

Volume 21b - Chromatin Structure and Function: Levels of Organization and Cell Function edited by Claudio A. Nicolini

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Chromatin Structure and Function Molecular and Cellular Biophysical Methods

PartA

Edited by

Claudio A. Nicolini Temple University Philadelphia, Pennsylvania

SPRINGER SCIENCE+BUSINESS MEDIA, LLC

Library of Congress Cataloging in Publication Data

Nato Advanced Study Institute, Erice, Italy, 1978. Chromatin structure and function.

(NATO advanced study institutes series: Series A, Life sciences; v. 21) lncludes bibliographical references and indexes. CONTENTS: pt. A. Molecular and cellular biophysical methods. - pt. B. Levels

of organization and .cel! function. l. Chromatin - Congresses. 2. Carcinogenesis- Congresses. 1. Nicolini, Claudio A.

Il. Title. III. Series. QH599.N37 1978 574.8'732 78-24268 ISBN 978-1-4684-0975-8 ISBN 978-1-4684-0973-4 (eBook) DOI 10.1007/978-1-4684-0973-4

First half of the Proceedings of the NATO Advanced Study Institute, held at Erice, Italy, Apri112-26, 1978

© 1979 Springer Science+Business Media New York Softcover reprint of the hardcover lst edition 1979 Originally published by Plenum Press, New York in 1979

AII righ ts reserved

No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microftlming, recording, or otherwise, without written permission from the Publisher

To

My Wife Julia

and

My Sons Davide and Christian

PREFACE

This book, divided in two volumes, is the result of a NATO­Advanced Study Institute, held at Erice during 1978 and aims to approach in a widely interdisciplinary fashion the problem of chromatin structure, both at molecular and cellular levels.

It has been edited in an organic and tutorial format, with the contributions of several leading scientists; to cite a few, authors range from physical chemists (as K. F. Van Holde and P. O. TS'o) biologists(as J. Bonner and J. Gilmour) molecular biophysicists(as M. E. Bradbury and D. Olins) to cytologists (as T. Casperson and M. Mendelsohn) and geneticists(as L. Sachs).

This first volume, following an introduction to the properties of isolated chromatin (section I), contains basic chapters which presents the theory and instrumentation of all techniques (section II) applicable to the study of nucleosome and chromatin (as electron microscopy, circular dichroism, hydrodynamics, Raman Spectroscopy, nuclear magnetic resonance, neutron and x-ray diffraction, equilibrium binding studies, flow birefringence, transcriptional assays) and of nuclei and chromosome (cytochemistry, automated image analysis, microfluorimetry, scanning and flow cytometry).

In order to make this volume comprehensive and accessible to a wider scientific community (particularly graduate and post­graduate students in physical biosciences) equal emphasis is placed in the presentation both for the breadth, going from molecular biophysics to biophysical cytology~ and the depth, where the numerous techniques are treated in detail.

Even if particular reference is made to the genetic apparatus and its constituents, an attempt is made to present the relevant advantages and disadvantages of each biophysical method in general terms, as applicable also to the study of any large biomolecule; towards this end, brief summaries of the relevant and fundamental physical principles are frequently given.

Claudio Nicolini

CONTENTS OF PART A

Introduction C. Nicolini

SECTION I: WHAT IS THE CHROMATIN?

Properties and Composition of Isolat.ed Chromatin

J. Bonner

Expressed and Nonexpressed Portions of the Genome: Their Separation and Their Characterization • • • • •

J. Bonner

Discussion

SECTION II: PHYSICAL, CHEMICAL AND BIOLOGICAL TECHNIQUES FOR STUDYInG NUCLEOSOME,

CHROMATIN, CHROMOSOME AND NUCLEI

Electron Microscopy: A Tool for Visualizing Chromatin

A. L. Olins

Transcriptional Control of Native Chromatin

R. S. Gilmour

Circular Dichroism of DNA, Protein and Chromatin • • • • • •

G. D. Fasman

Important Hydrodynamic and Spectroscopic Techniques in the Field of Chromatin Structure • • • • • • • • . • • •

D. E. Olins ix

xi

3

15

25

31

41

67

109

x

Preparation and Analysis of Core Particles and Nuc1eosomes: A Conveinient Method For Studying the Protein Composition of Nuc1eosomes Using Protamine-Release into Triton-Acid-Urea Gels • • • • • •

