ROLE OF RNA AND DNA IN BRAIN FUNCTION

TOPICS IN THE NEUROSCIENCES

Other books in the series:

Rami Rahamimoff and Sir Bernard Katz, eds.: Calcium, Neuronal Function and Transmitter Release. ISBN 0-89838-791-4.

Robert C.A. Frederickson, ed.: Neuroregulation of Autonomic, Endocrine and Immune Systems. ISBN 0-89838-800-7.

ROLE OF RNA AND DNA IN BRAIN FUNCTION

A Molecular Biological Approach

edited by

Antonio Giuditta Department of General and Environmental Physiology

University of Naples, IT AL Y

Barry B. Kaplan Western Psychiatric Institute and Clinic

University of Pittsburgh School of Medicine, USA

Claire Zomzely-Neurath Office of Naval Research

London Branch, UNITED KINGDOM

Martinus Nijhoff Publishing a member of the Kluwer Academic Publishers Group

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The picture on the cover originally appeared in KOS magazine, edited by Franco Maria Ricci (via Durini 19, 20122 Milano, Italy) in the issue of January I, 1986, and is reprinted with permission.

Library of Congress Cataloging-in-Publication Data

Role of RNA and DNA in brain function.

(Topics in the neurosciences) Based on the Symposium "Role of DNA in Brain Activity"

held in Ravello, Italy on May 27-29, 1985, as a satellite meeting of the 10th Meeting of the International Society for Neurochemistry, held in Riva del Garda, Italy on 19-24 May 1985.

Includes indexes. 1. Brain-Congresses. 2. Neurogenetics-Congresses.

3. Gene expression-Congresses. 4. Nerve proteins­Congresses. 5. Nucleic acids-Congresses. I. Giuditta, Antonio. II. Kaplan, Barry B. III. Zomzely-Neurath, Claire. IV. Symposium "Role of DNA in Brain Activity" (1985 : Ravello, Italy) V. International Society for Neurochemistry. Meeting (lOth : 1985 : Riva, Italy) VI. Series. [DNLM: 1. Brain-physiology-congresses. 2. DNA-physiology-congresses. 3. RNA-physiology­Congresses. QU 58 R7443 1985] QP376.R64 1986 591.1 '88 86-12525

ISBN-13: 978-0-89838-814-5 e-ISBN-13: 978-1-4613-2321-1 001: 10. 1007/978-1-4613-2321-1

Copyright © 1986 by Martinus Nijhoff Publishing Reprint of the original edition 1986

All rights reserved. No part of this publication my be reproduced, stored in a retrieval system, or transmitted in any form or by any means, mechanical, photocopying, recording, or otherwise, without the prior written permission of the publisher, Martinus Nijhoff Publishing, 101 Philip Drive, Assinippi Park, Norwell, Massachusetts 02061.

CONTENTS

LIST OF CONTRIBUTORS ix

PREFACE xi

ACKNOWLEDGEMENTS xiii

I. BRAIN GENE PRODUCTS

1. A COMPARATIVE STUDY OF THE DIVERSITY OF GENE EXPRESSION IN BRAIN 1 B.B. Kaplan. A.E. Gioio. C. Perrone Capano and A. Giuditta

2. MESSENGER RNA IN THE BRAIN 10 W.E. Hahn. N. Chaudhari. J. Sikela and G. Owens

3. GENE EXPRESSION IN THE MAMMALIAN BRAIN 23 J.G. Sutcliffe. R.D. McKinnon and A.-P. Tsou

4. EXPRESSION OF BRAIN-SPECIFIC PROTEINS R.J. Milner

5. MESSENGER RNA IS PRESENT IN THE AXOPLASM

32

OF SQUID GIANT AXONS 42 A. Giuditta. T. Hunt. C. Perrone Capano. L. Santella and B.B. Kaplan

II. CLONING AND EXPRESSION OF BRAIN GENES

6. MOLECULAR GENETICS OF TYROSINE HYDROXYLASE J. Mallet. A. Berod. F. B1anot. C. Boni. M. Buda. N. Faucon Biguet. B. Grima, Ph. Horellou, J. -F. Julien, A. Lamouroux and J. Powell

57

vi

7. ANALYSIS OF SYNAPSIN I AND G-SUBSTRA TE GENE EXPRESSION BY eDNA CLONING AND IN SITU HYBRIDIZATION HISTOCHEMISTRY 71 L.J. DeGennaro, M. W. Kilimann and C.A. Haas

8. THE EXPRESSION OF MICROTUBULE PROTEINS DURING THE DEVELOPMENT OF THE NERVOUS SYSTEM 81 1. Ginzburg and U. Z. Littauer

9. REGULATION OF EXPRESSION OF THE HUMAN PROENKEPHALIN GENE IN HETEROLOGOUS CELL SYSTEMS 90 E. Herbert, M. Comb, G. Thomas, D. Liston, A. Seasholtz, H. Rosen and B. Thorne

10. THE REGULATION OF PROOPIOMELANOCORTIN GENE EXPRESSION BY ESTROGEN IN THE RAT HYPOTHALAMUS 112 J.L. Roberts, J.N. Wilcox and M. Blum

III. THE MOLECULAR BIOLOGY OF NEUROLOGICAL DISEASES

11. DNA ANALYSIS OF DUCHENNE AND BECKER MUSCULAR DYSTROPHIES K.E. Davies, H.R. Dorkins, S. McGlade, S.P. Ball, S. J. Kenwrick, T. Smith, S. Forrest, L. Wilson, 1. Lavenir, A. Speer and Ch. Coutelle

12. GENETIC ANALYSIS OF THE FRAGILE X-MENTAL RETARDATION SYNDROME WITH POLYMORPHIC

123

DNA MARKERS 131 1. Oberli, G. Camerino, B. Arveiler, J. Boue, M. G. Mattei, J.F. Mattei and J.L. Mandel

13. BRAIN MESSENGER RNA IN ALZHEIMER'S DISEASE M.R. Morrison, W.S. T. Griffin and C.L. White, III

14. THE EFFECT OF TRISOMY-21 (DOWN'S SYNDROME)

142

ON BRAIN TRANSCRIPTION 160 L. Lim, C. Hall, T. Leung and S. Whatley

vii

IV. BRAIN DNA

A. Neuronal Chromatin Structure and DNA Content

15. NEURONAL CHROMATIN DURING DEVELOPMENT I.R. Brown

16. POST-TRANSLATIONAL MODIFICATIONS OF CHROMOSOMAL PROTEINS IN NEURONAL AND

174

GLIAL NUCLEI FROM DEVELOPING RAT BRAIN 182 I. Serra and A.M. Giuffrida

17. DNA CONTENT IN NEURONS O. Bernocchi and E. Scherini

B. Enzymes Related to DNA Metabolism

18. ENZYMOLOGY OF DNA REPLICATION AND REP AIR IN BRAIN C.c. Kuenzle

19. BRAIN DNases AND THEIR FUNCTIONAL IMPORTANCE K. Subba Rao

20. POLYADP-RIBOSE POLYMERASE AND ADP-RIBOSYLATION REACTION P. Mandel, C. Niedergang, M.E. IUel, H. Thomassin and A. Masmoudi

c. DNA Turnover

21. DNA SYNTHESIS AND CELL NUMBER HOMEOSTASIS IN THE BRAIN V. Mares

22. BRAIN DNA CHANGES DURING LEARNING S. Reinis

197

211

224

233

247

256

viR

23. ROLE OF DNA IN BRAIN INFORMATION PROCESSING A. Giuditta, M. V. Ambrosini, F. Morelli, C. Perrone Capano, T. Menna, C. Buono, C. Lamberti, A. Cerbone and A. Sadile

V. SPECIAL LECTURE

24. RNA AND LEARNING H. Hyden

VI. POSTERS

25. MOLECULAR GENETICS OF THE NERVE CELL ADHESION MOLECULE N-CAM: EVIDENCE FOR MULTIPLE, DEVELOPMENT ALLY REGULATED,

268

280

mRNA SPECIES 294 G. Gennarini, M.R. Hirsch, M. Hirn and C. Goridis

26. THE CHARACTERIZATION OF eDNAs ENCODING BRAIN-SPECIFIC AND UBIQUITOUS mRNA BY HYBRID-SELECTED TRANSLATION 296 C. Hall, T. Leung and L. Lim

27. OPIOID PEPTIDE PRECURSORS IN THE AMPHIBIAN XENOPUS LAEVIS G.J.M. Martens, O. Civelli and E. Herbert

28. SENSITIVE HYBRIDIZATION TECHNIQUES AS POWERFUL TOOLS IN MOLECULAR GENETICS

299

TO IDENTIFY BRAIN-SPECIFIC GENE PRODUCTS 303 T.A. Rhyner, A.A. Borbe/y and J. Mallet

29. MOLECULAR CLONING AND NUCLEOTIDE SEQUENCES OF eDNA AND GENOMIC DNA FOR THE RAT BRAIN S-I00 PROTEIN 308 Y. Takahashi, R. Kuwano, H. Usui, T. Maeda and T. Iwanaga

AUTHOR INDEX 313

SUBJECT INDEX 315

LIST OF CONTRIBUTORS

G. Bernocchi, Dipartimento di Biologia Animale, Universita di Pavia, Piazza Botta 10, 1-21700 Pavia, Italy

I.R. Brown, Department of Zoology, University of Toronto, West Hill, Ontario, MIC lA4 Canada

G. Camerino, Dipartimento di Genetica e Microbiologia, Universita di Pavia, Via S. Epifanio 14, 27100 Pavia, Italy

K.E. Davies, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, England

L.J. DeGennaro, Abteil ung Neurochemie, Max-Pl anck- Inst itut fUr Psi chi atrie, Am Klopferspitz 18A, D-8033 Planegg-Martinsried, FRG

G. Gennarini, Centre d' Immunologie INSERM-CNRS, Case 906, F-13288 Marseille, France

I. Ginzburg, Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel

A. Giudttta, Dipartimento di Fisiologia Generale e Ambientale, Universita di Napoli, Via Mezzocannone 8, 80134 Naploli, Italy

A.M. Giuffrida, Istituto di Chimica Biologica, Facolta di Medicina, Universita di Catania, Viale A. Doria 6, 95100 Catania, Italy

W.E. Hahn, Department of Anatomy, University of Colorado School of Medicine, 4200 East Ninth Avenue, Denver, Colorado 80262, USA

C. Hall, Department of Neurochemistry, Institute of Neurology, The National Hospital, Queen Square, London WCIN 3BG, England

Eo Herbert, Insti tute of Bi omedi cal Research and Department of Bi ochemi stry, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, Oregon 97201, USA

H. Hyden, Institute of Neurobiology, Faculty of Medicine, Faculty of Medicine, University of G6teborg, Fack S-400 33 G6teborg 33, Sweden

B.B. Kaplan, Molecular Neurobiology Program, Department of Psychiatry, Western Psychiatric Institute & Clinic, 3811 O'Hara Street, Pittsburgh, Pennsylvania 15213, USA

c.c. Kuenzle, Institut fUr Pharmakologie und Biochemie, Universitat ZUrich-Irchel, Winterthurerstr 190, CH-8057 ZUrich, Switzerland

x

L. Lim, Department of Neurochemistry, Institute of Neurology, The National Hospital, Queen Square, London WCIN 3BG, England

J. Mallet, Laboratoire de Neurobiologie, Cellulaire et Moleculaire, CNRS, F-9II90 Gif-sur-Yvette, France

P. Mandel, Centre de Neurochimie, CNRS, 5 rue Blaise Pascal, F-67084 Strasbourg, France

V. Mares, Institute of Physiology, Czechoslovak Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czechoslovakia

G.J.M. Martens, Department of Zoology, Faculty of Science, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands

R.J. Milner, Division of Preclinical Neuroscience and Endocrinology, Research Institute of Scripps Clinic, 10666 Torrey Pines Road, La Jolla, California 92037, USA

M.R. Morrison, Department of Neurology, University of Texas, Health Science Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235, USA

s. Reinis, Department of Psychology, University of Waterloo, Waterloo, Ontario, N2L 3Gl Canada

T .A. Rhyner, Laboratoire de Neurobiologie, Cellulaire et Moleculaire, CNRS, F-91190 Gif-sur-Yvette, France

J.l. Roberts, Center for Reproductive Sciences, Columbia University, 630 West 168th Street, New York, New York 10032, USA

K. Subba Rao, School of Life Sciences, University of Hyderabad, 500 134 Hyderabad, AP, India

J.G. Sutcliffe, Department of Molecular Biology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, California, 92037, USA

Y. Takahashi, Department of Neuropharmacology, Brain Research Institute, Niigata University, Asahimachi I, Niigata City 951, Japan

PREFACE

There was once an old Chinese man working on a hill with a boy. On the plain, near the sea, rested the village, the inhabitants eagerly engaged in their daily activities. Suddenly, the old man noticed that a huge wave, far distant in the sea, was approaching the shore endangering all. The only safe place was the hill. So, he began waving his hands and screaming aloud, to no avail. The villagers were too busy with their own work and paid little heed to the old man, who was considered a bit eccentric. But soon flames were on the hill, the wheat fields ablaze. The old man had resorted to this ultimate step to alert his fellow citizens. Now, they all went running towards him, angry about their burning crop, and in the process, avoided the imminent danger.

For some mysterious reason, my mind focused on this story prior to the Symposium on the Role of DNA, which took place in Ravello, Italy at the end of May 1985. Having made a call for people to meet and reflect for a few days, the analogy began to take shape. Ravello was indeed a hill, magically overlooking the sea from medieval quarters. True, its countryside is filled with vineyards, not wheat fields, but that is an improvement on the story. However, what was the wave? Perhaps, the growing amount of data on cloned brain genes that threatens to engulf neurobiologists. Perhaps the raising shadow of moral issues that are bound to emerge from the applications of the new tricks and technology. Perhaps, it was the overwhelming implications of the molecular neurobiology studies. Maybe, a combination of all these explanations. At that time it was hard to say, and still is.

This book of Proceedings may be considered an attempt to challenge a larger audience in the endeavor to understand the nature of the wave. After all, we all contribute, mostly unknowingly, to the shape and impetus of upcoming waves, and all should be given the opportunity to try and look at them from a safe hill, before they get too close.

Antonio Giuditta

ACKNOWLEDGEMENTS

This book contains most of the manuscripts of the lectures and posters presented at the Symposium "Role of DNA in Brain Activity" held in Ravello, Italy on May 27-29, 1985. The symposium was a satellite meeting of the 10th Meeting of the International Society for Neurochemi stry. Fi nanci a 1 support for the Symposi um was obtai ned from the Office of Naval Research (London branch), Universita di Napo1 i, Istituto Ita1 iano per gl i Studi Filosofici, Consig110 Naziona1e delle Ricerche, Banco di Napoli, International Society for Neurochemistry, Fidia and New England Biolabs. We are grateful to these Institutions for their generous help. We are also thankful to the Istituto Internaziona1e di Genetica e Biofisica, the Dipartimento di Fisiologia Genera1e e Ambientale and, in particular, to the Stazione Zoologica di Napoli for the many services and facilities provided.

The symposium was organized by A. Giuditta. To him and to the other members of the Organizing Committee (B.B. Kaplan, C. Perrone Capano and T. Menna), we extend our most sincere thanks. Special thanks are also due to C. Buono for his help in the organization of the Symposium and to J.-L. Knox for her indefatigable and highly professional service in the typing of the final mansucript.

ROLE OF RNA AND DNA IN BRAIN FUNCTION

1 A COMPARATIVE STUDY OF THE DIVERSITY OF GENE EXPRESSION IN BRAIN B.B. KAPLAN, A.E. GIOIO, C. PERRONE CAPAN0 1 AND A. GIUDITTA 1

Western Psychiatric Institute and Clinic, Pittsburgh, PA, USA and 1 International Institute of Genetics and Biophysics and Department of General and Environmental Physiology, Naples, Italy

ABSTRACT Results of previ ous RNA-DNA hybridi zat i on experiments have demon­

strated that the mammal ian brain expresses 2- to 5-fold more of the haploid genome than other somatic tissues or organs. The striking complexity of gene expression in brain raises fundamental questions regarding the ultimate function of this large amount of genetic information and the degree to which it participates in the development and maintenance of tissue-specific structure and function. Here, we revi ew our recent resul ts obtai ned from a comparative study of the diversity of gene expression in brain. In this work, RNA-DNA saturation hybridization was used to estimate the sequence complexity of nuclear and polysomal RNA from rat, goldfish and squid brain. Additionally, the data were compared to the complexity of RNA from a typical non-neural tissue of each of these animal species. Our findings suggest that, as is the case in mammals, the diversity of gene expression in the CNS of teleosts and cephalopod mollusks is greater than in non-neural tissue. Importantly, however, the differences in the complexity of goldfish and squid brain RNA relative to that of non-neural tissue is significantly less than that observed in several mammalian species.

INTRODUCTION The sequence complexity of RNA in various mammalian organs has been

est imated by RNA-DNA saturation hybridization. In these experiments, single-copy DNA (scDNA) is hybridized to large excesses of nuclear or cytoplasmic RNAs, yielding a direct measure of the amount of the genome transcribed. In general, the results of these studies show that the mammalian brain expresses 2 to 5 times more of the haploid genome than other somatic tissues or organs (for review, see ref. 1-3). For example, mammalian brain nuclear RNA hybridizes to 16 to 24% of the scDNA, whereas liver, kidney and spleen RNA are complementary to 4 to

2

10% of the single-copy genome (4-7). Assuming asymmetric transcription, the sequence complexity of rodent and sheep brain nuclear RNA is 5.9-6.3 x 10 8 nucleotides (nt), a value sufficient to code for 140,000 different nuclear RNAs averaging 4500 nt in length.

Consistent with the nuclear RNA findings, mammalian brain polysomal RNA is complementary to significantly more scDNA than mRNA from other organs (see for example, ref. 8,9). The striking diversity of gene expression in brain is generally interpreted as reflecting the extensive heterogeneity of cell types in the tissue. However, data obtained from the analysis of several clonal cell lines of neuroectodermal origin suggest that, like the brain itself, neural cells express an unusual amount of the genome (6,8).

In the past, comparisons have been made of the sequence complexity of nuclear RNA from several brain regions differing markedly in cell composition, structure, and function (6-8). Results of these studies show that the great majority of nuclear RNA transcribed in whole brain is also present in the major brain regions. However, unlike the regional distribution of nuclear RNA sequences, the diversity of brain cytoplasmic RNA seems to reflect the summation of regional RNA popula­tions of somewhat lower complexity (8,9). Taken together, these findings call attention to three important features of gene expression in brain. First, that each brain region, regardless of cell composition or function, utilizes a remarkable amount of genetic information. Second. that information necessary for region-specific function is encoded ina re 1 at i ve ly small mi nori ty of the total genes expressed. Third. that post-transcriptional regulatory mechanisms may play an important role in the elaboration of region-specific structure and function.

In view of the profound functional and evolutionary implications of the above observations. we have begun a phylogenetic comparison of RNA sequence complexity in neural tissue. As a working hypothesis, we postulated that the diversity of gene expression in brain would increase during phylogeny. correlating with the evolutionary development of the organ. It bears emphasis. however, that there is no a priori reason to believe that the evolution of the mammalian brain results directly from the expression of a large number of new gene "sets." Rather, evolution

3

could occur from alterations in the pattern of organization of a large number of intrinsically complex cellular units. In this instance, increase in the diversity of gene expression in the central nervous system (CNS) might not occur during vertebrate evolution.

In this chapter, we review our recent results on the sequence complexity of polysomal and nuclear RNA from the CNS of rat, goldfish and squid. Results of this study indicate that the diversity of genetic information expressed in the CNS of lower vertebrates and of inverte­brates is greater than in other somatic tissues. Importantly, however, the differences observed in goldfish and squid are significantly less than those reported in mammals.

RESULTS Characterization of genomic and scDNA.

Prior to comparing the sequence complexity of RNA, it proved neces­sary to first define the complexity of the different kinetic components of the rat, goldfish and squid genomic DNA. In this experiment, DNA was purified from nuclei isolated by sedimentation through dense sucrose and sheared to approximately 450 nt in length by sonication. The DNA fragments were heat-denatured and reassoci ated by i ncubat ion in 0.6 M Na+ at 680 • Under these conditions, denatured DNA will reanneal to form duplex structures nearly identical to native DNA with regard to their thermal stability and fidelity of base pairing (reviewed in ref. 1).

As shown in Fig. 1, the kinetics of renaturation of rat and goldfish genomic DNA are similar, consisting of approximately 25% rapidly reanneal ing, repetitive sequence elements. Approximately 10% of the total DNA is highly repetitive, reannealing at an equivalent Cot < 10- 1 M sec, the earliest values obtained in this experiment. The middle repetitive kinetic component of rat and goldfish DNA renatures with a Cot of 2.0 and 0.8, respectively. In contrast to these find­ings, the majority of the squid genome (70-75%) consists of repetitive sequences. The middle repetitive kinetic component of squid DNA renatures with a Cot of 3.5 and comprises 50% of the total DNA.

Genomic DNA was radiolabeled in vitro by nick-translation (13,14) and scDNA prepared from each animal species by several cycles of rena­turation and purification by HAP chromatography as previously described

4

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Fig. 1. Reassociation kinetics of squid, goldfish and rat genomic and scDNA. [3H]labeled scDNA (250-350 nt) was renatured in the presence of excess total genomic DNA (450 nt) in 0.6 M Na+ at 68 0 and DNA renatura­tion measured by hydroxylapatite (HAP) chromatography. The curves descri bi ng the reassoci at i on of scDNA are a computer-determi ned, best least-squares fit to the data using the program of Pearson et al. (10). Data reproduced with permission from ref. 11,12.

(6,11,12) . The kinetics of hybridization of the [3H]DNA probe prepared from

each animal species is shown in Fig. 1. Single-copy DNA from rat and goldfish contained < 3% repetitive sequence elements. The squid scDNA conta i ned somewhat greater 1 eve 1 s of repet it i ve DNA due to the re 1 a­tively high levels of this kinetic component present in the squid genome. In all cases, total DNA renatured to > 90% at termination of the reaction. The observed Cot ~ val ues, second-order rate constants, kinetic complexity and mean melting temperature (Tm) of the scDNA probe from each animal species is summarized in Table 1.

TABLE 1.

Animal Species

Rat

Goldfish

Squid

Characterization of the

Fraction of total DNA

(%)

70

75

25

Observed Cot

(M sec)

1900

350

600

5

scDNA hybridization

Second order rate constant (L mol-1sec-1)

5.3 X 10- 4

2.9 X 10-3

1.7 X 10-3

probes.

Kinetic complexity

(nt)

1. 9 X 109

3.3 X 10 8

5.6 X 10 8

a Tm b

(0 C)

85

85

80

a. Estimates made with reference to the renaturation of E. coli DNA (450 nt) reacted under identical conditions (15). b. Mean melting temperature as measured by thermal elution from HAP (16).

Sequence complexity of polysomal RNA. Total polysomal RNA was isolated from rat (17) and goldfish (11)

brain and hybridized to trace amounts of [3H]labeled scDNA as previously described (6,13). The amount of [3H]DNA in RNA-DNA hybrids was measured by HAP chromatography. Final values were corrected for the content of [3H]DNA-DNA duplexes and for the reactivity of each of the DNA probes. At saturation, rat and goldfish brain polysomal RNA hybridized to 7.2%

and 6.7% of the scDNA probe, respectively (Table 2). Hybridization values obtained for rat polysomal RNA are in good agreement with those previously reported for rodent brain by Chikaraishi (18) and Van Ness et al. (19).

Table 2 also gives the hybridization values and sequence complexity estimates for rat and goldfish kidney polysomal RNA. In contrast to the data obtained with brain RNA, rat and goldfish kidney polysomal RNA was complementary to only 1.9% and 3.1% of the DNA probe, respectively. Therefore, as in the case of mammals, the teleost brain expresses significantly more of the haploid genome than other somatic organs. Interestingly, however, the relative difference in the diversity of brain and kidney RNA populations was greater in the rat (brain/kidney = 4.0) compared to goldfish (brain/kidney = 2.2). Therefore, the complexity of brain RNA, relative to that of kidney, seems to increase approximately 2-fold during vertebrate evolution.

The general applicability of the above findings was investigated by extending the hybridization analysis to highly evolved invertebrates,

6

TABLE 2. A comparison of polysomal RNA sequence complexity.

Animal Organ scDNA Sequence Organ Ratio Species Hybridi zed a Complexity (brain/

(%) (nt)b somatic organ)

Brain 7.2 2.6 x 108 Rat 4.0

Kidney 1.9 6.5 x 107

Brain 6.7 4.4 x 107 Goldfish c 2.2

Kidney 3.1 2.0 x 10 7

Brain 8.1 9.1 x 10 7 Squid d 2.8

Gi 11 2.9 3.2 x 107

a. Hybridization values corrected for the reactivity of the [3H]DNA probe. b. Calculation based on the assumption that DNA transcription is asymmetric and that the sequence complexity of rat, goldfish and squid scDNA is 1.9 x 109 , 3.3 X 108 , and 5.6 x 108 nt, respectively. c. Data from ref. 11. d. Data from ref. 12.

using the squid, Loligo pealii, as a model system. Hybridization values and estimates of the sequence complexity of squid optic lobe and gill are given in Table 2. In this experiment, polysomal RNA from nervous tissue and gill was complementary to 8.1% and 2.9% of the scDNA, respectively. Similar to the situation observed in goldfish, the difference in the complexity of squid optic lobe and gill polysomal RNA was 2.8. The data in Table 2, therefore, call attention to the striking diversity of gene expression in the CNS of this species and extend to cephalopod mollusks the observation that nervous tissue expresses significantly more genetic information than non-neural tissue.

Since the complexity of mammal ian brain polysomal RNA is divided between two separate, non-overlapping populations of polyadenylated and nonadenylated RNA (18,19), we investigated the diversity of the squid poly(A)+mRNA population by RNA-DNA hybridization. Polysomal poly(A)+RNA was isolated from optic lobe total polysomal RNA by affinity chroma­tography on oligo(dT)-cellulose (20). Approximately 1.3% of the total polysomal RNA bound to oligo(dT)-cellulose after three passages. Poly(A)+mRNA was hybridized to sc[3H]DNA to an equivalent Cot of 8.7 x

7

10 3 M sec at an RNA:DNA mass ratio of 1500:1. At saturation, poly(A)+ mRNA was complementary to 6.7% of the scDNA probe, a value representing 83% of the sequence complexity of total polysomal RNA (12). This result differs from that in rodent where poly (A) + mRNA represents only 50% of the complexity of brain total polysomal RNA (18,19). The possibility exists, therefore, that the acquisition of a highly diverse nonadeny­lated polysomal RNA population is a relatively late evolutionary development. Interestingly, in mouse brain the transcription of non­adenylated RNAs is developmentally regulated, appearing in the polyribo­some fraction shortly after birth and requiring approximately 30 days to attain adult levels (21). Sequence complexity of nuclear RNA.

The di vers ity of the nucl ear RNA popul at i on from the CNS of rat, goldfish and squid is shown in Fig. 2. Here, data are expressed as the ratio of brain RNA complexity relative to that of a non-neural tissue, i.e. kidney or gill. Additionally, we have included data from mouse (19) and sheep (7), thereby summarizing all the available information on brain nuclear RNA complexity. Consistent with the findings obtained with polysomal RNA, the goldfish and squid brain transcribes approxi­mately twice the amount of scDNA than other somatic organs. Moreover, the data in Fig. 2 suggest that the ratio of brain RNA complexity relative to a non-neural tissue increases during phylogeny, the sequence complexity ratio approaching four in mammals.

CONCLUSION The sequence complexity of rat, goldfish and squid brain nuclear

and polysomal RNA was determined by RNA-DNA hybridization to single-copy genomi c DNA. To our knowl edge, thi sis the fi rst compari son of the diversity of gene expression in neural tissue of non-mammalian species. Consistent with the findings of others, the amount of genetic informa­tion expressed in brain far exceeds that of non-neural tissue. The comparat i ve data establ ish 1) that the di fference in the amount of the haploid genome transcribed in brain relative to other somatic organs is established relatively early in phylogeny, and 2) that the difference in brain RNA sequence complexity relative to non-neural tissue tends to increase during evolution. Finally, these initial results support the

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e (B) POL YSOMAL RNA o ~ « a: >­!:: x W ...J c.. :::!; o u w U Z w :::> o w (J)

5.0

4.0

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Fig. 2. Phylogenetic comparison of nuclear and polysomal RNA diversity. Data are expressed as the ratio of brain RNA sequence complexity relative to that of a non-neural tissue. Kidney served as the organ of comparison in all species except squid and sheep where the standard was gill and liver, respectively. Origin of the data for each animal is as follows: squid (12), goldfish (11), mouse (4,19), rat (5,18), sheep (7,9).

hypothesis that the striking amount of genetic information expressed in brain is related to the tissue's heterogeneity of structure and function.

9

ACKNOWLEDGEMENTS The authors wish to thank J.L. Knox for assistance in the prepara­

tion of this manuscript. We are also grateful to Drs. S.L. Bernstein and O.K. Batter for valuable technical assistance. This work was supported by NIH Grant HD 19440 and grants 82.02005.04 and 83.01456.04 from the Consiglio Nazionale delle Ricerche. BBK is the recipient of an ADAMHA Research Scientist Development Award (MH00518).

REFERENCES 1. Kaplan, B.B. In: Handbook of Neurochemistry, 2nd Edition (Ed. A.

Lajtha), Plenum Press, New York, 1982, Vol. 2, pp. 1-26. 2. Kaplan, B.B. and Finch, C.E. In: Molecular Approaches to

Neurobiology (Ed. I.R. Brown), Academic Press, New York, 1982, pp. 71-98.

3. Dokas, L.A. Brain Res. Rev. ~: 177-218, 1983. 4. Bantle, J.A. and Hahn, W.E. Cell!!: 139-150, 1976. 5. Chikaraishi, D.M., Deeb, S.S. and Sueoka, N. Cell 13: 111-120,

1978. 6. Kaplan, B.B., Schachter, B.S., Osterburg, H.H., de Vellis, J.S. and

Finch, C.E. Biochemistry 1I: 5516-5524, 1978. 7. Deeb, S.S. Cell. Molec. Biol. 29: 113-119, 1983. 8. Beckmann, S.L., Chikaraishi, D.M., Deeb, 5.5. and Sueko, N.

Biochemistry 20: 2684-2692, 1981. 9. Hitti, Y.S. and Deeb, 5.5. Cell. Molec. Biol. 30: 169-174, 1984. 10. Pearson, W.R., Davidson, E.H. and Britten, R.J. Nucleic Acids

Res. !: 1727-1797, 1977. 11. Kaplan, B.B. and Gioio, A.E. Compo Biochem. Physiol. (in press). 12. Perrone Capano, C., Gioio, A.E., Giuditta, A. and Kaplan, B.B.

J. Neurochem. (in press). 13. Colman, P.O., Kaplan, B.B., Osterburg, H.H. and Finch, C.E.

J. Neurochem. 34: 335-345, 1980. 14. Rigby, P.W.J., Dieckman, M., Rhodes, C. and Berg, P. J. Mol. Biol.

Ill: 237-251, 1977. 15. Hough, B.R., Smith, M.J., Britten, R.J. and Davidson, E.H. Cell

~: 291-299, 1975. 16. Martinson, H.G. and Wagenaar, E.B. Anal. Biochem. 61: 144-154,

1974. 17. Bernstein, S.L., Gioio, A.E. and Kaplan, B.B. J. Neurogen. 1:

71-86, 1983. 18. Chikaraishi, D.M. Biochemistry 18: 3250-3256, 1979. 19. Van Ness, J., Maxwell, LH. and Hahn, W.E. Cell 18: 1341-1349,

1979. 20. Aviv, H. and Leder, P. Proc. Natl. Acad. Sci. USA 69: 1408-1412,

1972. 21. Chaudhari, N. and Hahn, W.E. Science 220: 924-928, 1983.

2 MESSENGER RNA IN THE BRAIN W.E. HAHN, N. CHAUDHARI, J. SIKELA AND G. OWENS Department of Anatomy, University of Colorado School of Medicine, Denver, CO, USA

ABSTRACT Much of the protein encoding portion of the genome is expressed in

the mammalian brain. Hence, it may be expected that a large number of brain-restricted proteins are to be found. Most of these proteins are probably low in abundance as most brain-specific mRNAs are rare copy species. However, a variety of molecular cloning approaches provide the amplification required to identify and characterize individual, low abundance proteins. Alignment of rare copy mRNAs with their respective proteins is an important step in establishing the functional signifi­cance of rare copy mRNAs. Many brain mRNAs appear during the course of postnatal development. It is suggested that some of these late mRNAs encode for proteins involved in the last major developmental phase of the brain, namely, establishment of connectivity. Most of the postnatal brain mRNAs lack a 3'-poly(A) tract and appear to be transcriptionally regul ated. Some mRNAs are apparently bimorphi c with respect to the presence or absence of poly(A) tracts. The coupling of molecular genetics with anatomy and cytology should provide a powerful new approach toward gaining an understanding of brain function in the context of speci al ized cell popul at ions. Proteins, such as receptors and components of synaptic membranes, are attractive targets for investigation.

INTRODUCTION The messenger RNA population in the brain is about 4-5 times more

diverse than found in other complex mammalian organs (1-6). Considering the cell ul ar di vers i ty and mi croheterogenei ty wi thi n the categori es of cells in the brain, it is not surprising that the number of gene products is greater in this organ than in others. The sequence complex­ity of mRNA has been ascertained by saturation hybridization with single-copy genomic DNA (scDNA) or by kinetic measurements using comple-

11

mentary DNA (cDNA) made by reverse transcription of brain mRNA. Both methods yield similar estimates of sequence complexity (2,5). From sequence complexity, the number of different mRNAs can only be guessed since the size of most of the different individual messengers is of course not establ ished. Brain mRNA ranges in size from a few hundred nucleotides (nt) to over 5000 nt in length. If it is assumed that a mass average size of 2000 nt approximates that of most of the different mRNAs, then division of the estimated sequence complexity by 2000 yields a total number of different messages of about 110,000. Variation in processing of primary transcripts to yield multiple mRNAs adds further to the problem of estimating the number of mRNA species (7,8). However, this heterogeneity does not add to the linear sequence complexity. Whatever the case, in terms of simple linear sequence, the mRNA population of the mouse brain is approximately 50-60 times more complex than the RNA in the bacterium Escherichia coli (9).

The complexity of mRNA from the brain of adult mice was first estimated by analysis of polyadenylated RNA isolated from polysomal RNA. Measurement of this fraction of polysomal RNA was assumed to provide an estimate of total mRNA complexity as it was thought that almost all different messages contain poly(A) tracts at their 3' ends. Somewhat surprisingly, the complexity of polysomal RNA was found to be nearly twice that of the poly(A)+ mRNA fraction alone. Other experiments showed that preparations of polysomes were free of RNAs which may have leaked from nuclei during cellular fractionation, at 1 east to the extent of contri but i ng to measurable sequence complexity (5). Hence, a nonpolyadenylated fraction of high sequence complexity was indicated. These sequences were termed poly(A)-mRNAs, as the only RNAs of high sequence complexity which are components of polysomes are assumed to be messengers. Other experiments showed these sequences could be released from polysomes by disruption of ribosomal subunits (6). Fractionation procedures using benzoylated cellulose (see Fig. 1), which selectively binds RNA molecules with little secondary structure, permitted extensive purification of poly(A)-mRNA.

The following features and characteristics apply to brain poly­(A)-mRNA. This RNA is heterogeneous in size, and functions as template in a cell-free translation system (6). Poly(A)-mRNAs constitute about

B~und poly(A) mRNA

12

Polysoma1 RNA I

~ (repeated cycles)

Flowthrough poly(Af RNA

BehzoYlated cellulose

Bound ~thrOUgh poly(Af mRNA mostly rRNA l (-1.3") .

Primed mRNA with random oligonucleotides at 12:1 mass ratlo to obtain cDNA (150 - 1200 nt)

hYbridiJ w/kidney rRNA (BC flowthrough)

I 3 mI. 5ephacryl 5-400 column

excluded ~ included fraction fraction cDNA to poly(Af mRNA (6096)

rRNA complementary cDNA (4096)

Fig. 1. Preparation of eDNA from brain poly(A)-mRNA.

0.5 to 1.0% of the mass of the total polysomal RNA as shown by fraction­ation experiments using benzoylated cellulose. As is the case for poly (A)+mRNAs, these sequences are present in varying degrees of abundance. Species of abundant to intermediate copy frequency make up about 80% of the mass, with the remaining 20% being infrequent to rare copy species. These mRNAs, as a complex population, are apparently restricted to the brain. That is, the sequence complexities of total polysomal RNA and of the polyadenylated polysomal RNA are essentially equal in all other organs which have been examined (10,11). It should be emphasized that the presence of poly(A)-mRNAs is not a unique feature of the brain per

13

se, but the presence of a complex population of such sequences is. For example, poly(A}-mRNAs, such as those for protamines and actins, are known to exist in various tissues (I2,I4).

The brain poly(A)-mRNAs are developmentally regulated such that most of the sequences fi rst appear in the nuclear and pol ysoma 1 RNA during the course of postnatal development (15). Therefore, their

appearance is associated with the last major developmental event in the brain, namely the establishment of connectivity (process outgrowth, synaptogenesis, etc.).

Little is known about the biogenesis of poly(A)-mRNAs, but sequence complexity measurements on nuclear RNA during postnatal development suggest that these mRNAs ari se from nonpolyadenyl ated hnRNA mol ecul es (IS). In the case of brain poly(A}+mRNAs, the sequence complexity of polyadenylated hnRNA is 3 to 4 times greater than the sequence which is conserved as mature message (16). In the case of poly(A)- mRNA, the sequence complexity of respective nonpolyadenylated hnRNA is only about 1.5 times greater than the message itself. Hence, the post-transcrip­tional proceSSing of these two classes of mRNA species may be rather different. It is known from earlier studies that many, if not all, of the brain poly(A)+mRNAs arise from large polyadenylated hnRNA molecules containing intervening sequences (I7). That is, many of these mRNAs are encoded by genes in which the coding sequence is discontinuous. Similar analysis of the poly(A)-mRNAs has not been done, so the question remains open as to whether these mRNAs are encoded by split genes.

While it is a tacit assumption that RNAs of high sequence complex­ity that are components of polyribosomes are messengers, alignment of most of the poly(A}+ and poly(A}- mRNA sequences with specific poly­peptides in the brain has not been done, although this has been accom­plished for certain "unknown" brain mRNAs (I8). Therefore, it is of fundamental importance to associate specific poly(A}+ and poly(A}-mRNA sequences, especially rare class species, with respective polypeptides in the brain. This is particularly important in the case of poly(A}­mRNAs, as their existence is a departure from eukaryotic cell biology dogma and few examples of such mRNAs are documented. Therefore, we now present some initial efforts to clone the poly(A}-mRNA sequences with the intent of seeking their respective proteins in the brain.

14

RESULTS Isolation of polyCA)-mRNA.

Because poly(A)-mRNA sequences lack a homopolymer, a hybridization affinity procedure for isolation of these sequences cannot be used. However, owing to the high degree of efficiency of removing the poly­adenyl ated mol ecul es by repeated cycl es of di saggregat i on and passage through oligo(dT)-cellulose columns, it is possible to generate poly(A)­polysomal RNA relatively free of poly(A)+mRNA sequences, provided the RNA is intact. It is known from earlier studies that benzoylated cellulose (BC), under appropriate ionic conditions, will preferentially bind RNA molecules with little secondary structure such as mRNA, thus allowing separation from transfer and ribosomal RNAs which have extensive secondary structure (19). For example, we have observed that virtually all the sequence complexity of E. coli total RNA and of mammalian polysomal RNA can be retained on BC columns. Under the right conditions, BC-chromatography removes more than 99% of the rRNA leaving an mRNA fraction which is at least 50% pure and, therefore, suitable for generating cDNA. It should be noted that proper preparation of the BC is critical for obtaining discrimination against rRNA. To prepare BC, we have used a modification of the procedure of Gilliam et al. (20). Considerable variation can be encountered amongst preparations, and we recommend making small batches and testing the BC using a variety of labeled RNA molecules.

Analysis of the BC-bound fraction, which is about 1% of the total polysomal RNA, shows that rRNA sequences are still present in the range of 30 to 50% of the mass. Much of this rRNA is slightly degraded. This probably eliminates some of its secondary structure. Also present is a small amount of poly(A)+mRNA which is not removed from polysomal RNA during the oligo(dT)-cellulose purification. Although that process is very effect i ve, mol ecul es wh i ch have been deadenyl ated or sequences which are 5' to cleavage points, do not bind oligo(dT)-cellulose. We measured the extent of "contamination" of the poly(A)-mRNA with poly(A)+ mRNA sequences by hybridization kinetics of cDNA complementary to poly(A)+mRNA. The rate of hybridization to BC-bound RNA was 50 to 100 times slower than when purified poly(A)+ mRNA was used to drive the reaction. This indicates that poly(A)+mRNA sequences appearing in the

15

TABLE 1. Poly(A)-mRNA mini-library: Classification of 10,000 clones.

Category (Complementarity of Clone) Percent of Colonies

A. rRNA 9

B. Liver & kidney total mRNA, brain poly(A)+mRNA

23

C. Brain poly(A)-mRNA (brain specific by low 0.8 sensitivity hybridization)

D. Colony hybridization negative (many of these represent putatively brain-specific 65 poly(Ar mRNAs, see Fig. 3)

poly(A)-mRNA fraction constitute only about 1 to 2% of the mass. While the reaction was not taken to completion, we assume most, if not all, of the poly(A)+mRNA sequences are represented in the poly(A)-RNA prepara­tion at this low concentration. Hence, while the BC-bound poly(A)- RNA lacks the purity attainable with poly(A)+RNAs, the level of contamina­ting species is sufficiently reduced as to permit production of a cDNA suitable for producing libraries representing brain poly(A)-mRNA. Cloning of DNA from poly(Al mRNA.

Production of cDNA molecules from poly(A)-mRNA templates and the subsequent purification of these sequences, prior to cloning as double strand cDNAs, are outlined in Fig. 1. As indicated, further purifica­tion of the cDNAs can be obtained by hybridization steps aimed at removing cDNA molecules complementary to rRNA. Once double-strand cDNAs are cloned, screening the library allows discrimination of the remaining rRNA-complementary clones. A profile of the initial characterization of a mini-library constructed from poly(A)- sequences is given in Table 1. Note that the bulk of the colonies are complementary to unknown RNAs, but rRNA-complementary clones still constitute about 10% of the recombinants. Initial characterization of this library showed that 23% of the colonies hybridize with cDNA probes made from brain, liver and kidney poly(A)+RNA or liver and kidney poly(A)-RNA (i.e. shared mRNAs). Many of the colonies in this mini-library probably represent mRNA species which are infrequent class species as they are not detected by standard colony hybridization procedures.