B. R. Shaw and R. G. Richards

The Interaction of Histones with DNA: Equilibrium Binding Studies

D. R. Burton, M. J. Butler, J. E. Hyde, D. Phillips, C. J. Skidmore and I. O. Walker

Nuc1eosome Shape and Structure in Solution from Flow Birefringence

R. E. Harrington

Scattering and Diffraction by Neutrons and X-rays Study of Chromatin

J. F. Pardon

in the

Nuclear Magnetic Resonance Studies of Nucleic Acids and Proteins

P. O. P. Tslo and L.-S. Kan

Techniques for Cytochemical Studies of the Nucleus and its Substructures

T. Caspersson

Chromatin Study in Situ: I. Image Analysis F. Kendall, F. Be1trame and C. Nicolini

Chromatin Study in Situ: II. Static and Flow Microf1uorimetry

C. Nicolini, S. Parodi, S. Lessin,

CONTENTS OF PART A

125

137

167

187

217

251

265

293

A. Belmont, S. Abraham, S. Zietz and M. Grattaro1a

Chromatin Study in Situ: III. Differential Effects of Feu1gen Hydrolysis • •

W. A. Linden, S. M. Fang, S. Zietz and C. Nicolini

323

Scanning and Flow Photometry of Chromosomes • • • • • • •• 341 M. L. Mendelsohn

Discussion 357

Index • • • xxi

CONTENTS OF PART B

Introduction C. Nicolini

SECTION' III: VARIOUS LEVELS OF CHROMATIN ORGANIZATION AND MECHANISMS

FOR TRANSCRIPTIONAL CONTROL

Histones Assembly and Their Structural Role for Nucleosome Core

N. M. Maraldi, S. Capitani, L. Cocco and F. A. Manzoli

Nuclease Digestion and the Structure of Chromatin • • • • • • •

K. E. Van Holde, J. R. Allen, J. Corden, D. Lohr, K. Tatchell and W. O. Weischet

Reconstitution of Nucleosomes • • • K. Tatchell and K. E. Van Holde

Conformation of Polynucleosomes in Low Ionic Strength Solution

B. R. Shaw and K. S. Schmitz

Chromatin Structure: Relation of Nucleosomes of DNA Sequences •••••••••

A. Prunell

Histone Complexes, Nucleosomes, Chromatin and Cell-Cycle Dependent Modification of Histones • • • • • • • • • • • •

H. W. E •. Rattle, G. G. Kneale, J. P. Baldwin, H. R. Matthews, C. Crane-Robinson, P. D. Cary, B. G. Carpenter, P. Suau and E. M. Bradbury

xi

xiii

371

389

413

427

441

451

xii CONTENTS OF PART B

Evidence for Superstructures of Wet Chromatin ••••••

S. Basu

Chromatin Fractionation and the Properties of Transcriptionally Active Regions of Chromatin • • • • • • • • • • •

J. Gottesfeld

Chromatin Reconstitution and Non-Histone Proteins

R. S. Gilmour

Discussion

SECTION IV: STRUCTURE-FUNCTION OF THE GENETIC APPARATUS AND CELL CYCLE, AGING, NEOPLASTIC TRANSFORMATION,

DIFFERE1ITIATION, CHEMICAL CARCINOGENESIS

The Structure and Function of Chromatin in Lower Eukaryotes

K. E. Davies and I. O. Walker

Chromatin Structure from Angstrom to Micorn Levels, and Its Relationship to Mammalian Cell Proliferation

C. Nicolini

Chromatin Pattern in Situ: Dependence upon Cell Cycle, Preimplantation and Development, and Cellular Aging in Vitro .......... .

W. Sawicki

Neoplastic Transformation: The Relevance of in Vitro Studies for the Understanding of Tumor Pathenogenesis and Neoplastic Growth • • • • • • • • • • . . • . . .