16

Some of the clones obtained from this library represent "bimorphic" mRNAs. We have observed in brain poly{A)-mRNA libraries cDNAs that are complementary to abundant and infrequent mRNAs present in both poly{A)+ and poly{A)- forms (Fig. 2). We do not bel ieve such observations are attributable to the artifact of simple cleavage of poly{A)+ molecules, but instead that they point to the presence of the same or highly homologous mRNA species in both poly{A) + and poly{A) - forms. This is supported by the observat i on that a cDNA complementary to an abundant brain-restricted poly{A)+mRNA hybridizes to slot blots of brain poly{A)+ mRNA but hardly at all to brain poly{A)-mRNA. Since this mRNA is an abundant species, this is a fairly sensitive test showing that cleavage of the mRNA is not the explanation for the observed bimorphism, at least of this control sequence (Fig. 2). The existence of bimorphic mRNAs

Fig. 2. Slot-blot hybridization of poly{A)+ and poly{A)- mRNAs from brain, liver and kidney with several cloned cDNAs from the poly{A)-mRNA library (pB5:56, pBI-5, pBI-I). pB5:56 hybridizes to mRNAs of similar abundance present in both poly{A) + and poly{A)- mRNA fractions from bra in. Th is is also true for 1 i ver and ki dney, but abundance of the mRNA is lower. Clones pBI-5 and pBI-I hybridize to a barely detectable extent to poly{A)+ and poly{A) - brain mRNA (arrows), but not to liver and kidney. Clone pBI: 69 represents a rel at ively abundant poly{A)+ mRNA which is brain specific (also shown by Northern blots). Only faint hybridization is shown with brain poly{A)-mRNA indicating that extensive cleavage of this mRNA leading to a failure to bind to oligo{dT)-cellu­lose did not occur.

17

is not uniquely observed in the brain. Pulse-labeling experiments suggest the presence of a number of bimorphi c speci es in He La ce 11 s (14,21).

Further characterization of a number of randomly selected clones, many of which are not identified during colony hybridization, are being ana lyzed by more sens i t i ve screeni ng procedures. A number of clones complementary to rare class brain-specific polysomal RNA have been tentatively identified. An example is shown in Fig. 3. This mRNA was not detected by colony or Northern blot hybridization and, hence, is a rare class species. We speculate that rare class species are, in fact, abundant in some cells of the brain; their low abundance is due to restricted distribution. cDNA clones complementary to poly(A)-brain­specific mRNAs are suitable candidates for recloning into expression vectors with the intent of obtaining fusion proteins and generating antibodies to search for the respective brain polypeptides. Production of a fusion protein in expression vectors also verifies the presence of an open reading frame in the inserted cDNA. This approach should permit the al ignment of a variety of poly(A) -mRNA species with polypeptides present in the brain. This opens the door for developing probes for more systematic studies of the developmental appearance and the anatom-

Fig. 3. Hybridi zat ion of an RNA probe, produced by the pGEM-1 vector into wh i ch a cDNA was inserted, is i nd i cated by the arrow at 1 eft for lane "B", brain polysomal RNA. No hybridization was noted for liver polysomal RNA, "L" (or for kidney RNA, not shown). The length of protected (hybridized) probe is equal to the length of the synthesized probe (310 nt). The RNA probe hybridizes with a polysomal RNA sequence which was not detected by slot-blot or Northern blot procedures when the same cDNA was used as a ni ck-transl ated probe. Thi s probe does not hybridize to brain poly(A)+mRNA and putatively corresponds to a rare copy brain poly(A)-mRNA.

18

ical and cellular location of such proteins. Assessment of the cellular location of these polypeptides will provide interesting data, particu­larly in relationship to the connectivity aspects of postnatal brain development (process outgrowth and synapse formation), as most of these mRNAs appear in the brain after birth. Regulation of poly{Al+ and poly{Al-mRNA species in brain.

Not unexpectedly, much of the regulation of brain mRNA sequences appears to be under transcriptional control. While many of the poly(A)+ mRNAs found in brain are shared with other organs such as 1 iver and kidney, well over half of the poly(A)+ mRNAs (sequence complexity) appears to be restricted to the brain, as demonstrated by the hybridiza­tion of complex cDNA probes. This is also suggested by the screening of cDNA libraries (22,23). However, some of the poly(A)+mRNA species which appear to be restricted to brain polysomal RNA are, nonetheless, tran­scribed in other organs. This has been demonstrated using a cDNA probe complementary to poly(A)+ mRNA. In this case, cDNA complementary to whole brain poly(A)+mRNA from mouse was fractionated by hybridization procedures to yield a population of cDNA sequences which did not hybridize with polysomal RNA from liver and kidney. Therefore, this cDNA largely represented mRNA species present in brain polysomes but not in liver or kidney polysomes. This complex probe, when mixed with total nuclear or poly(A) hnRNA from kidney or liver, hybridized to about 30% (Table 2). The rate at which the reactions occurred indicated that the concentration of the molecules driving hybridization is roughly similar in the nuclear RNAs of brain, liver and kidney. Thus, while transcripts containing some of the putatively brain-specific mRNA sequences are present in liver and kidney, they are apparently restricted to the nucleus. Although the status of these transcripts is not known, they might be transcriptionally normal products which are regulated at the post-transcriptional level, as suggested from tissue comparisons in the developing sea urchin (24,25). Whatever the case, a substantial post­transcriptional regulatory component operates in controlling some species of poly(A)+mRNA, although control at the transcriptional level appears to predominate.

In contrast, poly(A)-mRNAs appear to be more tightly regulated at the transcriptional level. A complex cDNA probe complementary to brain

19

TABLE 2. Comparison of transcriptional level regulation of brain poly(A)+ and poly(A)- polysomal RNAs.

cDNA

Brain poly(A)+mRNAl

RNA

Brain nuclear RNA Brain polysomal Kidney polysomal Liver polysomal Liver nuclear Kidney nuclear

Brain nuclear Brain poly(A)-mRNA Liver polysomal Liver nuclear Kidney polysomal Kidney nuclear

Percent cDNA Hybridized

100* 100*

5 4

30 32

100** 100**

5 12

6 10

1. cDNA complementary to brain poly(A)+mRNA was "exhaustively" hybrid­ized with liver and kidney polysomal RNA and the unhybridized molecules were isolated. This cDNA is therefore largely devoid of species comple­mentary to 1 i ver and ki dney mRNA. The observed 4 to 5% hybridization with these mRNA, was considered a "background" reaction and subtracted from liver and kidney nuclear RNA values. 2. cDNA complementary to brain poly(A)-mRNA was first hybridized with rRNA and with liver, kidney and brain pol ysoma 1 RNA to low Cot (concentrat i on x time) to remove sequences complementary to highly abundant shared mRNAs and rRNA. Therefore, the cDNA represents largely infrequent class brain mRNA species which comprise most of the complexity. * Normalized from the 97% value determined using the S nuclease assay (31). Nuclear RNA of liver and kidney are adjusted relative to this slight normalization. **Normalized from the 93% observed value. The other values are accord­ingly adjusted.

poly(A)-mRNA, from which most of the sequences complementary to liver or kidney polysomal RNA had been removed, was incubated with nuclear RNA from these organs. As shown in Tabl e 2, cDNA representing, by mass, mostly the infrequent class of poly(A)- brain mRNA, hybridizes to a level of only about 10% with nuclear RNA from liver or kidney. Much of this hybridization was attributable to a fast reacting component probably representing RNA sequences common to the three organs. Hence, it appears that the vast majority of the genes encoding poly(A)-mRNA sequences are under transcriptional control. Additional experiments using individual cloned species are needed to further verify the extent

20

of this control. It is possible that the postnatal activation of these genes may have some bearing developmentally or evolutionarily on the nature of this regulation.

As of yet, we have no knowledge of any special regulatory elements which might, in fact, be different for genes producing poly(A}-mRNA in comparison to genes coding poly(A)+mRNA. Certainly, at the present time, there is little reason to suspect that these mRNAs are regulated in any remarkably different manner.

Recently, a short, repeated sequence, the so-called brain "identi­fier" (IO) sequence, has been proposed as a control element in the transcription of genes which are expressed specifically in the brain (26,27). While it is possible that some regulatory function might be performed by transcripts from thi s intronic sequence, recent measure­ments on nuclear RNA show that a similar representation of this sequence exists in the transcripts of several organs of rat and mouse (29). The copy number of the ID sequence, estimated by saturation hybridization, is 130,000 in the rat genome and about 19,000 in the mouse (28). This is reflected in the representation of the repeat element in nuclear RNA, as its abundance is roughly 4 to 7 times greater in the rat than in the mouse. The lack of tissue specificity and the abundance of the transcript in approximate proportion to the genomic copy number suggest a rather random distribution and transcription of this sequence. Hence, it is improbable that this repeat element, as it may appear in struc­tural genes, is a control element involved in the transcriptional regulation of genes expressed specifically in the brain (see ref. 29 for data relevant to this view). However, accumulation of small (110-160 nt) RNA species complementary to this repeat seems to be unique to the brain, at least quantitatively. While a function for this small RNA species has not been experimentally demonstrated, perhaps a post-tran­scriptional regulatory role exists. This should be explored, as little is known about tissue-specific regulation of mRNA populations.

CONCLUSION Now that a general overview of the population of mRNA molecules in

the brain is available and we have found its complexity not be inordi­nately greater than that of other mammalian organs, it appears that

21

characterization of various genes and gene products in the brain should prove to be a productive approach toward brain chemistry. It should be noted that additional heterogeneity and complexity can arise from

differential processing of messenger RNAs and differential processing of thei r respect i ve pol ypept ides. A 1 so, DNA sequence rearrangements can add to complexity beyond that which can be assayed by simple measure­ments of 1 i near sequence compl exity. Pl aci ng mol ecul ar genetics in context with cell biology and anatomy will obviously be of great help in understanding the role of many brain specific proteins. For example, in situ hybridization and the use of antibody probes should reveal areas of specialization relative to gene expression.

Important classes of brain protein on which to focus include components of synapses, proteins which function on the cell surface to modul ate mi grat i on and cell-celli nteract ion, and receptors. To thi s end, we have made use of expression libraries from which we have isolated several cDNA clones using antibodies against brain proteins. Among the first to be isolated are cDNA clones putatively representing calcineurin, the major postsynaptic density protein, fodrin and a number of as yet unidentified calmodulin-binding proteins in the brain (30).

REFERENCES 1. Young, B.D., Birnie, G.D. and Paul, J. Biochemistry 12: 2823-2829,

1976. 2. Van Ness, J. and Hahn, W.E. Nucleic Acids Res. ~: 4259-4269, 1980. 3. Grady, L.J., North, A.B. and Campbell, W.O. Nucleic Acids Res. ~:

697-712, 1978. 4. Kaplan, B.B., Schachter, B.S., Osterburg, H.H., DeVellis, J.S. and

Finch, C.E. Biochemistry 1I: 5516-5524, 1978. 5. Van Ness, J., Maxwell, I.H. and Hahn, W.E. Cell 18: 1341-1349,

1979. 6. Chikaraishi, D.M. Biochemistry 18: 3249-3256, 1979. 7. Rosenfeld, M.G., Lin, C.R., Amara, S.G., Stolarsky, L., Roos, B.A.,

Ong, E.S. and Evans, R.M. Proc. Natl. Acad. Sci. USA 79: 1717-1721, 1982.

8. King, C.R. and Piatigorsky, J. Cell 32: 707-712, 1983. 9. Hahn, W.E., Pettijohn, D.E. and Van Ness, J. Science 197: 582-584,

1977 . 10. Ouellette, A.J. and Ordahl, C.P. J. Biol. Chem. 256: 5104-5108,

1981. 11. Hahn, W.E., Van Ness, J. and Chaudhari, N. In: Molecular Genetic

Neuroscience (Eds. F.O. Schmidtt, S.J. Bird and F.E. Bloom), Raven Press, New York, 1982, pp. 323-334.

12. Latrou, K. and Dixon, G.H. Cell 10: 433-441, 1977. 13. Hunter, T. and Garrels, J.I. Cell 1£: 767-781, 1977.

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14. Kaufman, Y., Milcarek, C., Berissi, H. and Penman, S. Proc. Natl. Acad. Sci. USA 74: 4801-4805, 1977.

15. Chaudhari, N. and Hahn, W.E. Science 220: 924-928, 1983. 16. Bantle, J.A. and Hahn, W.E. Cell~: 139-150, 1976. 17. Maxwell, I.H., Maxwell, F. and Hahn, W.E. Nucleic Acids Res. ~:

5875-5894, 1980. 18. Sutcliffe, J.G., Milner, R.J., Shinnick, T.M. and Bloom, F.E. Cell

33: 671-682, 1983. 19. Roberts, W.R. Biochemistry 13: 3677-3682, 1974. 20. Gillam, J., Millward, S., Blew, D., Tigerstrom, M., Wimmer, E. and

Tener, G.M. Biochemistry §: 3043-3056, 1967. 21. Milcarek, C. Eur. J. Biochem. 102: 467-476, 1979. 22. Hahn, W.E., Chaudhari, N., Beck, L., Wilber, K. and Peffley, D.

Cold Spring Harbor Symp. Quant. Biol. 48: 456-476, 1983. 23. Milner, R.J. and Sutcliffe, J.G. Nucleic Acids Res. 11: 5497-5520,

1983. 24. Wold, B.J., Klein, W.H., Hough-Evans, B.R., Britten, R.J. and

Davidson, E.H. Cell 14: 941-950, 1978. 25. Lev, Z., Thomas, T.L., Lee, A.S., Angerer, R.C., Britten, R.J. and

Davidson, E.H. Devel. Biol. 76: 322-340, 1980. 26. Sutcliffe, J.G., Milner, R.J., Gottesfeld, J.M. and Lerner, R.A.

Nature 308: 237-241, 1984. 27. Sutcliffe, J.G., Milner, R.J., Gottesfeld, J.M. and Reynolds, W.

Science 225: 1308-1315, 1984. 28. Sapienza, C., submitted. 29. Owens, G.P., Chaudhari, N. and Hahn, W.E. Science 229: 1263-1265,

1985. 30. Sikela, J.M. and Hahn, W.E. Trans. Soc. Neurosci. ~: 35, 1985. 31. Maxwell, I.H., Van Ness, J. and Hahn, W.E. Nucleic Acid Res.

~: 2033-2038, 1978.

3 GENE EXPRESSION IN THE MAMMALIAN BRAIN J.G. SUTCLIFFE, R.D. McKINNON AND A.-P. TSOU Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA, USA

ABSTRACT We present an update in our ongoing studies to describe the

structures of rat brain mRNAs, elucidate the mechanisms of their tissue­specific control, and identify and characterize their protein products. Here we discuss 1) mRNAs encoding a probable precursor of a neurotran­smitter that functions in olfactory, somatosensory, limbic, auditory and motor systems, 2) mRNAs encoding myel in proteol ipid protein, and 3) a polymorphic family of 88 mRNAs transcribed from a single gene. We show that primate and rodent share many brain proteins and that approximately 5,000 to 10,000 genes are first expressed after birth. The developmental regulation of these genes may be controlled by identifier sequences, a neuronal enhancer.

I NTRODUCTI ON In an animal with differentiated tissues, what makes one cell

di fferent from another is the part i cul ar set of protei ns each cell contains. In the brain, cells are further distinguished by their individual morphologies and the other cells they contact; both of these result, in part, from particular protein sets. Since each protein is translated from a messenger RNA and the information contents of mRNAs and proteins are the same, we have studied copies (clones) of rat brain poly(AYmRNA. We have been particularly interested in clones of mRNAs detectable in the brain but not detectable in non-neuronal control tissues. Here we discuss the structures and products of these "brain­specific" so-called Class III RNAs (1) and possible mechanisms for the tissue-specific control of their expression.

RESULTS AND DISCUSSION mRNA for neuropeptide precursor.

One Class III 2500 nucleotide (nt) mRNA, called IB236, is expressed

24

in brain at a level of about 0.01% (1). Strand-specific hybridization experiments utilizing fragments from our partial cDNA clone allowed us to identify the sense strand of the mRNA (2). This Class III mRNA is more prevalent in the hindbrain, cerebellum, hypothalamus and caudate nucleus than in cortex or olfactory regions. Our nucleotide sequence of the cDNA clone revealed an open reading frame (ORF) whose translation gave us a novel (no analogous protein found by computer search) protein sequence from which we made peptides, then anti-peptide antibodies (2). The antisera to three nonoverlapping peptides react with a brain­specific 100 kD glycoprotein in Western immunoblotting experiments and the immunoreactivity is blocked in each case by preincubation of the immune serum with the appropriate, but not any other, peptide. The protein target is a glycoprotein because the size of the target in immunoblots is reduced from 100 kD to 70 kD by incubating brain extracts with endoglycosidase F, an enzyme which cleaves asparagine-linked sugars from proteins (3). Further analysis of this experiment revealed that there are at least two (and possible four) large molecular forms of the 18236 protein. Some of these do not react with antisera to the extreme C-termi nal pept ides. Immunocytochemi cal studi es usi ng 1 ight and el ec­tron mi croscopy show the 18236 protei n is in fi bers and associ ated with vesicles at terminals in a subset of neurons (3,4). Again, it is the coincident, blockable reactivity of the antisera to multiple non­overlapping peptides which allows this conclusion to be reached with certainty. The protein and its mRNA begin to appear in the brain 10 days after birth, earlier in the more primitive brain regions (5). In colchicine treated rats, the cellular sites of synthesis for the 18236 glycoprotein can be identified (2,4). The protein is synthesized in cell bodies and transported to terminals in 5 systems: olfactory, somatosensory, audi tory, 1 imbi c and extrapyramidal motor systems (4). These systems share the anatomi cal property that they are sequent i a 1 , focused (few diffuse outputs) pathways. Each of the pathways is punc­tuated with respect to 18236. That is, although several kinds of cells within a pathway contain this molecule, no two cells adjacent in the series contain 18236. These observations suggest that several cell types within each pathway are chemically (and perhaps epigenetically) related to one another, and that the 5 systems are chemically and

25

possibly evolutionarily related. The anatomical distribution of the

IB236 protein is distinct from that of any previously characterized

brain protein: thus, the antibodies to the IB236 protein not only

provide information about the distribution of this protein, but they

also act as a new anatomical staining reagent. The primary amino acid

sequence of the IB236 protein contains several sets of tandem basic

amino acid residues (lys, arg) near its carboxy terminus, suggesting

this molecule could possibly be proteolytically processed. Indeed,

radioimmunoassay studies using antisera to peptides corresponding to the

putative cleavage products show that a substantial portion of the

i mmunoreact i vi ty is present in brain as pept ides (6). The synthet i c

analogues produce electrophysiological responses when appl ied to anatomically relevant sites. Therefore, IB236 may be a precursor for

"neurotransmi tter-l ike" pept ides, a concept consi stent wi th its punc­

tuated distribution within a system and its subcellular localization in vesicles at terminals. Part of these experiments are discussed in much greater detail in this volume by Milner.

The single rat gene for the IB236 mRNA has been isolated and most

of its sequence determined. The gene contains at least 7 introns and

covers more than 15 kb. It contains two repetitive elements called

identifier sequences (10) (7). The gene gives rise to at least two mRNA

products which are matured from the primary RNA transcript by alterna­tive RNA splicing schemes. The two demonstrated mRNA variants differ in

their C-terminal coding capacity, and must give rise to some of the different endoglycosidase F products detected in Western blots (discussed above). Other large protein forms of IB236 may be processing

intermediates. The IB236 cDNA clone hybridizes in Northern blots to an equivalent-sized mRNA detected in brain, but not liver, of mouse and

monkey (8). The mouse IB236 gene has been mapped by restriction fragment length polymorphism (RFLP) analysis to mouse chromosome 7, very

near the quiverer locus (Cila Blatt, unpublished data). Thus, we are in the process of rigorously characterizing the IB236

gene, its two mRNA products and several molecular forms of the corre­

spondi ng protei ns (precursors and proteolytic cl eavage products). We are in a position to suggest that the IB236 protein, whose existence we inferred from a randomly selected brain mRNA, may have a role as

26

precursor of neuropept ides i nvo 1 ved in 0 lfactory, somatosensory,

auditory, limbic and motor functions. Two mRNAs for proteolipid protein.

A second Class III cDNA clone (plB208) hybridizes to extremely abundant (greater than 1%) brain mRNAs of 1600 and 3200 nt (1,9) which are first detected a wk after birth and also appear in C6 glioma cells. The two mRNAs, whose complete sequences we have deduced from full-length cDNA clones, are coextensive at their 5' ends, differing only by alternative sites for poly(A) addition in their 3' noncoding regions. Monkey and mouse exhibit only the larger of the two RNA species which has a noncoding region of more than 2000 nt. Southern blot analysis suggests that there is a single gene for IB208. ,The mouse IB208 gene is apparently located on the X-chromosome because Southern blots, using DNA prepared from females, always give a signal twice as intense as that of DNA from males. A common ORF is shared by these two glial mRNAs encoding an extremely hydrophobic protein of 277 amino acids, which is brain myelin proteolipid protein (PLP). The rat PLP is 99% homologous to the bovine protein and is inserted in the myel in membrane without benefit of a signal protein, as only the single N-terminal methionine is proteolytically removed post-translationally. A family of polymorphic mRNAs.

A third cDNA clone (pO-44) we have studied hybridizes to a large polymorphic family of mRNAs. Members of this family (which are all products of the same single rat gene) differ from other members at one or more of 3 sites along a common backbone. Figure 1 shows a cartoon representing these structures which may include 88 or more distinct RNAs (10). These structures have been worked out by sequence analysis of 9 cDNA clones (8 of which are probably full-length) and verified by further analysis (10). We find a nested set of 5' ends. One of 22, or possibly more sites, may serve as the first nucleotide of the RNA. Two techniques, primer extension and S1 nuclease protection, were used to demonstrate this. Many of these multiple 5' ends are only detected in brain RNA, but others are shared with non-brain tissues. Thus, the 0-44 gene is transcribed differently in brain than in other tissues (10). The tissue difference is already evident at prenatal day 14. The center portions of these polymorphic RNAs are of two forms that differ by the

27

presence or absence of 17 nt. These most probably arise by alternative processing of the 0-44 hnRNA precursor. The 3' poly(A} tail appears at either of two sites. These RNAs encode two putative proteins (novel in computer searches) which differ in C-terminal length.

The above descriptions demonstrate that the relationships between brain genes, mRNAs and proteins are not simple. However, because of the techniques available to define and measure these structures, the brain is not intractable to molecular study. Evolutionary conservation and developmental onset of brain mRNAs.

We investigated the expression of rat brain Class III mRNAs in adult and embryonic tissues from mouse and monkey (Table I) by high stringency (95%) Northern blot hybridization (8). Nine of ten rat brain-specific mRNAs are very close in sequence to analogous brain­specific mouse mRNAs, and half of them are close in sequence to analogous brain-specific monkey mRNAs. Thus, the primate and rodent central nervous systems probably share most proteins. In all cases, developmental regulation is maintained. Four of ten brain mRNAs first appear in the postnatal rat. Si nce the brain expresses about 30,000

mRNA species, if our previous measurements (I) of brain RNAs are accurate, and more than 60% of these are brain-specific, then the number of mRNAs which have postnatal onset may be between 5,000 and 10,000

species. Thus, the brain molecular hardware is not complete until after birth (8).

1 7 bp insertion

~ r----AAAAA _--+-t---~~l"""-I -----iLAAAII Open· Reading Frame AAAAA

Fig. I. Structure of a polymorphic family of brain mRNAs.

28

TABLE 1. Northern blot hybridization of rat brain-specific cDNA clones to other species.*

Clone Class Rat Mouse Monkey B L E B L E B L

IB15 I + + + + + + + + lA75 III + - + + - + 18236 III + + + -18208 III + + - - + -0-30 III + + - - + -2A355 III + - + + - + IB380 III + - + + - + + -0-40 III + - + + - + 181075 III + - + + - + 18213 III + - + + - + + -18207 III + - - - - -# Class III 10 0 6 9 0 6 5 0

*8, brain; L, liver; E, embryo.

Identifier sequence elements may regulate neuronal gene expression. In the course of these studies, we have found a repetitive DNA

element (called ID), which is transcribed in vivo by RNA polymerase III (Pol III) exclusively in neuronal cells. The Pol III transcription begins 1-3 days postnatally (11) and produces brain-specific RNAs 8CI and 8C2 (12,13). Clones of these RNAs are potent Pol III templates in vitro. As 8rown has discussed in this volume, cDNA clones of 8C1 and 8C2 have heterogeneous, but highly conserved sequences - each clone differing from others for at most a few nucleotides. All of such cDNA clones share a common 5' end, hence, 8C RNAs are transcribed by Pol III from many of the 10 5 rat genomic ID elements (7). Curiously, rodent cell lines, regardless of their tissue origin, also express 8C RNAs (8).

When linked to reported genes such as neomycin resistance or chl orampheni co 1 acetyltransferase, ID el ements st imul ate the rate of reporter gene expression in transfected cell lines (14). Since this effect is observed when the ID element is in either orientation, and in sites upstream, downstream and within the reporter genes, and because the ID elements and the SV40 enhancer element do not show multiplicative or even additive stimulatory effects, we conclude that ID elements are related to enhancer elements and act in cis to regulate Pol II gene

29

expression at the level of transcription. In experiments conducted thus far, the enhancer activity of 10 sequences is only observed in cells in which Pol III transcription of ID sequences occurs. The ID enhancer activity is not detected, for example, in primary kidney cells, although other enhancers (SV40, Moloney) work well in these cells. Thus, since Pol III transcription of 10 sequences is strictly exclusive to postnatal brain cells in vivo (although not in cell culture), ID elements may be enhancers for adult neuron-specific genes. The glial virus JC, the agent for progressive multifocal leukoencephalopathy, has a glia-specif­ic enhancer which contains a region 23/25 identical to 10 (15).

Further support for an association between 10 sequences and adult neuronal genes comes from studies on RNA isolated from neuronal nuclei. It is important to realize that 10 elements are likely to be associated wi th neuronal and not gl i a 1 gene regul at i on and that 85 to 90% of the cells in the brain are glia cells. One should not, therefore, study nuclear RNA from whole brain, but rather separate neuronal nuclei from gl ial nuclei prior to RNA isolation. As described by Brown in this volume, neuronal hnRNA contains 5.2-fold more 10 sequences than glial or kidney hnRNA. The developmental onset of the difference is between postnatal days 8 and 14. Thus, 10 elements are clustered within genes transcribed by Pol II that have postnatal onset and are neuron-specific.

Transcription patterns of isolated nuclei mimic in vitro what is found in vivo. Isolated brain nuclei incorporate precursors into RNA mostly in neurons (16). The RNA made by brain nuclei in vitro contains IS-fold more ID sequences than that made by 1 iver or kidney nucl ei (13). A large portion of the difference is due to the synthesis of BC2 RNA by Pol III exclusively by the brain nuclei. There are considerably more ID containing Pol II transcripts made by brain than 1 iver or kidney, possibly the 5-fold difference observed in vivo. Cumulatively, these experiments suggest that the 5,000 to 10,000 adult neuronal genes are greatly enriched in 10 sequences compared to genes not exclusive to postnatal neurons. These 10 sequences are mostly in intronic regions, since mature mRNA does not hybridize significantly to 10 probes. This set of ID el ements may represent only a small subset of the total 105

rat ID copi es. Most of them are probably located in nontranscri bed regions of the genome.

30

Because ID elements function as enhancers, are transcribed by Pol III postnatally only in neurons, and are clustered within neuronal Pol II genes, we have proposed that they may be involved in regulating the expression of this battery of an estimated 5,000 to 10,000 genes (11). Since (as discussed in Brown's chapter) neuronal chromatin undergoes a rearrangement of its nucleosomal structure coupled with a depletion of histone HI by the end of the first wk of rat postnatal life, and because the DNA in these neurons simultaneously becomes sensitive to nucleases (17,18), we imagine that the postulated enhancer effect of ID (which may be mediated by Pol III) acts by unmasking local regions, thus potenti­ating interactions of transcriptional specificity factors at promoters. The value of this model is that it is easily accessible to experimental test. Its particularly novel features are that a Pol III transcription event may work in enhancing Pol II transcription, that enhancers and promoters may respond to independent regulatory programs (in particular, enhancers may be related to cell determination), and that in gene activation there may be a temporal order in which enhancer activity precedes promoter activity in the process of transcription.

ACKNOWLEDGEMENTS We thank I. Brown, R. Milner, P. Danielson, M. Kiel, M.A. Brow,

C. La i, K. Nave and C. Bl att for co 11 aborat ions on unpub 1 i shed experi­ments and l. El der for ass i stance with the manuscri pt. Supported in part by NIH grant GM32355. R. McK. is a Fellow of the National Cancer Institute of Canada and A.-P.T. is a Fellow of the Leukemia Society of America.

REFERENCES 1. Milner, R.J. and Sutcliffe, J.G. Nucleic Acids Res. 11 (16):

5497-5520, 1983. 2. Sutcliffe, J.G., Milner, R.J., Shinnick, T.M. and Bloom, F.E. Cell

33: 671-682, 1983. 3. Sutcliffe, J.G., Milner, R.J. and Bloom, F.E. Cold Spring Harbor

Symp. Quant. Biol. 48: 477-484, 1983. 4. Bloom, F.E., Battenberg, E.L.F., Milner, R.J. and Sutcliffe, J.G.

J. Neurosci. Q: 1781-1802, 1985. 5. Lenoir, D., Battenberg, E., Kiel, M., Bloom, F.E. and Milner, R.J.

J. Neurosci. (in press). 6. Malfroy, B., Bakhit, C., Bloom, F.E., Sutcliffe, J.G. and Milner,

R.J. Proc. Natl. Acad. Sci. USA 82: 2009, 1985.

31

7. Milner, R.J., Bloom, F.E., Lai, C., Lerner, R.A. and Sutcliffe, J.G. Proc. Natl. Acad. Sci. USA 81: 713-717, 1984.

8. Fitting, T., Danielson, P., McKinnon, R.D., Tsou, A.-P. and Sutcliffe, J.G. (in preparation).

9. Milner, R.J., Lai, C., Nave, K., Lenoir, D., Ogata, J., Bloom, F.E. and Sutcliffe, J.G. Cell!Z: 931-939, 1985.

10. Tsou, A.-P., Lai, C., Danielson, P., Noonan, D. and Sutcliffe, J.G. J. Mol. Cell Biol. (in press).

11. Sutcliffe, J.G., Milner, R.J., Gottesfeld, J.M. and Reynolds, W. Science 225: 1308-1315, 1984.

12. Sutcliffe, J.G., Milner, R., Bloom, F.E. and Lerner, R.A. Proc. Natl. Acad. Sci. USA 79: 4942-4946, 1982.

13. Sutcliffe, J.G., Milner, R.J., Gottesfeld, J.M. and Lerner, R.A. Nature 308: 237-241, 1984.

14. McKinnon, R.D., Shinnick, T.M. and Sutcliffe, J.G. Science, submitted.

15. Kenney, S., Natarajan, V., Strike, D., Khoury, G. and Salzman, P. Science 226: 1337-1339, 1984.

16. Thomas, J.O. and Thompson, R.J. Cell 10: 633, 1977. 17. Brown, I.R. In: Handbook of Neurochemistry, 2nd edition (Ed. A.

Lajtha), Plenum Press, New York, Vol. 5, 1983, pp. 217-226. 18. Brown, I.R. and Greenwood, P.D. In: Molecular Approaches to

Neurobiology (Ed. I.R. Brown), Academic Press, New York, 1982, pp. 41-69.

4 EXPRESSION OF BRAIN-SPECIFIC PROTEINS R.J. MILNER Division of Precl inical Neuroscience and Endocrinology, Research Institute of Scripps Clinic, La Jolla, CA, USA

ABSTRACT The brain of an adult rat expresses approximately 30,000 different

mRNAs. To determine the structure, location and possible functions of their encoded proteins, we have selected cDNAs corresponding to mRNAs expressed only in brain. One of these mRNAs encodes a protein that defines a widely distributed neuronal system and may be the precursor for a family of novel neuropeptides. A second clone encodes rat brain myelin proteo-lipid protein. Both of these genes are expressed only during postnatal development.

INTRODUCTION The physiological properties of any tissue are largely determined

by the set of protei n mol ecul es made by the cell s of that tissue. Functions that are unique to a particular tissue or organ therefore depend largely on the expression of proteins, many of which are unique to that organ or tissue. Because many of the properties and functions of the nervous system are uni que, we may expect cells in the brain (neurons and glial cells) to express a wide variety of proteins that are expressed only in neural tissues. We believe that a more detailed understanding of the mammalian brain requires the isolation and charac­terization of protein molecules that are uniquely or predominantly expressed by the brain.

We can estimate the number of protein molecules expressed in the brain from measurements of brain mRNA complexity (1). These studies have indicated that approximately 100,000 different mRNA molecules are expressed in the rodent brain. Our own analysis (2) has suggested that 30,000 different mRNAs may be a more realistic number. Of these, 50 to 90% may be expressed predominantly or uniquely in the brain. Assuming that each mRNA encodes a particular protein molecule, we can expect that up to 30,000 different proteins are expressed in the brain. Our current

33

knowledge of these proteins, however, is extremely limited, although recombinant DNA techniques have allowed rapid progress in the past five years, as many of the papers in this volume can attest.

We have approached the characterization of brain proteins with a strategy (2, 3 and Sutcliffe et al., this volume) based on recombinant DNA technology, which allows us to define the problem at the level of mRNA rather than protein. A cDNA library was generated from rat brain poly(A)+mRNA and screened by Northern blotting for clones of mRNAs which were expressed in brain but not in liver or kidney. Selected brain­specific clones were analyzed by nucleotide sequencing, providing the partial sequences of the corresponding mRNA and protein. To identify the proteins corresponding to these clones, we made antibodies against synthetic peptides mimicking selected regions of these hypothetical sequences. This report will summarize our studies on two clones ident ifi ed and characteri zed by thi s approach, that is, those corre­sponding to the brain-specific protein 1B236, a potential neuropeptide precursor (3-5), and rat brain proteolipid protein (PLP) (6), the major component of central nervous system myelin.

RESULTS AND DISCUSSION The brain-specific protein 1B236.

The clone plB236 hybridizes to a mRNA which is present in brain with an abundance of approximately 0.01%, but is undetectable in liver or kidney. The nucleotide sequence of plB236 provided the 318 amino acid carboxy terminal sequence of the corresponding protein, which had no homology to any previously defined sequence. The relationships of the IB236 mRNA, the cDNA clone plB236 and the putative open reading frame for the protein lB236 are shown in Fig. lAo A notable feature of the IB236 amino acid sequence is the presence of several pairs of basic amino acids (ArgArg, LysLys, LysArg) in the carboxy terminal region (Fig. lB). Such sequences, particularly the dipeptide LysArg, have been found previously in sequences of neuropeptide or peptide hormone precursors and have been shown to be the sites of proteolytic processing to produce bioactive peptides (7). In many cases, the precursor sequence contains multiple basic dipeptide sites, often clustered at the carboxy termi nus, and these precursors are processed to a vari ety of

34

pept ide products. Based on thi s structural simil arity, we postul ated that the 18236 protein might be proteolytically processed to generate a number of different peptides with potential bioactivity (3).

Multiple molecylar forms of IB236. In order to detect the IB236 protein and to define its possible cleavage products in the brain, rabbit antibodies were generated against synthetic peptides P5, P6 and P7 (Fig. IA) which correspond to the most likely products of proteolytic processing at pairs of basic residues (Fig. IB) and which were derived from non-overlapping regions of the sequence. Brains were extracted under a variety of different conditions and the extracts centrifuged to remove insoluble material. Aliquots of each supernatant were fraction-

A. 500 1000 1500 2000

I I I I

mRNA -----------

eDNA ". ' . .............

".

--.., P5 P6 P;!

•• Ala lie Leu lie Ala lie Val Cvs Tvr lie Thr Gin Thr Arg P5

Arg LV' Lvs Asn Val Thr Glu Ser Pro Ser Phe Ser Ala GlV Asp

Asn Pro His Val Leu Tvr Ser Pro Glu PheArg lie Ser Glv AI.

Pro Asp Lvs TVr Glu Sor Glu Lvs Arg Leu GlV Ser Glu Arg Arg

P6 Leu Leu GIV LeuArg GIV Glu Pro Pro Glu Leu Asp Leu Ser Tvr

P7 Ser His SerAspLeu GlV Lvs Arg Pro Thr LvsAspSer TV' Thr

Leu Thr Glu Giu Leu Ala Glu Tvr Ala Giu lie Arg Val Lvs

/

Fig. I. Structure of the IB236 protein. A) The IB236 mRNA, the plB236 cDNA clone and the IB236 protein are schematically represented (2). The mRNA is approximately 2500 nt in length; the cDNA clone plB236 corre­sponds to the 3' 1500 nt. The open reading frame in the cDNA sequence is shown by the filled bar, the 3' non-coding region is shown by the open bar. Also shown are the positions of peptides P5, P6 and P7. B) The carboxy terminal sequence of the IB236 protein is shown, with peptides PS, P6 and P7 underlined (taken from ref. 4).

3S

ated by gel filtration on Sephadex G-7S and the column fractions assayed for 1B236 immunoreactivity by radi oimmunoassay (RIA), usi ng the anti­peptide antibodies. Where possible, the same column eluate was assayed in parallel for all three peptides. In these experiments, two types of immunoreact ive materi a 1 were detected: materi al which el uted in the void volume of the Sephadex G-7S column with a size greater than 13 kD (cytochrome c) and was defined as high molecular weight (HMW) material; material which eluted in the included volume of the column, with a size less than 13 kD and was defined as low molecular weight (LMW) material.

Brain extracts prepared with the nonionic detergent NP40 contained large amounts of PS, P6 and P7 HMW material. The elution patterns of the HMW material were very similar for each of the peptide immunoreac­tivities and are consistent with the presence of HMW components contain­ing all three peptide sequences. Using Western blotting, we have previ­ously detected HMW 1B236 in rat brain extracts solubilized with SDS (9). Thi s protei n has an apparent MW of 100 kD and has been subsequently found to be a membrane-bound glycoprotein (R.J. Milner, C. Chavkin, and B. Malfroy, in preparation). The coelution of the 1B236 protein detected by Western blotting and by RIA (Fig. 2) indicates that this

z o -I-U < tl::

500

~ 250 U1 w ..J o ::E lJ..

10 20 FRACTION NUMBER

30

Fig. 2. Radioimmunoassay and Western blot analysiS of HMW 1B236. An extract of brain in Tris/NaCl/NP40 was fractionated on Sephadex G-7S and fractions assayed for IB236 using RIA for PS. In parallel, aliquots of each fraction were assayed for 1B236 by Western blotting (8) using anti­PS antibodies: the photograph of the blot showing the 100 kD immuno­reactive band is aligned so that each gel slot corresponds to its appropriate fraction (taken from ref. 4).

36

species corresponds to the HMW form extracted by detergent and detected by RIA. In addition, there may be small amounts of a second HMW 1B236-like protein that appears to be soluble in the absence of detergent.

Detergent extracts also contained LMW P6- and P7-like material. Because peptides could be expected to be relatively sensitive to proteo­lysi s when extracted wi thout any part i cul ar precaution under neutral conditions, we tested extraction conditions designed to reduce or el iminate endogenous proteolysis, such as rapid heating of the brain by microwave irradiation before extraction and extraction under acidic conditions. When whole rat brains were extracted under either of these conditions, there was a large increase in the amount of extractable LMW P5- and P7-like material. On an HPLC protein analysis column, this material was resolved into peaks of P5 and P7 immunoreactivity, each eluting at the same position as the corresponding synthetic peptide, and a second peak of P5 immunoreactivity which eluted at the position of enkephalin (Fig. 3).

The multiplicity of IB236 immunoreactive forms suggests that this molecule undergoes extensive post-translational modification,

BSA P5P7ENK BSA P5P7ENK

• .. • • .. • 150 A B

z 0

;: 100 u < 0:: LL "-(J)

w ~ 50 0 ::E LL

r Jth. 0 1 40 60 BO 40 60 BO

FRACTION NUMBER

Fig. 3. HPLC analysis of LMW forms of IB236 in extracts of rat whole brain after microwave irradiation. A rat whole brain was subjected to microwave irradiation immediately after dissection, extracted in Tris/ NaCl, and a 500 ~l aliquot injected on two HPLC protein analysis columns mounted in series. The eluted fractions were assayed by RIA for P5 (A) and P7 (8) (taken from ref. 4).

37

including proteolytic processing, to generate potentially bioactive peptides. Isolation of these processed peptides, determination of their exact relationship to the HMW forms of IB236, and the demonstration that these peptides are physiologically relevant will be necessary for the further understanding of this system.

Immunocytochemi cal anal ysi s of IB236. In immunocytochemi cal experiments, antisera against each of the three non-overlapping peptide regions produced identical cytological maps of intense neuropil staining in adult rat brain which was blocked in each case by preincubation of the antibodies with the appropriate peptide. The immunoreactivity was distributed heterogeneously in the brain and was most pronounced within olfactory bulb and peduncle, specific hypothalamic and preoptic nuclei, the neostriatum, limbic and neocortical regions, particularly somato­sensory cortex (Fig. 4).

In electron microscopy studies, at least part of the immunoreac­tivity in cortex was found associated with synaptic vesicles. The

18236 IMMUNOREACTIVE NEURONS

Fig. 4. Neuroanatomical distribution of IB236: schematic overview of neuropil and perikaryon immunoreactivity patterns. Cell body-rich areas are symbolized by circles, neuropil-rich regions by dots; presumptive fiber trajectories are symbolized by solid lines and putative pathways by dotted lines. Abbreviations: AON, anterior olfactory nucleus; BNST, bed nucleus of the stria terminal is; CC, corpus callosum; DBB, diagonal band of Broca; DCN, deep cerebellar nuclei; ER, entorhinal cortex; MS, medial septal nucleus; MTz, medial trapezoid nucleus; PAG, periaque­ductal grey; PMN, premamillary nucleus; Str, striatum; V, spinal trigeminal nucleus; VP, ventral pallidal area; XII, hypoglossal nucleus.

38

data suggest that 1B236 is present in particular neuronal elements

within functionally related central systems and that these elements may have evolved from related progenitor lines.

Expression of 1B236 during brain development. To determine the time course of expression of 1B236 and other brain-specific gene products during brain development, we studied brain protein and mRNA extracts between embryonic day 14 to postnatal day (PD) 30, and adult. 1B236 mRNA was detected by RNA blotting; 1B236 protein was assayed by Western blotting and RIA. 1B236 mRNA is first detectable in extracts of whole rat brain at PD5 and increases to a maximum concentration at PD25 (Fig. 5). In extracts of dissected brain regions, 1B236 mRNA is first detectable at PD5 in hindbrain and cerebellum, at PD9 in midbrain, but not until PD13 in telencephalon. The appearance of 1B236 protein follows a very similar time course to that of its mRNA in both whole brain and dissected brain regions, suggesting that the expression of the protein is regulated largely by transcription of its mRNA.