L. A. Smets

Cell Differentiation and Malignancy in Leukemia • • • • • • •

L. Sachs

Cellular Morphometry in Transformation, Differentiation and Aging • •

S. Parodi, G. Brambilla, F. Beltrame, S. and C. A. Nicolini

Lessin

515

541

561

593

599

613

667

683

705

721

CONTENTS OF PART B

Basic Mechanisms in Chemical Carcinogenesis • • • • • • • • P. O. P. Ts'o

Carcinogen Induced Alteration in Gene Packing and Its Possible Significance in Carcinogenesis

P. M. Rao, S. Rajalakshmi and D. S. R. Sarma

Covalent Binding of a Carcinogen to DNA as a Probe of Chromatin Structure

F. X. Wilhelm, M. L. Wilhelm and G. Metzger

Carcinogenesis, DNA Repair and Chromatin W. G. Verly and L. Thibodeau

Electromagnetic Induction of Electrochemical Information at Cell Surfaces: Application to Chromatin

xiii

751

771

781

803

Structure Modification • • • • •• 811 A. Chiabrera, M. Hinsenkamp, A. A. Pilla and C. Nicolini

Discussion

SECTION V: REVIEW AND SUMMARY OF THE GENETIC APPARATUS

Session I: Basic Components of the Genetic Apparatus • • • • • • • • • • •

E. M. Bradbury, S. Bram, G. Fasman, D. Olins, J. Pardon, A. Prunell, R. Sperling, K. E. Van Holde and I. Walker

Session II: The Second Level of Organization -Chromatin . . . . • . . . . . • . . •

E. M. Bradbury, G. Fasman, S. Gilmour, J. Gottesfeld, C. Nicolini, D. Olins, J. Pardon, B. Shaw and F. X. Wilhelm

Session III: The Third Level of Organization E. M. Bradbury, S. Bram, J. Gottesfeld, F. Kendall, C. Nicolini and I. Walker

Session IV: Generalized Biological Effects •••• A. Chiabrera, W. Linden, C. Nicolini, S. Parodi and W. Sawicki

Index ••

841

849

855

861

867

871

INTRODUCTION

During April 12-26, 1978, the eighth course of the International School of Biophysics, a NATO - Advanced Study Institute, was held at the "Ettore Majorana Center for Scientific Culture" in Erice, Sicily, co-sponsored by the North Atlantic Treaty Organization, National Science Foundation (USA), The Italian Government and the European Molecular Biology Organization.

The subject of the course was "Chromatin Structure and Function" with 91 participants (from 15 different countries) selected world­wide.

The current high level of interest in the structure and function of chromatin"is adequately testified by the thousands of manuscripts which have appeared in the literature during the past five years which have pertained to areas directly related to these subjects. The scope and depth of knowledge and range of disciplines which have been brought to bear in the study of chromatin structure and its relation to cell function are indicated in several recent review articles.

One of the objectives that the Erice course has successfully accomplished has been to promote the close communication and colla­boration among scientists active in this field of "chromatin" with different backgrounds and expertise, such as: biologists, physicists, biophysicists, biochemists, engineers, and physicians toward an advancement of knowledge in this basic and interdiscipli­nary field of life sciences.

The implications of a definite characterization of chromatin structure and function are now obvious since they bear directly on the mechanisms of cancer, aging, medical genetics, chemical carcinogenesis, and cell proliferation.

During the Advanced Study Institute and consequent proceedings, now published by PLENUM, we adopted a stpuctured, organic and comprehensive appPOach to the pPObZem of chromatin structure and function (both at the moZecutar and ceZZutar ZeveZ) with focus on

xv

xvi INTRODUCTION

the methodoZogies3 techniques and on the various ZeveZs of chpomatin opganization3 stpessing theip impZications fop ceZZ function.