The pattern of 1B236 expression was confirmed by immunocytochemical localization of 1B236 protein. Immunoreactive material can be detected first in spinal cord at PD3-PD5 and then appears in progressively more rostral brain regions in increasingly older animals, occurring last in cerebra 1 cortex. Severa 1 brain regi ons, however, that do not contain 1B236 immunoreactivity in the adult, such as optic nerve and somatic

Fig. 5. Developmental expression of 1B236 mRNA detected by RNA blot­ting. Al iquots (2 119) of poly(A)+ RNA extracted from rat brains of the indicated embryonic and postnatal ages and from adult (A) were separated by electrophoresis, blotted to nitrocellulose and hybridized with [32p]_ labeled p1B236.

39

efferent cranial nerve nuclei, show transient expression of IB236 during postnatal development. The postnatal expression of IB236 indicates that this protein is a marker for the terminal differentiation of particular neurons and suggests that the IB236 protein probably mediates functions specific to the adult nervous system. Rat brain myelin proteolipid protein.

A second cDNA clone (plB208) hybridized to two highly abundant, brain-specific mRNAs, 3200 and 1600 nt in length, which were also expressed in C6 glioma cells. Using plB208 as a probe, we isolated additional clones, corresponding to both mRNAs, from a cDNA 1 ibrary favoring full-length cDNA inserts. Nucleotide sequence analysis of these clones indicated that the two mRNAs are coextensive at their 5' ends but differ in the 3' site of polyadenylation. Preliminary evidence suggests that a single gene encodes the two mRNAs in the rat and that they are derived by differential polyadenylation of the same primary RNA transcript. The finding that both mouse and monkey express the 3200 but not the 1600 nt mRNA suggests that there is no functional necessity for two PLP mRNAs in the rat.

Both mRNAs encode identical proteins, 277 amino acids in length (Fig. 6), that can be unambiguously identified as myelin PLP, also known as 1 ipophil in, by homology with the known sequences of rat and bovine PLP (10-12). The amino acid sequence translated from the nucleotide sequence is identical in length with the mature protein, as defined by protein sequencing studies, with the exception of the amino terminal methionine, which is presumably removed after synthesis of the poly­peptide. This indicates that PLP does not require a signal peptide sequence for insertion into the myelin membrane. There are at least two and possibly up to seven amino acids different in the complete sequences of rat and bovine PLPs (97 to 99% homology), indicating that the sequence of proteolipid protein has been highly conserved during evolution. The developmental expression of the mRNAs encoding PLP follows a postnatal time course similar to that for IB236 and consistent with the time course of myelinogenesis.

Myelin PLP is one of the most abundant brain-specific proteins. As the major protein component of CNS myelin, it clearly plays a critical role in forming and maintaining that unique biological structure.

40

AMINO ACID SEQUENCE OF RAT PROTEOLIPID PROTEIN

20 [MetlGlyLeuLeuGluCysCysAlaArgCysLeuValGlyAlaProPheAlaSerLeuValAlaThrGlyLeUCys

• 40 • PhePheGlyValAlaLeuPheeysGlyCysGlyHisGluAlaLeuThrGlyThrGluLysLeUIleGluThrTyrPhe

60 • SerLysAsnTyrGlnAspTyrGluTyrLeUIleAsnValIleHisAlaPheGlnTyrValIleTyrGlyThrAlaSer

80 100 PhePhePheLeuTyrGlyAlaLeuLeuLeuAlaGluGlyPheTyrThrThrGlySerValArgGlnIlePheGlyAsp

Tyr 120

TyrLysThrThrIleCysGlyLysGlyLeuSerAlaThrValThrGlyGlyGlnLysGlyArgGlySerArgGlyGln

• 140 HisGlnAlaHisSerLeuGluArgValCysHisCysLeuGlyLysTrpLeuGlyHisPrOAspLysPheValGlyIle

160 180 ThrTyrAlaLeuThrValValTrpLeuLeuValPheAlaCysSerAlaValProValTyrIleTyrPheASnThrTrp

200 ThrThrCysGlnSerIleAlaPhePrOSerLysThrSerAlaSerIleGlySerLeuCysAlaAspAlaArgMetTyr

Ala Thr 220

GlyValLeuPrOTrpAsnAlaPheProGlyLysValCysGlySerAsnLeULeuSerIleCysLysThrAlaGluPhe

240 GlnMetThrPheHisLeuPheIleAlaAlaPheValGlyAlaAlaA1aThrLeuValSerLeuLeuThrPheMetI1e

Val 260

AlaAlaThrTyrAsnPheAlaValLeuLysLeuMetGlyArgGlyThrLysPhe

Fig. 6. The amino acid sequences of rat and bovine brain PLPs. The sequence of the rat protein derived from nucleotide sequence analysis is shown. Residues different from the published complete sequences of bovine PLP (11, 12) are shown below the line. Residues are numbered from the amino terminal Gly of the mature protein.

Furthermore, recent evidence that injection of pure PLP into rabbits can induce a chronic allergic encephalomyelitis implicates this protein in demyelinating disease (13). Availability of cDNA clones for PLP will enable a rapid characterization of the human protein and assessment of its involvement in the etiology of degenerative diseases such as multiple sclerosis.

ACKNOWLEDGEMENTS I recognize the contributions of my collaborators J.G. Sutcliffe,

F. Bloom, B. Malfroy, C. Bakhit, D. Lenoir, C. Lai, K. Nave and E. Battenberg whose studies I have reviewed here. This work was supported,

41

in part, by NIH grants NS 20728, NS 21815 and GM 32355, NIAAA Alcohol Research Center grant AA 06420 and grants from McNeil Laboratories.

REFERENCES 1. Bantle, J.A. and Hahn, W.E. Cell~: 139-150, 1976. 2. Milner, R.J. and Sutcliffe, J.G. Nucleic Acids Res. 11: 5497-5520,

1983. 3. Sutcliffe, J.G., Milner, R.J., Shinnick, T.M. and Bloom, F.E. Cell

33: 671-682, 1983. 4. Malfroy, B., Bakhit, C., Bloom, F.E., Sutcliffe, J.G. and Milner,

R.J. Proc. Natl. Acad. Sci. USA 82: 2009-2013, 1985. 5. Bloom, F.E., Battenberg, E.L.F., Milner, R.J. and Sutcliffe, J.G.

J. Neurosci. ~: 1781-1802, 1985. 6. Milner, R.J., Lai, C., Nave, K.-A., Lenoir, D., Ogata, J., Bloom,

F.E. and Sutcliffe, J.G. Cell 42: 931-939, 1985. 7. Douglass, J., Cive11i, O. and Herbert, E. Ann. Rev. Biochem. 53:

665-715, 1984. 8. Towbin, H., Staehe1in, T. and Gordon, J. Proc. Nat1. Acad. Sci. USA

79: 4540-4544, 1982. 9. Sutcliffe, J.G., Milner, R.J. and Bloom, F.E. Cold Spring Harbor

Symp. Quant. Bio1. 48: 477-484, 1983. 10. Jo1les, J., Nussbaum, J.L. and Jo11es, J. Biochim. Biophys. Acta.

742: 33-38, 1983. 11. Laursen, R.A., Samiu11ah, M. and Lees, M.B. Proc. Nat1. Acad. Sci.

USA 81: 2912-2916, 1984. 12. Stoffel, W., Hillen, H. and Giersiefen, H. Proc. Nat1. Acad. Sci.

USA 81: 5012-5016, 1984. 13. Cambi, F., Lees, M.B., Williams, R.M. and Macklin, W.B. Ann.

Neuro1. 13: 303-308, 1983.

5 MESSENGER RNA IS PRESENT IN THE AXOPLASM OF SQUID GIANT AXONS A. GIUDITTA, T. HUNT l , C. PERRONE CAPANO, L. SANTELLA2 AND B.B. KAPLAN 3

Department of General and Environmental Physiology and International Institute of Genetics and Biophysics, Naples, Italy; 1 Department of Biochemistry, Cambridge, UK; 2Zoological Station, Naples, Italy and 3Western Psychiatric Institute and Clinic, Pittsburgh, PA, USA

ABSTRACT Using a translation assay it has been shown that the axoplasm

extruded from the squid giant axon contains mRNA. The translation pattern ofaxoplasmic mRNA, as resolved on SDS-polyacrylamide gels, is different from the translation patterns of stellate nerve and giant fibre lobe (nerve cell bodies) RNA, although many protein bands are the same in axoplasm and the nerve cell bodies, as might be expected. Comparison of the axoplasmic proteins reveals a significant degree of correspondence. Axop 1 asmi c mRNA is associ ated wi th the "mi crosoma 1 " fract i on of the axopl asm. Using reverse transcri ptase, axopl asmi c RNA can be transcribed into cDNA. The results of hybridization kinetics of axoplasmic cDNA with its template RNA show that the diversity ofaxo­plasmic RNA is considerable higher than indicated by the translation assays. On the other hand, the comp 1 exi ty ofaxop 1 asmi c RNA is about 30-fold lower than that of total giant fibre lobe RNA and about 150-fold lower than that of optic lobe polysomal poly(A)+ mRNA. The latter results strongly supports the view that the high complexity of brain mRNA reflects the marked cellular and subcellular heterogeneity of the tissue.

INTRODUCTION According to current views, neuronal protein synthesis occurs only

in cell bodies and in proximal dendrites and is lacking in the axons and synaptic terminals (I). Two main observations support this conclu­sion. Ribosomes are largely confined to the cell body, and have not been detected by electron microscopy of the axon, except for a few reports (2,3). In support of these morphological data, axoplasmic RNA has been found to be of the 4S type (4,5). However, it is very diffi­cult to obtain pure axonal fractions for biochemical analysis, except in

43

the case of giant axons such as occur in the squid. Here a large body of evidence suggests that the synthesis ofaxoplasmic proteins by the isolated giant axon (6,7) is due to the activity of peri axonal glia cells which export newly-made proteins to the axon (8,9). Protein synthesis has not been detected in extruded axoplasm.

Despite the general consensus denying the possi bil i ty of protei n synthesis in the squid giant axon, it has proven possible to detect many of the components of the protein synthesis machinery in extruded axoplasm. These include the soluble factors (10), all species of tRNA and thei r respective synthetases (10,11), the e1 ongat i on factors (10) and minor amounts of rRNA (12). The latter component may be present in limiting concentrations. The latest and most intriguing addition to the list concerns the demonstration that squid axoplasm also contains a discrete subset of messenger RNA whose translation products are largely but not entirely overlapping with those directed by cell body and glial mRNA (13).

MATERIALS AND METHODS Tissue preparation.

Pure samples of axoplasm were obtained from adult specimens of the squid, Loligo pea7ii, available during the summer months at the Marine Biological Laboratory in Woods Hole, MA, USA. The most medial stellate nerves were dissected out. The proximal end of the giant axon was freed of the surrounding thin axons and axoplasm was extruded using a tiny roller. Axoplasm and remaining stellate nerve were frozen at -80°. The latter sample contains the sheath of the giant axon and the thin axons which surround the giant axon. Apart from connective tissue cells, its mai n structural components are the peri axonal gl i a1 cell s associ ated with the thin axons and the giant axon, as well as the axoplasm of the thin axons and the outer layers of the giant axon. The giant fibre lobe (cell bodies of the giant axon) and the optic lobes (head ganglia) were also dissected out and stored at -80°. RNA extraction.

For the translation assays, RNA was purified by extraction with phenol and chloroform and added to an RNase-treated rabbit reticulocyte lysate supplemented with [35S]methionine (Amersham International; 1400

44

Ci/mM) (14). The radioactive translation products were separated on ID SDS acrylamide gels (15,16). Samples of RNA to be used in the hybrid­ization analyses were purified using a comparable procedure. Polysomal poly(A)+ RNA from squid optic lobes was purified by three cycles of affinity chromatography on oligo(dT) cellulose, as previously described (17,18).

RESULTS Translation activity ofaxoplasmic RNA.

Incubation of the reticuloctye cell-free translation mixture in the absence of exogenous RNA yielded a background incorporation of [35S]meth­i oni ne of approximately 1000 cpm for an al i quot correspondi ng to one tenth the total volume. A considerable incorporation (several hundred thousand cpm) was obtained wi th a saturating concentration of tobacco mosaic virus RNA. Addition ofaxoplasmic RNA stimulated incorporation 2- to 5-fold over background values. In these experiments, RNA was extracted from approximately 100 mg axoplasm and one tenth of the extracted RNA was added to the assay mixture. The same results were obtained in several preparations of axoplasm using two different batches of lysate during three consecutive summers. In two analyses, axoplasm was extruded from isolated giant axons, that is following complete dissection of the surrounding small axons. This additional precaution insured that no material derived from the stellate nerve could contami­nate the axoplasmic sample during the extrusion step. Under the latter conditions, the overall activity of the axoplasmic sample was comparable to that obtained with the routine method of preparation, while only a barely detectable overall activity was found in the axonal sheath remaining after the extrusion step. This structure contains the gl ia cell layer surrounding the giant axon. The result implied that the mRNA activity of the axoplasmic sample could not be attributed to mRNA pinched off from glial processes during the extrusion step.

For comparison, we also assayed the translation activity of RNA extracted from the optic lobe, the giant fibre lobe and the remaining stellate nerve. The optic lobe was taken as representing the mixture of cellular and subcellular elements present in squid brain. It gave a substantial increment in incorporation, several times larger than with

45

axoplasmic RNA. An even stronger stimulation, up to 10 to 50-fold over background, was given by RNA extracted from the giant fibre lobe. As already mentioned, the giant fibre lobe contains the cell bodies of the giant axon system, thus providing a unique opportunity to compare the transl at i on act i vi ty ofaxopl asmi c RNA wi th that of the correspondi ng

perikarya. On a wet wt basis, the giant fibre lobe is 10 to 20-fold more active than axoplasm (Table 1), as expected on the basis of its markedly higher content of rRNA and, by inference, of actively trans-1 at ing polysomes. On the other hand, the transl at i on act i vi ty of the remaining stellate nerve (after axoplasm extrusion from the giant axon) is comparable to that of axoplasm. The activity of the stellate nerve is largely attributable to peri axonal glia cells, although the contribu­tion of axoplasm associated to the thin axons and to the outer layers of the giant axon cannot be neglected. The similar degree of translation activity of axoplasm and stellate nerve RNA confirmed the lack of contamination of the axoplasmic sample by material derived from the nerve during the extrusion step. Electrophoretic separation of the cell-free translation products.

Definite proof of the presence of mRNA in squid axoplasm was provided by gel electrophoretic analysis of its translation products.

TABLE 1. Cell-free translation assay of RNA from squid axoplasm and related regions of the nervous system.

Tissue

Axoplasm

Stellate Nerve

Giant Fibre Lobe

Protein Radioactivity (cpm/mg wet wt)

(1)

15,600

25,000

386,800

Experiment

(2)

4,600

5,100

27,200

The incorporation of [35S]methionine into protein was determined follow­i ng i ncubat i on of the rabbi t ret i cul ocyte lysate wi th exogenous RNA (14-16).

46

As shown in Fig. 1, several radioactive bands were separated ranging in size from less than 20 kD to considerable more than 100 kD. Some of the most intense bands resemble actin and the tubulin subunits in electro­phoretic mobility (dashes to the left; see also Fig. 3). Other axoplasmic bands, notably at about 116 kD appear to be lacking in the translation patterns of the giant fibre lobe and of the remaining stellate nerve. Another axoplasmic protein (18 kD) is absent in the lobe pattern, but is present in the nerve pattern.

Fig. 1. Electrophoretic patterns of the radioactive proteins synthe­sized in the rabbit reticulocyte lysate following addition of RNA extracted from the giant fiber lobe (1), the axoplasm (a) and the remaining stellate nerve (n); p, radioactive protein markers whose MW (kD) is indicated on the right side; b, blank without added RNA. Patterns of comparable intensity were obtained by appropriate dilution of the samples corresponding to the giant fibre lobe and the remaining stellate nerve with unlabeled lysate mixture. Exposure time, 68 hr at -80 0 on Fuji RX X-ray film.

47

Si nce axopl asmi c mRNA speci fi es polypeptides that are not present

in the translation products obtained with mRNA from the stellate nerve,

it seems that the mRNA in axoplasm cannot arise by contamination from

the surrounding nerve. This conclusion is further supported by the

great differences between the two patterns. It is more surprising that

there are at least two fairly prominent polypeptides made by axoplasmic

mRNA that are not at all abundant (if they are present at all) in the

mRNA from the cell bodies of the giant axon. Presumably, the mRNA in

axoplasm originates from the cell body, and although most of the mRNA

is probably bound to ribosomes which are restrained form entering"

axoplasm, it is not unexpected to find that some of it escapes. But the

foregoing observation suggests that some may actually be designed for

export. Unfortunately, we cannot be certain at what rate this mRNA

travels down the axon. If it were associated with the vesicular

component of the fast transport system, it might be del i vered to the

nerve terminals. In some ways, this is an attractive idea, because it

woul d be cons i stent wi th the fail ure to detect protei n synthes is in

extruded axoplasm. However, we do not know whether this mRNA is trans­

lated in vivo, and if so, where this occurs.

Axoplasmic mRNA is not derived from mitochondria.

On theoretical grounds, mitochondrial mRNA cannot be translated in

the reticulocyte lysate (19). Nonetheless, we considered the possibil­

ity that axoplasmic mRNA was derived from mitochondria in experiments

of subcellular fractionation of an axoplasmic homogenate prepared in 1M

sucrose. Of the three fractions obtained by differential centrifuga­

t ion, only the "mi crosoma 1" fract ion (170, OOOxg for 3 hr) yi e 1 ded an

overall activity comparable to that of whole axoplasm (4200 cpm/mg wet

wt). Neither the mitochondrial fraction (13,000xg for 20 min) nor the post-mi crosomal II supernatant n fraction di spl ayed activity. These results were confirmed by analysis of the corresponding translation products by gel electrophoresis (not shown). We concluded that

axoplasmic mRNA is largely present in association with cytoplasmic

particles requiring higher centrifugal forces than mitochondria to be

sedimented. In accord with these results, we failed to detect mRNA

act i vi ty ina puri fi ed fract i on of synaptosomal mi tochondri a prepared from the optic lobe (courtesy of Dr. R. Cohen; synaptosomal mitochon-

48

dria are analogous to axonal mitochondria and may be readily obtained in larger yields). On the other hand, other subcellular fractions of the optic lobe gave good stimulation of [35S]methionine incorporation. This result was confirmed by electrophoretic analysis of the translation products (Fig. 2). Comparison ofaxoplasmic translation products with bulk axoplasmic proteins.

The discovery ofaxoplasmic mRNA raised the question of the correspondence of its translation products wi th bul k axopl asmi c pro­teins. The latter proteins were separated by electrophoresis in the

116

68

56

42 40

35

n m b s t

Fig. 2. Translation patterns obtained using RNA extracted from various subcellular fractions of the squid optic lobe. n, nuclear-mitochondrial fraction containing free mitochondria; s, synaptosomal fraction; m, mitochondrial fraction; t, synaptosomal mitochondria; b, blank without added RNA. The MW of marker proteins are indicated on the left side.

49

same gel used to fractionate cell-free translation products of axoplasmic RNA. The gel was stained with Coomassie blue before fluor­ography. In Fig. 3 the stained pattern of bulk axoplasmic proteins is shown side by side with the translation pattern ofaxoplasmic RNA. It is of interest that a significant degree of correspondence is present between the translation pattern ofaxoplasmic RNA and the pattern of bulk axoplasmic proteins, particularly in the intermediate and high MW range. Complexity ofaxoplasmic mRNA.

The electrophoretic method of separation of the translation products on 10 gels provided a minimal estimate of the number of different mRNAs present in the axoplasm, as it revealed only the most abundant species. A more accurate estimate had to rely on RNA-DNA hybridization techniques, which are better able to detect mRNA species

116

68

56 42 40

35

17

b r p t

Fig. 3. Comparison of the translation pattern ofaxoplasmic RNA (r) with the electrophoretic pattern of bulk axoplasmic proteins (p; stained with Coomassie blue). b, translation mixture without added RNA; t, tubulin fraction purified from a molluskan species (tubulin subunits at 60 and 56 kD). The MW of the marker proteins is shown on the left side.

50

in low abundance. Our experimental approach required the preparation of

radiolabeled cDNA using axoplasmic RNA as template in the oligo(dT)­primed reaction catalyzed by reverse transcriptase (20). In this experiment, the radi 01 abel ed dexoyri bonucl eot i de was incorporated into cDNA fragments 200 to 2000 nt in length, as judged by alkaline sucrose density gradient centrifugation (not shown). [32p]cDNA and the homolo­gous RNA were subsequently incubated and the extent of hybridization determi ned at i ncreas i ng Rot values by the S 1 nucl ease/DEAE fi lter method (21). For comparative purposes, we determined the RNA complexity of the giant fibre lobe and optic lobe, using total poly(A)+ RNA and poly(A)+mRNA, respectively. Estimates of the sequence complexity of each of the RNA populations was derived from an analysis of the kinetics of hybridization (for review, see ref. 22). The complexity of optic lobe poly(A)+mRNA, giant fibre lobe poly(A)+RNA and axoplasmic RNA was

TABLE 2. Complexity of poly(A)+RNA from axoplasm and related regions of squid nervous system.

Source of Frequency Fraction Cot la Sequence No. Diff. RNA Class DNA Obs. Cor.a Comp 1 exi ty b Sequences c

Hybrid. (M.sec) (M.sec) (nt)

OL Abundant 0.18 0.6 0.10 2.4 x 10 6 1,580 poly(A)+

10 7 mRNA Rare 0.82 50.0 41.0 9.0 x 60,000

GFL Abundant 0.28 0.16 0.04 8.8 x 10 4 590 poly(A)+

10 7 RNA Rare 0.72 13.0 9.4 2.1 x 14,000

AXO Abundant 0.56 0.004 0.002 5.3 x 10 3 4 total

RNA Rare 0.44 0.7 0.31 6.2 x 10 5 410

Data derived from analysis of the hybridization kinetics of cDNA to its homologous (template) RNA. a, calculated from observed Cot~ , assuming the kinetic component was pure and comprised all the reacting RNA sequences (Cot la x fraction of cDNA hybridized);b, the hybridization of rabbit globin cDNA to its template RNA was used as a kinetic standard (Cot la, 6 X 10- 4 M. sec; K = 1.4 kb); c, calculated assuming that the number average size of mRNA is 1500 nt. OL, optic lobe; GLF, giant fibre lobe; AXO, Axoplasm.

51

9.2 X 10 7 , 2.1 X 10 7 , and 6.2 x 10 5 nt, respectively (Table 2). The diversity of the axoplasmic RNA population was sufficient to code for about 414 different mRNA species, averaging 1500 nt in length. These data indicate that the complexity ofaxoplasmic RNA represents about 3% that of the poly(A}+RNA population present in the neuronal cell bodies. The complexity of optic lobe poly(A)+mRNA estimated by the cDNA kinetic method was identical to that obtained independently by RNA-DNA satura­tion hybridization analysis (23). Interestingly, the RNA complexity of a homogeneous population of neurons (giant fibre lobe) is considerably lower than that of the optic lobe, a brain region characterized by sub­stantial cellular heterogeneity. These results support the belief that the large diversity of gene expression in brain (22,24) is related to the structural and funct i ona 1 heterogeneity of the tissue (see also chapter by Kaplan et al., and by Hahn et al.). The relatively low complexity ofaxoplasmic RNA is in line with this view, since it corre­lates quite well with the high degree of structural homogeneity of the axoplasmic domain.

In absolute terms, however, the number of different mRNA sequences present in axoplasm is surprisingly high. Most of these sequences are not detected as transl at i on products on 10 gel s, whi ch separate, at most, 50 protein bands. It may be predicted that many more components would be resolved on 2D gels, provided the abundance of the corres­ponding axoplasmic mRNAs is not below the limit of sensitivity of the translation method.

Further evidence of the presence of poly(A)+ mRNA in axopl asm was obtained by analysis of its poly(A)+ content using a [3H]poly(U) assay (25). Similar analyses were made in the giant fibre lobe, in the remaining stellate nerve and in the optic lobe. The results of these assays indicated that the axoplasm contains a significant amount of poly(A) (0.24 ng/~g RNA), albeit lower than in the giant fibre lobe (1.4 ng/~g RNA) and in the optic lobe (1.3 ng/~g RNA). An intermediate value is present in the stellate nerve (0.54 ng/~g RNA). In terms of wet wt, the concentration of poly(A) is highest in the giant fibre lobe (3.5 ng/mg) and lowest in the axoplasm (0.017 ng/mg), with intermediate values in optic lobe (0.78 ng/mg) and in the stellate nerve (0.096 ng/ mg). Assuming an average length of 100 nt for the poly(A) tail and

52

1500 nt for the whole mRNA, axoplasmic poly(A)+mRNA can be calculated to represent 0.36% of the total RNA mass, corresponding to a concentration of 0.26 ng/mg wet wt. The corresponding values for the giant fibre lobe are 2.1% and 52.5 ng/mg wet wt. Intermediate values are present in the optic lobe (1.9% and 11.6 ng/mg wet wt) and in the stellate nerve (0.8% and 1.4 ng/mg wet wt).

DISCUSSION The occurrence of mRNA in the axoplasm of the squid giant axon is

demonstrated by several lines of evidence. In the first place, axoplas­mic RNA stimulates incorporation of [ 35S]methionine in the reticulocyte lysate assay well above background values. Its translation products can be resolved by ID gel electrophoresis into at least 50 different bands ranging in MW from less than 20 kD to considerably more than 100 kD. Some of the radioactive bands have the same mobility as known proteins, such as actin, the tubulin subunits, and some of the neurofilament proteins. These proteins are present in axopl asm as bul k proteins. Secondly, significant amounts of poly(A)+have been detected in axoplasm usi ng a [3H]poly(U) hybridi zati on assay. Thi rdly, axopl asmi c RNA has been shown to direct the synthesis of cDNA by avian reverse transcrip­tase, thus demonstrating the presence of poly(A)+mRNA. Hybridization of the resulting [32p]cDNA with its homologous RNA has provided a higher estimate of the number ofaxoplasmic mRNA sequences. The data indicate that approximately 400 different sequences of 1500 nt may be present in axoplasm, mostly in low abundance. The kinetic data also indicate that a small number « 10) of mRNA are present in axoplasm in relatively high abundance. These may correspond to structural proteins, such as actin and tubulin.

The possibility that these findings may be attributed to contamina­tion of the axoplasmic sample by extraneous material derived from the stellate nerve during the extrusion step may be ruled out. Axoplasm was always prepared with the utmost care to prevent contamination. More compell ingly, the translation ofaxoplasmic RNA is clearly different from the translation pattern of stellate nerve RNA. A few radioactive bands present in the axoplasmic pattern are lacking in the nerve pattern. In addition, when giant axons were thoroughly cleaned of their

53

surrounding thin axons before the extrusion step, the degree of stimula­tion of [35S]methionine incorporation and the corresponding translation pattern ofaxoplasmic RNA remained essentially the same as under routine preparation conditions. Conversely, the mRNA activity of the axonal sheath (containing peri axonal gl ia cells) was considerable lower than that of the axoplasmic sample. On the basis of this evidence, it may be safely concluded that mRNA is present in squid axoplasm.

Of the several questions raised by this finding perhaps the most pressing one regards the origin and role ofaxoplasmic mRNA. In view of the function generally ascribed to this class of RNA, an obvious possi­bility is to relate it to a process of protein synthesis presumably occurring in the axon itself (26). This hypothesis is supported by the presence of other components of the protein synthesis machinery in squid axoplasm (10,12) and by the limited but definite degree of correspon­dence existing between the axoplasmic translation pattern and the pattern of bulk axoplasmic proteins. The hypothesis is, however, strongly contradicted by the inability of extruded axoplasm to sustain protein synthesis and by a number of other observations which support the glia-transfer hypothesis (8,9). In addition, the electrophoretic pattern of labeled axoplasmic proteins seen when isolated giant axons are incubated with [ 35 S]methionine is different from the transl ation pattern obtained with axoplasmic mRNA (data not shown). This observa­tion might imply that the contribution ofaxoplasmic mRNA translation to the synthesis ofaxoplasmic proteins is at most small, although the lack of post-translation modifications in the translated proteins might also account for the difference. Following an alternative hypothesis, axoplasmic mRNA may be though of being en route to a different site of protein synthesis. Assuming a perikaryal origin ofaxoplasmic mRNA and its distal movement with the axoplasmic flow, this site might be located at the nerve terminal or even in the post-synaptic neuron, provided a mechanism for trans-synaptic passage of mRNA exists. If the latter assumption is accepted, a less likely but still possible alternative would imply a post-synaptic origin ofaxoplasmic mRNA and its proximal transport towards the protei n synthetic site of neuronal peri karyon. Additional variants of the hypothesis consider periaxonal glia cells as sources or destination sites ofaxoplasmic mRNA. These questions will

54

be addressed in future work. It may be added that isolated squid giant axons are capable of synthesizing axoplasmic RNA, presumably in their peri axonal glial cells (27). Finally, it is also possible that mRNA may not be translated in axoplasm and be present merely as a "leakage" product form the cell body. This possibility has been discussed in the previous section and considered an unlikely alternative on the basis of the distinct differences between the axoplasmic translation pattern and the pattern of the corresponding nerve cell perikarya. We may add that while the concentration ofaxoplasmic mRNA is much lower than in the cell bodies, its overall content represents a relevant fraction of the peri karya 1 mRNA content. Synthes i zi ng and exporting into the axonal compartment mRNAs devoid of physiological meaning would impose a considerable burden on the nerve cell. The burden would become greater if one takes into consideration the other components of the protein synthesis machinery which are present in squid axoplasm and which, according to the prevailing view, may also be devoid of function. Confronted with these considerations, one wonders whether a certain degree of protein synthesis may not be occurring in the axonal periphery j n vj vo but be lost duri ng the preparat i ve procedures, due to the degradation of a key component or to the release of a powerful inhibitor.

Foll owi ng the demonstration of a di screte axopl asmi c subset of neuronal mRNA, it has become possible to compare its sequence complexity with that of the corresponding cell bodies and of the whole brain. The results of the hybridization kinetic analyses carried out between cDNA and its homologous RNA template have shown that the degree of RNA complexity is strongly related to the cellular and subcellular hetero­geneity of the sample. This conclusion is in accord with the idea that nerve cells or at 1 east certa in types of nerve ce 11 s may contain di sti nct and non-overl appi ng subsets of mRNAs which may refl ect and identify their functional relationship with other cells. New perspec­tives on the significance of these "tagging" mRNA species may be opened by the discovery of the axonal family of neuronal mRNA. Elucidation of its role, origin and fate should bring new light in this respect. It is easy to forecast that the giant axon system of the squid will prove of great value in these investigations.

55

ACKNOWLEDGEMENTS This paper is dedicated to Dr. C. Lalor Burdick whose open-minded

support contributed to the early studies made by A.G. on the squid giant axon. A.G. acknowledges financial support by NATO grant 18781, by the CNR Progetto Finalizzato di Medicina Preventiva e Riabilitativa and by the Ministero della Pubblica Istruzione. We also wish to thank C. Di Fusco and J.-L. Knox for preparation of the manuscript.

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162-165, 1973. 5. Ingogl ia, N.A. Science 206: 73-75, 1979. 6. Fischer, S. and Litvak, S. J. Cell. Physiol. 70: 69-74, 1967. 7. Giuditta, A., Dettbarn, w.O. and Brzin, M. Proc. Natl. Acad. Sci.

USA 59: 1284-1287, 1968. 8. Lasek, R.J., Gainer, H. and Pryzblyski, R.J. Proc. Natl. Acad. Sci.

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241-252, 1973. 16. Laskey, R.A., and Mills, A.D. Eur. J. Biochem. 56: 335-341, 1975. 17. Kaplan, B.B., Schachter, B.S., Osterburg, H.H., de Vellis, J.S and

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S6

25. Bantle, J.A., Maxwell, I.H. and Hahn, W.E. Analyt. Biochem. 72: 413-427, 1976.

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Neurochem. Res. ~: 1621-1634, 1983.

6

MOLECULAR GENETICS OF TYROSINE HYDROXYLASE

J. MALLET, A. BEROD1, F. BLANOT, C. BONI, M. BUDA1, N. FAUCON BIGUET, B. GRIMA, PH. HORELLOU, J.-F. JULIEN, A. LAMOUROUX AND J. POWELL

Laboratoire de Neurobiologie Cellulaire et Moleculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France. 1 Institut National de la Sante et de la Recherche Medicale, Hopital Ste-Eugenie, Saint­Genis-Laval, France

ABSTRACT

The i so 1 at i on of tyros i ne hydroxyl ase (TH) cDNA clones h as allowed

the determination of the entire amino acid sequence of this previously

uncharacteri zed protei n. Ami no aci d sequence compari son with phenyl­alanine hydroxylase indicates that these enzymes share a high degree of

homology. The human TH gene maps to the short arm of chromosome 11.

The changes in TH mRNA and enzyme after reserpine injection have been

analyzed. Their possible significance as to the mechanism of TH induc­

tion is discussed. Finally, new developments have concerned the

detection of TH mRNA in tissue sections and the analysis of the TH nuclear gene.

INTRODUCTION Tyrosine hydroxylase (TH), the rate-limiting enzyme in the

synthes is of catechol ami nes, has been i ntens i vely investigated because

of its key role in the physiology of adrenergic neurons. The regulation

of its expression is under developmental control (1) and its synthesis

can be induced in vivo by nerve stimulation (2,3) or by treatment with

reserpine (4-6) or steroids (7). Multiple kinase activities may be

involved in the short-term regulation of catecholamine biosynthesis by

afferent activity (8-13).

The advent of mol ecul ar genetics greatl y facil itates the study of

mo 1 ecul es such as TH whi ch are present at a low abundance and play an important role in neurotransmission. Taking advantages of these new

developments, we wi 11 present data concerni ng the structure of the TH gene and protein and discuss the use of the corresponding probes in human genetics and in the analysis of the induction of this enzyme.

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RESULTS AND DISCUSSION Isolation of TH cDNA clones and determination of the complete coding sequence of TH mRNA.

To isolate TH cDNA clones, we took advantage of the PC12 cell line which is derived from a rat pheochromocytoma tumor and which contains relatively high levels of TH enzyme (14). In a first series of experi­ments, 5 TH clones were identified by screening 350 recombinant clones constructed from an enriched fraction of mRNA, prepared by sucrose gradient centrifugation. A rapid and powerful screening of the recombinant clones was carried out by differential colony hybridization, taking into account that TH is a tissue-specific protein. The final se 1 ect i on re 1 i ed upon the abil i ty of the cDNA inserts to hybri d i ze specifically to TH mRNA as judged by cell- free translation and immuno­precipitation (14). Blot hybridization analysis of mRNA derived from PC12 cells indicated a major mRNA species of 1.9 kb.

The longest cDNA insert, pTH-l contained only 742 bp and could not include the complete coding sequence of TH mRNA. To obtain longer clones, a second cDNA library containing 50,000 bacterial transformants was constructed. Recombinant plasmids were then tested by in situ hybridization for their ability to hybridize with the nick-translated pTH-l DNA insert under stringent conditions. A total of 160 colonies gave a positive signal. Stepwise, screening allowed us to isolated the clone pTH-51 harboring the longest insert of 1786 bp including 15 deoxyadenosine residues at its 3' end (15). Allowing for about 100 deoxyadenosine residues in TH mRNA, this clone was almost full length.

-10 10

Rat GGA CCA CCA GCT TGC ACT ATG CCC ACC CCC AGC GCC

+++ +++ + MET Pro Thr Pro Ser Ala

Beef CCG GAC CTC GCC GGC ACC ATG CCT ACC CCC AAC GCC MET Pro Thr Pro Asn Ala

Fig. 1. Comparison between rat and beef nucleotide sequences in the region of the ATG codons located at the 5' end of the corresponding mRNAs. In the coding region, the deduced amino acids are indicated. Nucleotides determined from a rat genomic clone are indicated by crosses. Other nucleotides from the rat sequence could also be deduced from the genomic clone, and the sequence was in perfect agreement with that deduced from pTH-51.

59

In fact, recent primer extension experiments indicate that it lacked only 24 residues at its 5' end.

The sequence of pTH-51 showed that an ATG codon is located 12 nucleotides from the 01 igo (dG) tail at the 5' end (Fig. 1). We have initially established that this ATG corresponds to the initiation codon from the following two arguments. First, the CAC nucleotides preceding the ATG in positions 4, 3, and 2, respectively, and especially the highly conserved purine, three nt upstream from the initiation codon correspond to a consensus sequence that contri butes to recognition of the initiation site in most eukaryotic mRNAs. This consensus sequence is not associated with the other ATG codons present in pTH-51. Second, we have compared the 5' nucleotide sequences of pTH-51 with those of a beef TH cDNA clone (Fig. 1). This clone also contains near its 5' end an ATG codon preceded by an initiation site consensus sequence. Strik­ingly, the two consensus sequences constitute the only homologous sequences upstream from the ATG codon, whereas the downstream sequences bear strong homology. That untrans 1 ated sequences di verge much more rapidly than coding sequences is well established.

To confirm that pTH-51 contains the complete coding sequence of rat TH mRNA, the 01 igo (dG and dC) tail residues were removed, and the remaining cDNA was subcloned into a vector that contains the specific promoter found in Salmonella typhimurium phage SP6. Since the corres­ponding RNA polymerase efficiently initiates transcription only at SP6 phage promoter, mi crogram amounts of RNA coul d be produced. After microinjection into frog oocytes, this mRNA directs the synthesis of a protein which comigrates with native PC12 TH. Furthermore, this protein has the ability to promote the synthesis of dopa from tyrosine (Horellou et al., in preparation).

Initial Northern blot experiments (14) indicated that rat cDNA clones recogn i ze human TH mRNA puri fi ed from a human pheochromocytoma tumor. A cDNA library was generated from such a tissue and the corresponding human TH cDNA clones were isolated. The longest clone containing 1526 bp was sequenced. Its 3' end extends to the poly A residues. The 3' untranslated sequence has a size similar to that of the rat and, assuming that the initiation codon aligns with that of the rat, it is lacking 279 bp at the 5' end. The alignment of the corres-

60

ponding amino acid sequence with that of the rat is shown in Fig. 2. A wealth of information can be obtained from the analysis of the

complete rat TH amino acid sequence. The most striking observation concerns the charge distribution for which two domains are clearly dis-

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50

Fig. 2. Alignment of the amino acid sequence of rat TH (top), a frag­ment of human TH (middle), and human phenylalanine hydroxylase (PH, bottom) . The sequences for rat TH and human PH have been taken from references 15 and 23, respectively. The one-letter amino acid notation is used. Identical residues between the three sequences are enclosed in boxes.

61

tinguishable. The NH2 -terminal part, until about amino acid 300, is positively charged, whereas the eOOH-terminal portion has a high density of negative charge. An i soel ectri c poi nt of 6.4 has been cal cul ated from the sequence shown in Fig. 2. This value agrees closely with that reported for beef TH (16). When this enzyme is treated with chymotryp­sin, the resulting protein that possesses TH activity has a MW of 34 kD and an isoelectric point of 4.9 (16). Interestingly, the calculated isoelectric point corresponding to the last 258 amino acids is 5.48. Clearly then, this fragment should include the eOOH terminus. Partial sequence data obtained in our 1 aboratory from a beef cDNA clone show that beef and rat TH are very homologous (unpublished data). We may then infer from this discussion that the enzymatic site is located in the eOOH-terminal part of the molecule.

Inspection of possible sites of cAMP-dependent protein kinase phosphorylation leads to a similar conclusion. It is generally found that serine residues, which are good candidates for such phosphoryla­tion, are preceded in positions -2, -3 or -3, -4 by the positively charged amino acids Arg-Arg or Lys-Arg, respectively (17,18). Such serine residues are present at positions 40 and 153. After treatment with chymotrypsin, the enzyme can no longer be phosphorylated, which confirms that phosphorylation sites are located in the N-terminal domain. Homology between phenylalanine and tyrosine hydroxylases.

Sequence comparison between TH and PH confirms the above findings. Both enzymes are mixed-function oxidases that share many characteristic biochemical and immunological properties (19,20). The cloning and sequencing of cDNAs encoding rat and human PH, respectively, have recently been reported (21-23). Figure 2 clearly indicates that TH and PH share a high degree of homology in the central and carboxyl terminus of the proteins. For example, rat and human TH residues 238-400 and human PH residues 192-354 exhibit over 75% identity with no gaps.

Interest i ngly, the phosphoryl ated site of PH is near the ami no terminus and the active proteolytic fragment which is estimated to be 35 kD is also believed to include the carboxyl end of the protein (24). This finding is consistent with the hypothesis that the common determi­nants for enzymatic activity lie within the highly homologous sequences.

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In contrast, the NH2 -terminal domains share no homology and contain determinants that contribute to the regulation of the two enzymes such as the cAMP-dependent phosphorylation sites.

It will be of interest to compare the sequences of TH and PH with that of tryptophan hydroxylase which also utilizes molecular oxygen to oxidize simultaneously an aromatic acid substrate and tetrahydrobio­pterin (19). Further, it has been suggested that the catecholamines synthesizing enzyme genes TH, dopamine-B-hydroxylase (DBH) , and phenyl­ethanol amine-N-methyl transferase, originate from a common ancestral gene (25). Sequence analysis should help resolve this question. Chromosome 11 and human genetics.

The human TH gene was first assigned to chromosome 11 by the analysis of DNA from a panel of human - mouse cell hybrids using the human cDNA probe (25a). Analysis of silver grain distribution, follow­ing in situ hybridization with the same probe on both male and female chromosome preparations, confirmed the assignment and provided a region­al localization to I1p15 (S.P. Craig et al., submitted for publication).

In view of the strong homology between TH and PH, it is of interest to note that PH has been localized to the long arm of chromosome 12 (26). Similarly, other homologous genes, such as insulin-like growth factors I and II, have been assigned to the short arm of chromosome 11 and to the long arm of chromosome 12, respectively (27). These findings are at first surprising, but are not irrational. They add further weight to the speculation, originally based on similarities of morph­ology and banding pattern, that chromosomes 11 and 12 result from an ancient tetraploidisation, and support the suggestion by Brissenden et al. (28) that the relative order of loci on the two chromosomes may have been disrupted by a pericentric inversion.

Digestion of human cellular DNA with the restriction endonuclease EcoRI revealed a high frequency restriction fragment length polymorphism which constitute a suitable marker for future linkage studies involving the TH gene. More particularly, there is now little doubt that psychia­tri c di sorders exhi bit famil i al segregation. Depressi on is generally related to a deficiency of catecholamine (usually norepinephrine) at functionally important central adrenergic receptors. Mania and schizo­phrenia, on the other hand, are thought to result from an excess of

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catecholamines, more particularly dopamine in the latter case. It is then of importance to establish whether TH gene markers segregate with these diseases in large families where homogeneity and high penetrance of the trait has been carefully established. Effect of reserpine treatment on TH mRNA in rat adrenal. locus coeruleus and substantia nigra.

The administration of reserpine has been shown to increase the activity of TH (and also of DBH) up to 2 to 3-fold in both peripheral and central noradrenergic neurons (4-6,29). Enzyme activity reaches a peak 48 to 72 hr after injection of reserpine and returns to control values after about 3 wk. This effect can be abolished by interruption of the preganglionic sympathetic fibers and is, therefore, referred to as trans-synaptic induction. The increase in TH activity that occurs in adrenal medulla and locus coeruleus following administration of reser­pi ne has been shown by immunopreci pitat ion studi es to resul t from an increase in the amount of TH and not from an activation of pre-existing enzyme molecules (5).

To analyze the profiles of TH mRNA and enzyme changes after reserpine treatment, we have developed a quantitative and sensitive Northern blot procedure to assay TH mRNA in small amounts of brain tissue (30). The sensitivity of the technique, clearly illustrated in

Fig. 3. Analysis of TH mRNA from rat locus coeruleus. Total RNA was extracted from the locus coeruleus of one rat, electrophoresed, blotted and hybridized with TH cDNA as described by Faucon Biguet et al. (30). Lanes a and b contained 1 ].1 g and 2 ].1 g RNA (1/5 and 2/5 of the total RNA of one locus coeruleus) of the control animal; lanes c and d contained 1 ].1g and 2].1g RNA from a reserpine-treated animal.

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Fig. 3, indicates that it is possible to detect TH mRNA in a sample con­taining about 600 noradrenergic cells of the locus coeruleus. Many samples can be processed simultaneously, and this approach is practical for routine pharmacological tests. Rats were injected with a single

DAYS

Fig. 4. Time course of changes in TH mRNA and TIl activity in the locus coeruleus, substantia nigra and adrenals of rats following administra­tion of a single dose of reserpine (10 mg/kg, s.c.). The results are expressed as percentage of control rats injected with the appropriate vehicle and sacrificed at the same time. Results of TH activity and TIl mRNA are means ± SEM of 3 to 5 independent experiments.

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doses of reserpine and changes in mRNA and enzyme activity were analyzed at various times in three different structures.

In locus coeruleus, substantia nigra and rat adrenals the TH cONA probe hybridizes to a single species whose size is identical to that of TU mRNA from PC12 cells.

The time course analysis shown in Fig. 4 reveals that the increase in enzyme activity elicited by reserpine in adrenals and locus coeruleus is preceded by a shan) increase in Tii mRNA that reaches a maximum level 2 days after the drug injection. These results, as I'le 11 as those obtained from actinomycin D experiments (31) suggest that the increase in enzyme act i v tty refl ects an enhanced transcri pt i on of the Til gene. No effect was observed in substantia nigra in agreement with the initial work of Reis et a1. (32).

Surprisingly. Fig. 4 also indicates that both in the adrenals and locus coeruleus, the maximum relative increase of TU mRNA is much higher than that of enzyme activity. Also, TU mRNA levels decrease sharply after day 2, suggesting that the available pool of Tfl mRNA has not been effici ently processed to increase enzyme act i v tty . The decay profil es of 111 mRNA and enzyme activity are, however, quite different in adrenals and locus coeruleus. Clearly, reserpine elicits a much longer lasting effect in the brain nuclei than in the peripheral catecholaminergic ce 11 s . I n the adrenal s, TU mRNA 1 eve 1 has almost returned to the initial value at day 4, whereas it is still 3-fold higher in the locus coeruleus. In fact, in this latter structure, after day 4 both Tii mRNA and enzyme 1 eve 1 s dec 1 i ne slowly and the effect of the drug is st ill Significant at day 18, confirming earlier 111 activity studies by Zigmond (33). At this stage, it is attractive to speculate that the difference in the ampl itude of the effects observed after day 4, between locus coeru1eus and adrena1s, results from a difference in the stability of the Til mRNA. Experiments on isolated nuclei should allow liS to test this hypothesis.

Interestingly, this study also revealed that the 3,000 catechol­aminergic cells of the rat locus coeruleus contain about the same amount of Tfl mRNA as 57,000 chromaffin cells, indicating that a loclls coeruleus cell contains approximately 20 times more Til mRNA than one chromaffin cell. This ratio is probably oven higher for substantia nigra. Tho HI

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mRNA level represents a much higher percentage of total mRNA in

catecho 1 ami nerg; c ce 11 s of the bra; n than i n adrenal s. Furthermore, locus coeruleus and substantia nigra contain approximately 4 times more TH mRNA per unit of enzyme activity than adrenals. These results can easily be rationalized by considering that the cell bodies of catechol­aminergic brain cells have to synthesize large amounts of enzyme protein which are transported to the terminal areas. A high level of mRNA is necessary to sustain the considerable amount of enzyme activity distributed throughout the terminal field. In contrast, in the adrenal medulla where the enzyme is not transported, both the amount of TH mRNA per cell and the ration of TH mRNA to enzyme activity are lower.

Little is known about the sequence of events linking the increase in neural activity with rat TH mRNA. Clearly, how external influences induce long lasting changes in neuronal properties have only begun to be explored. Detection of mRNA in tissue sections.

Because of the great heterogeneity of nervous tissue, it is desirable that specific mRNA hybridizations be detected at the cellular level. In situ hybridization histochemistry is now being developed in various laboratories and Fig. 5 shows initial experiments in which the expression of the TH gene was analyzed in tissue sections of bovine adrenal glands (34). A reproducible selective labeling of the medulla was obtained with single-stranded cDNA probes from M13 templates

a b c

Fig. 5. Autoradiograms obtained after incubation of a transverse section of a beef adrenal gland with a [32P]labeled TH-cDNA or a control probe. Sections a and b are adjacent. The photograph in c represents a cresyl violet staining of the autoradiographed section shown in a. MS, adrenal medulla; CS, adrenal cortex (M x 4).

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(Fig. 5a). In contrast, no signal was obtained with a control probe not complementary to the message strand (Fig. 5b).

This methodology, which can be optimized by using various isotopes such as [ 1251], [35S], and [3H], will provide new information about gene expression in specific cell populations. More particularly, this approach should help define the molecular characteristic underlying the plasticity of catecholamine expression. At what time during development do putative catecholaminergic neurons start to transcribe TH mRNA? Are there neurons, in the developing or adult rat, that possess TH mRNA which is not translated into TH enzyme molecules? If so, could these mRNA be translated in response to perturbations? Do, for example, chol­inergic cells possess permanently or at a given time in their 1 ife TH mRNA? In other words, can a correla-tion be established between the transcription of an a priori unexpected mRNA and the plasticity of a given phenotype? Analysis of the rat TH gene.

A rat genomic 1 ibrary was constructed using a cosmid vector that contains, as a dominant selectable eukaryotic marker, the neomycin resistance gene under the control of the Herpes Simplex virus thymidine kinase promoter sequences (35). From the initial screening of 400,000 recombinant clones, two clones were isolated which hybridized positively with the rat cDNA probe. The sequences contained in these two clones span approximately 55 kb of chromosomal DNA, and clone cosTH-l was shown to contain the complete TH gene which is about 12 kb.

In a first series of experiments aimed at analyzing the functional role of sequences preceding the coding portion of the TH gene, CosTH-l was introduced by the calcium phosphate precipitate method (36) into a mouse neurobl astoma and a hamster gl i al cell 1 ine that do not produce detectable levels of TH mRNA. In both instances, neomycin resistant clones were visible after 2 to 3 wk. Several clones were isolated and tested for the presence of TH mRNA. About half of the selected clones expressed an mRNA species that was recognized by the TH probe. On Northern blots, the mRNA bands were identical in size with that of mRNA from PC12 cells. The relative abundance of this hybridizing species with respect to the total amount of total mRNA, was variable in different clones and represented, in one glial cell transformant, about

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one fifth the amount of TH mRNA present in PC12 cells. S1 mapping experiments are now being conducted to confirm that transformants express mRNA coding for TH, which is identical to that of TH producing cells. The expression of TH protein was first tested with a TH anti­body. A positive reaction could only be observed with the transformed glial cell line that produced the highest amount of TH like mRNA. The corresponding enzyme activity could, however, not be detected. Clearly, DNA-mediated gene transfer represent a powerful approach to dissect out the events necessary for the expression of enzymatically active TH molecules, and the above results open the way to a detailed analysis of sequences required for TH gene transcription.

CONCLUSION This article illustrates some recent applications of molecular

genetics to the study of TH, which is the key enzyme in catecholamine biosynthesis. Many open questions can now be approached experimentally. The availability of a cDNA containing the full coding sequence will fac il itate, by mutagen i c stud i es, the i dent i fi cat i on of the res i dues that are essential for TH activity. The isolation of the human gene and the search for polymorphic DNA markers genetically 1 inked to various psychiatric disorders, may prove clinically important. It is now also possible to investigate at the molecular level, using TH as a paradigm, the mechanism of the regulation of neurotransmitter expression and plasticity. Clearly, the impact of molecular genetics on the neuro­sciences is only in its infancy, but the perspectives opened are extremely promising.

ACKNOWLEDGEMENTS We wish to thank D. Samolyk for efficient technical assistance.

This work was supported by grants from the Centre National de la Recherche Scientifique, the Fondation pour la Recherche Medicale Francaise, the Institut National de la Sante et de la Recherche Medicale, the Association pour la Recherche sur le Cancer and Rhone­Poulenc Sante.

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7 ANALYSIS OF SYNAPSIN I AND G-SUBSTRATE GENE EXPRESSION BY cDNA CLONING AND IN SITU HYBRIDIZATION HISTOCHEMISTRY L.J. DeGENNARO, M.W. KILIMANN AND C.A. HAAS Department of Neurochemistry, Max-Planck-Institute for Psychiatry, Martinsried, FRG

ABSTRACT Synapsin I and G-substrate are two neuron-specific phosphoproteins.

We have employed various molecular genetic methods in order to investigate the mechanisms regulating the expression of their genes. To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated by immunoadsorption from rat brain polysomes. A fragment (1600 nt) from the longest cDNA clone hybridized with two RNA species, 5,800 and 4,500 nt long, present in polyadenylated RNA from rat brain and PC12 cells. G-substrate gene expression has been assayed by in situ hybridization using oligonucleotide probes predicted from the primary sequence of the protein. These probes specifically labelled Purkinje cell bodies in slices of rat cerebellum.

INTRODUCTION Synapsin I is a phosphoprotein which is specifically expressed in

neurons and is found throughout the central and peripheral nervous system (I). It is composed of two polypeptides of 86 kD and 80 kD termed, respectively, synapsin Ia and Ib (2). Immunohistochemistry and the preparation of highly purified synaptic vesicles have local ized synapsin I, associated with the membranes of synaptic vesicles, to the synaptic terminal region of most, if not all neurons (3,4). The protein is phosphoryl ated by both cAMP-dependent and Ca++ Ical modul in-dependent protein kinases, and the state of phosphorylation is altered by condi­tions that affect neuronal activity (for a review, see 5).

The G-substrate is a 23 kD protein substrate for cGMP-aependent protein kinase (6). The amino acid sequences surrounding the sites of phosphorylation have been determined (7). G-substrate has been local­ized specifically in the Purkinje cells of the cerebellum by examination of mutant mice missing specific types of cerebellar neurons (8).

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The biosynthesis of synapsin I and G-substrate is under develop­mental control. The level of polysomal mRNA in rat brain that can direct the synthesis of synapsin I in vitro rises sharply during the first wk of postnatal life and goes through a maximum between days 10-16. This correlates well with the time course of synapse formation during ontogenesis (9). Similar studies measuring the level of G-sub­strate mRNA activity in rat cerebellum show that this activity has a distinct maximum at a time corresponding to the formation of synaptic connections between the Purkinje cell dendrites and the axons of climbing and parallel fibers (10).

A full understanding of the tissue-specific and developmental regulation of neuron-specific gene expression requires the analysis of these genes and the RNA transcripts they encode. We report here our first steps toward this analysis for the genes coding for synapsin I and G-substrate.

MATERIALS AND METHODS RNA purification.

Polysomes were prepared from the brains of 10 to 14 day-old rats using the Mg++precipitation method (11). Synapsin I mRNA was enriched by immunoadsorption of polysomes (12). Total poly(A)+RNA was prepared from whole brain by extraction with guanidinium isothiocyanate (13) or from polysomes by phenol extraction and subsequent passage over an oligo(dT)-cellulose column. Globin mRNA was from an in vitro transla­tion kit purchased from New England Nuclear (NEN). In vitro translation and immunoprecipitation.

In vitro translation in the rabbit reticulocyte lysate system was carried out for 3 hr at 37° using commercial kits from NEN or Bethesda Research laboratories (BRl) and [ 35S]methionine as the label. The immunopreci pitat i on procedure was that descri bed by Ivari e and Jones (14) .

cDNA synthesis. cloning and screening. cDNA was prepared according to the method of Gubler and Hoffman

(15). The cDNA was oligo(dC)-tailed, inserted into commercially available oligo(dG)-tailed pBR322 (NEN) and introduced into E. co7i C 600 using the method of Hanahan (16). Tetracycline-resistant colonies

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were replica-plated on nitrocellulose filters, amplified with chloram­phenicol (340 )lg/ml), lysed (5 min each on filter paper saturated with 0.5 M NaOH; 1.5 M NaCl, 0.5 M Tris, pH 7.4; and 2 M ammonium acetate),

and hybridized with [32 P]labeled single-stranded eDNA probes. Purification of plasmids and hybrid selection.

Plasmids were purified using alkaline lysis and CsCl gradient centrifugation (17). For hybrid selection, 20 g plasmid DNA was nicked and denatured by boil i ng for 90 sec in 100 mM NaOH, neutral i zed by bringing to 1.5 M ammonium acetate and applied to 9 mm nitrocellulose discs. Before the first use, filters were soaked in prehybridization buffer overnight, boiled 3 min in water and washed extensively. Pre­hybridization and hybridization were carried out as described previously (18) . Probes and conditions for hybridization.

Single-stranded cDNA probes for colony hybridization were prepared by oligo(dT)-primed reverse transcription of mRNAs (15) in the presence of [32p]dATP (10 )lM). Hybridization was carried out with 5 x 10 5 cpm of probe (derived from 10-20 ng mRNA) at 55° for 40 hr. The hybridization buffer was 50% formamide, 5 x Deinhardt's solution, 0.6 M NaCl, 40 mM NaH2P04 (pH 7.4), 4 mM EDTA, 0.1% SDS, 100 )lg/ml herring sperm DNA, and 100 )lg/ml poly(A). Prehybridization was in the same buffer at 55°over­night. Fragment 5E2, the probe used in blot-hybridization experiments, was labeled with [u32 p]dCTP by nick-translation (19). Prehybridization and hybridization were each carried out as described above with 3 x 10 7

cpm/ml (3 x 10 8 cpm/)lg) of nick-translated probe. Oligonucleotides.

A mixture of 16 different 14-base oligonucleotides was synthesized with an Applied Biosystems DNA synthesizer. The oligomers were purified by HPLC and their actual base sequences were verified by DNA sequencing according to Maxam and Gilbert (20). The synthetic oligomers were 5'­end-labeled with T4 polynucleotide kinase and [y32p]ATP to a specific activity of 1 x 10 8 cpm/)lg. In situ hybridization histochemistry.

Cerebella of 30 day-old Sprague-Dawley rats were fixed in 80% ethanol, 4% formaldehyde, 10% acetic acid for 2 hr, and then washed with phosphate buffered saline/30% sucrose at 4° overnight. Cryostatic

74

sections (10 ~m) were prepared and stored at -20 0 • Immediately before use, the sections were pretreated as described by Brahic and Haase (21). The final hybridization solution contained [ 32P]5'-labeled oligonucleotide 14-mers at a final concentration of approximately 1 ng/ pl, 50% formamide, 10% dextran sulfate, 4 x SSC (SSC equals 0.15 M NaCl, 0.015 M Na-citrate), 1 mM EDTA, and 1 x Deinhardt's solution. Slices were incubated in humid chambers at 28 0 for 24 hr. After hybridization, preparations were washed extensively in 50% formamide, 2 x SSC, 1 mM EDTA at 40 , dehydrated sequentially in 70%, 80%, and 95% ethanol containing 0.3 M ammonium acetate, and air dried. Autoradiography was carried out with Ilford K2 photoemulsion (22).

RESULTS AND DISCUSSION Enrichment of synapsin I mRNA by polysome immunoadsorption.

Total polysomes were prepared from the brains of 10 to 14 day-old rats. Synapsin I containing polysomes were selectively enriched from total rat brain polysomes by immunoadsorption. When poly(A}+ RNA prepared from these enriched polysomes is translated in an in vitro translation system, synapsin I is found to be the main translation product with a relative abundance of at least 10% (Fig. 1, lane 2).

Fig. 1. Autoradiogram of in vitro translation products. Lane 1, translation mixture without added RNA. Lanes 2 and 3, 1 ng purified synapsin I mRNA was translated in vitro, half of the reaction mixture was applied directly to the gel (lane 2) and the other half applied after immunoprecipitation (lane 3). Exposure time, 3.5 days.

75

The newly-synthesized synapsin displays the typical doublet of poly­peptides Ia and Ib which comigrate with authentic synapsin I (not shown) and are immunoprecipitated with anti-synapsin I antibody (Fig. 1, lane 3) .

A second mRNA species that is enriched in some preparations codes for a protein we have tentatively identified as Protein III (23), another neuronal phosphoprotein to which anti-synapsin I antibodies

often display cross-reactivity (24). We believe that protein III mRNA is enriched due to this cross-reactivity. Construction and screening of a cDNA library.

cDNA was prepared from 400 ng of total poly(A)+RNA from 10 to 14 day-old rat brain polysomes supplemented with 50 ng of enriched synapsin I mRNA, and was inserted into the Pst! site of pBR322. Sixteen thousand bacterial clones were obtained and screened by differential colony hybridization (Fig. 2). The hybridization probes were single-stranded cDNAs derived from synapsin I-enriched mRNA, total polysomal poly(AtRNA, and globin mRNA by oligo(dT)-primed reverse transcription. About 50 colonies were detected which gave distinctly stronger hybridization signals with the synapsin I probe than with the other two probes.

pSyn1- "'e

pSyn1t­pSyn2-

synapsill I probe

Fig. 2. Differential colony hybridization with single-stranded cDNA probes derived from purified synapsin I mRNA and total polysomal poly­(A)+RNA from 10 to 14 day-old rat brains. The three colonies in this figure that gi ve stronger hybri di zat ion signals with the synaps in I probe were designated as pSyn 1, pSyn 2, and pSyn 16. Exposure time, 3.5 days.

76

Identification of synapsin I cDNAs by hybrid-selected translation. P1asmids identified by colony hybridization and designated pSyn 1,

pSyn 5, pSyn 13, pSyn 16, and the longest EcoRI fragment from pSyn 5 (termed 5E2) were used in hybrid selection experiments. Figure 3 shows that all these molecules select, from total po1y(A)+ RNA, mRNA which directs the in vitro synthesis of synapsin I. pSyn 1 and pSyn 5, which contain po1y(dT) tracts, display some unspecific background retention of the comp1 ete spectrum of po1y(A)+ RNA species (1 anes 3 and 4), whereas pSyn 13, pSyn 16, and fragment 5E2, which are free of po1y(dT) tails, give essentially no background except that of the translation assay itself (lanes 5 to 7). Controls, employing pBR322 and various recombi­nant plasmids free of poly(dT) tracts, do not select synapsin I mRNA (lanes 1, 2, 9 and 10).

Figure 3 demonstrates that the cDNAs descri bed above se1 ect mRNA which, upon in vitro translation, gives rise to the typical polypeptide

kDa

-~ ~-- --

Fig. 3. Identification of synapsin I cDNAs by hybrid selection. Trans­lation products of hybrid-selected mRNA were analyzed on SDS/po1yacry1-amide (10%) gels directly (lanes 1 to 10) or following immunoprecipita­tion (lanes 11 to 22). Nitrocellulose-immob1ized DNAs used in the hybrid selection were: lanes 1,11, unidentified cDNA (0.6 kb) from rat brain in pBR322; lanes 2,12, a human procol1agen cDNA (1.8 kb) in pBR322 (11 ].1g); lanes 3,13, pSyn 1; lanes 4,14, pSyn 5; lanes 5,15, fragment 5E2 (2.5 ].1g); lanes 6,16, pSyn 13; lanes 7,17, pSyn 16; lanes 9,19, unidentified cDNA (0.1 kb) from rat brain in pAT 153; lanes 10,20, pBR322; lanes 8,18, translation products of 1 ng-enriched synapsin I mRNA; lane 21, translation products of a 5% aliquot of a hybridization mixture (ca. 1 ].1g poly{A)+RNA + 3 ng synapsin I mRNA), and lane 22, products of mRNA selected from this mixture by pSyn 16, both after immunoprecipitation.

77

doublet of synapsin la and lb. As stated above, anti-synapsin I anti­body crossreacts with protein III. However, protein III mRNA, which is present in the hybridization mixture (lane 21), does not hybridize to the synapsin I cDNA in pSyn 16 (lane 22). This suggests that the simi­larity between these proteins breaks down at the level of their mRNA sequences, at least for the sequence covered by this clone. Northern blot analysis of rat RNA from different tissues.

PolY(A)+RNA from various rat tissues was subjected to agarose gel electrophoresis, transferred to nitrocellulose, and hybridized to nick­translated fragment 5E2 (Fig. 4). This probe hybridized to two RNA species, a minor band of 5,800 nt and a major band of 4,500 nt.

1 2 3 4 5 6 7 8 9 10

21200-

5000-

3500- - 6330

- 3570

2030- - 2370 1710- -1780

947-

564-

Fig. 4. Northern blot analysis of rat RNA. RNA samples were glyoxal­denatured, resolved on a 1% agarose gel, transferred to nitrocellulose (26) and hybridized to nick-translated fragment 5E2. Lane 1, 10 ]1g poly(A)+ RNA from heart ventricle; lane 2, same from skeletal muscle; lane 3, same from liver; lane 4, 40 ng purified synapsin I mRNA; lanes 5-7, polysomal poly(A)+RNA from 10 to 14 day-old brain (lane 5, 40 ng; lane 6, 200 ng; lane 7, 1 ]1g); lane 8, 3.75]1g poly(A)+RNA from 10 day­old whole brain; lane 9, 2.5]1g poly(A)+RNA from PC12 cells; lane 10, ).lg poly(A)-RNA from 10 to 14 day-old brain polysomes. Exposure time, 2 hr. Molecular size standards (in bases, 27) are given in the margins. Glyoxal-denatured ribosomal RNAs from rat and E. co7i (right) and a nondenatured EcoRI/Hi ndII I di gest of bacteri ophage (1 eft) were run on the same gel and visualized by ethidium bromide staining.

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These RNA species were detected in RNA from 10 day-old rat brain prepared either from polysomes by phenol extraction or from whole tissue by extraction with guanidinium isothiocyanate (lanes 5 to 8), in approx­imately the same abundance. In immuno-puri fi ed synapsi n I mRNA, they are enriched by more than two orders of magnitude (lane 4). The two RNA species are also present in poly(A)+RNA from PC12 cells, although less abundant by one order of magnitude (lane 9). Small amounts of what appears to be their degradation products are found in brain poly(A)-RNA flow-through after two passages over oligo(dT)-cellulose (lane 10). The tissue specificity of the two RNA species was tested by hybridization of the probe to RNA from non-neural tissues. They cannot be detected in large quantities of poly(A)+ RNA from 1 iver, skeletal muscle and heart ventricle (lanes 1 to 3), even after exposure of the autoradiogram for a much longer time (18 hr, not shown).

Both RNA species are clearly long enough to accommodate the coding sequence required for either polypeptide (2,000 to 2,500 nt) and, in addit ion, seem to contain long un translated sequences. The apparent length difference between the two RNA molecules (1,300 nt) far exceeds the additional coding sequence (150 nt) required to account for the observed difference in MW between the two synapsin I polypeptides (6 kD) . Thi s data confi rms for a specifi c, well-characteri zed neuronal protein the observation that brain-specific mRNAs tend to display exceptional nucleotide length (25). Detection of G-substrate mRNA by in situ hybridization histochemistry.

A mixture of 14-base oligonucleotides (16-fold degenerate) was predicted from the amino acid sequence of the two phosphorylation sites of the G-substrate protein (shown below in Table 1). The oligonucleo-

TABLE 1. Predicted nucleotide sequences for G-substrate protein phosphorylation sites.

Amino Acid Sequence

mRNA Sequence (predicted)

cDNA Sequence (predicted)

N, all four bases possible

LYS - ASP - THR - PRO - ALA

AA~ - GAg. - ACN - CCN - GCN

TTT - CTG - TGN - GGN - CGN

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Fig. 5. In situ hybridization of G-substrate mRNA. A 10 \lm section of rat cerebellum was probed with a [32P]labeled oligonucleotide mixture specific for G-substrate mRNA (see text). The figure shows, under dark­field optics, the deposition of silver grains over the soma of cere­bellar Purkinje cells.

tide mixture was synthesized and 5'end-labeled with [32p]phosphate as described in Methods. In RNA blots under stringent hybridization conditions, the mixture hybridized specifically to a 2,700 nt RNA found in cerebellar poly(A) RNA, but not in poly(A) RNA from forebrain or liver (Haase and De Gennaro, not shown). This probe was used to detect G-substrate mRNA sequences in slices of rat cerebellum by in situ hybridization histochemistry. Figure 5 shows that the probe labeled Purkinje cell bodies significantly above background.

CONCLUSION The isolation of cDNA sequences encoding synapsin I will now allow

direct determination of synapsin I mRNA concentrations at different stages of development. Such sequences will also make it possible to deduce the primary structure of synapsin I, and open up approaches for the further characterization of its function. Finally, these sequences are a prerequisite for the isolation of the synapsin I gene, and for the study of the molecular basis of the developmental control, as well as for the tissue specificity of its expression. The use of oligonucleo­tide probes for the detection of G-substrate mRNA in the Purkinje cells of the cerebellum will allow the direct in situ assay of G-substrate gene expression during Purkinje cell differentiation.

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ACKNOWLEDGEMENTS We are indebted to E. Erhard for technical assistance, and to R.

Heumann and U. Benedum for gifts of enzymes, [32P]labeled oligo(dT) and poly(A)+RNAs from skeletal and cardiac muscle and from PC12 cells.

REFERENCES 1. Greengard, P. The Harvey Lectures. Series 75: 277-331, 1981. 2. Ueda, T. and Greengard, P. J. Biol. Chem. 252: 5155-5163, 1977. 3. DeCamilli, P., Cameron, R. and Greengard, P. J. Cell Biol. 96:

1337-1354, 1983. 4. DeCamili, P., Harris, S.M.Jr., Huttner, W.B. and Greengard, P. J.

Cell Biol. 96: 1355-1373, 1983. 5. Nestler, E.J. and Greengard, P. Protein Phosphorylation in the

Nervous System. J. Wiley & Sons, New York, 1984. 6. Aswad, D.W. and Greengard, P. J. Biol. Chem. 256: 3487-3493, 1981. 7. Aitken, A., Bilham, T., Cohen, P., Aswad, D.W. and Greengard, P. J.

Biol. Chem. 256: 3501-3506, 1981. 8. Schlicter, D.J., Detre, J.A., Aswad, D.W., Chehrazi, B. and

Greengard, P. Proc.Natl. Acad. Sci. USA 77: 5537-5541, 1980. 9. DeGennaro, l.J., Kanazir, S.D., Wallace, W.C., Lewis, R.M. and

Greengard, P. Cold Spring Harbor Symp. Quant. Biol. 48: 337-345, 1984.

10. Lewis, R.M., Wallace, W.C., Kanazir, S.D. and Greengard, P. Cold Spring Harbor Symp. Quant. Biol. 48: 347-354, 1984.

11. Palmiter, R.D. Biochemistry~: 3606-3614, 1974. 12. Shapiro, S.Z. and Young, J.R. J. Biol. Chem. 256: 1495-1498, 1981. 13. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J.

Biochemistry 18: 5294-5299, 1979. 14. Ivarie, R.D. and Jones, P.P. Anal. Biochem. 97: 24-35, 1979. 15. Gubler, U. and Hoffman, B.J. Gene 26: 263-269, 1983. 16. Hanahan, D. J. Mol. Biol. 155: 557-580, 1983. 17. Maniatis, T., Fritsch, E.F. and Sambrook, J. Molecular Cloning:

A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982, pp. 86-94.

18. Kilimann, M.W. and DeGennaro, L.J. EMBO J. !: 1997-2002, 1985. 19. Rigby, P.W.J., Dieckmann, M., Rhodes, C. and Berg, P. J. Mol. Biol.

111: 237-241, 1977. 20. Maxam, A.M. and Gilbert W. Meth. Enzymol. 65: 499, 1980. 21. Brahic, M. and Haase, A.T. Proc. Natl. Acad. Sci. USA 75:

6125-6129, 1978. 22. Gall, J. and Pardue, M. Meth. Enzymol. 21: 470-480, 1977. 23. Huang, C.-K., Browning, M.D. and Greengard, P. J. Biol. Chem. ~:

6524-6532, 1982. 24. Browning, M.D., Huang, C.-K. and Greengard, P. Trans. Soc.

Neurosci. 8: 794, 1982. 25. Milner, R.J. and Sutcliffe, J.G. Nucleic Acids Res. 11: 5497-5520,

1983. 26. Thomas, P.S. Proc. Natl. Acad. Sci. USA 77: 5201-5205, 1980. 27. Minter, S. and Sealey, P. In: Gel Electrophoresis of Nucleic Acids

(Eds. D. Rickwood and B.D. Hames), IRL Press, Oxford/Washington, D.C., 1983, pp. 229-232.

8 THE EXPRESSION OF MICROTUBULE PROTEINS DURING THE DEVELOPMENT OF THE NERVOUS SYSTEM I. GINZBURG AND U.Z. LITTAUER Department of Neurobiology, The Weizmann Institute of Science, Rehovot, Israel

ABSTRACT Microtubules, a major cytoskeleton element, are involved in many

cellular functions such as cell division, cell migration, cell shape and secretion. In the neuronal system, they are intimately involved in cell differentiation and synaptic transmission. Recent biochemical and immunological evidence proves that different microtubule structures exist within a single cell and may be involved in various functions. This diversity may be the result of microtubule assembly from different tubul in i soforms. Tubul in is encoded by a mult igene family, and in this report, we present the nucleotide sequence of two rat a-tubul in cDNA clones which show high divergence in their 3' untranslated region. However, each of the clones shares high interspecies homology when compared with a tubul in i sotype from human and chi nese-hamster, thus suggesting a common, regul atory mechanism. Using the a-tubul in cDNA probe for the in situ hybridization studies of rat cerebellum, we have shown differential expression of tubul in mRNA level in identified cell types.

INTRODUCTION Microtubules are ubiquitous cytoskeletal elements found in all

eukaryotic cells. They participate in the formation of a large variety of structures such as cytoplasmic networks, mitotic spindles and highly ordered organelles such as centrioles and flagella axoneme. Function­ally, microtubules are involved in a number of dynamic processes (1-3) such as chromosomal movement, cell migration, intracellular transport, cell shaping and secretion. In the nervous system, microtubules are present in high concentration and playa unique role in the development and maintenance of the asymmetric shape of the nerve cell that governs the function of the neuronal network and its connectivity. Synapse formation in the developing brain is an integral part of neuronal

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differentiation. As such, it depends not only on the establishment of axon terminals and synaptic junctions but also on the extent and degree of branching ofaxons and dendrites that provide the framework on which synapses are made. All these elements are formed by a process in which morphologically and functionally distinct domains are progressively established within the neuroblast. The basic neuronal microdifferenti­ation ofaxons and dendrites has been shown to depend on the redistri­bution of microtubules. This stage occurs when the nerve cells, in the developing brain, stop dividing and enter the phase of terminal differ­ent i ati on whi ch cul mi nates in axon outgrowth and the development of axoplamsic transport (4-8).

The dynamic nature of microtubules and the broad spectrum of cellular processes in which they are involved suggests that their assembly and elongation is controlled temporally as well as spatially. The detailed mechanisms by which microtubules regulate these processes are not yet well understood. The great variety of microtubule functions can only be explained by the large number of interactions between tubulin and other proteins, such as microtubule associated proteins (MAPs), and is possibly governed by the microheterogeneity of the microtubule proteins. Microtubule heterogeneity can arise in the process of assembly from its various components, i.e. the diverse tubul in i soforms and the family of MAPs. The 1 atter i ncl ude the HMW components MAPI and MAP2 and a number of closely related proteins of 56 to 62 kD designated as TAU factors (9-11). The availability and the change in composition of the set of these components determine their interaction. Thus, during rat brain development there is an increase in number as well as a change in the distribution of the various tubulin isotypes, TAU and HMW MAPs (5,12,32).

The major differences in isotubulin and TAU forms that arise during brain development are controlled at the mRNA level (1,12). Thus, the genes coding for microtubule proteins exhibit a brain-specific pattern of expression which is characterized by a wider variety of isoforms than in other tissues as well as by a speci fi c time of act i vat i on. In addition, post-translational modifications such as phosphorylation and detyrosylation contribute to a greater isotype heterogeneity of tubulin and MAPs (13-18).

83

To study the involvement of the different isotubulins and MAPs at the molecular level, specific probes are required. Since microtubule proteins are evolutionarily highly conserved, it is in principle difficult to obtain probes that will specifically hybridize with a s i ngl e i sotype. For thi s purpose we have constructed cDNA 1 i braries derived from newborn and adult rat brain as well as from various neuroblastoma and glioma cells (19). The nucleotide sequences of the DNA clones were determined and unique divergent regions specific to a single isotype were either subcloned or chemically synthesized.

The specific DNA probes together with specific antibodies are being used for immunocytochemistry and in situ hybridization studies which will determine the expression of specific microtubule components within individual cells as well as in various regions of the central nervous system.

RESULTS AND DISCUSSION Isolation and sequence analysis of two rat a-tubulin cDNA clones.

From the limited data available it appears that the various isotubulin mRNAs are distinguished by their highly divergent 3'-untrans­lated regions while the coding regions show extensive sequence homology (20-22). It was clear that only high efficiency cloning techniques might yield tubulin cDNA clones derived from the less abundant isotub­ulin mRNA species. For this purpose, several cDNA libraries were constructed in AgUO phage. These i ncl ude cDNA 1 i brari es from rat brain at different developmental stages and cDNA libraries from various neuroblastoma and glioma cell lines. The mRNAs were isolated from rat brain or cell cultures and converted into double-stranded DNA (ds cDNA) according to the method of Gubler and Hoffmann (23). This method yields large size cDNA molecules with an intact 3' -noncoding region. The ds cDNA molecules thus derived were cloned in the high efficiency phage AgtlO cloning system using EcoRI linkers. In contrast to genomic libraries which also contain pseudogenes, the cDNA libraries score only sequences derived from mRNA transcripts. The tubulin cDNA clones were identified by in situ colony hybridization with [32P]labeled a-tubul in eDNA. By these methods over 100 tubulin eDNA clones were identified. Since we were interested in clones containing the 3'-ends, tubulin eDNA

84

clones were further hybridized to short synthetic oligonucleotide sequences (20 nt long) that are homologous to the tubul in 3' -coding regions. By this selection, we limited our analysis to putative clones that also contain the adjacent 3'-noncoding regions.

Figure 1a and 1b show the sequence of the 3'-noncoding regions of two rat a.-tubul in clones. pTa26 is a cDNA clone identical to our previously described pT25 clone coding for a.-tubulin (20) except that it contains the complete 3'-noncoding region up to and including the poly(A) tail. The second clone isolated and sequenced is pTal which is similar to the clone previously described by Lemischka et al. (21).

While there is close homology along the coding region between these clones, a high degree of divergence occurs in the 3'-noncoding regions. The 3' -noncoding region of clone pTa26 contains 156 bp and its poly­adenyl at ion signal is AAGTAAA which precedes by nine bases the poly(A) tract. On the other hand, the 3'-noncoding region of clone pTa 1 is longer and contains 194 bp. Its polyadenylation signal is AATAAA and is located 14 bases before its poly(A) tract. Interspecies homology of g-tubulin genes.

It has been previously noted that while individual isotubulin genes are totally dissimilar within a given species, in turn each gene shares very high interspecies homology (21,22,25). The homology is evident when the polyadenylation signal is used as an alignment point for mammalian isotubulin sequences. Clearly, the rat pTa26, the human ka.1 (22) and the hamster i genes (25) are closely related, having AAGTAAA as the polyadenylation signal, and are probably derived from a common ancestral gene. A high degree of homology is also present in the 3'­noncoding region of rat pTa1, human ba.1 and hamster II genes, all of which contain AATAAA as the polyadenylation signal. It is interesting to note that the length of the 3'-noncoding regions of the mRNAs coding for isotypic species is similar within the groups. The length of human ked, hamster I and rat pTa26 3' -noncoding regions is 172, 172 and 156 bp, respectively, and the homology between the species ranges between 64 and 69%. On the other hand, the length of the human ba.1, hamster II and rat pTal 3'-noncoding regions is 216, 202 and 194 bp, respectively, and the homology ranges between 75 and 88%. Moreover, the changes in nucleotide sequence along the 3'-noncoding regions seem to be nonrandom.

85

Human- k,(,l:TTA C T C TTT G C G Te CCAG T T T TTA HamsterI CT T T T T C G CTAG T TAT Rat-pT26 _ TTCACTCCTGCA GTCCC TGTA TCA TGTCAAACTC_AACTCCAGCTCCAGCACTAGC

Human_ ko\l: A ATG TA A T GGTTA ATTGT A G_ T Hamster I : AT T T C TG TCATC A A TG Rat-pT26 : TG_CAGGCATCGATGCTT _CTATGCTG __ TTTCCCTTCTGTGATCATGTCTT _CTCC

Hurnan- kD(.1: G_ TAT C TA G_T A C TC(A) HamsterI:_ GC G ATG TC T A CACTT~A)n Rat-pT26 : A TGT GTACCTCTTAAG _ TTTTCCA TGACGTC TCAAAGTAAAA _GCTTTAAG (A) n

10

Hur.l aOO '" 1: G ACT A C TT TA T Ham4terII: TAT A T A RatpTAl :A_TT~AATGTCAC_AAGGTGCTGCTTTCACAGGGATGTTTATTCTG_GTCCAACATAGA

HumaOO.l. 1: AT TAG A CGC C T CAA HamsterII: C T Rat pTAl : AA_GTTGTGGGCTGATCAGTTAATTTGTATGTGGCAATGTGTGCTTJCATACAG_TT

Humar>bol.l: G C ATGCTC A AC CC T CAT T TG HamsterII: ATGGTTG Ai ACT A T Rat,.r.l : A_CTGACTT __ TAAG_TGTGAATGAJTTGTCAGAGACCCGAGCCGTCCACTTCAC

HumaOO.,( 1: G G AATG(A)n

Hamster II: T RatrTAl : TGATGGGTTTTAllilliATACTCCCTGTCTT (A)n

Ib

Fig. 1. Sequence of the 3'-noncoding regions of rat a.-tubulin cDNA clones pT26 and pTal.

A hi gher degree of conservati on is observed towards the 3' -end of the noncoding region around the polyadenylation signal. Whether this extensive interspecies homology is related to any function remains to be determined. Both a.-tubul in genes were found to encode an identical size mRNA of 1.8 kb. Somewhat different results were observed fors­tubulin. We have recently determined the nucleotide sequence of a rat brain a-tubulin. The coding region shows a high degree of homology when compared to chicken and human S-tubulin sequences. However, the 3'­termin.al coding end shows high divergence, and no homology is observed at the 3'-noncoding regions (26,27). These differences appear to contribute to changes in tubulin binding sites towards its various

86

1 igands, thus generati ng functionally di fferent microtubul es (19). At least three ~-tubulin mRNA species are present in rat brain. A dominant neural I.B kb species and two minor species of 2.6 and 2.9 kb, respec­tively. The 2.9 kb and 2.6 kb mRNAs represent an early and a late neural species, respectively. By using oligonucleotide probes, we have demonstrated that the three mRNAs are distinct species which are developmentally regulated.

Using a subclone of the 3'-noncoding regions of a.-tubulin pT25, namely pT2, we were able to detect its homologous gene. Only one hybridization fragment was observed in DNA isolated from rat, rat C6 glioma, mouse, mouse NIB neuroblastoma and human SK-N-AS neuroblastoma. Us i ng a s imil ar approach, subclones of the 3' -untransl ated regi ons or synthetic nucleotide sequences specific for a given subtype may aid in the identification of the expressed genes of the tubulin gene family. In situ hybridization studies.

We have previously shown that the tubulin microheterogeneity found in bra in is deve 1 opmenta 11 y determi ned, i ncreas i ng from fi ve to six i soforms prenatally, to seven i sotubul ins postnatally and reachi ng a value of nine to eleven distinct components during early brain matura­tion (28,29). The question, therefore, arises as to whether changes occur in the relative proportion of the isotubulins upon assumption of different roles within the same nerve cell. Alternatively, the increase in tubulin microheterogeneity might arise from developmental changes in the brain cell population which, unlike that of other organs, is composed of many cell types. Experiments with neuroblastoma cells (30) and with single neuronal cells in culture (31) have demonstrated that some degree of tubulin heterogeneity exists at the single cell level. The only way to differentiate between the above possibilities is to use in situ hybridization techniques which measure both aspects at the cellular level, that is changes in gene expression, cell type and number. Figure 2 shows the in situ hybridization of the [3H]labeled pT25 rat a-tubulin cDNA clone to cerebellar slices of 10 day-old rats. Relatively more a-tubulin mRNA is present in mitotically active external granule layer cells than in the internal granule layer cells. These results show that migration and differentiation of granule cells is accompanied by a decrease in their a.-tubulin mRNA level. Furthermore,

87

Fig. 2. In situ hybridization of [3Hllabeled pT25 cDNA in a section from 10 day-old rat cerebellum. A) focus on cells. B) focus on hybridization grains.

the relative levels of a.-tubulin mRNA in prenatally-formed Purkinje cells and in postnatally-formed stellate cells is less than that of the differentiated granule cells. We are now developing methods that will allow the use of synthetic deoxyoligonucleotides for in situ hybridiza­tion experiments. These specific probes will enable us to study changes in the expression of various isotubulin and MAP genes during normal brain development and to compare them to neurological disorders.

ACKNOWLEDGEMENTS Supported in part by grants from the United States-Israel

Binational Science Foundation, from the Forchheimer Center for Molecular Genetics, from the National Council for Research and Development, Israel, and from the Deutsches Krebsforschungzentrum, Heidelberg, Germany.

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(Eds. C. Zomzely-Neurath and W.A. Walker), J. Wiley & Sons, New York, 1985. pp. 125-156.