Today new knowledge, not only in biophysics which is at the cpossing of severuZ ''hapd'' and "soft" sciences, is frequently produced by deeply interdisciplinary interactions among scientists of different backgrounds. In this respect, chromatin constitutes a unique example since we may identify at least three dimensions where research is conducted: one (X-axis), al~ng the level of chromatin organization studied from the Angstrom (histone protein octamers and the nucleosome) through the multimeters and solenoid, up to the micron level, i.e. intact interphase nuclei and metaphase chromo­some; the second (Y-axis), along the methodology and technology utilized, from biology through chemistry up to physics and engineer­ing; the third one (Z-axis), along the specific biological system or mechanism, approached from the concept of the cell cycle, through aging and carcinogenesis, up to differentiation. Each investigator, has his own X-Y-Z coordinates in such a '~hpee dimensionaZ configu­pation" and frequently conducts his search in an isolated environ­ment with occasional and superficial contacts with the remaining "scientific space". As occurs also in all other human endeavors, this frequently leads to an acritical intellectual inertia or at best to self-perpetuating inner circles, whose primary functions are to produce an avalanche of "papers", some of which do fulfill a need for exchange of new findings, but some of which are generated to satisfy personal, academic or economic imperatives. Looking at the rate at which the scientific "literature" is growing, one wonders whether knowledge is growing at the same rate, or whether intellectual energy and economical resources (of finite amount in any society) are wasted because proper "vaZue cpitepia and channeZs of communications" are not open among scientists active in parallel approaches toward the solution of the same problems. Need exists, therefore, for the adoption of an absolute reference system where findings and efforts are to be judged and/or compre­hensive approaches developed. This should also help to decrease the so frequently encountered intellectual arrogance (due to cultural "isolation" or lack of sophistication) and increase the sense of self-criticism and humility (in terms of a more open attitude toward new technology or ideas) in studying the complex mechanisms determining the structure and function of living systems. In the twentieth century any significant conquest of the human race (as splitting the atom or reaching the moon) has been the pigopous (step-by-step, without mirucuZous shoptcuts, as attempted unsuccess­fully over the past 20 years in cancer research) and anaZyticaZ 'WOpk of teams of scientists with diffepent ''hard science" backgpounds and expeptise. Even if knowledge is transmitted to younger generations (in the University) through traditionally separated disciplines such as engineering, physics, chemistry, biology or medicine, this surely does not correspond to the way new knowledge is acquired in all fields of sciences, and particularly in life science.

INTRODUCTION

To contribute toward the filling of such gaps, participants and lecturers of the Erice Advanced Study Institute and contribu­tions to this book on chromatin have been chosen in such a manner as to warrant spherically isotropic distribution in the three­dimensional space outlined above.

xvii

The simultaneous contribution of several outstanding scientists, each one a world-wide leader in his own specialization, has per­mitted me to edit this comprehensive book, which hopefully respects such interdisciplinary aims. Several books are available in the area, but they usually cover specific topics, focusing mostly either on a given technique, biological problem, chromatin constituents, or level of organization, but few are covering the extremely broad field in an organic and tutorial format (i.e. comprehensive and accessible with profit to a wider scientific community) from histone proteins to intact nuclei, from molecular to cytological approaches.

Within the inherent limitations of any conference proceeding (such as this) I have attempted to structure the entire book in an organic and tutorial format, such as to have not a scattered collec­tion of research papers, incoherent and with frequent unnecessary overlap, but a sequential series of chapters dealt in depth, from the basic properties of chromatin throughout all the numerous techniques employed (occasionally treated in details, including a brief summary of their basic physical principles), through the various levels of chromatin organization, up to their implications for cell function.

The Institute's content did not reflect the volume of literature pertaining to a particular technique or chromatin component, but how they are uniquely useful in providing additional and complemen­tary information on chromatin structure and its relation to cell function.

Specifically the book consists of four parts, each one followed by a chapter on the pertinent discussion which occurred at the time of oral presentation.

I) an introduction to the physical, chemical and biological properties of isolated chromatin and their relationship to chromatin of living cells (Janes Bonner, USA).

II) basic chapters which present the theory and instrumenta­tion of all the numerous physical, chemical, functional, morphological techniques and methodology applicable to the study of chromatin, both IN SITU and isolated from living cells (Stuart Gilmour, UK; G. Harrington, USA; Gerald Fasman, USA; Ada Olins, USA; Donald Olins, USA; Ian Walker, UK; Frank Kendall, USA; John Pardon, UK; Edwin M. Bradbury, UK; Tobjorn Caspersson, Sweden; Claudio Nicolini, USA; Mortimer Mendelsohn, USA: B. Shaw, USA; Paul Ts'o, USA).

xviii

III)

IV)

INTRODUCTION

various levels of chromatin organization as determined by the above techniques, i.e. nucleosome, multimers, chromatin, chromosomal proteins and their enzymatic modifications, such as acetylation, methylation, and phosphorylations in determining gene expression and chromatin organization (Kensel Van Holde, USA; I. O. Walker, UK; A. Prunell, USA; John Ploem, The Netherlands; Joel Gottesfield, UK; S. Bram, France; G. Dixon, Canada; Donald Olins, USA; B. Shaw, USA; S. Gilmour, UK).