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R.B. Neurosci.!!: 819-846, 1984. 7. Daniels, M.P. J. Cell Bio. 53: 164-176, 1972. 8. Marchisio, P.C., Weber, K. and Osborn, M. In: Tissue Culture in

Neurobiology (Eds. E. Giacobini, A. Vernadakis and A. Shahar), Raven Press, New York, 1980, pp. 99-109.

9. Murphy, D.B. and Borisy, G.G. Proc. Natl. Acad. Sci. USA 72: 2696-2700, 1975.

10. Weingarten, M.D., Lockwood, A.H., Hwo, S.-Y. and Kirschner, M.W. Proc. Natl. Acad. Sci. USA 72: 1858-1862, 1975.

11. Kirschner, M.W., Williams, R.C., Weingarten, N.D. and Gerhart, J.C. Proc. Natl. Acad. Sci. USA 72: 1159-1163, 1974.

12. Ginzburg, I., Scherson, T., Giveon, D., Behar, L. and Littauer, U.Z. Proc. Natl. Acad. Sci. USA 79: 4892-4896, 1982.

13. Gard, L.D. and Kirschner, M.W. J. Cell. Biol. 100: 764-774, 1985. 14. McKeithan, T.W., LeFebure, P.A., Silflow, C.D. and Rosenbaum, J.L.

J. Cell Biol. 96: 1056-1063, 1983. 15. Raybin, D. and Flavin, M. Biochem. Biophys. Res. Commun. 65:

1088-1095, 1975. 16. Sloboda, R.D., Rudolph, S.A., Rosenbaum, J.L. and Greengard, P.

Proc. Natl. Acad. Sci. USA 72: 177-181, 1975. 17. Vallee, R.B., DiBartolomeis, M.G. and Theurkauf, W.E. J. Cell Bio1.

22: 568-576, 1981. 18. Lindwa11, G. and Cole, D. J. Bio1. Chern. 259: 5301-5305, 1984. 19. Ginzburg, I. and Littauer, U.Z. In: Molecular Biology of the

Cytoskeleton (Eds. G.G. Borisy, D.W. Cleveland and D.B. Murphy), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1984, pp. 357-366.

20. Ginzburg, I., Behar, L., Givol, D. and Littauer, U.Z. Nucleic Acids Res. 2: 2691-2697, 1981.

21. Lemischka, I.R., Farmer, S., Racanie110, V.R. and Sharp, P.A. J. Mol. Biol. ~: 101-120, 1981.

22. Cowan, N.J., Dobner, P.R., Fuchs, E.V. and Cleveland, D.W. Mol. Cell Bio1. ~: 1738-1745, 1983.

23. Gubler, U. and Hoffman, B.J. Gene Z: 263-269, 1983. 24. Lemischka, I. and Sharp, P.A. Nature 300: 330-335, 1982. 25. Elliot, E.M., Okayama, H., Sarangi, F., Henderson, G. and Ling, V.

Mol. Cell Biol. ~: 236-241, 1985. 26. Sullivan, K.F. and Cleveland, D.W. J. Cell Biol. 99: 1754-1760,

1985. 27. Ginzburg, I., Teichman, A., Bodemont, H.J., Behar, L. and Littauer,

U.Z. EMBO J. (in press). 28. Gozes," I. and Littauer, U.Z. Nature 276: 411-413, 1978.

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29. Littauer, U.Z., de Baetselier, A., Ginzburg, I. and Gazes, I. In: Neurotransmitters and Their Receptors (Eds. U.Z. Littauer, Y. Dudai, I. Silman, V. Teichberg and Z. Vogel), J. Wiley & Sons, New York, 1980, pp. 547-557.

30. Gazes, I., Saya, D. and Littauer, U.Z. Brain Res. 1Il: 171-174, 1979.

31. Gazes, I. and Sweadner, K.J. Nature 294: 477-480, 1981. 32. Mareck, A., Fellous, A., Francon, J. and Nunez, J. Nature 284: 353-

355, 1980.

9 REGULATION OF EXPRESSION OF THE HUMAN PROENKEPHALIN GENE IN HETEROLOGOUS CELL SYSTEMS E. HERBERT, M. COMB, G. THOMAS, D. LISTON, A. SEASHOLTZ, H. ROSEN AND B. THORNE Department of Chemistry, University of Oregon, Eugene, OR, USA

ABSTRACT Gene transfer studies allow for different aspects of the regulation

of neuropeptide gene expression to be studied. Stable integration of the human proenkephalin gene into the genome of a mouse pituitary cell line (AtT-20) results in the accurate transcription of the proenkephalin gene. Protei n analysi s reveal ed that the transl ated proenkephal in is accurately cl eaved to mature Met-enkephal in peptides. A second gene transfer approach used to study the processing pathway of precursor proteins is to transiently express the proenkephalin mRNA directly into the cell cytoplasm. The unique biology of vaccinia virus allows for this type of study. Infection of a variety of cell lines with a recom­binant vaccinia virus containing the human proenkephalin gene lead to rapid expression and secretion of human proenkephalin. Of all cell lines tested, only AtT-20 cells possessed the capacity to cleave human proenkephalin to small enkephalin peptides.

I NTRODUCTI ON A milestone in the field of neuroscience has been the discovery of

a vast repertoire of neuropeptides that mediate specific behavior patterns in animals. Upon rel ease at the synapse, neuropept ides, 1 ike other kinds of neurotransmitters, combine with receptors on postsynap­tic neurons or target cells to initiate an intracellular cascade of events that leads to an electrical or chemical response. All of these processes (neuropeptide producti on and release, receptor function and intracellular cascades) must be defined at the molecular level to achieve an understanding of behavior. Molecular biology has provided us with methods and tools necessary to define these processes.

The impact of molecular biology on neuroscience is no more apparent than in the study of opioid peptides. Since the discovery of Met- and Leu-enkephalin in 1975 by Hughes et al. (I), a large family of peptides

91

exhibiting opioid activity have been isolated and subsequently shown by recombinant DNA techniques to be derived from three different precursor proteins (Fig. 1). Proopiomelanocortin (POMC) gives rise to B-endor­phin, ACTH and melanocyte stimulating hormones (MSH) (2-4). Proenkeph­al in (proenkephal in A) contains six Met-enkephal in sequences and one Leu-enkephalin sequence and gives rise to Met- and Leu-enkephalin as well as a variety of larger enkephalin-containing peptides (5-S). Prodynorphi n (proenkepha 1 in 8) contains three Leu-enkepha 1 in sequences and gives rise to a- and B-neoendorphin and dynorphin A and dynorphin 8 related peptides (9). Each opioid precursor undergoes proteolytic processing to generate biologically active peptides during its maturation and transport through the secretory pathway to storage vesicles (10,11). Recent evidence suggests that all three opioid precursors can be cleaved to different sets of biologically active peptides in the different tissues in which they are expressed (10-12). For example, POMC is cleaved in the anterior lobe of the pituitary to produce ACTH, B -LPH, a joining peptide and an N-terminal fragment, while in the intermediate lobe of the pituitary this same molecule is processed to B-endorphin, a-MSH and a C-terminal fragment of ACTH which have different biological activities and target tissues (13). Simi-1arly' proenkephalin is cleaved to free enkephalins in several brain regions while processing in the adrenal medulla yields a variety of 1 arge enkephal in-containing peptides and very 1 ittle free enkephal in (11,14).

Regulation of synthesis and release of peptides derived from opioid precursors is also tissue-specific. Synthesis and release of POMC peptides, for example, is stimulated by corticotropin releasing factor (CRF) and vasopressin and inhibited by glucocorticoids (15-1S) in the anterior lobe of the pituitary, whereas in the intermediate lobe synthesis and secretion of POMC peptides are not affected by these agents but rather by input from dopaminergic neurons with cell bodies in the hypothalamus.

The neuroactive peptides derived from the three opioid precursors mediate very diverse kinds of behavior in animals including responses to stress, pain, anxiety, feeding behavior and analgesic states through their interactions with the nervous, endocrine and immune systems (19).

92

STRUCTURE OF PRECURSORS TO THE OPIOID PEPTIDES

Precursor

POMC

Pro-en kephalin (Human)

Pro-j3-neo-end -dynorphin

(Porcine)

SIG IN-TERMINAL 26

cn(f)(I) (f)t/)(/)

(J(J (J (J (J (J

II I I I I SIG IN-TERMINAL 26

~ ~~~~~ ~ u uUU U U U

I III I I I I SIG I

20

ACTH I/I-endl ~

j3-LPH

.. I I I II I met-enkephalin

D leu-enkephalin

II

o fi-neo-end

II II

c::::::J Dynorphin

I

no. of amino aCids

267

267

256

Fig. 1. Comparison of the structure of the three opoid peptide precursors: upper, POMC; middle, proenkephalin A; lower, proenkephalin B (prodynorphin).

It is not surprising, therefore, to find that in the animal, opioid peptides and ACTH related peptides (including ~-MSH) are secreted from peptidergic neurons in specific regions of the brain where they have been implicated in the facilitation of learning and memory during stressful situations (20). Receptor mediated gene regUlation. The intracellular cascade.

Neurotransmi tters and regul atory agents (neuromodul ators and hormones) combi ne wi th receptors on post-synapt i c neurons and hormone target tissues to initiate an intracellular cascade often involving cAMP and Ca++ mediated protein phosphorylation by protein kinases (21,22). These proteins play a role in electrical transmission, secretion of hormones or other cell products, and in the regulation of synthesis of proteins, by turning the transcription of specific genes or sets of genes on and off. To understand how neurotransmi tters and modul ators might be involved in learning and memory, it is necessary to chemically

93

define all of the steps involved in the cascade leading to gene activa­t i on or i nhi bi t ion. We are attempting to defi ne these steps for the genes that code for the opioid peptide precursors because the peptides that are derived from these precursors play an important role in learning processes. The regulation of these genes is very interesting because it occurs at several levels of gene expression. For example, secretion of POMC peptides and transcription of the POMC gene are both stimulated by CRF and catecholamines and inhibited by glucocorticoids in anterior pituitary cells. Cyclic AMP derivatives have the same action as CRF in culture systems. The work descri bed in thi s paper is an initial step in the direction of defining the steps involved in the cascade of regulation of expression of opioid peptide genes by the techniques of gene transfer. Gene transfer approaches to the studv of gene expression.

The availability of cloned genes of known sequence makes it possible to study control of expression by gene transfer techniques. If the regions flanking the genes (5' and 3' flanking regions) are present, one can identify elements that regulate transcription such as promoters, enhancers and receptor binding sites. Once these regulatory elements have been identified, they can be used as probes for the isolation and characterization of intracellular factors (protein kinases, receptor binding complexes, etc.) that interact with the gene to control its activity.

Transfer of cloned genes into recipient cells can be performed in a number of ways. For example, one can introduce a plasmid that contains the gene of interest along wi th a gene that codes for a res i stance factor to a lethal antibiotic into host cells. Transformed cells can be selected by their resistance to the antibiotic. The cells that survive form colonies and can be grown in large quantities. A very high proportion of these colonies express the gene of interest and can be transformed into stabl e cell 1 i nes, that is the trans fected genes can be integrated into the recipient cell chromosomes and passed on to succeedi ng generations. Once expressi on of the transfected gene is established in the host cells, one can modify the sequence of the gene by in vitro mutagenesis prior to transfection and then determine the effect of these mutations on transcription, translation and processing

94

of the precursor protein.

Another method of gene transfer involves vaCClnla virus (VV) as a vehicle for introducing cDNA directly into a foreign cell. In this

case, the transfected DNA is expressed only in the cytoplasm and is not

integrated into the recipient cell DNA. This approach has some advan­

tages over other methods of gene transfer because VV infects a very

wide range of host cells. Thus, use of this vector now makes it

possible to introduce genes into a number of different types of cell

(neurons or endocrine cells, for example) and study the mechanisms that

regulate cell or tissue specific expression of these genes.

In this paper, we describe the use of mammalian cell lines that

have been transformed by pl asmi d vectors or VV to study regul at i on of

expression of opioid peptide genes.

RESULTS

The expressi on of human proenkepha 1 in in mouse pi tuitary tumor cells that synthesize and process POMC.

We have introduced and expressed the human proenkepha 1 in gene in

mouse anterior pituitary (AtT-20) cells in order to study transcrip­

tiona 1 regul at i on of thi s gene and to el ucidate the mechani sms under­

lying processing of proenkephalin. The AtT-20 cell line was chosen

as the host system for these studies because the cells possess well

deve loped secretory ves i c 1 es, and store and release 1 arge amounts of

ACTH and S-LPH. They also respond to physiological regulators and

process POMC ina manner simil ar to that of anteri or pitui tary cells.

The end products of POMC processing in these cells are the same as in

anterior pituitary cells, that is ACTH l - 39 , S-LPH, N-terminal fragment

and joining peptide (10). Hence, these cells provide a well character­

ized secretory pathway in which to examine the proteolytic processing

and chemical modifications of neuropeptide precursors introduced by gene

transfer.

In the brief description of results below, we show that a recombi­

nant plasmid carrying the human proenkephalin gene can be introduced and

expressed in AtT-20 cells. The transfection of AtT-20 cells was per­

formed by a co-transformation procedure (23) (see Fig. 2) using two

kinds of plasmids, one containing the human proenkephalin gene with its

promoter region and regulatory elements intact but containing a 2.5 kb

95

deletion in intron III (pHEnk 5.5) and the other containing the pRSVneo gene which codes for a neomycin resistance factor (24). Cells that take up the pRSVneo plasmid grow into colonies that are resistant to neomycin (see Fig. 2). Cells that do not take up and express this plasmid are killed by the antibiotic G418 (neomycin derivative) thus providing a selection procedure for isolating transformed cells.

Southern analysis indicates that 50% of the G418-resistant colonies carry at least one copy of the human proenkephalin gene (25). Although the majority of the colonies contained between one and four copies of the gene, several colonies incorporated between 10 and 30 gene copies. Northern blot analysis shows that several clones produce a 1.45 kb RNA which hybridizes to proenkephalin cDNA (Fig. 3). This is the same size proenkephalin mRNA produced by human pheochromocytoma tumors (adrenal medulla tumors) indicating that the proenkephal in gene is transcribed accurately and that processing of the RNA occurs normally. There ap-

pBR322 I 0.1

TRANSFORMING VEHICLES

ExonllZ:

co-transform AtT-20cells

[Ca++P04 ] preci pitate of DNA

Measure Enk mRNA and Enk peptides in +colonies

rmm LTR-Long terminal repeat

Select for antibiotic resistance (G-418)

Detect multiple copies of Human pro-enkephalin gene-

Fig. 2. Strategy for the introduction of the human praenkephalin gene in the mouse anterior pituitary cell line AtT-20. The structure of pRSVneo is described by Gorman et al. (24).

96

pears to be little correlation between the number of integrated gene copies (gene dosage) and the level of proenkephal in mRNA transcripts found in the cell, as the clone that exhibits the highest levels of mature proenkephalin mRNA contains the lowest gene copy number (M. Comb, unpublished).

The conclusion that the 1.45 kb RNA transcript found in transformed AtT-20 cells is human and not mouse proenkephalin RNA is based on two observations. First, only the transformed AtT-20 clones exhibit the 1.45 kb transcript, as shown in Fig. 3, and second, the hybridization and wash conditions are stringent enough to eliminate almost all cross hybri di zat i on to mouse proen kepha 1 in mRNA as determi ned by Southern analysis (25).

wtabc d e f 9 h

.e-1.4kb Human Proenk

Fig. 3. Northern blot analysis of RNA isolated from AtT-20 clones transformed with the human proenkephalin gene. Total RNA was isolated as described in M. Comb et al. (25), denatured with glyoxal, size fractionated by electrophoresis through a 1.7% agarose gel, transferred to nitrocellulose and hybridized with a labeled 918 nt HincII human proenkephalin cDNA probe. All lanes contained 25]1g total RNA except lane f (19 ]1g) and lane h (17 119). Control AtT-20 RNA is designated wt. Autoradiography was performed for 4 days at -70 0 in the presence of an intensifying screen.

97

Comparison of the level of proenkephalin mRNA produced by the AtT-20 clones with that of POMC mRNA found in the same RNA samples indicates that proenkephalin transcripts range between 20% and 50% the level of POMC transcripts, and contain about 0.3 to 1% of the total poly(A)+RNA. This is at least an order of magnitude higher than that observed for proinsulin cDNA which has also been introduced into AtT-20 cells (26). Thus, the results indicate that the human proenkephalin promoter functions efficiently in AtT-20 cells. Furthermore, since the plasmid contained only pBR322 and human proenkephalin gene sequences, all of the proenkephal in control signal s such as the promoter, poly{A) addition sites and RNA splicing signals must be utilized by the recipient cells to express mature mRNA. Because the plasmid pHENK 5.5 contains a 2.5 kb deletion in intron III, it would appear that this deleted DNA is not essential for correct RNA processing.

The results show that stable transformants of AtT-20 cells expres­sing both human proenkephalin mRNA and mouse POMC mRNA have been identi­fied {25}. In view of the high level of expression of proenkephalin mRNA relative to POMC mRNA, it was of interest to determine how much proenkephalin was synthesized and the extent to which this precursor is

TABLE 1. Cellular levels of Met-enkephalin-IR and ACTH-IR in AtT-20 clones containing the human proenkephalin gene.

Clone

WT a b c d f g h

Met-enkephalin ACTH

1.4 5.0

19.3 11.8

122.6 10.0 16.0 69.6

{pmole IR/mg protein}

53.5 73.5 96.0

127.5 78.5 88.0 26.5

135.0

PE/POMC (%)

0.4 1.1 3.3 1.5

26.0 1.9

10.1 8.6

Levels of Met-enkephalin and ACTH are the average of two separate cultures, each assayed in duplicate. The molar ratio of proenkephalin to proopiomelanocortin (PE/POMC) was estimated by first dividing the moles of Met-enkephalin by six (for the number of copies of Met-enkeph­lin in proenkephalin). This value was divided by the moles of ACTH (one copy per mole of POMC) to arrive at the ratio PE/POMC.

98

processed in AtT-20 cells. Protein extracts from the stable transfor­mants were assayed for expression of enkephal1n products by radioimmuno­assay (RIA) for Met-enkephal in before and after sequential digestion with trypsin and carboxypeptidase B. This proteolytic treatment releases peptides such as Met-enkephalin from larger precursors in which they are flanked by basic amino acid residues (27). As expected, wild type AtT-20 cells exhibit very little Met-enkephalin RIA, near the 1 imit of detection (Table 1). In contrast, many of the transformed, G418-resistant clones that express proenkephalin mRNA contain signifi­cant amounts of immunoreactive (IR) Met-enkephalin, in some cases

REVERSE PHASE HPLC OF LOW MOLECULAR WEIGHT PEPTIDES PRODUCED BY AtT-20 CELLS TRANSFECTED WITH PROENKEPHALIN GENE

35

z o >= U

~25 LL a: T ~ z W I I-

~15 ~ o ::t; a.

5

o

B

~ I

~ ~ I I

• " II II I I I I I I I I I I I I I I I I

r--! " I I

r-------------' \ I I r----------------- _J \

I I I I

I I

I 50 :

I I I

~ Z

25 ~ w u « ;J1.

I I I I 0

20 40 60 80 FRACTION NUMBER

Fig. 4. Reverse phase HPLC of low MW peptides present in AtT -20/hENK clone d. An acid extract of clone d was fractionated on a Sephadex G-100 column (25) and material eluting in the position of free Met-enkeph­alin (before digestion with trypsin and carboxypeptidase B) was injected onto an Altex octyl column eluted with a discontinuous gradient of acetoni tril e (dashed 1 i ne) (25). Met-enkepha 1 i n- IR was determi ned in the resulting fractions by RIA. The major peak of IR (70% of total) eluted with the same retention time as authentic Met-enkephalin. The recovery of Met-enkephal in-IR from the column was 88%. Cal ibration standards were: a, Met-enkephalin sulfoxide; b, Met-enkephalin-Arg6-Arg7; c, Met-enkephalin-Arg6; d, ArgO-Met-enkephalin; e, Met-enkephalin; f, Leu-enkephalin; g, Met-enkephalin-Arg6-Gly7-Leu8; h, Met-enkephalin­Arg6-Phe7.

99

comparable to the levels of ACTH (Table 1). The Met-enkephalin-IR material in the protein extracted from several clones was further characterized by gel-filtration analysis on G-100 Sephadex columns (25). Most of the IR material migrated in the position of free Met­enkephalin on this column prior to digestion with proteolytic enzymes. Further analysis of the Met-enkephalin-like material from the gel-fil­tration column by reverse phase HPLC (25) showed that 80 to 90% of this material is present as authentic Met-enkephalin (Fig. 4).

Hence, AtT-20 cells are capable of cleaving almost all of the pairs of basic amino acid residues in human proenkephal in (Fig. 5). This is of considerable interest because these cells cleave only four of the eight pairs of basic amino acid residues in mouse POMC to produce ACTH, 8-LPH, and an N-terminal fragment (Fig. 5).

0: 0: 0:

'f 0: Of POMC

I

PROCESSING IN ANTERIOR PmJlTARY I AND ATT-20 CELLS

II N-Terminal

Proenkephalin

~o: ~o:

II

ACTH

O:~ :X:::X:: I I •

0: ~ I

"

0:

Of

B-LPH

0: 0: 0: 0: 0: 0: ~ ~ ~o: ~~ 1.1 I.' 1.1

0: ~ I -,

Fig. 5. Schematic comparison of the primary protein structure of mouse POMC and human proenkephal in. Only two of the eight pairs of basic amino acids appearing within the sequence of POMC are cleaved by AtT-20 cells at a significant rate.

100

Because the human proenkephalin introduced into these cells appears to be transcribed, translated and processed in a conventional manner, we have asked whether secretion of Met-enkephalin is similar to that of the endogenous peptides derived from POMC. Table 2 shows the amounts of ACTH- and Met-enkephal in-IR released under basal conditions and after treatment of cells with CRF or dexamethasone for 45 min. CRF stimulated secretion of both ACTH- and Met-enkephal in-IR approximately two-fold above the basal release level. Short-term treatment with dexamethasone did not induce or inhibit secretion of either peptide, in agreement with results reported by others (28). The molar ratio of the secreted peptides remained relatively constant with varying degrees of release, suggesting that the two peptides may be contained within the same population of secretory vesicles.

TABLE 2. Secretion of Met-enkephalin-IR and ACTH-IR by AtT-20/hENK Clone d.

Control

CRF (l0-8M)

Dex (10-6M)

ACTH Met-enkephal in

2.56

5.40

2.80

(pmole IR/mL media)

0.98

2.61

1.15

Ce 11 s were grown in 24-we 11 plates. Va 1 ues represent the average of three well s, each assayed in dupl i cat ion. Incubation was carri ed out for 45 min in the presence or absence of regulators.

Finally, we wanted to know how levels of human proenkephalin mRNA are regulated in the transformed cell s. Previ ous studies have shown that the level of POMC mRNA in AtT-20 cells is elevated by treatment of these cells with CRF or cAMP and depressed by long-term treatment with dexamethasone. Proenkephalin is normally expressed in chromaffin cells from bovine adrenal medulla as well as in a number of sites in the brain and reproductive tract. In bovine chromaffin cells, it has been shown that proenkephalin mRNA is elevated by agents that increase cAMP levels such as forskolin and by nicotinic cholinergic agonists (29). We have

101

found that cAMP and CRF increase the levels of human proenkephalin mRNA several-fold in transformed AtT-20 cells (M. Comb, unpublished). Hence, the transfected proenkephalin gene contains the sequences that are required for regulation of expression of this gene by cAMP and CRF.

Another interesting question that is raised by the transformation experiments is why the transfected human proenkephalin gene is expressed in the same cellular environment in which the mouse proenkephalin gene is silent. One possibility is that chromosomal location dictates which gene is expressed. A second possibility is that structural differences in the two genes determine whether they are expressed. A third possi­bil i ty is that the state of methyl at i on of cytos i ne res idues in CpG sequences in the two genes determines whether they are expressed in the AtT-20 cells. It is known that methylation of CpG sequences suppresses the expression of many structural genes (30). Since the cloned human proenkephalin gene used for the transfection studies described here is completely unmethylated, we thought the last possibility might be worth testing.

The human proenkephalin gene has clusters of CpG residues in the 5' flanking region of the gene, in the coding region and in the 3' flanking sequence. Seven CpG sequences in the 5' region of this gene were methylated using the HhaI enzyme. When a plasmid containing the HhaI methylated gene was injected into Xenopus oocytes, expression was markedly suppressed as measured by the amount of human proenkephal in mRNA produced in the oocytes (Rosen and Seasholts, unpublished). Methylation of all CpG sites in this gene with the enzymes HhaI and HpaII reduced expression of the gene to the same extent as HhaI methyl at i on of CpG sequences. Thi s and other studi es have suggested that the methylation of CpG sequences in the 5' flanking region of the gene is important for control of expression of this gene.

Further experiments are now being conducted with fusion gene constructs to pinpoint the regulatory regions of the proenkephalin gene. Plasmids containing a 403 bp element of this gene which includes 200 bp of the 5' flanking region have been fused to a reporter gene-the bacteri al gene that codes for the enzyme chl orampheni col acetyltrans­ferase (CAT). Since plasmid DNA itself does not contain promoter or enhancer elements, the level of CAT activity in transformed cells is an

102

assay for the presence of transcriptional regulatory elements in the 5' flanking region of the proenkephalin gene. One can pinpoint regulatory regions by determining the effect of mutations and deletions in the regulatory region on expression of CAT in cells transformed by mutant plasmids. In this way, it has been possible to show that methylation of CpG sites in the 200 bp 5' flanking region of the proenkephalin gene regulates expression required for the unpublished).

of the proenkephalin gene and that this region is cAMP effects referred to earlier (M. Comb,

Use of vaccinia virus as a transformation vehicle. The above method of gene transfer leads to integration of

transfected genes into the host genome and creates stable cell lines which are very useful for the study of transcriptional regulation of gene expression. If one wishes to study regulation of expression of a gene product in the cytoplasm of a cell, it is eas i er to i nt roduce cDNA directly into the cytoplasm of a cell rather than into the host nuclear genome. Cells remain viable for many hours or days after infection. This approach is now possible because of the development of VV as a cloning and expression vehicle, as mentioned in the previous section. Use of this virus as an expression vector has several major advantages: (a) the virus has a very broad host cell range, and (b) unlike other DNA viruses, the infectious cycle occurs entirely in the cytoplasm of the host cell. Insertion of cDNA into the virus downstream from the early viral promoter leads to rapid transcription of the cDNA in the host cell (within minutes of infection) and efficient production of protein from the mRNA. Although the use of VV as an expression vector is relatively new, several viral coat proteins have already been produced by this approach (31-35). Vaccinia virus construction.

The construction of the recombinant VV containing human proenkeph­alin cDNA (VV:PE) has already been described as well as the titer of virus required to produce maximum various mammalian cell lines (36). in Fig. 6.

expression of Met-enkephalin in This construction is illustrated

Expression of human proenkephalin in different cell types. A salient feature of the VV expression vector system is the ability

hiHHil

~ ............... ~~ ~

pVV3 (4.11<1>1

103

.).

\. / , .... .. --........./ T

pHR5 (40Kb)

Fig. 6. Construction of the pVV3:PE recombinant vector plasmid. Bold lines indicate VV DNA sequences. The VV 7.5 K promoter is represented by the P. The orientation of the PE cDNA insert is indicated.

to infect a wide spectrum of cell types and to allow the observation of the production of a foreign protein in an expedient manner. To this end, five different cell lines were infected with VV:PE. After 24 hr of infection, the cells were harvested and extracted with acetic acid. The acetic acid was removed by lyophil ization and the extract was sequen­tially digested with trypsin and carboxypeptidase B to release enkeph­ali n from 1 arger pept ides and then assayed for Met-enkephal in immuno­activity by RIA. The results, presented in Table 3, show that although Met-enkephalin-IR was detected in each cell type, the level of Met­enkephalin-IR varied over 5-fold in both the cellular and secreted levels depending on the cell type infected. Cell-specific processing of human proenkephalin.

Although infect i on of each cell type wi th VV: PE resulted in the product ion of Met-enkephal i n- IR, it was not known to what extent each cell type was capable of processing human proenkephalin into smaller

104

TABLE 3. Met-enkephalin-IR in different cell lines.

BSC40 AtT-20 GH4 L P388Dl DI6v

MI

VV:WT 0.1

VV:PE (cell ) 19.4 10.1 43.6 7 18.3

(secreted) 180.0 36.0 79.0 43 70.0

Parallel plates of each cell line were either mock infected (MI), infected with VV:WT or VV:PE at a titer of 5 pfu/cell, except for BSC40 which was 0.5 pfu/cell. After 24 hr of infection, cells were processed for the Met-enkephalin RIA. Values are given as pmoles Met-enkephalin­IR/I06 cell s (average of two separate experiments). See ref. 36 for definition of plaque forming units (pfu).

peptides. To answer this question, acetic acid extracts from cells and culture medium were lyophilized and resuspended in 0.25 M triethyl ammonium formate. Each sample was then applied to a TSK-125 HPLC sizing column. Following peptide separation, each fraction was assayed for Met-enkephalin-IR. As shown in the lower chart in Fig. 7, analysis of BSC-40 cells infected with VV:PE reveals two prominent peaks of Met­enkephalin-IR. The faster migrating peak 1 elutes with an apparent MW of 28 kD, the expected size of human proenkephalin. The slower migrating peak 2 elutes with an apparent MW of 16 kD. Similar results were obtained for GH4Cl, LtK and P388Dl cell lines (macrophages; data not shown). To determine whether material in peaks 1 and 2 is secreted, a small aliquot of the culture medium from BSC-40 cells was also analyzed. As shown in the lower chart in Fig. 7, only one prominent peak of Met-enkephalin-IR is secreted. This peak coeluted with peak 1 from the cell extract.

In contrast, AtT-20 cells infected with VV:PE exhibit five major peaks (Fig. 7, upper part) of Met-enkephalin-IR. Peaks 1 and 2 coelute with peaks 1 and 2 of Met-enkephalin-IR from VV:PE infected BSC-40 cells. Peaks 3 and 4 elute with apparent MW of 4.5 kD and 2.5 kD, respectively. The slowest migrating peak 5 coelutes with purified Met-

Fig. 7. Processing of human proenkephalin in AtT-20 cells (upper) and BSC-40 cells (lower) transformed by VV. A small aliquot of either acetic acid cell extracts (.--.) or culture medium (0--0) was dried by rotary evaporation and resuspended in 100 ~l of 0.25 M TEAF pH 3.0. The resuspended sample was sonicated and insoluble material was removed by centrifugation. Each sample was next applied to a TSK-125 Bio-Sil HPLC sizing column. The column was run in 0.25 M TEAF pH 3.0 at a flow rate of 0.5 mljmin. Fraction volumes were 300 ~l. Following removal of the TEAF, each fraction was digested with trypsin and carboxypeptidase B. An aliquot of each digest was assayed for Met-enkephalin-IR.

enkephalin. Analysis of the media from VV:PE infected AtT-20 cells reveals two major peaks of secreted Met-enkephalin-IR. The faster migrating peak coelutes with peak 1 of the cell extract and the slower migrating peak coelutes with peak 5 and purified Met-enkephalin. Hence, both methods of gene transfer described here result in the production of free Met-enkephalin by AtT-20 cells.

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A recombinant VV containing POMC cDNA has also been constructed and used to transform the same cell lines listed in Table 3. Processing of POMC was analyzed in the cells and the medium and the same results were obtained as for proenkephalin, that is, the only cell line that showed extensive processing of POMC was the AtT-20 cell line.

DISCUSSION The mouse anterior pituitary cell 1 ine AtT -20016v which synthe­

sizes, stores and releases 1 arge amounts of POMC-deri ved pept i des has been programmed by DNA-mediated gene transfer to express the human proenkephalin gene. In this study, we have addressed three questions: (a) can the proteolytic enzymes present in AtT-20 cells process human proenkephalin, a protein closely related to the endogenous protein (POMC) in both structure and function, to smaller bioactive peptides; (b) can the transfected cell s secrete enkephal in peptides under the control of regulators; and (c) is the production of proenkephalin mRNA under regulation by secretogogues? We were particularly interested in precisely characterizing the products of proteolytic processing, with the aim of uncovering any subtle differences in the manner in which these two precursors were cleaved by the endoprotease and exoprotease activities in AtT-20 cells.

The results presented here i ndi cate that a pl asmid carryi ng the human proenkephal in gene can be efficiently introduced and accurately expressed in mouse AtT-20 cells by cotransformation with the plasmid pRSVneo followed by G418 selection to identify stable transformants. The stable transformants produced a human proenkephalin mRNA identical in size to human pheochromocytoma proenkephal in mRNA, suggesting that AtT-20/human proenkephalin cells correctly transcribe the exogenous gene and process the RNA transcript to mature human proenkephalin mRNA. The introduced gene contained only 200 nt of 5' flanking DNA, demon­strating that 200 bp or less of 5' flanking DNA are sufficient for accurate and efficient transcription. This result agrees with the findings of Terao et al. (37) who have shown that no more than 170 nt 5' to the CAP site of the human proenkephalin gene are required for effi­cient transcription in monkey kidney COS cells.

Analysis of protein extracts from AtT-20/human proenkephalin clones

107

using an RIA specific for Met-enkephalin indicates that these cells synthesi ze proenkephal in protei nand proteolyti ca lly process the precursor to free Met-enkephalin. Gel-filtration chromatography and reverse phase HPLC of Met-enkephalin-IR in extracts from various clones show that about 50% of the total Met-enkephalin-IR is present as free Met-enkephalin. This surprising result suggests that the endoproteo­lytic processing enzymes present in AtT-20 cells can also recognize and efficiently process all (or almost all) of the paired basic amino acid cleavage sites flanking Met-enkephalin in the precursor. Subsequently, a carboxypeptidase B-like exoprotease removes the C-terminal arginine or lysine residue to produce the fully active peptide.

AtT -20 cell s serve as a useful model system for endocri ne cell secretion in general and for anterior pituitary corticotrophs in particular. The observation that AtT-20/human proenkephalin cells secrete Met-enkephalin in parallel with ACTH suggests that the two peptides are localized within the same secretory vesicle. However, two populations of vesicles that are under similar control mechanisms cannot be ruled out. Experiments are in progress to resolve this question.

The observation that the level of human proenkephalin mRNA in AtT-20 cells is elevated by treatment of these cells with cAMP indicates that cis-regulatory element or elements necessary for the cAMP response is contained in the DNA of the transfected proenkephalin gene. Trans­fection experiments performed with hybrid genes (M. Comb, unpublished) made up of the 5' flanking region of the proenkephalin gene fused to the CAT gene have shown that the cAMP control element is contained within the 200 bp 5' flanking region of the human proenkephal in gene. These experiments also demonstrate that the state of methylation of CpG sequences in this region of the gene is critical in controlling expres­sion of this gene. Hence, the techniques of gene transfer have enabled us to localize DNA sequences required for two major kinds of regulatory responses of the proenkephal in gene. In vitro mutagenesi s techni ques should allow us to further define these sequences. It may then be possible to use DNA fragments containing these sequences as probes to help identify regulatory proteins that interact with these sequences and to further define the events involved in the intracellular cascade.

In this paper, we have also attempted to show that a recombinant vv

108

can be used as a transient expression vector for studying the post­translational maturation of specific protein. The salient features of this system are: (a) the ease of constructing recombinant VV (Fig. 6); (b) vaccinia's cytoplasmic mode of replication; and (c) the broad host range of VV (Table 1) and the short infection time needed to detect sufficient quantities of protein (36). The abil ity of VV to infect a wide spectrum of cells allows for a novel method of identifying prote­ases which are responsible for the processing of the precursor protein into bioactive peptides. By screening a wide variety of cell types, we should now be able to correlate the extent of precursor maturation with the presence or absence of a specific protease in the cell. This infor­mation will be extremely useful when correlated with the characteriza­tion of protease specificity in vitro.

In Fig. 7, it is shown that human proenkephalin is processed differently in different transformed cell types. The size distribution of Met-enkephalin-IR in BSC-40 cells reveals two prominent peaks of immunoreactivity. The faster migrating peak 1 elutes with an apparent MW of 28 kD. A peak of identical size is observed to be secreted into the culture medium. The coelution of a cellular and secreted 28 kD protein suggests that peak 1 contains bonafide human proenkephalin. In contrast, peak 2 is observed solely in the cytoplasm and is not secreted into the culture medium. The apparent size of this peak (16.5 kD) suggests that it is a derived form of peak 1, but further studies are required to establish this relationship.

It is interesting to note that GH4C1 cells (rat pituitary) and P388D (mouse monocyte) are unable to process human proenkephalin (data not shown). In contrast, AtT-20 cells (mouse anterior pituitary) are fully capable of processing VV:PE encoded human proenkephalin into mature peptides (Fig. 7, upper part). The fact that peaks 1 and 2 were observed in each infected cell type, argues that the modification responsible for their separation on the sizing column is common to many mammalian cells. However, of the five cell lines studied, only AtT-20 cells possess the post-translational machinery necessary for the compl ete maturation of human proenkephal in. The si ze di stri but i on of Met-enkephalin-IR, depicted in the lower part of Fig. 7, is in excellent agreement with the size distribution of Met-enkephalin-IR in AtT-20

109

cells stably transfected with the human proenkephalin gene. This argues that the VV infection does not alter the cellular protein maturation pathway. Purification and identification of each of the processing intermediates will be necessary in order to determine the proteolytic maturation pathway of human proenkephalin. Pulse-label and pulse-chase techniques with radiolabeled amino acids will be required to determine the time course of production of each of the processing intermediates.

Although GH4Cl cells and P388Dl cells do not process proenkephalin to Met-enkephalin as do AtT-20 cells, they are capable of converting other precursor proteins to mature bioactive peptides. For example, GH4Cl cells that have been transformed with proparathyroid hormone cDNA (38) can convert this precursor to mature parathyroid hormone. P388Dl cells secrete interleukin 1 in the form of an inactive precursor protein (31 kD precursor; 39) which is cleaved during or shortly after secretion to form mature interl euki n. In both precursors, proteolytic cl eavage sites exist N-terminal to the sequence of the mature peptide. This cleavage sequence is Lys-Lys-Arg in proparathyroid hormone and Lys-Lys­Arg-Arg in the interleukin 1 precursor. If these sequences are actually the sites of cleavage in these precursors, then the enzyme systems involved must require more than pairs of basic amino acid residues as cleavage recognition signals, otherwise they would process proenkephalin to enkephalin peptides. This suggests that endoproteolytic processing enzymes have a high degree of specificity and that a few enzymes with broad specificity might not be sufficient to process the wide variety of prohormones that exist.

ACKNOWLEDGEMENTS We thank N. Gay and S. Engbretson for expert manuscript prepara­

tion. A.S. was supported by Postdoctoral Fellowship F32-DA05261 from the National Institute on Drug Abuse and D.L. by Postdoctoral Fellowship 5F32NS07637-02 from the National Institutes of Health. G.T. was supported by a Damon Runyon-Walter Wi nche 11 Cancer Fell owsh i p DRG-797. The research performed in the E.H. lab was supported by Research Grants AM-16879 and AM-30155 from the National Institutes of Arthritis, Diabetes, Digestive and Kidney Diseases, and Research Grant DA-02736 from the National Institute on Drug Abuse.

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S. Nature 295: 206-208, 1982. 7. Comb, M., Seeburg, P.H., Adelman, J., Eiden, L. and Herbert E.

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12. Seizinger, B.R., Grimm, C., Ho11t, V. and Herz, A. J. Neurochem. 42: 447-457, 1984.

13. Herbert, E. Trends Biochem. Sci. 2: 184-188, 1981. 14. Liston, D.R., Vanderhaeghen, J-J. and Rossier, R. Nature 302:

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39. Lomedico, P.T., Gubler, U., Hellmann, C.P., Dukovich, M., Giri, J.G., Pan, V-C.E., Collier, K., Semionow, R., Chua, A.O. and Mizel, S.B. Nature 312: 458-462, 1984.

10 THE REGULATION OF PROOPIOMELANOCORTIN GENE EXPRESSION BY ESTROGEN IN THE RAT HYPOTHALAMUS J.L. ROBERTS, J.N. WILCOX AND M. BLUM Center for Reproductive Sciences, Columbia University, New York, NY, USA

ABSTRACT Estrogen is known to the decrease the level of 8-endorphin, a pro­

opiomelanocortin (POMC) derived peptide, in the rat hypothalamus. We have used a POMe cDNA probe to measure the levels of POMC mRNA in the arcuate region of the brain in ovariectomized female rats with and without estrogen replacement. Our results show that estrogen causes a time dependent decrease in POMe mRNA of approximately 40% in rats ovari­ectomized for 2 wk. Preliminary studies using a nuclear transcription system have i ndi cated that estrogen may decrease POMC mRNA 1 evel s by having an inhibitory effect on transcription of the POMC gene. It is not clear from these results, however, whether estrogen is acting directly on POMe neurons, or whether it is producing it inhibitory effect through an interneuron system.

INTRODUCTION POMC is a large precursor protein from which 8-lipotropin, ACTH,

the MSHs, and 8 -endorphi n are deri ved (1; see also the chapter by Herbert et al. in this volume). Immunocytochemical staining using ACTH or endorphin antibodies indicates that POMC is present in the cell bodies of the peri arcuate region of rat hypothalamus extending from the retrochiasmatic area to the premammilary nucleus (2-9). It was ini­t i ally thought that the POMC pept ides in the brain were deri ved by transport and uptake of these proteins from the pituitary, a tissue rich in ACTH and 8 -endorphi n. However, it has recently been shown that the hypothalamus actually synthesizes POMC (10), contains POMe mRNA (11,12), and releases S-endorphin into the portal blood (13,14). This suggests that the hypothalamic POMC cells synthesize POMC.

Recently, evidence has been presented that POMe synthesized in the brain is related to the reproductive function of the animal. Beta endorphin administration has profound influences on pituitary secretion,

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resulting in a decrease of plasma luteinizing hormone and follicle stimulating hormone, and an increase of plasma growth hormone and prolactin levels (reviewed in 15). Estrogen administration has been shown to decrease endorphi n 1 evel sin the rat bas a 1 hypothalamus, as determi ned by radi oammunoassay (16), although it is not cl ear whether this represents a direct effect on endorphin synthesis. Beta endorphin levels decrease in the peri arcuate nucleus and increase in the median eminence on the afternoon of proestrus in rats (17), suggesting that the ovarian hormones may alter the release and/or the transport of this neuropeptide from the cell bodies in the arcuate nucleus (18,19). In order to resolve this controversy, we set out to study the effects of ovari ectomy and estrogen replacement on hypothalami c POMC mRNA 1 eve 1 s. While mRNA levels closely parallel the synthesis of neuropeptides, there is no evidence that mRNAs are axonally transported in brain, so they are not subject to the same problems of interpretation as neuropeptide levels. Thus, changes in POMC mRNA levels after steroid replacement may be interpreted as reflecting alterations in POMC biosynthesis. In addi­tion, we used an in vitro nuclear run-on transcription assay in order to measure the number of RNA polymerase complexes on the POMC gene in the arcuate POMC neurons as a function of estrogen treatment. These studies have allowed us to begin to interpret the molecular mechanism by which estrogen acts to alter POMC mRNA levels.