structure and function of the genetic apparatus in the mammalian cell, stressing their relationship to neoplastic transformation, aging, cell cycle, medical genetics differentiation, and chemical carcinogenesis (Edwin Bradbury, UK; Louis Smets, The Netherlands; Silvio Parodi, Italy; W. Sawicki, Poland; Walfried Linden, West Germany; Leo Sachs, Israel; Paul Ts'o, USA; Claudio Nicolini, USA; D. S. Sarma, Canada; Ian Walker, UK; Ferruccio Ritossa, Italy; F. X. Wilhelm, France; G. Verly, France).

At the end of the book, (part V) I have included a final review and synthesis of the genetic apparatus dealing with clarifications of specific topics, or focusing on controversial issues as models for chromatin structure and in new avenues as biophysical cytology or neutron diffraction. The course was of such interdisciplinary nature that the scientists specialized in one field have been teaching scientists highly qualified in a different area. The role of lecturer and student was frequently interchanged during the meeting as the theme of common interest (chromatin study) was developed from the viewpoint of different sciences, in a beautiful small town on top of a mountain overlooking the Mediterranean (that, according to a legend, was founded by Erice, son of Venus, more than three thousand years ago). In synthetic analytical terms we could say, with L. Sachs, that se + AA = LE, that is Science in Ch:r>omatin pZus Art in ArchaeoZogy equaZ Life in Erice. It is not paradox then to state that the Chromatin Institute was held in the same geographical region where a few thousand years before the Greek Leucippus and Democritus and Zater on the Roman Lucretius (in his poem "De Rerum Natura") gave the foundation of biophysics, describing how the atoms, after various interactions, acquire stabZe configura­tions, cOI'I'esponding to the Ziving and inanimate worZds. This simple and unitary theory, which brings Zife science into the reaZm of physicaZ science, remarkably maintains its validity even after several centuries of alternative vicissitudes.

To follow the evolution of such fundamental ideas in successive steps, is quite impossible in such context: I like however only to recall that the content of this Erice Institute (and therefore of this book) which relates chromatin structure to cell function, represents one of the most recent developments of that old idea.

INTRODUCTION xix

Following the earlier discovery of the direct reZationship between spatiaZ structures of such moZecuZes such as methane and benzene and chemicaZ activity, the discovery in 1953 of the struc­ture of the doubZe heZix of DNA represents the turning point for a simiZar reZationship between three dimensionaZ structure and bioZogy. It is indeed this relationship that emerged as one of the most intriguing "take home messages" from the institute: the reZation­ship between ceZZ jUnction and tertiary (nucZeosomeJ and quaternary (soZenoid or other form of superpackingJ structures of chromatin DNA, as moduZated by interaction with histone and non-histone proteins (and their enzymatic modifications) during the ceZZ cycZe, ceZZ transformation, aging, and differentiation. In addition to affirm a more dynamic view of DNA organization in isolated chromatin, the Erice Institute raises the question as to whether tertiary -quaternary structures are specificaZZy Zinked to a higher order (quinternaryJ organization which can now be detected "In situ" by means of recent technological advancements in the area of biophysicaZ cytoZogy, to an extent up to now impossible to any human observer or biochemical assay.

In conclusion, I hope that this book will constitute a useful and stimulating guideline to doctoral and post-doctoral students as well as to senior scientists, interested in the most recent developments in the wide interdisciplinary approach to structure and function of the genetic apparatus and its constituents and their relationships to cell function.

Finally, I would like to express my graditude to Professor Antonio Borsellino for giving me the opportunity to direct the eighth course of the International School of Biophysics (which have seen in previous years the active participation also of several Noble-Prize winners, such as Wald, Eccles, Katz) and to Ms. Pinola and Dr. Grabriele of the Majorana Centre for coupling high efficiency and courtesy in a unique cultural setting. My last, but not least, acknowledgement is to my wife Julia and my Uncle Luigi for their constant advice and dedication, considering that to realize and operate within a "three-dimensional scientific space" was a quite difficult and absorbing experience, even if challenging, not only in purely scientific terms, but also for its profound social implications.

Claudio Nicolini