The POMC neurons of the peri arcuate hypothalamus have an extensive set of projections to a variety of brain regions. For example, they project forward into the preoptic region, laterally to the amygdaloid nucleus, and posteriorly down the brain stem. Recent studies coupling POMC peptide immunohistochemistry with retrograde tracing of axonal projections indicate that peri arcuate POMC neurons may be divided into discrete subgroups according to the brain region to which they project. For example, the POMC neurons from the middle region of the peri arcuate nucleus are, in general, the ones that project to the preoptic area of the rat. If one were able to functionally subdivide the POMC neurons, it would become easier to interpret the changes in POMC gene expression. Unfortunately, this has not been possible even with such techniques as Palkovits' punch dissection of the peri arcuate region. In an alterna­tive approach, we have used a single cell mRNA assay which we have

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developed from the in situ cDNA:mRNA hybridization procedure. By coupling our semiquantitative in situ hybridization technique with fluorescent dye retrograde transport techniques, we have been able to identify and quantitate the amount of POMC mRNA in neurons which project to the preoptic area of rat hypothalamus. We feel this type of tech­nique will allow neurobio10gists to begin to measure the effects of various types of environmental, behavioral, and hormonal influences on the expression of specific genes in discrete populations of neurons in the brain.

MATERIALS AND METHODS Animals.

Female Sprague-Dawley rats, 150-200 g body weight, were housed with a 14/10 light/dark cycle (lights on at 0600), and given food and water ad 7ibitum. Animals were bilaterally ovariectomized under ether anesthesia 2 or 3 wk prior to experimentation. Animals receiving estrogen were implanted with silastic capsules containing 262 ~g estra­diol 17- a/m1 in sesame oil (10 m1/100 g body weight) according to the method of Moreines (20). The capsul es were impl anted subcutaneously in the lower abdomen under light ether anesthesia. In our laboratory, these implants produce mean serum estrogen levels of 100 pg/m1 measured 1 or 4 days after implantation. Control animals received similar si1as­tic implants containing sesame oil alone. Brain dissection.

At selected times after hormonal treatment, animals were sacrificed by decapitation, and the brain was removed and dissected on a cold glass plate as follows: a coronal sl ice, 1 imited rostrally by the posterior border of the optic chiasm and caudally by the anterior border of the mammilary bodies, was removed. The arcuate median eminence region (Arc-ME) was dissected from the coronal section by making two diagonal cuts extendi ng from the hypothalami c fi ssures to the th i rd ventri c 1 e, about 3 mm from the central surface of the brain. The Arc-ME weighed approximately 5 mg and represented the smallest dissectab1e region containing all the hypothalamic POMC neurons as determined by immunocy­tochemistry.

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Isolation of RNA. Nucleic acids were isolated from the Arc-ME sample by proteinase

K/SOS/phenol extraction (21). After dissection, the tissue was immediately placed in SET buffer (0.5% SOS, 10 mM EDTA, 20 mM Tris pH 8.0) containing 65 ].Ig/ml proteinase K, and homogenized by vigorous pipetting with a large bore micropipette. The homogenate was then incubated for 2 hr at 42°, 3 III 3% PMSF (a protease inhibitor) and alcohol were added, and the samples were frozen in liquid nitrogen, and stored at -70°. After all the samples had been collected, the homo­genates were thaw-mounted and extracted twice with phenol/chloroform, followed by a single ethanol/NaCl precipitation overnight at -20°. Nu­cleic acids were collected by centrifugation at 15,000 x g at 4°. The pellet was redissolved in 300 III sterile 1 x TE (10 mM Tris pH 8.0, 1 mM EOTA) and aliquots were taken for the estimation of RNA concentration by the ethidi urn bromide/agarose assay descri bed previ ously (21). Indi vi d­ual Arc-ME dissections yielded approximately 15 to 20 Ilg total RNA by this procedure. Ouantitation of mRNA.

POMC mRNA was determined in pooled Arc-ME samples by filter hybrid­ization/dot blot (22). Aliquots of each total nucleic acid sample containing approximately 2 to 4 Ilg total RNA were diluted in sterile 1 x TE to 100 III total volume. The samples were heated at 65° for 10 min to denature RNA, and diluted with 100 III cold 20 x SSC, (SSC is 0.15 M NaCl, 0.015 M sodium citrate pH 7.2). Samples were spotted on nitro­cellulose filters with a manifold dot blot apparatus (Schleicher and Schuell) using gentle vacuum and rinsed with 100 III 10 x SSC. The nitrocellulose filters were air-dried and baked at 80° under vacuum for 3 hr.

Prehybridization and hybridization was carried out according to the method of Wahl et al. (23). Hybridization was performed using a [32P]_ 1 abe 1 ed cDNA conta in i ng the 0.55 kb cod i ng sequence of the POMC gene (21). After hybridization, the unhybridized material was removed by rinsing and the nitrocellulose filter dried and exposed to X-ray film. The intensities of the different dots were measured by quantitative autoradiography. Normalization of all dots for the total amount of mRNA spotted was made using a [32P]labeled oligo(dT) probe, as previously

116

described (24). Such normalization greatly enhanced our ability to measure small changes in POMC mRNA levels. Nuclear transcription assays.

The transcription assay was performed using a previously published procedure (25). The only significant modification was the inclusion of creatine phosphokinase and creatine phosphate as an ATP regenerating system. Nuclei were isolated from the Arc-ME of animals treated 3 wk post-ovariectomy with similar silastic tubing implants as discussed above. POMC mRNA transcripts from the in vitro transcription reaction were quantitated by hybridization for 48 hr to a DNA fragment encoding the entire protein region of exon region 3 of the POMC rat gene (26). All data was corrected for 100% hybridization using an internal hybrid­ization standard of tritiated POMC mRNA (25,26). Background hybridiza­tion was measured using a pBR322 DNA control filter. Data is expressed relative to the total amount of incorporation in the in vitro transcrip­tion assay. In situ cDNA:mRNA hybridization.

The in situ cDNA:mRNA hybridization procedure has been described previously in great detail (27). Briefly, the procedure involves perfusing an animal with 4% formaldehyde for 20 min, removing the brain, immersing it in a sucrose phosphate buffer saline solution for 1 hr, freezing the tissue in OTC embedding compound, and taking 10 ~m frozen sect ions wi th a cryostat. The sections were thaw-mounted on subbed slides and immediately stored at -70 0 with dessicant. To prepare for hybridization, the tissue was thawed in a 10 ~g/ml proteinase K solution to inhibit ribonucleases as well as to make the tissue permeable. The proteinase K solution was rinsed off and replaced with a prehybridiza­tion solution, which was replaced 2 hr later with a hybridization solution containing the same components plus 10,000 to 20,000 counts of the tritiated cDNA probe for the POMC gene. After 24 hr of hybridiza­tion, the unhybridized probe was washed off by overnight rinsing with 0.5 x SSC, and the tissue was dried and coated with Kodak NTB2 emulsion. After exposure for 2 to 6 wk, the sections were developed and counter­stained with eosin and hemotoxalin.

In experiments where retrograde axonal tracing was performed, the procedure was essentially the same with the exception that the animals

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were injected stereotaxically with 100 nl of fast blue dye in the pre opt i c area 3 days pri or to perfus i on with paraforma 1 dehyde. The location of the fast blue dye was identified by fluorescence microscopy.

RESULTS We examined the effect of estrogen or oil implants left in place

for 1 or 3 days on Arc-ME POMe mRNA levels in 2 wk ovariectomized rats (28). The nucleic acid extracts originating from the Arc-ME of each group of animals were combined into a single sample and spotted five times on a nitrocellulose filter. The filter was hybridized to the [32PJDNA probe and normalized to the total poly(A)+RNA, as described in the Methods section. There was no observable effect of estrogen treat­ment on the amount of total RNA isolated or on the amount of poly(AtRNA detected in the dot blot assay, relative to the nonestrogen-treated, castrated controls. One day of estrogen treatment did not significantly affect the Arc-ME POMe mRNA levels relative to oil controls (Table 1). However, three days of estrogen treatment sign ifi cant ly reduced POMe levels in the Arc-ME to 61% of oil treated controls (t = 5.21, df = 8, P < 0.001). Similar results were obtained by repeating the same experi­ment.

TABLE 1. Relative POMe mRNA levels in Arc-ME.

Oil

Estrogen

1 Day

1.00 ± 0.11

0.93 ± 0.06

3 Day

0.82 ± 0.03

0.50 ± 0.06

Nucleic acid samples were pooled from each group of rats (N=5), and 2 ~g total RNA spotted five times on nitrocellulose filters. Relative amounts of POMe mRNA are expressed as mean ± SD ~g equivalents POMe mRNA /~g equivalent poly(A)+RNA, as described in Methods.

The effect of estrogen on POMe gene transcri pt ion in castrated animals was also analyzed in a similar type of experiment. Female rats were ovariectomized for 3 wk and then given silastic implants as described for the mRNA studies. The animals were subsequently sacri-

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ficed and the nuclei of the peri arcuate region of the hypothalamus were isolated. These nuclei were shown to specifically transcribe the POMC gene at a rate corresponding to 0.012% of total transcription. Animals treated with estrogen-containing implants were shown to have a decreased level of POMC gene transcription. Pre1 iminary experiments show that estrogen elicited an approximately 18% decrease in the level of POMC gene transcription after 60 min, and 34% after 4 days.

In situ hybridization analysis with the POMC cDNA probe allows the identification of the neurons which contain POMC mRNA (Fig. 1). By coupling this procedure with retrograde axonal tracing, three classes of neurons can be identified. Some cells contain the blue dye, but no POMC autoradiographic grains; in other cells, there are autoradiographic grains only, showing the presence of POMC mRNA; and in a group of cells, there is both blue dye and POMC autoradiographic grains. The latter cell s appear to express the POMC gene and to project to the preopt i c area. Only the middle areas of the peri arcuate region showed substan­tial numbers of POMC neurons also stained with the retrogradely trans­ported dye, in agreement with previous immunohistochemical studies.

Fig. 1. In situ cDNA:mRNA hybridization in the peri arcuate region of rat hypothalamus. Coronal sections (10 ~m) from a paraforma1ehyde per­fused rat hypothalamus were hybridized with a tritiated POMC cDNA and processed for in situ hybridization. Exposure time, 3 wk. Note the abundance of autoradiographic grains over a discrete subset of neurons.

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DISCUSSION We have shown that three days of estrogen treatment of a 2 wk

ovari ectomi zed rat, reduces hypotha 1 ami c mRNA 1 eve 1 s by 40%. These resul ts are consi stent with those of Wardl aw and coll eagues (16), who observed a reduction in S -endorphin in the hypothalamus of ovariecto­mized rats after estrogen treatment. It was unclear from their studies whether estrogen was acting on the synthesis or release of POMC peptides from the hypothalamus. Since neuropeptides are transported away from their site of synthesis, the observed decrease in neuropeptide at the cell body may not reflect reduced synthesis, but may instead be due to an enhanced transport of the neuropeptide to other regions of the brain. Now that the decl ine in POMC peptide has been associated with a decline in POMC mRNA levels, the hypothesis that estrogen acts to reduce the synthesis of POMC peptides in the brain is strongly supported.

The effect of estrogen on POMC mRNA levels requires more than 24 hr to become apparent. Indeed, the effects of estrogen on other neural processes, such as facilitation of lordosis behavior (29), and induction of progesterone receptors (30), do not become maximal before 24 hr as well. Since S-endorphin has been shown to be inhibitory to the display of female sexual behavior in the rat (31), it is of interest that the estrogen treatment that reduces POMC mRNA 1 evel s, is simil ar to that which facilitates lordosis (20).

The changes in POMC mRNA content el i ci ted by estrogen treatment appear to be mediated, at least in part, by similar effects of the hormone on transcri pt i on of the POMC gene in the peri arcuate regi on. Indeed, POMC gene transcription was inhibited within 60 min after estrogen administration. We hypothesize that the reduced transcription of the POMC gene eventua 11 y causes a reduct ion in the 1 eve 1 of POMC mRNA, which would then reduce the synthesis of POMC peptides. The fact that the changes in POMC gene expression induced by estrogen were detected first at the level of transcription is not surprising when compared with other steroid-responsive gene expression systems. Since a single gene is often responsible for producing the thousands of copies of mRNA in the cell, changes in gene transcription often require many hours to days to induce a 1 terat ions in the 1 eve 1 of 1 arge pools of

120

specific mRNAs. The small changes in POMC mRNA levels in the Arc-ME after estrogen

treatment could result from either small changes in all POMC neurons, or from a large change in a particular subset of the POMC system. The presence of a large unresponsive population of cells would dilute the effect of estrogen on POMC mRNA levels. This would be analogous to the effects of glucocorticoids on POMC gene expression in the anterior and intermediate lobes of the rat pituitary gland. It had originally been reported that glucocorticoid treatment of adrenalectomized rats caused a 3- to 4-fold increase in whole pituitary POMC mRNA level s (32). How­ever, when anterior and intermediate lobes were separated and assayed independently, a 30- to 50-fold decrease was observed in the anterior lobe, while no change was detected in the intermediate lobe (33,34). Since less than 10% of the POMC cells in the arcuate nucleus accumulate radioactive estrogen (36), the possibility that a large population of non-responsive cells reduce the overall effect of estrogen in the brain cannot be discounted. Complete delineation of this problem awaits the study of POMC gene expression in the arcuate neurons by in situ cDNA: mRNA hybridization coupled with retrograde axonal tracing techniques.

Preliminary results have shown the feasibility of coupling the POMC mRNA single cell quantitation assay with retrograde axonal tracing tech­niques. We were able to identify the POMC neurons which project to the preoptic area using retrograde tracing methods coupled with in situ hybridization histochemistry on the same section. We are currently using this technique to examine the effect of estrogen on the POMC cells projecting to the preoptic nucleus of the rat.

ACKNOWLEDGEMENTS We would like to thank C. Ippolitto for technical assistance, and

E. Kupsaw for secretarial assistance. Preliminary retrograde dye traci ng experiments were performed in coll aborati on wi th T. 0' Donohue and B. Chronwall of NINCDS. This research was supported by grant AM-27484 to JLR and grant HD-07093 to JNW.

REFERENCES 1. Eipper, B.A. and Mains, R.E. Endocrin. Rev. 1: 1-27, 1980.

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2. Bloch, B., Bugnon, C., Fellmann, D., Lenys, D. and Gouget, A. Cell Tiss. Res. 204: 1-15, 1979.

3. Bloom, F., Battenberg, E., Rossier, J., Ling, N. and Guillemin, R. Proc. Natl. Acad. Sci. USA 75: 1591-1595, 1978.

4. Finley, J.C.W., Lindstrom, P. and Petrusz, P. Neuroendocrin. 33: 28-42, 1981.

5. Joseph, S.A. Am. J. Anat. 158: 533-548, 1980. 6. Pelletier, G. and Leclerc, R. Endocrin. 104: 1426-1433, 1979. 7. Watson, S.J., Richard, C.W. and Barchas, J.D. Science 200: 1180-

1182, 1978. 8. Watkins, W.B. Cell Tiss. Res. 207: 55-80, 1980. 9. Zimmerman, E.A., Liotta, J.A. and Krieger, D.T. Cell Tiss. Res.

186: 393-398, 1978. 10. Liotta, A.S., Gildersleeve, D., Brownstein, M.J. and Kreiger, D.T.

Proc. Natl. Acad. Sci. USA 76: 1448-1452, 1979. 11. Civelli, 0., Birnberg, N. and Herbert, E. J. Biol. Chern. 257:

6783-6787, 1982. 12. Gee, C.E., Chen, C.L.C., Roberts, J.L., Thompson, R. and Watson,

S.J. Nature 306: 374-376, 1983. 13. Wardlaw, S.L., Wehrenberg, W.B., Ferin, M., Carmel, P.W. and Frantz,

A.G. Endocrin. 106: 1323-1326, 1980. 14. Sarkar, D.K. and Yen, S.S.C. 7th Int. Congr. Endocrin., Abs. #2062,

1984. 15. Meites, J., Bruini, J.F., Van Vugt, D.A. and Smith, A.E. Life Sci.

24: 1325-1336, 1979. 16. Wardlaw, S.L., Thoron, L. and Frantz, A.G. Brain Res. 245: 327-331,

1982. 17. Barden, N., Merand, Y., Rouleau, D., Garon, M. and Dupont, A. Brain

Res. 204: 441-445, 1981. 18. Knuth, U.A., Sikand, G.S., Casanueva, F.F., Havlicek, V. and

Friesen, H.G. Life Sci. 33: 1443-1447, 1983. 19. Petraglia, F., Penalva, A., Locatelli, V., Cocchi, D., Panerai,

A.E., Genazzani, A.R. and Muller, E.E. Endocrin. lll: 1224-1229, 1982.

20. Moreines, J.D.K. Ph.D. Thesis, Univ. of Michigan, 1980. 21. Chen, C.L.C., Dionne, F.T. and Roberts, J.L. Proc. Natl. Acad. Sci.

USA 80: 2211-2215, 1982. 22. Thomas, P.S. Meth. Enzymol. 100: 255-266, 1977. 23. Wahl, G.M., Stern, M. and Stark, G.R. Proc. Natl. Acad. Sci. USA

76: 3683-3687, 1979. 24. Murphy, D., Brickell, P.M., Latchman, D.S., Willison, K. and Rigby,

P.W.J. Cell 35: 865-871, 1983. 25. Evans, M.I., Hager, L.J. and McKnight, G.S. Cell 25: 187-193, 1981. 26. Eberwine, J.H. and Roberts, J.L. J. Biol. Chern. 259: 2166-2170,

1984. 27. Wilcox, J.N., Gee, C.E. and Roberts, J.L. Meth. Enzymol. (in

press). 28. Wilcox, J.N. and Roberts, J.L. Endocrin. (in press). 29. Quadagno, D.M., McCullough, J. and Langan, R. Horm. Behav. ~:

175-179, 1972. 30. Moguilewsky, M. and Raynaud, J.P. Endocrin. 105: 516-522, 1979. 31. Sirinathsinghji, D.J.S. Neuroendocrin. 39: 222-230, 1984. 32. Nakanishi, S., Kita, T., Taii, S., Imura, H. and Numa, S. Proc.

Natl. Acad. Sci. USA 74: 3283-3286, 1977.

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33. Schachter, B.S., Johnson, L.K., Baxter, J.D. and Roberts, J.L. Endocrin. llQ: 1442-1444, 1982.

34. Birnberg, N.C., Lissitzky, J.D., Hinman, M. and Herbert, E. Proc. Natl. Acad. Sci. USA 80: 6982-6986, 1983.

35. Morrell, J.I., McGinty, J. and Pfaff, D.W. Trans. Soc. Neurosci. 2: 90, 1983.

n DNA ANALYSIS OF DUCHENNE AND BECKER MUSCULAR DYSTROPHIES K.E. DAVIES, H.R. DORKINS, S. McGLADE, S.P. BALL, S.J. KENWRICK, T. SMITH, S. FORREST, L. WILSON, I. LAVENIR, A. SPEER 1 AND CH. COUTELLEl Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford, UK and 1 Department of Cell Differentiation, Central Institute of Molecular Biology, Academy of Science, Berlin, GDR

ABSTRACT The 1 i nkage analys is of DNA markers in famil i es segregat i ng for

Duchenne muscular dystrophy and Becker muscular dystrophy localizes these mutations to the same region of the human X chromosome at Xp21. Several restriction fragment length polymorphisms bridging the loci have been characterized which permit the carrier detection and antenatal diagnosis of these disorders in informative families. Methods being developed to identify the defects at the DNA level should lead to a better understandi ng of the mol ecul ar bas is of these muscul ar dystrophies.

I NTRODUCTI ON It is now relatively straightforward to analyze the molecular

defect in a genetic disease where the mutated product is known. Gene­specific probes can be isolated from the knowledge of a small stretch of protein sequence or from the immunological identification of the protein product produced in the bacterial cell by the cloned DNA. DNA probes which identify restriction fragment length polymorphisms (RFLPs) can be used as genetic linkage markers in family studies (1-3). This type of analysis enables the carrier status determination of individuals at risk for a monogenic disorder as well as antenatal diagnosis even before the underlying biochemical mechanism has been elucidated (4,5).

Genetic maps of chromosomes at 10 centimorgan (cM) interval s are now being constructed by the isolation of polymorphic sequences from individual chromosome libraries (6-8). Once a phenotype has been assigned to a specific chromosomal region by genetic linkage analysis, several methods can be used to identify more closely linked markers and the basic defect. One approach involves the cross-screening of cDNA libraries with chromosome-specific genomic libraries in order to isolate

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the expressed genes which are involved in the particular genetic disease of interest. Alternatively, chromosome walking techniques may be employed. It is also possible to compare DNA from patients possessing deletions with DNA from normal non-deleted individuals. Assuming that the deletion has resulted in the phenotype, this should lead to the identification of the DNA sequences at, or very close to, the defective locus.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder affecting 1 in 3000 males. The boys are often wheel chair bound by the time they are twelve years old and usually die in their teens. No antenatal diagnosis for this disorder was available before the advent of DNA recombinant technology. The only evidence for the localization of the mutation was the observation that females manifesting the disease possessed balanced X/autosome translocations with the breakpoint at Xp21 (for review, see 9).

RFLPs have now been characterized which bridge the DMD locus and which confirm the assignment of DMD to the middle of the short arm of the X chromosome and provide markers for carrier detection and antenatal diagnosis. Becker muscular dystrophy (BMD) , a clinically milder dis­order, has been localized to the same region of Xp21. In this review, we outl i ne the strategi es used for the mappi ng of the mutations and describe the methods used for the identification of the basic defect in these disorders.

RESULTS AND DISCUSSION Characterization of X chromosome-specific sequences.

We constructed an X chromosome enriched genomic library from human X chromosomes purified on a fluorescence activated cell sorter after staining of the chromosomes with ethidium bromide (6). Sorted DNA was cloned into Agt.WES.AB after complete digestion with the restriction enzyme EcoRI. The est i mated purity of the 1 i brary, from the sort i ng profile and detailed characterization of sequences, is 90% X chromosome sequences with 10% contamination with sequences derived from chromosomes 7 and S.

Human X chromosome single-copy sequences were selected after pre­screen i ng of clones from the Xli brary wi th [32 P]l abe 1 ed total human

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DNA. Under these conditions, only sequences repeated 100 to 150 times or more in the human genome give a positive hybridization signal (10). Seventy percent of the recombinants gave a strong, positive signal and hybridized as highly repetitive sequences. Many of the recombinant phages also hybridized to total lymphocyte poly(A) RNA illustrating that some of the repetitive sequences are expressed in human lymphocytes. This demonstrates the difficulty in isolating chromosome-specific expressed sequences by the di rect screeni ng of a chromosome- specifi c 1 i brary wi th mRNA because of expressed repet it i ve elements di spersed throughout the human genome.

Localization of the recombinant DNA sequences to the human X chromosome was demonstrated by hybridizing the clones to a Southern blot of human female DNA. DNA was i so 1 ated from a human/mouse hybri d ce 11 line containing the X chromosome as its only human component, and total mouse genomic DNA was digested to completion with EcoRI (6). Single­copy X-specific clones were assigned to regions of the X chromosome by hybridization to a human/rodent hybrid panel containing human X chromosome translocations (11). The clones were also assigned to chromosome regions by direct hybridization in situ to metaphase spreads (12). The latter technique is invaluable for the regional assignment of highly conserved sequences which hybridize in a similar manner to rodent cell line DNA and human DNA. In situ hybridization is also very useful for locating clones to regions of the chromosome where suitable trans­locations and somatic cell hybrids are not available. Identification of restriction fragment length polymorphisms.

X chromosome-specific clones were used to identify DNA sequence variants in the normal population. As DNA sequence polymorphisms occur approximately once in every 200 bp in the human genome (13), it should be possible to isolate enough closely linked markers to map the whole length of the human X chromosome. Drayna et al. (14) published the first map of the human X chromosome made from the study of the segregat i on of ei ght RFLPs i so 1 ated from an X chromosome 1 i brary and conventional markers. The total genetic length was estimated to be approximately 200 cM. Linkage studies in Duchenne muscular dystrophy families.

The first markers shown to be linked to the DMD locus were the

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probes RCS localized at Xp22.3 and LI.2S localized at Xpll.3. Both show linkage at a recombination fraction of approximately 0.17 and the segregation pattern is consistent with the localization of the mutation at Xp21 between them (15). Further studies using these markers in BMD families showed a similar pattern of inheritance of the RFLPs with the BMD mutation (16,17). BMD and DMD are thus localized to the same region of the human X chromosome and may be allelic.

Eleven RFLPs have been reported that bridge the DMD locus at Xp21 (15,18-20) . Although none of these alone is close enough genet i cally for accurate predictive diagnosis, the use of informative bridging markers is very valuable (5,20-22). Since the chance of double recombination between markers in Xp21 is small, it is possible to use the two more closely 1 inked bridging markers, C7 and 754, for the prenatal diagnosis of DMD (23). C7 is estimated to be approximately 10 cM from the locus on the oppos i te side from the centromere to the mutat ion (24). The marker 754, 1 oca 1 i zed on the oppos i te side of DMD from C7 nearer the centromere, is estimated to be 1 inked at 10 cM (25,26). Thus, the probability of double crossover between these loci is very small and the predictive diagnosis in informative famil ies can be carried out with greater than 99% accuracy. The physical assignments of probes used in the antenatal diagnosis of DMD are given in Fig. 1.

The probes have been localized physically by somatic cell hybrid genetics (27,28,34).

The segregation of C7 and 754 have been studi ed in BMD famil i es. The data are very similar to those reported for DMD families and support the hypothesis that these two mutations lie within the same region of the human X chromosome at Xp21 and may be allelic (24).

The markers C7 and 754 lie within 3000 kb of each other in Xp21 assuming random condensation of the DNA along the chromosome length. Also, 754 is localized within deletions in patients suffering from the di sease and other cl i ni ca 1 di sorders such as chroni c granulomatosus disease (27) and glycerol kinase deficiency (29). Thus, if 754 and C7 are 10 cM away from DMD, a high frequency of recombination occurs in this region of the human X chromosome compared to physical distance. Identification of the basic defect.

Thirty percent of DMD patients presenting at clinics are new muta-

DIU

2l3l

22.32

22 l1

222

22.13

2212

2211

21l

212

211

11.4

112l

1122

1121 111 cen

127

Fig. 1. Local ization in the X chromosome of RFLP probes used in the antenatal diagnosis of oMo.

tions. This is due to the very high mutation rate which appears to occur equally in the male and female gametes (30). These births cannot be prevented by prenatal diagnosis without a method of screening every pregnancy for the defect. Thus there is a great need to i dent i fy and understand the mutations giving rise to the phenotype.

One strategy for the isolation of the defect depends on the identi­fication of the junction fragments bridging the breakpoints in the affected females with X/autosome transl ocat ions. The female wi th the X/21 translocation is most amenable to this approach because the break in chromosome 21 occurs in ribosomal cistrons. Theoretically, if a library made from the derivative X chromosome is screened with ribosomal DNA (rONA) sequences, one of the positives should not only contain rONA sequences but also X chromosomal DNA. This strategy has been described by Worton et al. (31).

An alternative approach to the localization of the gene is based on

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patients who possess deletions 'at Xp21 (27). Excess DNA from the deleted patient is exhaustively hybridized to normal DNA. The DNA sequences within the deletion can be, selectively cloned allowing the isolation of a library of sequences enriched for sequences that lie within the deleted region of interest. This technique was used by Lamar and Palmer to characterize Y chromosome sequences (32) and promises to be a very useful approach to genetic diseases characterized by deletions.

Finally, methods have been developed to compare expressed sequences in different populations (33). This approach to DMD is complicated by the fact that the tissue in which the defect is expressed (if it is expressed) is far from clear.

CONCLUSION Several RFLPs bridging the DMD and BMD loci are now available such

that accurate predictive diagnosis is possible in many families. New techniques for comparing cDNA and genomic libraries have now been developed such that the mutant sequences themselves should soon be identified.

ACKNOWLEDGEMENTS We are very grateful to R. Kitt (Oxford) for patient typing of the

manuscript. We would like to thank The Medical Research Council, The Muscular Dystrophy Group of Great Britain and The Muscular Dystrophy Association of America. Part of this work was supported by a Wellcome Trust grant to St. Mary's Hospital Medical School allowing a three month working visit for CC at its Biochemistry Department.

REFERENCES 1. Botstein, D., White, R.l., Scolnick, M.H. and Davis, R.W. Am. J.

Hum. Genet. ~: 314-331, 1980. 2. Davies, K.E. Hum. Genet. ~: 351-357, 1981. 3. Solomon, E. and Bodmer, W.F. Lancet 1: 923, 1979. 4. Kan, Y.W. and Oozy, A.M. Proc. Natl. Acad. Sci. USA~: 5631-5635,

1978. 5. Harper, P.S., O'Brien. T., Murray, J.M., Davies, K.E., Pearson. P.L.

and Williamson, R.J. Med. Genet. zq: 252-254, 1983. 6. Davies, K.E., Young, B.D., El1es, R.G., fli11. M.E. and Will iamson,

R. Nature~: 374-376, 1981. 7. Kunkel, l.M., Tantravahi, U., Kurnit, D.M., Eisenhardt M., Bruns,

G.P. and Latt. S.A. Nucleic Acids Res. 11: 7961-7979, 1983.

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8. Krumlauf, R., Jeanpierre, M. and Young, B.D. Proc. Natl. Acad. Sci. USA 12: 2971-2975, 1982.

9. Moser, H. Hum. Genet. §.§.: 17-40, 1984. 10. Crampton, J.M., Davies, K.E. and Knapp, J.F. Nucleic Acids Res. 2:

3821-3824, 1981. 11. Wieacker, P., Davies, K.E., Cooke, H.J., Pearson, P.l., Williamson,

R., Southern, E., Zimmer, J. and Ropers, H-H. Am. J. Hum. Genet. ~: 265-276, 1984.

12. Hartley, D.A., Davies, K.E., Drayna, 0., White, R.L. and Williamson, R. Nucleic Acids Res. 1': 5277-5285, 1984.

13. Jeffreys, A.J. Cell~: 1-10, 1979. 14. Drayna, D., Davies, K.E., Hartley, D.A., Williamson, R. and White,

R. Proc. Natl. Acad. Sci. USA~: 2836-2839, 1984. 15. Davies, K.E., Pearon, P.L., Harper, P.S., Murray, J.M., O'Brien, T.,

Sarfarazi, M. and Williamson, R. Nucleic Acids Res. 11: 2303-2312, 1983.

16. Kingston, H.M., Sarfarazi, M., Thomas, N.S.T. and Harper, P.S. Hum. Genet. §1: 6-17, 1984.

17. Kingston, H.M.,·Thomas, N.S.T., Pearson, P.L., Sarfarazi, M. and Harper, P.S. J. Med. Genet. ZQ: 255-258, 1983.

18. Aldridge, J., Kunkel, L., Bruns, G., Lalande, M., Tantravahi, U. and Latt, S. Ital. J. Neurol. Sci. ~: 39-46, 1984.

19. Davies, K.E. J. Med. Genet. ZZ: 243-249, 1985. 20. Pearson, P.l. and Van Ommen, G.B. In: Research Into the Origin and

Treatment of Muscular Dystrophy (Eds. l.P. Ten Kate, P.l. Pearson and A.M. Stadhouders), Excerpta Medica, Amsterdam, 1984, pp. 91-100.

21. Davies, K.E., Briand, P., Ionasescu, V., Ionasescu, G., Williamson, R., Brown, C., Cavard, C. and Cathe1ineau, l. Nucleic Acids Res. 11: 155-165, 1985.

22. Pembrey, M.E., Davies, K.E., Winter, R.M., E11es, R.G., Williamson, R" Fazzoni, T.A. and Walker, C. Arch. Dis. Child. Q2: 208-216, 1983.

23. Bakker, E., Hofker, M.H., Goorl, N., Mandel, J.l., Davies, K.E., Kunkel, l.M., Willard, ILF., Fenton, W.A., Sandkuyl, l., Majoor­Krakauer, 0., Van Essen, A., Jahoda, M., Sachs, E.S., Van Ommen, G.J.B. and Pearson, P.l. lancet 1: 655-658, 1985.

24. Dorki ns, H. R., Mandel, J. -l. I Wrogemann, K., Moi son, J. P., Junien, C" Martinez, M., Old, J.M., Bundey, S., Schwartz, M., Carpenter, N.J., Hill, 0., lind10f, M., De 18 Chapelle, A., Pearson, P.l. and Davies, K.E. Hum. Genet. (in press).

25. Davies, K.E., Speer, A., Herrmann, F., Spiegler, A.W.J., McGlade, S., Hofker, M.H., Brfand, P., Hanke, R., Schwartz, M., Steinbicker, V., Szibor, R., Korner, H., Sommer, 0., Pearson, P.l. and Coutelle, C. Nucleic Acids Res. 11: 3419-3426, 1985.

26. Brown, C.S., Thomas, N.S.T., Sarfarazi, M., Davies, K.E., Kunkel, l., Pearson, P.l., Kingston, H.M., Shaw, D.J. and Harper, P.S. Hum. Genet. (in press).

27. Francke, U., Ochs, H.D., De Martinville, B., Giacalone, J., lindgren, V., Disteche, C., Pagon, R.A., Hofker, M.H., Van Ommen, G.J.B., Pearson, P.l. and Wedgwood, R.J. Am. J. Hum. Genet. ll: 250-268, 1985.

28. De Martinville, B., Kunkel, t.M., Bruns, G., Morle, F., Koenig, M., Mandel, J.l., Horwich, A., Latt, S.A., Gusella, J.F., Housman, D. and Francke, U. Am. J. Hum. Genet. ll: 235-249, 1985.

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29. Dunger, D.8., Davies, K.E., Williams, D., Pembrey, M., Lake, B., Pearson, P., Whitfield, T. and Dillon, M.J.D. New England J. Med., submitted.

30. Moser, H. In: Research into the Origin and Treatment of Muscular Dystrophy (Eds. L.P. Ten Kate, P.L. Pearson and A.M. Stadhouders), Excerpta Medica, Amsterdam, 1984, pp. 41-52.

31. Worton, R.G., Duff, C., Sylvester, J.E., Schmickel, R.D. and Willard, H.F. Science 224: 1447-1449, 1984.

32. Lamar, E.E. and Palmer, E. Cell 37: 171-177, 1984. 33. Davis, M.M., Cohen, D.I., Nielsen, E.A., Stenmetz, M., Paul, W.E.

and Hood, L. Proc. Natl. Acad. Sci. USA 81: 2194-2198, 1984. 34. Hofker, M.H., Wapenaar, M.C., Goor, N., Bakker, B., Van Ommen, G.B.

and Pearson, P.L. Hum. Genet. 70: 148-156, 1985.

12 GENETIC ANALYSIS OF THE FRAGILE X-MENTAL RETARDATION SYNDROME WITH POLYMORPHIC DNA MARKERS

I 1 '2' 3 I. OaERLE, G. CAMERINO , B. ARVEILER, J. BOUE , M.G. MATTEI, J.F. MATTEI3 AND J.L. MANDEL Laboratoi re de Genet i que Mol ecul a ire des Eucaryotes du CNRS et Unite 184 de l'INSERM, Faculte de Medecine, Strasbourg, France. 1 Dipartimento di Genetica e Microbiologia, Universita di Pavia, Italy. 2 Unite 732 de l'INSERM, Chateau de Longchamp, Paris and 3Unite 242 de l'INSERM, Hopital de la Timone, Marseille, France

ABSTRACT We have performed a linkage analysis in 16 families between the Fra

X locus and two polymorphic DNA markers that correspond to the anonimous probe St14 and to the coapulation factor IX gene. Our results indicate that the order of loci is centromer - FIX - Fra X - St14 - Xqter, with est imate of the recombi nat i on fraction for the 1 i nkage FIX- Fra X of 0.12 and Fra X-Stl4 of 0.10. Used in conjunction with cytogenetic analyses, the segregation studies with both probes should improve the genetic counseling for the Fra X syndrome and should be useful for the formal genetic analysis of this unique disease.

I NTRODUCTI ON The fragile X-mental retardation syndrome (Fra X) accounts for

about one quarter to one third of families with X-linked mental retardation, and it is present in approximately 1/2000 newborn males. It may also account for 3 to 4% of all mental retardation in otherwise normal females. The Fra X syndrome is a pleiotropic trait consisting of 1) the presence of a fragil e site on the X chromosome at the q27 -q28 interface, induced in vitro by conditions which impair thymidylate synthesis (growth of peripheral lymphocytes in media with low concen­trations of thymidine and folic acid or in the presence of FUdR or methotrexate), 2) a variable (moderate to severe) degree of mental retardation in hemizygous males, usually accompanied by characteristic physical features (macroorchidism and a typical facies), and 3) a 35% risk of mental impairment in heterozygous females (for review, see 1-4).

The genetics of the Fra X syndrome departs from classic X-linked inheritance in several respects. The Fra X gene does not appear to be

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fully penetrant in males. Apparently, normal males (cytogenetically and/or cl i ni cally) can transmit the di sease, as fi rst suggested from retrospective analysis of large pedigrees (reviewed in ref. 1 and 2). On the other hand, the percentage of clinically expressing females is much higher than in other sex-linked diseases. Furthermore, a segre­gation analysis suggested that the mutation rate at this locus is very high (7.2 x 10-4 ) but that mutations occur only in sperm (5). The gene seems to be more penetrant in the offspring of daughters of transmitting males than in the offspring of mothers of transmitting males (6).

The diagnosis of carrier females is difficult since only about one half of females who carry the Fra X mutation can be detected by their phenotype (mental retardation and/or fragile site expression) (5). The proportion of females showing the fragile site appears negatively correlated with both IQ and age (2,4). Prenatal diagnosis can be performed by assaying for the presence of the fragile site in fetal cells obtained by amniocentesis (7), fetal blood sampling (8) and, more recently, by microvill i biopsy (9). The first two techniques can be performed in only a few centers due to the difficulty of detection of the fragile site in amniocytes or the greater obstetrical complexity of fetal blood sampling compared to amniocentesis. The reliability of fragile site detection in trophoblast cells is not yet documented. Thus, because of the prevalence of the disease and of the problems encountered for genetic counseling, it is important to improve the methods for carrier detection and prenatal diagnosis.

Segregation analysis with restriction fragment length polymorphism (RFLP) linked to the disease locus might be very useful in investigating these problems since such markers allow the assessment of the genotype of individuals. We have performed a 1 inkage analysis in 16 famil ies between the Fra X locus and two polymorphic DNA markers that correspond to the anonymous probe St14 (10) and to the coagulation factor IX gene (FIX) (11,12). Our results indicate that the order of loci is centromere-FIX-Fra X-St14-Xqter. The estimate of the recombination fraction for the linkage FIX-Fra X is 0.12 (90% confidence limits: 0.044 -0.225) and 0.10 for Fra X-Stl4 (90% confidence limits: 0.040-0.185) (13) . The two fl anki ng probes can be used for carri er and prenatal

133

diagnosis with an accuracy of about 98% in 40% of the families, and for detection of transmission of the disease through phenotypically normal males (13,14). In conjunction with the cytogenetic analysis, segrega­tion studies with both probes should improve the genetic counseling for the Fra X syndrome, and should be useful for the formal genetic analysis of this unique disease. It should also be possible to assess whether similar forms of X-linked mental retardation which lack the character­i st i c marker chromosome mi ght be genet i call y related to the frag il e X syndrome.

RESULTS Linkage analysis.

We have analyzed DNA from 169 individuals belonging to 16 families with Fra X. The DNAs were digested with the restriction endonuclease TaqI, fractionated by electrophoresis on agarose gel, blotted onto a reusable support (diazobenzyloxymethyl paper; 15) and analyzed with nick-translated probes. Coagulation Factor IX probes detect two useful two-allele RFLPs in human DNA digested with the restriction enzymes TaqI and DdeI (12,16). About 60% of the females are heterozygous for one or the other of these two polymorphisms, and are thus informative in a linkage analysis. The Stl4 probe is a DNA fragment with no known function which detects a very polymorphic region in the q28 region of the human X chromosome, closely linked to hemophilia A (10,17). In human DNA digested with the restriction enzyme TaqI, St14 detects a polymorphic system with at least 10 alleles, while in DNA digested with other enzymes, it detects additional two-allele polymorphisms. The combined heterozygosity at this locus is about 90% in caucasian popula­tions.

El even famil ies were i nformat i ve for both FIX and Stl4 probes. Several large families exhibited no recombination at meiosis between the Fra X locus and either the FIX or the St14 probes (Families 13 and 9, Fig. 1). In contrast, one family showed several recombination events with the two probes (Family 5, Fig. 1).

In order to estimate the genetic distance between loci (expressed as the fraction of meioses showing recombination between two loci), we analyzed the segregation data using the LINKAGE computer program (18).

134

It is important in this case to take into account the problem of incomplete penetrance, since a proportion of males or females who carry the mutation will not express it clinically or cytogenetically. Because only rough estimates of penetrances are available, we have considered the influence of varying penetrance values for males and females. Although the odds in favor of linkage vary with the various modes of calculation, the recombination fraction estimate remains quite stable (13). Part of the results obtained are shown in Fig. 2, where the relative probability of linkage is plotted as a function of the recombination fraction B. The maximum lod score (i.e. the log of odds

• AA 7 48

1\1

FAMILY 13

FAMILY 9 IV FAMILY 5

Fig. 1. Inheritance of the TaqI pol ymorph i sms detected by the probe St14 and by the coagulation factor IX gene (FIX) in families with the fragil e X - menta 1 retardation syndrome. Probe Stl4: all el es 1 to S; probe FIX: alleles A (1.8 kb) and a (1.3 kb). Genotypes between parenthesis were deduced ,Jromn<the genealogy. 0 ,0, phenotypically norma 1 but not exami ned; JLl , }U , deceased; in the fo 11 owi ng symbols, the left half denotes the clinical status and the right half the cyto­genetic analysis: OJ ,aD, normal with no fragile site in cells (0% Fra X cells); m , @ , dull (with 1 to 2% Fra X cells);. ,. , mentally retarded (with more than 2% Fra X cells). In generation III of family 13, there are no recombinants between factor IX and the fragile X locus, and two recombi nants between St14 and Fra X (i n II IS and II 19) . The allelic combination a7 found associated with the fragile X is derived from the cytogenetically and clinically normal grandfather. In genera­t i on II of family 9, there are no recombi nants between St14 and Fra X and two recombinants between FIX and Fra X (in 113 and lIS). In family 5, several recombination events took place especially between FIX and Fra X in generation III.

135

in favor of linkage) for the linkage between FIX and Fra X is 6.18 at a recombination fraction of 0.114 with 90% confidence limits of 0.044 to 0.225. The Stl4 locus also shows about 10% recombination with Fra X (90% confidence limits of 0.040 to 0.185), with a maximum lod score of 9.5.

The three point linkage analysis, as well as simple examination of large nuclear pedigree (Fig. 1), places the Fra X locus between the two marker loci. Since a similar conclusion has been reached by in situ hybridization of the FIX and St14 probes to mitotic chromosome display­ing the fragile site at Xq27 (19), this supports further the notion that the mutation is located in the same region as the cytogenetically demonstratable fragile site (24). Linkage analysis does not provide

1.5

CD I 0

>< >-I-...J

co 1.0 « co 0 II: a. w > i= « ...J

0.5 w II:

FIX-FRA X

Lod score=6.18 8=0.114

5 10 15 20 25 30 35

Ol I o ~

>< >­I-;! co « co o II: 0.. W > i= « ...J w II:

3

2

St14-FRA X

Lod score =9.50 8=0.098

5 10 15 20 25 30

RECOMBINATION FRACTION (%)

Fig. 2. Estimation of the recombination fraction between the Fra X and the polymorphic DNA markers St14 and FIX. The relative probability of linkage (RPL) is the ratio of the probability of obtaining the data if the two loci are linked with a recombination fraction B to the proba­bility that there is no linkage ( 8= 0.5). The lod score is the log of the maximum RPL obtained for the recombination factor e . The 90% limits for the recombination fractions (dotted lines) are the values of B for which the RPL is one tenth of the maximum.

136

evi dence for heterogeneity among famil i es as tested fo 11 owi ng Morton (20), suggesting that the mutations reside in the same region in the different families. Diagnostic applications.

Our results establish the validity of the Stl4 and FIX probes as tools in the genetic analysis in Fra X families. These markers flank the disease locus and can thus be used in conjunction with cytogenetic tests for prenatal diagnosis and carrier detection in families informa­tive at both loci. Given the recombination fraction between each test locus and the Fra X locus, double recombinants should occur in only about I to 2% of the meioses in the StI4-FIX interval. The heterozy­gosity for the combined TaqI and DdeI RFLPs detected by the FIX probe is 60% in caucasians (16), while that of the TaqI and MspI RFLPs at the Stl4 locus is about 90% (10). Thus, 54% of the families would be doubly informative. However, in 25 to 30% of the cases, a single recombination event will occur between the two marker loci, which will prevent diagnosis. Therefore, we can estimate that about 40% of the caucasian cases could benefit from a segregation analysis with the two probes. An example is shown in Fig. 3, where the sister (IVIO) of an affected male asked for genetic counseling on the occasion of a pregnancy. She was clinically and cytogenetically normal, with thus a 30% chance only of being a carrier (assuming a penetrance of 0.56). She has received from her doubly heterozygous mother FIX and StI4 alleles which are different from those of her affected brother or cousins. Risk calculation shows that her final risk of being a carrier is only 1.3%.

The marker study can also help to detect new mutat ions (as shown for hemophilia A, in preparation) and/or those families in which the disease was transmitted through normal males. Out of the 16 families analyzed, two were already known to have normal male transmitters, based on the pedigree alone (a conclusion only possible for large pedigrees). In family 13, the FIX segregation data were sufficient to establish male transmission beyond doubt (11), and the StI4 data confirm this conclusion. In family 1, the FIX segregation data also suggested male transmission, which was confirmed by further pedigree analysis (Fig. 3). Suggestive evidence for male transmission (or for a new mutation arising in a male) has also been obtained in two other families.

III

IV

v

Aa 44

137

(a) AA (4) 43

2-8

Fig. 3. Carrier detection and transmission by a phenotypically normal male in a family with Fra X. Legends as in Fig. 1.

These results would be in agreement with the high percentage of pheno­typically normal male carriers estimated by Sherman et al. (6), although it is possible that an ascertainment bias exists in favor of such famil i es. It must be emphas i zed that detect i on of male carri ers is of great importance in a genetic counseling context. Finally, the segrega­tion analysis with linked markers could help in testing the hypothesis that penetrance might be different among sibships of normal male carriers and sibships of affected males (6) by allowing us to infer the genotype of clinically and cytogenetically normal males. Does the Fra X mutation affect recombination?

The fragile site is visualized as a region which is not properly packaged for mitosis, probably as a result of inadequate DNA replication since it is induced by conditions which affect the synthesis of a DNA precursor. One might wonder whether the presence of the mutation has an effect on another chromosomal process, i.e. meiotic recombination. The region which includes the fragile site seems to exhibit a high frequency of recombination in normal families, since the two probes 52A and FIX, which map in q27, are at 0.30 recombi nat ion uni t from the cl uster of loci in q28, which includes probes DX13 and Stl4, and the G6PD and Hemophilia A loci (10,19,21). The physical distance between the two groups of loci can be estimated cytogenetically to correspond to about

138

5% of the X chromosome length (22) and a recombination fraction of 0.12 would be expected if the genetic distance was proportional to the physical distances. On the other hand, Szabo et al. (22) have suggested that the recombination fraction between FIX and G6PD cluster is much decreased in Fra X families. Our data do not support the hypothesis of Szabo et al. since the recombination fraction between St14 and FIX in Fra X families, as estimated from three locus analysis, is about 0.19 (or 0.22 when estimated from two point linkage data), while there was 28% recombi nat i on in normal famil i es. A x2 test showed that these differences are not significant. X-linked mental retardation without the fragile site.

The fragile X syndrome might account only for 1/3 of the families with "nonspecific" X-linked mental retardation. In the study of Fishburn et al. (3), the fragile X was detected in 12 out of 45 pairs of brothers with nonspecific mental retardation consistent with X linkage. Six had macroorchidism and no fragile X, and the others had none of the above signs. It would be of interest to determine whether such forms of mental retardation correspond to allel ic variations at the fragile X locus or are due to mutations at different loci on the X chromosome. An approach to this problem is to perform segregation analysis with the same markers that show linkage to the fragile X. We have started such a study with a large family from Hawaii, initially described by Proops et al. (23). With the St14 probe, we found 5 recombinants in 15 meioses. The factor IX marker was uninformative, but two markers located in q27, and corresponding to probes 45d and 52A (with the order Fra X-FIX-[45d, 52A]) show a minimum of 3 recombinants (8. Arveiler, unpublished results). A marker located in Xq13 (DXYS1) does not show 1 inkage. Thus, these preliminary results, although compatible with a localization of the putative mental retardation mutation in the region of the fragile site, do not give strong support for this hypothesis. On the other hand, results from Fi 1 i ppi et a 1. (24) suggested that mental retarda­tion' with macroorchidism but without fragile X, shows more recombina­tion with the G6PD locus (located in Xq28 close to St14) than the fragile X locus. One problem in such studies is that in the absence of a real pathognomonic sign, one might be dealing with heterogeneous entities, which would render a linkage study very difficult.

139

DISCUSSION

Although the presently available probes represent useful tools, it

is des i rab 1 e to fi nd other markers closer to the Fra X locus and to

increase the number of families informative for probes on the proximal

side of the Fra X locus. The families already investigated are an

extremely useful material to quickly map new polymorphic probes with

respect to the fragile site, Stl4 and FIX, since it is necessary to

analyze only those meioses showing recombination between the Fra X and

one of the test loci in order to know on which side of the fragile X

locus is the new marker and whether it is closer than the already

available ones. This multipoint linkage approach is much more efficient

in ordering loci than two point linkage data performed on different sets

of families. By this way, we have recently mapped a probe which detects a TaqI RFLP on the proximal side of the fragile site (K. Wrogeman et

al., in preparation). However, if the Fra X region is a hot spot for recombination as has been suggested (IO,22), it might be difficult to

find random probes genetically closer unless they are physically in the

immediate vicinity of the mutation. The fragile X is still a very mysterious disease. We can list some

questions which remain unanswered. What is the structure (normal and mutated) of the region which constitutes the fragile site? The high mutation rate suggests that this region might have a special organiza­tion (tandem or inverted repeats, for instance). Is the region a hot

spot for meiotic recombination in normal individuals? Does the mutation directly affect a specific gene (or genes) responsible for the pheno­type? An alternative hypothesis would be that induction of the fragile site occurs in vivo only in regions or cells which have a limiting supply of folate or of DNA precursors such as thymidylate, and that as a

result, expression of all the genes distal to the fragile site would be decreased. However, one shoul d bear in mi nd that many other frag il e

sites exist on autosomes with the same pattern of induction but with no

clinical consequences (25). Since the mutation appears to function in

cis with respect to the fragile site, why is the expression generally so

low in females compared to males? Preferential X chromosome inactiva­

tion due to cell selection does not seem to be the explanation. Is the non-penetrance of the mutation in some males due to the action of

140

modifying genes, or of environmental conditions, or due to a two step generation of the full mutation (the premutation hypothesis, see ref. 6)?

Ultimately, the cloning of the mutated region (and of its normal homologue) might give important clues to answer some of these ques­tions. Even with the help of new cloning techniques which are being developed, such as jumping cloning vectors (26) and microdissection of mitotic chromosomes (27), this will be an extremely difficult task.

ACKNOWLEDGEMENTS We wish to thank Drs. P. Jacobs (Honolulu, Hawaii), U. Froster­

Iskenius (Lubeck), DeGrouchy (Paris), J. Fraisse (St. Etienne) and R. Walbaum (Roubaix) for referring us families and C. Wer16 and C. Aron for help in the preparation of the manuscript. This work was supported by grants from the Ministere de l'Industrie et de la Recherche (C0671), the Fondation pour la Recherche Medicale and from Agir (to J.-L. Mandel) and the Progetto Finalizzato Ingegneria Genetica e Basi Molecolari delle Malattie Ereditarie of CNR (to G. Camerino).

REFERENCES 1. Mattei, J.F., Mattei, M.G., Auger, M. and Giraud, F. J. Genet. Hum.

32: 167-192, 1984. 2. Turner, G. and Jacobs, P.A. In: Advances in Human Genetics (Eds. H.

Harris and K. Hirschorn), Plenum Press, 1983, pp. 83-112. 3. Fishburn, J., Turner, G., Daniel, A. and Brookwell, R. Am. J. Med.

Genet. 14: 713-724, 1983. 4. Sutherland, G.R. Trends in Genet. 1: 108-112, 1985. 5. Sherman, S.L., Morton, N.E., Jacobs, P.A. and Turner, G. Ann. Hum.

Genet. 48: 21-37, 1984. 6. Sherman, S.L., Jacobs, P.A., Morton, N.E., Froster-Iskenius, U.,

Howard-Peebles, P.N., Nielsen, K.B., Partington, M.W., Sutherland, G.R., Turner, G. and Watson, M. Hum. Genet. 69: 289-299, 1985.

7. Jenkins, E.C., Brown, W.T., Duncan, C.J., Brooks, J., Ben-Yishay, M., Giordano, F.M. and Nitowsky, H.M. Lancet 11: 1292, 1981.

8. Webb, T., Butler, D., Insley, J., Weaver, J.B., Green, S. and Rocek, C. Lancet 11: 1423, 1981.

9. Tommerup, N., Sondergaard, F., Tonnesen, T., Arveiler, B. and Schinzel, A. Lancet: 870, 1985.

10. Oberle, I., Drayna, D., Camerino, G., White, R. and Mandel, J.L. Proc. Natl. Acad. Sci. USA 82: 2824-2828, 1985.

11. Camerino, G., Mattei, M.G.,lMattei, J.F., Jaye, M. and Mandel, J.L. Nature 306: 701-704, 1983.

141

12. Camerino, G., Grzeschik, K.H., Jaye, M., De la Salle, H., Tolstoshev, P., Lecocq, J.P., Heilig, R. and Mandel, J.L. Proc. Natl. Acad. Sci. USA 81: 498-502, 1984.

13. Oberle, I. Heilig, R.~Moisan, J.P., Kloepfer, C., Mattei, M.G., Mattei, J.F., Boue, J., Froster-Iskenius, U., Jacobs, P.A., Lathrop, G.M., Lalouel, J.M. and Mandel, J.L. Proc. Natl. Acad. Sci. USA (in press).

14. Oberle, I., Mandel, J.L., Boue, J., Mattei, M.G. and Mattei, J.F. Lancet: 871, 1985.

15. Alwine, J.C., Kemp, D.J., Parker, B.A., Reiser, J., Renart, J., Stark, G.R. and Wahl, G.M. Meth. Enzymol. 68: 220-242, 1979.

16. Winship, P.R., Anson, D.S., Rizza, C.R. and Brownlee, G.G. Nucleic Acids Res. 12: 8861-8872, 1984.

17. Oberle, I.,-Camerino, G., Heilig, R., Grunebaum, L., Cazenave, J.P., Crapanzano, C., Mannucci, P.M. and Mandel, J.L. New England J. Med. 312: 682-686, 1985.

18. Lathrop, G.M., Lalouel, J.M., Julier, C. and Ott, J. Proc. Natl. Acad. Sci. USA 81: 3443-3446, 1984.

19. Mattei, M.G., Baeteman, M.A., Heilig, R., Oberle, I., Davies, K., Mandel, J.L. and Mattei, J.F. Hum. Genet. 69: 327-331, 1985.

20. Morton, N.E. Cytogenet. Cell. Genet. 22: 15-36, 1978. 21. Drayna, D., Davies, K., Hartley, D., Mandel, J.L., Camerino, G.,

Williamson, R. and White, R. Proc. Natl. Acad. Sci. USA 81: 2836-2839, 1984.

22. Szabo, P., Purrello, M., Rocchi, M., Archidiacono, N., Alhadeff, B., Filippi, G., Toniolo, D., Martini, G., Luzzatto, L. and Siniscalco, M. Proc. Natl. Acad. Sci. USA 81: 7855-7859, 1984.

23. Proops, R., Mayer, M. and Jacobs, P.A. Clinical Genet. 23: 81-96, 1983.

24. Filippi, G., Rinaldi, A., Archidiacono, N., Ricchi, M., Balazs, I. and Siniscalco, M. Am. J. Med. Genet. ~: 113-119, 1983.

25. De la Chapelle, A. and Berger, R. Cytogenet. Cell. Genet. 37: 274, 1984.

26. Collins, F.S. and Weissman, S.M. Proc. Natl. Acad. Sci. USA 81: 6812-6816, 1984.

27. Rohme, D., Fox, H., Herrmann, B., Frischauf, A.M., Estrom, J.E., Mains, P., Silver, L.M. and Lehrach, H. Cell 36: 783-788, 1984.

13

BRAIN MESSENGER RNA IN ALZHEIMER'S DISEASE M.R. MORRISON, W.S.T. GRIFFIN! AND C.l. WHITE, 1112 Departments of Neurology, ! Cell Biology and 2 Pathology, University of Texas, Dallas, TX, USA

ABSTRACT The lack of available animal models for most neurological diseases

restri cts analys is of neuropathology to postmortem human brai n. The ability to quantitate alterations in specific mRNA levels in postmortem brain would better define the neuropathology with respect to brain region and to specific transcriptional or post-transcriptional changes. We have shown that postmortem mRNA degradation is essentially random, thus allowing meaningful quantitation of relative mRNA levels. Preliminary results suggest that analysis of mRNA content in individual cells by in situ hybridization is also feasible. We have found that RNA isolated from Alzheimer's brain is much more susceptible to degradation during the initial stages of isolation than is RNA from control brain. This is true for areas of the brain with both a low and a high content of plaques and tangl es. Th is suscept i bi 1 i ty to degradat i on cannot be explained by alterations in the levels of free or bound alkaline RNase, suggesting that the intracellular distribution of active RNases, rather than their absolute levels, may be altered. When care is taken to minimize RNase action during RNA isolation, the levels of total RNA and translationally active mRNA from both uninvolved and involved areas of Alzheimer's brain are similar to control values.

I NTRODUCTI ON There are no animal models for most neurological diseases. This

restricts biochemical and histological analysis of specific neuropath­ologies to postmortem human brain. Parameters such as agonal state, hypoxia, postmortem interval and temperature, and storage conditions, may affect the 1 eve 1 s of bi ochemi ca 1 const i tuents in postmortem human bra in. Appropri ate controls must be employed to account for poss i b 1 e variations in biochemical and histological measurements.

Regional variations in enzyme and neurotransmitter levels are

143

characteristic of many neurological disorders. Measurement of pertinent mRNA levels in different brain regions and cell types would define the neuropathology at the mol ecul ar 1 eve 1. Ana lysi s of mRNA 1 evel s woul d also determine the brain region and cell in which the protein is synthesized. This is in contrast to regional measurements of substances that are axonally transported; variations in these levels may be due to altered protein synthesis in cells of a distant region.

Our initial idea was that postmortem disruption of cellular metabolism might result in rapid mRNA degradation, precluding the quan­titation of specific mRNA levels in human brain. We found this not to be the case. In thi s chapter, we summari ze our evi dence that post­mortem mRNA degradation in normal brain tissue is relatively slow (1-3). Degradation of abundant mRNAs, encoding proteins of up to 56 kD, is random for at least the first 16 hr. This is important for quantita­tion of specific mRNA levels (1-5). Isolation of undegraded, transla­tionally active mRNA (6) and detection of poly(A)+mRNAs in specific cell types by in situ hybridization (7-10) allows specific mRNAs to be quantitated in postmortem tissue. Patterns of mRNA transcription in normal and diseased brain can now be compared.

In the studies reported here, we used tissue from age-matched controls with similar postmortem histories to compare to neuropatholog­ically defined Alzheimer's disease (AD) cases. We found that AD RNA is abnormally susceptible to degradation during the initial stages of the isolation procedure. When appropriate precautions were employed, the recovery, integrity, and translational activity of AD RNA were similar to those of RNA isolated from control brain.

RESULTS Control studies in the rat.

In order to characterize postmortem mRNA degradation under controll ed conditions, we analyzed mRNA degradation in rat cerebellum (1). Cerebella were maintained for different postmortem time intervals and temperatures. Total RNA was isolated from gram amounts of tissue by homogenization in guanidinium chloride and centrifugation through cesium chloride (1). For milligram quantities of tissue, RNA was isolated by two guanidinium!ethanol precipitations followed by a proteinase K diges-

144

tion and phenol extraction (6). This microisolation procedure allows us to rapidly isolate undegraded translationally active RNA from small amounts of tissue. There was 1 itt 1 e change in recovery of total RNA after cerebella were left at room temperature for 4 hr (Table 1). Eighty-three percent of the total RNA was recovered after 16 hr at room temperature and ninety percent if the tissue was stored at 4°. Electro­phoresis of the RNA on denaturing agarose gels showed that some 18S and 28S RNAs were still intact even after 16 hr at room temperature (Fig. 1). The ratio of 28S and 18S RNA was no longer 2:1, indicating partial degradation of rRNA. In vitro translation of the RNA in a wheat germ cell free protein synthesizing system showed that translationally active mRNA was present (Table 1). The translational activity decreased with increasing postmortem interval and postmortem storage temperature. Despite this decrease, two dimensional gel analysis of the in vitro translation products showed that the translationally active mRNAs present were representative of those present in vivo (1). This conclu­sion is valid for the abundant mRNAs encoding proteins of up to 60 to 70 kD.

TABLE 1. Recovery and translation activity of total RNA isolated from rat and human cerebellum.

Species

Rat

Human

Postmortem Storage

4 hr, 25° 16 hr, 4° 16 hr, 25°

4 hr, 25° 16 hr, 25° ,

RNA Recovery (l1g/mg) (% Control)

0.61 96 0.57 90 0.53 83

0.46 73 0.33 53

Translation Activity (cpm~g RNA) (% Control)

63,000 80 74,000 94 56,000 71

42,000 53 36,000 46

Total RNA was isolated in guanidinium chloride and translated at subsat-2.5 11Ci PSS] urating (2 119) inputs in a wheat germ cell-free system (2).

methionine were added/50 111.

RNA isolated from postmortem human tissue. We analyzed the mRNA content of human ti ssue after postmortem

intervals of up to 16 hr at room temperature (1-3) (Table 1). The total

145

2

-285

-185

Fig. 1. Denaturing gel electrophoresis of total RNA isolated from rat cerebellum after different postmortem intervals. Two~g RNA were loaded in each lane. The formaldehyde gels were run and stained according to Maniatis et al. (51). lane 1, fresh rat cerebellum; lane 2, rat cere­bellum left at room temperature for 16 hr.

RNA/g wet wt isolated from human material after 4 hr was 24% less than the comparable rat control. Between 4 hr and 16 hr postmortem, there is a 28% loss of total RNA. This rate of decay is also higher than in rat (13%). This may indicate that postmortem RNA degradation is more rapid in human brain, perhaps because the rate at which the body temperature decreases in human must be slower than in rat. Quantitation of RNA in human biopsy samples will determine the extent of RNA degradation in the immediate postmortem interval. Some 18S and 28S RNAs are still intact (see next section). The translational activity of the RNAs from human brain is also lower (33%) after 4 hr postmortem than that of rat. The decrease in translational activity between 4 hr and 16 hr postmortem was similar in rat and human (Table 1). Despite the lower translational activity of the human RNA samples, the two dimensional pattern of their in vitro translation products was similar to that of RNA isolated from rat brain immediately postmortem (Fig. 2).

The data summarized above differ markedly from those obtained when RNA is adventitiously degraded during isolation. In that case, higher MW mRNAs are selectively degraded and there is a decrease in transla­tional activity (Morrison, unpublished observations). This decrease is accompanied by a marked reduction in the in vitro synthesis of higher MW proteins. In contrast, the postmortem degradation of mRNA is random. The mRNAs that remain are present in the same relative amounts as in vivo.

146

- 100

- 70

-so - 40

Fig. 2. Two dimensional gel analysis of [35S]methionine-labeled pro­teins synthesized in vitro by total cytoplasmic RNA isolated from human cerebellum stored at room temperature for 4 hr (A) and 16 hr (8). Expo­sure time 13 days. A, actin; TBI, BI-tubulin; TB2' B2-tubulin; "hI ,0.1-

tubulin; Ta.2, a.2-tubulin; p, protein p.

147

This random pattern of degradation allowed us to study the levels of specific mRNAs in postmortem human brain stored for intervals of up to 16 hr prior to freezing. We have shown that the mRNA levels for glial fibrillary acidic protein increase concomitant with gliosis in the cerebellum of patients with Joseph disease, a form of spinocerebellar degeneration (11). We have also shown that the developmental changes in mRNAs encoding different tubulin isoforms in human cortex and cerebellum are similar to those in rat (2,3,12,13). In our developmental studies, we isolated poly(A)+ mRNAs from postmortem human brain (2,3,13). The same relative amount of poly(A)+mRNA (1.5 to 2%) is present in the human isolates as is present in total RNA isolated from fresh rat brain (13). It is important to note that the postmortem human poly(A)+mRNAs are very similar in MW to those isolated from rat; their complementary DNAs (cDNAs) have a normal size distribution on denaturing agarose gels (2). cDNA libraries have now been constructed from poly(A)+RNAs isolated -from different regions of postmortem human brain (2,14,15).

We have used the technique of in situ hybridization to determine whether the poly(A)+mRNAs in postmortem human tissue a) can be quanti­tated, and b) are st i 11 present ina 11 ce 11 types. Postmortem human brain tissue was frozen, fixed in Bouin's solution and sectioned prior to in situ hybridization with [3H]poly(U) (8-10). The postmortem levels of total poly(A)+mRNA in AD cerebellum are sufficient for the analysis of autoradiographic grains above the cell types and comparison to age­matched controls. RNA levels in Alzheimer's disease.

Alzheimer's disease (16,17) is characterized by a progressive dementia. Diagnosis is confirmed postmortem by the abundance of extra­cellular senile plaques and intracellular neurofibrillary tangles in AD hippocampus and neocortical areas (18). The origin of the insoluble amyloid-like proteins in plaques and tangles is unknown. Antibodies have been generated against the puri fied protei ns (19-21) and short amyloid peptides are being sequenced (22). A reduction in the levels of specific neurotransmitters and their synthesizing enzymes in hippo­campus, cortex and in nuclei projecting to these areas is also charac­teristic of AD (23-26). The etiology of these changes is also unknown.

There is a decrease of nucleolar volume in AD neurons, perhaps

148

indicating a decrease in ribosomal RNA transcription. The incidence of the decrease of nucleolar volume is controversial. Is this decrease found ina 11 cort i cal neurons (27-29) or is it 1 imited to the neurons with neurofi bri 11 ary tangl es (30) or hi ppocampa 1 granul ovacuol ar degeneration (31)? Direct RNA measurement in dissected single cell bodies shows that the RNA content of neurons in the prefrontal cortex of demented individuals does not change (32) while cells with neurofibril-1 ary tangl es contained 1 ess than half the normal compl ement of RNA (33). Bowen et al. (34,35) found that AD cortex had a lower RNA/DNA ratio than normal cortex, but only when hypoxic brain was used as a control. Early studies showed that the euchromatin content (36) and micrococcal nuclease sensitivity (37) of AD cortex chromatin were both lower, suggesting that AD chromatin may be less transcriptionally active.

In vivo measurements of cerebral blood flow show a decrease in AD cortex (38,39). Positron emission tomography scans show a reduction in cerebral glucose utilization (40) and in protein synthesis (41). The decrease in protein synthesis could be a consequence of an energy charge decrease (42), or of a decrease in translationally active mRNA.

In order to determine whether AD is characterized by a general loss of a 11 mRNAs or by altered 1 eve 1 s of spec i fi c mRNAs, AD RNA must be isolated and its content of total and specific mRNAs compared to that of controls. In the first attempts at such comparisons, only 50% as much total RNA was detected per wet wt in AD cortex compared to the cortex of age-matched controls. The po1y(A)+mRNA content of the total RNA in AD cortex was 3- to 4-fo1d reduced (43,44). The AD mRNA isolated by Marotta's group also translated 4-fold less efficiently and the proteins synthesized in vitro were deficient in the tubu1ins, actin, and most of the other abundant proteins visualized by 20 gel electrophoresis. The authors conclude that AD is characterized by an extensive in vivo loss of functional mRNA in affected cortex, likely correlated with the presence of plaques and tangles (43). They also found that there is an increased free alkaline RNase activity in AD cortex and speculated that a defect in the endogenous alkaline RNase inhibitor-RNase complex was responsible for the dramatic mRNA loss (44).

In the present study, we compared total RNA recovery and integrity

149

in areas of AD brain that have a high content of plaques and tangles (cortex) as well as in areas free of light microscopic neuropathology (cerebellum). The corresponding areas of control brain were also compared. We found that the method of RNA isolation routinely used on control brain samples resulted in low yields of degraded RNA from both affected (cortex) and unaffected (cerebellum) areas of AD brain. Relatively undegraded RNA could be isolated from AD brain only if additional precautions were taken during tissue dissection and homogen­ization to minimize RNA degradation by endogenous RNases.

Documentation of neuropathology. The ages of the 5 individuals with AD ranged from 63 to 86 yr (Tabl e 2). The ages of the control s ranged from 56 to 100 yr. Postmortem intervals were from 4 to 16 hr in both groups. The AD group had a clinical history of progressive dementia. All brains were sliced and the slices were stored at -70 0 •

The d i agnos is of AD was confi rmed postmortem by the presence of an increased number of plaques and tangles in hippocampus and cortex of

TABLE 2. Plaque counts on control and Alzheimer's disease brain.

Diagnosis Age Cerebellum Inferior Temporal Occipital Cortex Pole

(mean plaques/field)

Normals

C1 56 0 0 0 C2 unknown 0 0 0 C3 69 0 0 0 C4 65 0 0 0 C5 100 0 2.7 0.1

AD

Al 76 0 11.5 1.3 A2 63 A3 75 0 15.0 10.5 A4 86 0 6.5 2.3 AS 86 0 10.2 2.6

Frozen tissue was thawed, immersion-fixed in 10% phosphate-buffered neutral formalin and paraffin embedded. Six micron sections were stained with Sevier-Munger silver stain (52). Ten 1.4 mm diameter (100x) fields were counted per slide. The mean number of senile plaques per field was calculated for each slide.

150

formalin-fixed, silver-stained tissue samples compared to controls (Table 2). No plaques were found in AD cerebellum. The occipital pole contained some plaques and the inferior temporal cortex contained the most. The only positive plaque count in the controls was in the inferior temporal cortex of the 100 yr-old individual (Table 2).

Isolation of degraded RNA from AD brain. In initial experiments, total RNA was isolated from AD brain following the microisolation procedure we developed for isolating undegraded RNA from rat and human brain (6). Approximately 80 mg of frozen tissue was dissected onto a preweighed piece of aluminum foil, weighed, and stored in liquid nitrogen prior to RNA extraction. The process of dissecting and weighing samples of frozen tissue unavoidably results in partial thawing. The recoveries of total RNA per mg wet wt for 4 different brain regions from the first two control brains (C1 and C2) and two AD brains (AI and A2) are shown in Table 3. Recovery of total RNA was 40

TABLE 3. Recovery of total RNA from control and Alzheimer's disease brain.

Brain Region

Cerebellum Parietal cortex Anterior temporal cortex Orbital frontal cortex

RNA Recovery (~g RNA/mg wet wt)

Controls AD

0.30 0.40 0.39 0.31

0.26 0.16 0.25 0.18

% Control

87 40 64 58

Total RNA was extracted using the unmodified isolation procedure descri bed in text. Resui ts are the average yi e 1 ds for three separate isolates from each brain area.

to 64% less for AD cortex than for controls. RNA recoveries from cere­bell urn showed more correspondence (13% loss in AD cerebell urn). The third AD brain (A3) did not show as dramatic a loss of RNA (results not shown). Denaturing gel electrophoresis of the total RNA showed that the RNA isolates from AD brains Al and A2, including the cerebellar isolates, were degraded. In contrast to the AD samples, 28S and 18S rRNAs were still present in the control RNAs although the ratio of 28S

151

1 2 3 4 5 6 7 8 9 10 11

-285

-18s

Fig. 3. Denaturing gel electrophoresis of total RNA isolated from different regi ons of thawed control and Al zheimer' s di sease brai ns. Lanes 1 and 8, adult rat cerebellum; lane 2, orbital frontal cortex and lanes 3 and 4, hippocampus of AD brain 1; lane 5, orbital frontal; lane 6, anterior temporal and lane 7, frontal parasaggital cortex of AD brain 2; lanes 9 to 11, orbital frontal cortex of control brain 2. The dis­sected regions were partially thawed before homogenization in guanidin­ium chloride and RNA isolated as described (6). Two \1g of denatured total RNA were loaded on each lane.

to 18S RNA was no longer 2:1. These profiles were similar to those for postmortem rat (Fig. 1). Representative RNA profiles are shown in Fig. 3. RNAs isolated from seve"ral areas of the third AD brain were more similar to the controls (results not shown).

TABLE 4. In vitro translation of control and Alzheimer's disease RNA.

Brain Region

Cerebellum Parietal cortex Anterior temporal cortex Orbital frontal cortex

[35S] Incorporated (cpm/\1g RNA x 10-3 )

Control AD

66 50 78 87

4 6

15 14

% Control

6 12 19 16

In order to determine the relative proportion of translationally active mRNAs in each RNA sample, the RNAs were translated in a wheat germ cell-free protein synthesizing system. Table 4 shows that the RNAs isolated from all 3 AD brains were 81 to 94% less active than the

152

2 3 4 5

Fig. 4. ID gel electrophoresis of the translation products of total RNA isolated from control and Alzheimer's disease cortex. RNA was isolated and translated as described in Table 1. Translation products were frac­tionated on a 5 to 17% gradient polyacrylamide gel. Exposure time, 4 days. Lanes 1 to 4, pari eta 1 cortex of brain Cl, C2, Al and A2, respectively; lane 5, cerebellum of adult rat. T, tubulins; A, actin; E, endogeous translation product of the wheat germ lysate.

RNAs isolated from control brains. Mixing experiments showed that the RNA isolated from AD brain did not contain an inhibitor of the in vitro translation system. The rRNA degradation and low translational activity of the AD samples were indicative of mRNA degradation. This was shown by separation of the in vitro translation products on ID gels (Fig. 4). Equal numbers of TCA precipitable counts were loaded on the control and AD lanes. The AD translation products were of a uniformly low MW (lanes 3 and 4), whereas the control human translation products (lanes 1 and 2) had an MW range equivalent to that of the rat control (lane 5). There were no tubulin translation products in the AD samples, although small amounts of actin and a protei n wi th the MW of gl i a 1 fi brill ary acidi c protein were synthesized. This sort of profile is characteristic of RNA

153

samples in which the higher MW mRNAs are degraded, and is not unique to AD. The loss of tubulin mRNA sequences in the AD samples was confirmed by slot blot hybridization of total RNAs to a [ 32P]nick-translated tubulin DNA probe (3). Hybridization does not discriminate between part i a 11 y degraded and intact mRNAs, yet hybri d i zat i on to the AD RNAs was many-fold less than to controls (results not shown).

I so 1 at i on of undegraded RNA from AD bra in. The low recovery and degradation of mRNA in AD brain reported by Marotta's group was taken as evidence for a dramatic in vivo RNA loss in AD brain (33,44). However, degradat i on of AD RNAs coul d also occur postmortem or duri ng the RNA isolation procedure itself. For example, the RNA degradation we observed in AD brain might be a consequence of RNase activity in partially thawed tissue (see the methods outlined in the previous section). In order to eliminate this possibility, we analyzed the RNA content of control and AD tissue which was not thawed prior to homogen­ization in guanidinium chloride. In this experiment, approximately 80 mg of tissue was dissected from 3 regions (cerebellum, occipital lobe and inferior temporal cortex) of each frozen brain. These regions had characteristic degrees of neuropathology (Table 2). The unweighted tissue was immediately stored in liquid nitrogen. Three isolates were processed simultaneously from each brain area. The recovery of RNA was reproducible to within 25% for the 3 separate isolates from each brain area. Table 5 shows the recoveries of total RNAs in 4 control and 5 AD brains. In contrast to the results described in the previous section, there was no significant difference in the RNA content of any of the AD brain areas compared to controls, except for a decreased recovery of RNA in the inferior temporal cortex of the A5 brain. Denaturing gel elec­trophoresis showed that the AD RNAs were no more degraded than those isolated from control brain (Fig. 5). The translationally active mRNA content of the total RNA preparations was also very simi 1 ar in each brain area analyzed (Table 6). When tissue samples from the same brain area were deliberately allowed to thaw prior to extraction, the AD RNAs were degraded and had low translational activity. This result conclu­sively demonstrates that the degree of RNA degradation in these AD brains depends upon whether the tissue remained frozen or was allowed to thaw.

154

TABLE 5. Recovery of total RNA from control and Alzheimer's disease brain.

Brain Region

Cl

Cerebellum 0.36 Occipital pole 0.53 Inferior temporal cortex 0.69

Al

Cerebellum Occipital pole 0.43 Inferior temporal cortex 0.64

Brain Sample (~g RNA/mg wet wt)

C3 C4

0.59 0.59 0.31 0.76 0.65

A2 A3 A4

0.46 0.40 0.48 0.46

0.48 0.70 0.55

C5

0.30 0.30

A5

0.29 0.46 0.24

RNA was extracted using the modified procedure (see text). Results are the average of three separate isolates from each brain region.

Ribonuclease levels in AD and control brain. Marotta's group have found that the 1 evel s of free al kal i ne RNase in affected areas of AD cortex are higher than in controls (43,44). In their brain samples,

1 2 3 4

-28s

-18s

Fig. 5. Denaturing agarose gel electrophoresis of total RNA isolated from different regions of frozen control and Alzheimer's disease bra ins. Lane I, adult rat cerebellum; 1 ane 2 and 3, i nferi or temporal cortex of control brain 3; lane 4, inferior temporal cortex of AD brain 3. The di ssected regi ons were not thawed pri or to homogeni zati on in guanidinium chloride (6). Two ~ of denatured total RNA was loaded on each lane.

155

this increase correlates with either decreased levels or activity of the

brian alkaline RNase inhibitor (45-47). They postulate that the increase in free a 1 ka li ne RNase is the cause of the mRNA degradat ion they descri be in AD cortex (44). We measured free and bound RNase in the 3 AD and control brain areas. The levels of free RNase in the controls were similar to those reported by Marotta et al. (44) but the levels of free RNase in the AD samples were no different from control values. We also assayed for total RNase activity in the presence of p­hydroxymercuribenzoate, the inactivator of the alkaline RNase inhibitor (45,46). Again, in contrast to previous findings (44), we found that there was as much inhibitor bound to RNase in the AD samples as in the controls (results not shown).

TABLE 6. In vitro translation of control and Alzheimer's disease RNA.

Brain Region

Cerebellum Inferior temporal cortex Occipital pole

[35S] Incorporated (cpm/llg RNA x 10-3)

Control AD

22 15 21

29 28 20

Total RNA was translated in a wheat germ cell-free system as described in Table 1. Results are the average of two determinations.

CONCLUSION Our comparisons of translationally active mRNAs, isolated from

postmortem brain, show that the relative levels of specific mRNAs in these i so 1 ates were proport i ona 1 to the 1 eve 1 s present in the brains immediately postmortem. Some of the factors influencing the rate of postmortem RNA loss have not yet been fully analyzed. Isolation of RNA from biopsied human brain would give a baseline value for the amount of total RNA present in vivo. This would allow a more exact determina­tion of the rate of postmortem RNA degradation and show if the rate of degradation is higher immediately postmortem when the body temperature remains near normal values. Another parameter which must be considered

156

is the effect of freezing the brains postmortem. Our preliminary studies suggest that RNA recovery and degradation are more variable after freezing, possibly due to lysosomal breakdown and release of lysosomal RNases.

It is also possible that in some diseases, the rate of RNA decay might be accelerated postmortem, especially if RNase leakage from lyso­somes was abnormally high. Agonal state and drug regimens prior to demise might also be variables. One study concludes that since AD patients are usually hypoxic at death, the levels of brain constituents should be compared to those in hypoxic controls (35). These authors found that the postmortem levels of most substances did not change with hypoxia (34,35) but that total RNA levels were 43 to 49% elevated in the temporal lobe (35). Total RNA levels were not significantly reduced in temporal lobe of AD patients compared to normal controls, but were reduced 54% when compared to those with hypoxia (35). This conclusion needs to be carefully evaluated both for the total mRNA population and for individual mRNAs.

We found that RNA from AD brain was more easily degraded during the isolation procedure than the RNA in control brains. This susceptibility to degradation was not confined to brain areas with a high content of plaques (inferior temporal cortex) but was also observed in the occipi­tal pole (few plaques) and in the cerebellum (no plaques). RNA degrada­tion invariably took place if AD tissue was even partially thawed prior to homogenization in guanidinium chloride. One possible explanation for this degradation is that the levels of free RNase in AD brain is higher than in controls (44). Our preliminary results indicated that there was no change in the total alkaline RNase levels or in the activity of the alkaline RNase inhibitor (45) for any area of the AD brains we tested. We have not yet assayed the activity of the lysosomal acid RNases (46) in AD cytoplasm. The increased susceptibility of AD RNA to degradation implies that decreased RNA levels in AD cannot be taken as evidence that RNA degradation occurs in vivo. This is particularly true for mRNAs encoding proteins of relatively high MW such as tubulin, since these mRNAs are always preferentially degraded by endogenous RNases.

When care is taken to keep AD tissue frozen, the recoveri es of total RNA from each area of AD brain are similar to those of controls.

157

Since the extractions in these experiments were done on unweighed aliquots, we cannot conclude that the yields are not 10 to 20% lower, a result reported by Taylor et al. (49). However, in our first RNA extraction of weighed cortex from AD brain 3, the yields were not lower than control cortex (results not shown). Our results also showed that the in vitro translational activity of the isolated RNAs was nearly identical for each area of control and AD cortex. Thus, the content of translationally active RNA per unit of AD total RNA was not different from controls. The analysis of the in vitro translation products on ID gel s showed simil ar patterns between affected and unaffected areas of AD brain or between the same brain areas in AD and controls.

The presenile form of AD reportedly shows greater severity of neuropathological change than that of late onset (48). Only one of the brains in our study (A2) came from a patient who had presenile dementia. The only area of this brain analyzed was the inferior temporal cortex. This sample had the lowest RNA recovery and translational activity. Additional presenile cases will have to be analyzed to assess whether there is a great loss of RNA in this population.

ACKNOWLEDGEMENTS We thank S. Pardue, R. Ison and M.A. Alejos for their technical

assistance, E. Miller for editing, and C. Beisert for preparing the manuscript. This work was supported by NIH grants HD 14886 (MRM) and AG 05537 (WSTG), and by the Leland Fikes Foundation (MRM).

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14 THE EFFECT OF TRISOMY-21 (DOWN'S SYNDROME) ON BRAIN TRANSCRIPTION L. LIM, C. HALL, T. LEUNG AND S. WHATLEY Department of Neurochemistry, Institute of Neurology, London, UK

ABSTRACT Messenger RNA from foetal;;brains of normal and Down's syndrome

subjects was translated in vitrui Trisomy-21 resulted in a significant increase in the mRNA for a 68 j ;kD microtubule-associated protein (68K MAP) . Th is prote in. whose genedhus putat i ve 1 y maps to chromosome 21, is a component of synaptic structures and membranes. An imbalance in its synthesis 'during brain deve]opment could contribute to the abnormal brains characteristic of Down's~syndrome. The 68K MAP is homologous to a constitutively synthesized 7h~O member of the heat-inducible family of proteins (heat-shock or strj'.J proteins). However, in human fibro­blasts, the amount of 68K MAY'fjsynthesized was not dependent on the content of chromosome 21, in con~rast to that of another heat-inducible, but constitutive 74 kD protein. n :These results indicate that the effect of trisomy-21 on mRNA levels is tissue-specific and that chromosome 21 may be involved in stress responses in addition to cytoskeletal functions.

INTRODUCTION The genetic bases for most of the familial neurological disorders

are at present poorly understood. There are a few which are proving amenable to analYSiS, including certain muscular dystrophies and Huntington's chorea. In the latter, the genetic locus has now been narrowed to chromosome 4 (1). Even when the genetic abnormal ity has been discovered in a disease, it remains a major problem to determine how this results in the characteristic symptoms. In the case of Down's syndrome, the main cause of the disorder is due to an extra chromosome 21, or at least parts of it, with a minority of cases resulting from translocation involving chromosomes 21 and 14 (2,2a). Mental handicap is the most common feature and brains of affected individuals are smaller and invariably exhibit abnormal morphology. However, there are other pathological changes not involving the brain which are associated

16.1

with Down's syndrome, including cardiac abnormalities and leukemia. Apart from tissue specificity, other factors to be considered

include secondary effects resulting ultimately from increased trans­cription of genes on chromosome 21. Although it is fairly certain that there are dose-dependent changes in proteins which are coded for by the extra chromosome 21, such as superoxide dismutase 1 (SOD) (3-3b), there are increases in other proteins which do not map to this chromo­some (4). The differences that do 'occur are Slight. For example, comparison of the protein composition of normal disomic- and trisomic-21 fibroblasts has not revealed major j;>lterations (5). This is not surprising when allowing for the rela1!'ive contribution of the genes on the small chromosome 21. Neverthele-ss, these differences, however sma 11, coul d 1 ead 'to del eteri ous effect.s if they occurred at cruc i a 1 periods in organ differentiation. The 'ammalian brain is known to be particularly vulnerable to metabolic ( ects early during development and thus, relatively small changes OCC" Ing during this critical period could result in permanently altered st Icture and function (6). This may explain why mental deficiency is thd:l11ajor symptom of this disease.

We have accordingly investigated the effects of trisomy-21 on mRNA levels in human foetal brains, to determine, on the one hand, the meta­bolic consequences of a well established abnormal genetic constitution and, on the other, the nature of the genes on chromosome 21 which are expressed in brain. In man, the major development of the brain occurs prenatally, whereas in the rat, it occurs postnatally (6). This has made the rat a convenient model for investigating mammalian brain development, and our initial studies on the characterization of brain mRNA were conducted in rats. Studies with rodents have shown that brain transcripts are the most diverse of all organs (which reflects the complexity and the multiplicity of proteins required for specialized functions), and that this diversity is developmentally regulated (7).

RESULTS Analysis of brain mRNA~ in the rat.

The analysis of mature rat brain mRNA involved its translation in reticulocyte lysates in vitro and analysis of the translation products by 2D polyacrylamide gel electrophoresis (PAGE) and fluorography (8).

162

(01- + 200K

92K

69K

46K

30K

"" 14K

Fig. 1. Cell-free synthesis of proteins directed by rat brain mRNA. Rat brain mRNA was isolated from free (a) or membrane-bound (b) polysomes, translated in a reticulocyte lysate, and newly-synthesized [ 35 S]methi oni ne-l abel ed proteins analyzed by fl uorography of 2D gel s. The protein bands whose position is shown, were characterized by co­migration with purified proteins and peptide analysis. A, actin; B, endogenous lysate protein; C, calmodulin; CK, creatine kinase; NSE, neuron-specific enolase; T, tubulin. Full details in Hall et al. (8).

The source of mRNA was poly(A)+RNA (9) isolated from either free- or

membrane-bound polysomes (10). The latter were examined because of our

interest in the synthesis of proteins contributing to brain membranes.

In our initial studies, we have concentrated mainly on acidic proteins

(Fi g. 1), such as the more abundant structural protei ns of the cyto­

skeleton, like the various isoforms of tubulin (T) and actin (A).

Other major translation products included a variety of brain-specific

enzymes, for example the brain form of creatine kinase (CK), neuron­

specific enolase (NSE), calmodulin (C), the neuron-specific protein 14-

3-3 and 68K MAP. The identity of the above products was confirmed by

co-migration with purified proteins and by comparison of the peptide

maps generated by S. aureus V8 protease.

The presence in membrane-bound polysomes of mRNA for some enzymes

generally considered to be cytosol ic led to an examination, using 2D

PAGE, of the cellular composition of membranes. We found these brain

enzymes to be present only in synaptic plasma membranes (SPM) together

with actin, tubulin and the 68K MAP (shown in Fig. 3) (11). Confirma­

tion of the identity of the enzymes (and also the more basic pyruvate

kinase) was achieved by comparison of tryptic fingerprints of these

membrane-bound protei ns wi th known puri fied enzymes. The enzymes, all

163

involved in ATP generation, were cryptic on the membranes, but could be activated by a variety of means which explains their previously undetec­ted presence. Two dimensional PAGE is thus a powerful tool for analy­zing brain gene expression and demonstrating that synaptic membranes contain ATP-generating enzymes.

The mRNAs for the structural protei ns tubul ins and act ins were found to be relatively more abundant in neonatal rat brain, as previ­ously shown by others (quoted in 10). There was a developmental increase in the mRNA for these enzymes, as well as in the number of detectable translation products as expected from the established developmental increase in transcriptional diversity of the brain. Foetal human brain mRNAs and the effects of trisomy-21.

These methods were then appl ied to an analysis of foetal human brain mRNAs in normal and Down's syndrome subjects (12). The source of mRNA was poly(A)+ RNA isolated from total cellular RNA. On visual comparison of the 20 translation patterns obtained from normal and Down's syndrome subjects, less than a dozen differences were observed out of the more than 400 peptides whose synthesis was detected. Eight acidic peptides were consistently involved (Fig. 2). An analysis of the fluorographic intensities of these peptides in 5 pairs of normal and Down's syndrome subjects showed increases in the synthes is of a 68 kD and a 49 kD protein and decreases in the synthesis of 6 proteins of 65, 37, 35, 25.5, 24.5 and 23 kD, respectively. The changes in the fluoro­graphic intensity of these peptides were assumed to result from similar changes in the concentration of the corresponding mRNAs.

To supplement this visual comparison, the relevant portions of the gel containing the involved peptides were excised and subjected to radioassay. The relative contribution of each of the peptides to the radioactivity of the total translation products was then calculated (Table 1) (12). The increase in the 68 kD and 49 kD peptides and the decrease in the 23 to 37 kD peptides were statistically significant. The decrease in the 65 kD peptide was not significant, probably because of the high variability encountered. The extent of the increase in the 68 kD and 49 kD peptides in Down's syndrome brains was compatible with a dose-dependent increase resulting from trisomy-21, which putatively assigns their genes to chromosome 21.

164

'----/

PI 18 P

.... \' :: ·HK

-46K

,-SOK

Fig. 2. The effects of trisomy-21 on human brain mRNA. Brain mRNA from 2nd trimester foetuses of (a) normal and (b) Down's syndrome subjects was translated in a reticulocyte cell-free system. The positions of tubulin (T) and actin (A) are shown. Arrows indicate translation products wi th altered synthes is in Down's syndrome wi th boxed areas containing those consistently affected (12).

Detailed analysis of the cellular proteins from foetal brains of normal (6) and Down's syndrome subjects (5) showed that most of the translated products had their cellular protein counterparts, except for the 49 kD and the 23-37 kD peptides whose translation was affected in

165

TABLE 1. The effects of trisomy-21 on brain mRNA-directed protein synthesis.

Molecular Approx. pI Normal Down's syndrome Mass (kD)

1 68 5.6 4.66 ± 1.90 9.23 ± 2.32***

2 65 5.8 27.6 ± 18.8 12.8 ± 13.2

3 49 5.7 2.82 ± 0.40+ 4.02 ± 0.98*

4 37 5.6 9.13 ± 3.14 5.15 ± 0.69*

5 35 5.6 10.1 ± 1.86 4.64 ± 2.04***

6 25.5 5.3 5.28 ± 1.50 2.49 ± 1.55**

7 24.5 5.3 8.10±3.10 3.80 ± 0.98*

8 23 5.2 3.50 ± 1.20 1.85 ± 0.29*

Areas of gels corresponding to peptides of interest were excised and their radioactivity determined (12). Results (mean ± SO) are expressed in terms of the percentage of the total radioactivity of the translation products loaded on the gels. Normal, n = 6; +, n = 4; Down's syndrome n = 5. *, P < 0.05 (Student's t test); **, p < 0.02; ***, p < 0.01.

Down's syndrome. The latter peptides could represent precursors which were modified in vivo or proteins present in such small quantities to remain undetected in our 20 gel but endowed with high turnover rates. The analysis also revealed no consistent changes in the gross cellular composition of proteins in Down's syndrome brains except for an increase in SOD, whose gene has already been mapped to chromosome 21 (3). The absence of major changes in the cell ul ar concentration of the 68 kD protein, despite changes in its synthesis, implies that its turnover is altered, although there may be alternative explanations (12). Identification of the 68 kO human brain protein as the 68K MAP.

The position on 20 gels of the 68 kO radioactive peptide is identical to that of rat brain 68K MAP. When the human radioactive peptide was electrophoresed with MAP isolated from rat brain, the two 68 kO protei ns were found to co-mi grate. Thei r co- i dent ity was further established by subjecting the human brain 68K MAP, the rat brain 68K MAP and the 68 kO human radioactive peptide to digestion with S. aureus V8

166

protease (12). Identical peptides were generated from all three proteins. Tryptic fingerprint analysis also confirmed the co-identity of the human and rat brain 68K MAP. Thus, trisomy-21 results in increased brain levels of the mRNA for the 68 kD brain MAP, putatively mapping the 68K MAP gene to chromosome 21.

The 65 kD radioactive peptide of human brain was also found to be identical to its cellular counterpart by co-migration and peptide analysis of the two proteins. These analyses also showed that the human brain 65 kD protein and the 68K MAP were different proteins, although some common peptides were noted (12), also on tryptic fingerprints. In rat brain, unlike human brain, the 65 kD protein was not normally found, either as a translation product of mRNA or as a cellular constituent. Presence of 68K MAP in synaptic structures.

Us i ng rat brain preparat ions, the 68K MAP was found to occur not only in microtubules, in association with tubul in from which it can be isolated by phosphocellulose chromatography, but also in synaptosomes. In the latter, it was a component of synaptic vesicles, synaptic plasma membranes (SPM), as well as of post-synaptic densities derived from the SPM (Fig. 3) (13). The SPM extract, obtained by Triton X-I00/high salt extract ion, contained most of the 68K MAP. A proportion of the 68K MAP was bound to a calmodulin-cellulose affinity column. Using agents

+

(a)

PURIFIED MAP ~

+

.. (c)

SPM -

Fig. 3. Identification of 68K MAP in synaptic structures. The arrow indicates the presence of 68K MAP in (a) microtubule-associated proteins obtained by phosphocellulose chromatography, (b) synaptic vesicles (SV), (c) synaptic plasma membranes (SPM), and (d) post-synaptic densities (PSD) derived from (SPM). Details in Lim et al. (13).

167

like trifluoroperazine and melittin, which disrupt calmodulin interac­tions, much of the 68K MAP was released from the SPM which still retained virtually all their tubulin. The association of 68K MAP with other SPM components (most probably tubulin) thus appears to be calmod­ulin-dependent. Relationship of 68K MAP with heat-shock proteins.

The position of the 68K MAP on 20 gels was identical to that of a mammal ian 70 kD heat-shock protein (HSP 70) (14), suggesting a great degree of s imil ari ty between the two protei ns. The HSPs are a multi­gene family of proteins whose synthesis is induced on exposure to elevated temperatures (heat-shock), as well as to a variety of metabolic agents (15). The heat-shock response was originally observed in Droso­phila and found to be common to all species and tissues studied so far, including human brain. The composition of the induced proteins can vary with either the type of cell and/or the nature of the stress. It has been reported that some of these proteins are constitutively synthe­sized in mammalian cells, particularly the HSP 70 (14). There are, however, quite a few other acidic HSPs of approximately 70 kD present in varying degrees in either unstressed or stressed cells. Different investigators have assigned various MW to the family of HSPs.

To determine the relationship of the 68K MAP to the HSP 70, advan­tage was taken of the availability of the well characterized Drosophila HSP 70 gene (16). Rat brain mRNA was hybridized to plasmid DNA contain­ing the Drosophila gene. Unbound and bound mRNA was translated subse­quently (Fig. 4). The 68K MAP, which is one of the major translation products of rat brain mRNA, was found to be absent from the products of the unbound brain mRNA. The mRNA hybridized to the Drosophila HSP 70

gene was found to direct the exclusive synthesis of the 68K MAP. This specific hybridization under stringent conditions indicates a high degree of homology between the two coding sequences and, consequently, between the two proteins. The effect of i ncreas i n9 doses of chromosome 21 on the synthes is of heat-shock proteins.

Since the synthesis of the 68K MAP was affected in human brains as a result of trisomy-21, we next examined protein synthesis in human fi­broblasts with mono-, di- and trisomy-21. The 86K MAP was indeed syn­thesized in normal (disomy-21) fibroblasts, in keeping with reports of

•

168 (e)

-,

Fig. 4. The brain mRNA for 68K MAP is complementary to the gene for the HSP 70. The translation products of rat brain mRNA are shown before hybrid-selection (a) and following hybridization to the Drosophila HSP 70 gene linked to plasmid pBR322 (courtesy of H. Pelham; 17): bound mRNA (b) and unbound mRNA (c). (d) shows that brain mRNA does not bind to plasmid lacking the HSP 70 gene. The arrow points to the position of 68K MAP which is also that of HSP 70. Details in Lim et al. (13). Symbols as in Fig. 1.

the const itut i ve synthes is of HSP 70 i n mammal ian ce 11 s. However, we were unable to detect any dose-dependent effect of chromosome 21 on the constitutive synthesis of the protein. Surprisingly, we were able to detect a dose-related synthesis of yet another HSP, a more acidic 74 kD form (HSP 74). In these fibroblasts, progressively more HSP 74 was syn­thesized with increasing doses of chromosome 21 (Fig. 5). In support of this putative location of the HSP 74 gene on human chromosome 21, we observed an increased const i tut i ve synthes is of HSP 74 in mouse- human hybrids containing only chromosome 21 as the human complement (3-3b), in comparison with the parent mouse cell line. The synthesis of this pro­tein was increased in the mouse cells by heat-shock. In these, as well ·as in human fibroblasts and lymphocytes, we have found that the major HSPs induced was a 65 kD protein (HSP 65). This heat-inducible protein has identical co-ordinates on 2D gels to the human brain 65 kD transla­tion product, whose position is proximal to that of HSP 70, the synthe-

(a) MONOSOMY-2

DISOMY-21

TRISOMY-21

169

(el I-SHOCKED

J>ARENT CELlS

Fig. 5. Effect of chromosome 21 on the synthesis of HSP. (a) Monosomic-, disomic- and trisomic-21 fibroblasts were labeled with [35S] methionine, and newly-synthesized radioactive proteins were analyzed on 2D gels. The positions of actin (A) and HSP 70 are shown. The arrow points to the constitutively synthesized HSP 74. Note the increase of this HSP 74 with increasing content of chromosome 21. (b) Newly-synthe­sized [ 35S]labeled proteins obtained from mouse-human hybrid cells containing only human chromosome 21 and from parent mouse cells. Note the increased constitutive synthesis of HSP 74 in hybrids. (c) On incu­bation of mouse parent cells at 45° for 20 min, the HSPs include HSP 74, HSP 70 and HSP 65. HSP 65 is the major component and is virtually absent in unshocked cells.

sis of which is increased in these cells to a much smaller extent by heat-shock.

CONCLUSION A major characteristic of trisomy-21 is an increased level of brain

mRNA coding for a 68 kD MAP. This experimental finding suggests that the gene may map to chromosome 21. This protein exhibits a great homology to the HSP 70 protein constitutively synthesized in mammalian cells. The brain protein affected is a constituent of the cytoskeleton

170

and of synaptic structures and appears to be similar to a 68 kD cyto­skel eta 1 protei n associ ated with a number of i ntermedi ate fil aments, including microtubules and neurofilaments (18,18a). Interestingly, an apparent homology between this cytoskeletal protein and the constitutive highly conserved HSP 70 has previously been commented on (19), although we were not aware of this when we undertook our comparative studies on the 68K MAP and HSP 70.

Based on 2D gel analysis, the 68K MAP occurs in higher quantities in brain compared to a similar protein present in other tissues. Unlike the brain, in human fibroblasts a changed content of chromosome 21 does not apparently affect synthesis of the protein. Some explanations for this difference are: (a) in fibroblasts, unlike the developing brain which is accumulating structural proteins, there are compensatory mechanisms which maintain constant the synthesis of HSP 70 when the number of genes is altered; (b) there are at least two different genes for HSP 70, the one on chromosome 21 being expressed only in brain and the other(s) on a different chromosome responsible for HSP 70 synthesis in other tissues; (c) the 68K MAP and the HSP 70, though closely related, are not identical, and are the products of two different genes, the 68K MAP gene only being located on chromosome 21; (d) the increase in 68K MAP mRNA is a secondary effect of trisomy-21 exclusive to the brain. We are unable to distinguish between these and other possibil­it i es until the genes for these two protei ns are characteri zed, and their chromosomal location determined. Confirmation on the location of the 74 kD HSP on chromosome 21 will also have to await the character­ization of the relevant gene and chromosomal mapping studies.

The functions of the HSP are not, at present, established, although a cytoskeletal role has been proposed. Heat-shock is only one of the many forms of metabol ic stress that result in the induction of thei r synthesis. The exact composition of this family depends on the stress appl ied and the tissue affected (15). We have found that in human fibroblasts and lymphocytes, the 65 kD is the most prominent form induced by heat-shock, while the 74 kD and the 70 kD proteins are minor components. Exposure to heavy metals results in synthesis of the 65 kD, as well as of several smaller proteins of MW around 30 kD.

The brain 65 kD translation product whose synthesis, though

171

variable, is consistently lower in Down's syndrome brains, is probably the same as the 65 kD major heat-shock or stress protein, since they co-migrate on 2D gels and have been found to possess a similar peptide composition. Some of the approximately 30 kD proteins, whose synthesis is also lower in Down's syndrome brains, appear to be stress proteins, judging from their 2D gel co-ordinates. A plausible explanation for the lower synthesis of these stress proteins is that Down's syndrome brains have a diminished response to stress compared to normal brains. The stress may have been manifest during the prolonged process of prosta­glandin-induced abortion through which all the foetuses in this investi­gation were obtained. In support of this suggestion, the 65 kD protein was found to be virtually absent from translation products of brain mRNA of one normal foetus obtained through hysterectomy, a less stressful procedure. It is also absent from brain mRNA of rats which were decapitated. How the presence of an extra copy of chromosome 21, which may contain several members of the heat-shock genes, results in aberrant expression of these genes, involving an increase in some and a decrease in others, remains to be established.

In Down's syndrome brains, some of the structural alterations include microcephaly and lowered dendritic arborization (2,2a). An imbalance in the synthesis of the cytoskeletal and synaptic 68K MAP could interfere with the normal process of morphogenesis and synapto­genesis during brain development, particularly in view of the contribu­tion of the cytoskeleton to plasma membrane functions, including control of cell morphology. It is interesting to note that the 68K MAP is also associated with neurofilaments, the organization of which is pathologi­cally altered in Alzheimer's disease which results in pre-senile dementia. Premature aging can. also be one consequence of Down's syndrome. In certai n cases of Down's syndrome, there are neuropatho­logical changes similar to Alzheimer's disease, including the presence of neurofibrillary tangles. A familial relationship of Down's syndrome and Alzheimer's disease has also been reported (20). In this context, it would be useful to determine whether altered stress responses occur in Alzheimer's disease. The availability of a recently isolated human gene for the major heat- i nduci bl e protei n shoul d prove of great val ue in studies on Down's syndrome and Alzheimer's disease (21).

172

ACKNOWLEDGEMENTS We thank A. Davison for his valued counsel, the Wellcome Trust, the

Brain Research Trust, the Medical Research Council and the Worshipful Company of Pewterers for their generous support.

REFERENCES 1. Gusella, J.F., Wexler, N.S., Conneally, P.M., Naylor, S.L.,

Anderson, M.A., Tanzi, R.E., Watkins, P.C., Ottina, K., Wallace, M.R., Sakaguchi, A.Y., Young, A.B., Shoulson, l., Bonilla, E. and Martin, J.B. Nature 306: 234-238, 1983.

2. Crome, L. and Stern, J. Pathology of Mental Retardation, Churchill­Livingstone, London, 1962.

2a. Zellweger, H. and Simpson, J. Chromosomes of Man, William Heinemann Ltd., London, 1977.

3. Tan, Y.H., Tischfield, J. and Ruddle, F.H. J. Exp. Med., 137: 317-330, 1973.

3a. Brown, T.W., Dutrowski, R. and Darlington, G.J. Biochem. Biophys. Res. Commun. 102: 675-681, 1981.

3b. Sherman, L., Dafni, N., Lieman-Hurwitz, J. and Groner, J. Proc. Natl. Acad. Sci. USA 80: 5465-5469, 1983.

4. Kaback, M.M. and Bernstein, L.H. Ann. N.Y. Acad. Sci. lZl: 526-536, 1970.

5. Van Keurin, M.L., Goldman, D. and Merril, C.R. Ann. N.Y. Acad. Sci. 396: 55-67, 1982.

6. DaVison, A.N. and Dobbing, J. Applied Neurochemistry, Blackwell Scientific Publications, 1968.

7. Kaplan, B.B. In: Molecular Approaches to Neurobiology (Ed. l.R. Brown), Academic Press, 1982, pp. 71-98.

8. Hall, C., Mahadevan, L., Whatley, S., Biswas, G. and Lim, L. Biochem. J. 219: 751-761, 1984.

9. Lim, L. and Canellakis, E.S. Nature 227: 710-712, 1970. 10. Hall, C. and Lim, L. Biochem. J. 196: 327-336, 1981. 11. Lim, L., Hall, C., Leung, T., Mahadevan, L. and Whatley, S.A. J.

Neurochem. 41: 1177-1181, 1983. 12. Whatley, S.A., Hall, C., Davison, A.N. and Lim, L. Biochem. J. 220:

179-187, 1984. 13. Lim, L., Hall, C., Leung, T. and Whatley, S.A. Biochem. J. 224:

677-680, 1984. 14. Bensaude, 0., Babinet, C., Morange, M. and Jacob, F. Nature 305:

331-333, 1983. 15. Schlesinger, M.J., Ashburner, M. and Tissieres, A. In: Heat Shock

from Bacteria to Man, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982.

16. Torok, I. and Karch, F. Nucleic Acids Res. §: 3105-3123, 1980. 17. Pelham, H. Cell 30: 517-528, 1982. 18. Wang, C., Asai, D.J. and Lazarides, E. Proc. Natl. Acad. Sci. USA

77: 1541-1545, 1980. 18a.Granger, B.L. and Lazarides, E. Science, 221: 553-556, 1983. 19. Wang, C., Gomer, R.H. and Lazarides, E. Proc. Natl. Acad. Sci. USA

78: 3531-3535, 1981. 20. Ann. N.Y. Acad. Sci. 396: 1982.

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21. Wu, B., Hunt, C. and Morimoto, R. Mol. Cell Biol. ~: 330-341, 1985.

15 NEURONAL CHROMATIN DURING DEVELOPMENT I.R. BROWN Department of Zoology, University of Toronto, West Hill, Ontario, Canada

ABSTRACT Genes expressed in neurons of the adult rat brain have been

reported to contain a common intron element called the "identifier" or ID sequence which may be a neuronal-specific regulator of gene expression (1). In this report, the nucleosome structure of bulk chromatin and chromatin regions containing ID sequences have been compared in neurons of the rat cerebral hemispheres. Developmental changes in the transcription of ID sequences in neurons have also been analyzed in relation to the timing of the appearance of the atypically short nucleosomal DNA repeat length. Probing neuronal nuclear RNA with labeled ID sequence demonstrated a greater than a 5-fold developmental increase in ID sequence in hnRNA during the first two weeks after birth. At 15 days in rat, neuronal hnRNA contains much higher levels of ID sequence compared to glial or kidney nuclear RNA.

I NTRODUCTI ON Chromatin in eukaryotic organisms is organized in a series of

repeating nucleoprotein particles which are termed nucleosomes (2-4). Each nucleosome is composed of a core particle consisting of an octamer of the four histones (H2A, H2B, H3 and H4) with 146 bp of DNA coiled around the outside. Each nucleosome also has a linker region made up of hi stone HI and 1 inker DNA. The repeating nucl eosome structure gi ves chromatin the beads-on-a-string structure which has been observed in the electron microscope, particularly in chromatin depleted of histone HI.

Most higher eukaryotic cells exhibit a nucleosomal DNA repeat length of approximately 200 bp which refers to the average length of DNA associated with the nucleosomal core particle plus the 1 inker region (2-4). In neurons of the cerebral hemispheres of several mammals, an atypical short nucleosomal DNA repeat length of 160-170 bp is present while glial cells, from the same brain region, exhibit a repeat length of approximately 200 bp as found in liver and kidney (5-10). Since both

175

neuronal and gl ial nuclei demonstrate a DNA fragment of 146 bp in the nucleosomal core particle, the short DNA repeat length appears to be associated with a decrease in the length of DNA in the linker region of the nucleosome (for review, see ref. 11).

RESULTS AND DISCUSSION Developmental change in nucleosomal DNA repeat length in cortical neurons.

Chromatin, in neurons of the cerebral hemisphere, converts to the short nucleosomal DNA repeat length during the first wk of postnatal development in the mouse, rat and rabbit (6-9). As shown in Table 1, a typical DNA repeat length of 195-197 bp was present in neuronal and kidney nuclei of the rat, which were isolated 1 day after birth. By 8 la days, neuronal nuclei exhibited an atypically short nucleosomal DNA repeat length of 159 bp while the repeat length in kidney nuclei remained unchanged.

Rat neuronal nuclei have been examined before and after the change in chromatin conformation in order to investigate the possible func­tional significance of the short DNA repeat length. Neuronal nuclei become more sensitive to digestion with DNase I when isolated at a developmental stage after the shi ft to the short DNA repeat 1 ength (12). Since this enzyme is known to preferentially digest decondensed and transcri pt i ona 11 y active chromat in re 1 at i ve to condensed chromat in (13,14), this may suggest that the appearance of short repeat chromatin is correlated to a relative uncoiling of neuronal chromatin. Neuronal nuclei in the cerebral hemispheres also exhibit a 50% reduction in the

TABLE 1. Developmental change in nucleosomal DNA repeat 1 ength in neurons of the rat.

Days after birth 1 2 5 8la

Repeat length in base pairs

neuronal nuclei 195 185 174 159 kidney nuclei 197 190 201 199

(Reprinted with permission from ref. 9).

176

ratio of HI to core histone (15-17). The reduced levels of histone HI in neuronal nuclei may be related to the increased levels of transcrip­tional activity in these nuclei, relative to glial nuclei (5). Chromatin enriched in transcriptionally active DNA sequences has been reported to be deficient in histone HI (18). Developmental changes in the svnthesis of nonhistone nuclear proteins.

Nonhistone nuclear proteins may play important roles in the structural organi zat i on of chromatin and, perhaps, the specifi city of gene expression (19). The synthesis of this class of nuclear protein has been analyzed before and after the shift to the short nucleosomal DNA repeat length in rat cerebral hemisphere neurons (20). Following a one hr pulse with [35S]methionine, the appearance of labeled proteins in highly purified nuclei was analyzed by 2D gel electrophoresis. As shown in Fig. 1, the most important developmental change in the syn­thesis of nonhistone nuclear proteins was the synthesis of an abundant acidic protein of MW 41 kD (pI 4.8), which appeared in short repeat neuronal chromatin seven days after birth but was not present at day one (compare Fig. 1, panels A and B). This protein appeared to accumulate rapidly in the nucleus since it was not detectable in the cytoplasm.

IF- +

'" _,."" ~'_ro

.",

';:_,8.8

Fig. 1. Analysis of [ 35S] methionine incorporation into total non­histone nuclear proteins from rat neuronal nuclei isolated one day after birth (A) or at seven days (B) following a 60 min in vivo pulse-labeling period. Numerals on the autoradiogram indicate relative MW. Ac, actin (43 kD); Tu, tubulin (55 kD). (Reprinted with permission from ref. 20).

177

The 41K protein, which is not observed in gl ial nuclei, may play an important role in the structure or functioning of neuronal chromatin. Analysis of identifier sequences in neuronal chromatin.

Recently, we have become interested in investigating whether sets of neuron-specific genes are activated in temporal association with the appearance of the atypically short nucleosomal DNA repeat length in cerebral hemisphere neurons. Genes, expressed in neurons of the adult rat brain, have been reported to contain a common intron element called the identifier or 10 sequence, which has been proposed to be a neuron­specific regulator of gene expression (21-23). A gene activation model has been proposed by Sutcliffe et al. (1), which suggests that a brain­specific RNA polymerase III (Pol III) transcription factor binds to 10 sequences in brain-specific genes, causing a chromatin conformational change which opens up brain genes for potential Pol II transcription when the appropri ate brain Pol II factors are present. Brain -spec i fi c RNA molecules of unknown function, which are termed BC1 and BC2, appear in the cytoplasm as a result of Pol III transcription of the 10 sequence. During the past year, I have spent my sabbatical leave from the Universi ty of Toronto in the Sutcl i ffe 1 aboratory. Some results obtained during this period will be discussed below.

The first question was to analyze the structure of ID sequences in chromatin from neuronal and kidney nuclei. Are 10 sequences in neuronal chromatin nucleosome free? Highly pure nuclei were obtained from rat cerebral hemisphere, liver and kidney tissue by sedimentation through dense sucrose (20). The cerebral hemisphere nuclei were then fract i onated into neuronal and nonastrocyt ic gl i al popul at ions (20). The purified nuclei were digested with micrococcal nuclease, and nucleo­somal DNA fragments were i sol ated and analyzed by el ectrophoresi s on agarose gels (9). By staining these gels with ethidium bromide, the nucleosomal DNA repeat length of the bulk chromatin could be calculated as previously described (6,10). Thereafter, the DNA fragments in the same gel were transferred to BioDyne membranes, and the resulting Southern blot was hybridized with a [32P]labeled 10 probe (23). The DNA repeat length of chromatin regions containing 10 sequences was then cal­culated from the resulting autoradiogram.

This experiment revealed that the 10 probe hybridized to monomer

178

DNA and DNA in multimers, indicating that ID sequences in cerebral hemi sphere neurons are organi zed ina nucl eosome conformat i on. For

neuronal nuclei, the atypically short nucleosomal DNA repeat length was apparent in bulk chromatin and in chromatin regions containing ID sequences, at both early and advanced digestion times. In kidney and liver nuclei, bulk chromatin had a typical 190 to 200 bp repeat length at all digestion times. However, an interesting situation was apparent for chromatin regions containing 10 sequences. The most sensitive and moderately nuclease sensitive ID-containing chromatin fractions exhib­ited a typical 200 bp repeat length. However, a more resistant or "buried" 10 component in liver and kidney, which was released at late digestion times, exhibited a short repeat length similar to neuronal chromatin.

In the rat, Mi 1 ner and Sutcl iffe have cal cul ated that there are 105 10 sequences (22). They have pointed out that in the rat, there are more ID sequences than brain-specific genes and that some ID sequences have transposed to other locations in the genome. Hence, different classes of 10 sequences may be present, based on their location in the genome. The "buried" ID sequence in liver and kidney chromatin, which exhibits the atypically short nucleosomal DNA repeat length, may represent the 10 class which is situated in brain-specific genes which are transcriptionally inactive in these tissues. The Sutcliffe gene activation model (1) proposes that 10 sequences in brain­specific genes exhibit a unique chromatin conformation in all tissues, not just in brain tissue, but only in brain is a unique Pol III trans­cription factor present to bind to it. Developmental analysis of identifier sequences in hnRNA.

In the next set of experiments, 10 sequences in heterogeneous nuclear RNA (hnRNA) were analyzed in neurons and in kidney tissue during early postnatal development of the rat. Highly purified nuclei were isolated and utilized for the preparation of nuclear RNA free of DNA employing the guanidine thiocyanate/CsCl method (24). Northern blots of the hnRNA were then hybridized with a [32P]labeled 10 probe to detect Pol II nucl ear transcri pts which contai ned 10 sequences. Probes and blots were prepared as previously described (23). Controls included a) digestion of hnRNA with NaOH to test for DNA contamination in the

179

RNA preparations, and b) analysis of brain cytoplasmic poly(A) RNA to demonstrate specificity of the ID probe (2A120), based on its ability to bind to brain-specific BCl and BC2 (1,23).

These experiments were speci fi ca 11 y des igned to analyze cerebral hemisphere neuronal nuclear RNA, not total brain nuclear RNA. We were interested in sets of genes which might be turned on in neurons in early postnatal development. Use of a mixed population of nuclei from the total brain would cloud this issue. The same class of neuronal nuclei was used in these hnRNA studies, as was used in our previously described analysis of ID sequences in neuronal chromatin and in our earlier studies on the developmental appearance of the short nucleosomal DNA repeat length (6,9,11,16). This is an important point since neuronal and gl ial nuclei exhibit quite divergent chromatin conforma­tions and transcription activities (5-9,12).

Conclusions which can be drawn from this experiment are as follows. There was a developmental increase in ID sequences in hnRNA in cerebral hemisphere neurons from the newborn stage to day 14 in the rat, wi th a marked increase between day 8 and 14, that is after the appearance of the atypically short nucleosomal DNA repeat length. ID sequences were detectable in kidney hnRNA, although the amount was less than in neuronal nuclei. In fact, the 10 Signal was 5.2-fold greater in neuronal hnRNA compared to kidney hnRNA at 14 days. During the period from birth to 14 days, there was a 5.6-fold developmental increase in ID sequences in hnRNA in neurons, but little if any, developmental change in kidney.

ID sequences were also compared in hnRNA from neuronal and glial nuclei isolated 14 days after birth from rat cerebral hemispheres. The level of ID sequences in neuronal hnRNA was 5-fold greater than that found in glial nuclei.

From these experiments, it can be concluded that transcription of ID sequences was quantitatively greater in cerebral hemisphere neuronal nuclei compared to glial or kidney nuclei when measurements were made after the appearance of the atypi cally short nucl eosoma 1 DNA repeat length (6-9). At one day after birth, the level of ID sequence in hnRNA appeared to be similar in neuronal and kidney nuclei. These results reveal an interesting surge of transcription of ID sequences in neuronal

180

hnRNA during the first two wk of postnatal development in the rat. As

mentioned previously, these Northern blot experiments detect ID

sequences in Pol II nuclear transcripts. In previous work, Sutcl iffe

et al. (1) have reported on the postnatal appearance of the cytoplasmic

brain-specific RNA molecules BCI and BC2 which are Pol III transcripts

of ID sequences. They fi nd that BCI and BC2 appear after bi rth wi th

a large increase between days 5 and 10.

Sequencing of identifier clones derived from BCI and BC2.

Experiments were designed to investigate whether the brain-specific

transcripts BCI and BC2 are derived from many genes. These cytoplasmic

RNA molecules, which have been reported to be Pol III transcripts of

ID sequences, are 160 and 100 to 110 nt in length and contain a poly(A)

sequence (1,23). Rat brain poly(A) RNA was cloned in pUC18 by a modi­

fied Okayama-Berg procedure (25). Five thousand clones were screened

with the ID probe 2AI20 and 75 positives were isolated. Ten clones,

with small inserts, were then selected and subjected to sequence

analysis (26).

The ten independently isolated cDNA clones exhibited inserts of

approximately 95 nt comprising the ID sequence. Of these ten clones,

three had exactly the same sequence wh il e the sequence of each of the

other seven differed one from another . All the ID clones exh i bited

limited sequence heterogeneity (less than 3% nt changes), compared to a

consensus sequence deri ved from ali st of known ID sequences (1,22).

The present ID clones all served as highly efficient templates in a Pol

III transcription assay (27). They exhibited conservation of the Pol

II I promoter sequences and had di screte 5' -termi ni commenci ng at the

first nt of the ID consensus sequence (1). It would appear that

flanking sequences are not required in these ID cDNA clones for highly

efficient transcription. In summary, these results suggest that the ten

cDNA clones were derived from many genes, reflecting that the BCl and

BC2 RNA populations in the rat are transcribed from many ID sequences

rather than being the products of one or two active sequences.

ACKNOWLEDGEMENTS

I thank J.G. Sutcliffe, P. Danielson, C. Lai, J.M. Gottesfeld

and S. Rush for di scussi ons and coll abo rat ions on unpubl i shed experi-

181

ments; and L. Elder for manuscript assistance. Supported by grants from NSERC, Canada (to IRB) and NIH (GM32355 to JGS).

REFERENCES 1. Sutcliffe, J.G., Milner, R.J., Gottesfeld, J.M. and Reynolds, W.

Science~: 1308-1315, 1984. 2. Kornberg, R.D. Ann. Rev. Biochem. 46: 931-954, 1977. 3. Horgen, P.A. and Silver, J.C. Ann. Rev. Microbiol. 32: 249-284,

1978. 4. McGhee, J.D. and Felsenfeld, G. Ann. Rev. Biochem. 49: 1115-1156,

1980. 5. Thomas, J.O. and Thompson, R.J. Cell 10: 633-640, 1977. 6. Brown, I.R. Biochem. Biophys. Res. Commun. 84: 285-292, 1978. 7. Ermini, M. and Kuenzle, C.C. FEBS Lett. 90: 167-172, 1978. 8. Whatley, S.A., Hall, C. and Lim, L. Biochem. J. 196: 115-119, 1981. 9. Greenwood, P.O., Heikkila, J.J. and Brown, I.R. Neurochem. Res. I:

525-539, 1982. 10. Brown, I.B. Biochim. Biophys. Acta 698: 307-309, 1982. 11. Brown, I.R. In: Handbook of Neurochemistry, 2nd edition (Ed. A.

Lajtha) Plenum Press, New York, Vol. 5, 1983, pp. 217-226. 12. Greenwood, P.O. and Brown, I.R. Neurochem. Res. I: 965-975, 1982. 13. Burkholder, G.D. and Weaver, M.G. Exp. Cell. Res. 92: 518-522,

1975. 14. Jalouzot, R., Briane, D., Ahlenbusch, H.H., Wilhelm, M.L. and

Wilhelm, F.X. Eur. J. Biochem. 104: 423-431, 1980. 15. Greenwood, P.O., Silver, J.C. and Brown, I.R. J. Neurochem. 37:

498-505, 1981. 16. Brown, I.R. and Greenwood, P.O. In: Molecular Approaches to

Neurobiology (Ed. I.R.Brown), Academic Press, New York, 1982, pp. 41-69.

17. Pearson, E.C., Bates, D.L., Prospero, T.D. and Thomas, J.O. Eur. J. Biochem. 144: 353-360, 1984.

18. Pederson, T. Int. Rev. Cytol. 55: 1-22, 1978. 19. Elgin, S.C.R. and Weintraub, H. Ann. Rev. Biochem. 44: 726-744,

1975. 20. Ivanov, T .R. and Brown, I.R. Neurochem. Res. 2: 1323-1337, 1984. 21. Sutcliffe, J.G., Milner, R.J., Bloom, F.E. and Lerner, R.A. Proc.

Natl. Acad. Sci. USA 79: 4942-4946, 1982. 22. Milner, R.J., Bloom, F.E., Lai, C., Lerner, R.A. and Sutcliffe, J.G.

Proc. Natl. Acad. Sci. USA 81: 713-717, 1984. 23. Sutcliffe, J.G., Milner, R.J., Gottesfeld, J.M. and Lerner, R.A.

Nature 308: 237-241, 1984. 24. Chirgwin, J.M., Przbyla, A.E., McDonald, R.J. and Rutter, W.J.

Biochemistry 18: 5294-5299, 1979. 25. Okayama, H. and Berg, P. Molec. Cell. Biol. ~: 280-289, 1983. 26. Maxam, A.M. and Gilbert, W. Proc. Natl. Acad. Sci. USA 74: 560-564,

1977 • 27. Weil, P.A., Luse, D.S., Segall, J. and Roeder, R.G. Cell 18:

469-484, 1979.

16 POST-TRANSLATIONAL MODIFICATIONS OF CHROMOSOMAL PROTEINS IN NEURONAL AND GLIAL NUCLEI FROM DEVELOPING RAT BRAIN I. SERRA* AND A.M. GIUFFRIDA Institute of Biochemistry, Faculty of Medicine, University of Catania, Italy; *deceased August 25, 1985

ABSTRACT Post-translational modifications of nuclear proteins may induce

changes in DNA-protein interactions, which modify DNA conformation and affect the binding of enzymes and proteins involved in gene regulation. Age-specific changes of acetylation, methylation and phosphorylation of histone and non-histone proteins have been found in cerebral hemispheres and cerebellum of rats at 10 and 30 days of age. Age-specific differ­ences between neuronal and glial nuclei have also been noted, especially duri ng the fi rst peri od of postnatal development. These observati ons may be related to neuronal and glial cell differences in a) patterns of differentiation, b) chromatin structures, and c) transcription rate.

INTRODUCTION It is well known that mammalian brain undergoes extensive cellular

proliferation and differentiation during fetal and postnatal develop­ment. These processes must involve the activation and/or repression of different sets of genes. The regul at i on of gene express i on occurs at various levels, although predominant control is exerted at the level of transcri pti on. The DNA associ ated proteins of eukaryoti c chromosomes are thought to regul ate gene expressi on in both di fferent i at i ng and mature cells. This chapter summarizes some of the available information on chromatin-bound protei ns and on thei r modi fi cat ions duri ng brai n development. Brain histones.

Brain histones exhibit little or no tissue-specific features (1). The el ectrophoret i c patterns of the di fferent hi stone fractions are similar in cerebral hemispheres and cerebellum of rat and no qualitative di fferences between 10 and 30 days of age were observed (23). No changes in the electrophoretic properties of histones in different brain regions or at various stages of brain development have been reported

183

(1). The turnover of all five histone fractions in brain occurs at different rates and the average half-life of histones changes consid­erably during brain development. It has been reported that from 6 to 40 days, the half-l ife of total histones in rat brain is 19 days, whereas after 2 mo, the half-life of brainhistones is 132 days (4).

Studies focusing on neuronal and glial nuclei have revealed no qualitative differences in the electrophoretic mobilities of the different histone fractions. No quantitative differences were observed between neuronal and glial histones associated with the nucleosome core particle, H2A, H2B, H3 and H4. A striking quantitative difference was noted, however, on histone HI. Cortical neuronal nuclei, compared to glial or kidney nuclei, show a 50% reduction in the amount of histone HI per mg DNA and a similar reduction in the ratio of HI to core histones. The ratio of neuronal histone Hlo:HI increases at the time of birth in the rat and this may be related to the postnatal arrest of neuronal cell division (5). Developmental changes in histones associated with the nucleosome core particle were not apparent. This study suggests that in cortical neurons the synthesis of histones during development accompanies the synthesis of neuronal DNA. Other studies with isolated neuronal perikarya demonstrated a transient period of synthesis of core and linker histones, which initiates just prior to the conversion to the short nucleosome DNA repeat length (6). The transient postnatal ability of neuronal perikarya to incorporate labeled amino acids into histones may be correlated with a possible requirement for additional histone, since the number of nucleosomes per unit length of DNA increases during the developmental changes in neuronal chromatin structure. Brain non-histone proteins.

Non-histone chromosomal proteins (NHP) represent a class of highly heterogeneous protei ns which pl ay structural, enzymi c and regul atory roles in the chromatin complex (7). Non-histone proteins are currently viewed as the major regulators of gene expression during cell differen­tiation, although evidence for such a role remains largely circumstan­tial (8). In addition, some NHP may play an enzymatic role contributing to DNA replication, repair and genetic recombination, and catalyze various modifications of proteins (histone and non-histone) and DNA (7). The class of NHP is considerably more complex than the histone class.

184

Up to 450 different species of NHP are observed in SDS-polyacrylamide gels, depending on the source of chromatin and the resolving power of the method employed.

Different authors have reported tissue-specific differences of brain NHP when compared electrophoretically to the NHP from other tissues. In particular, it has been reported that the electrophoretic pattern of brain NHP is the most heterogeneous and includes an excep­tional number of unique high MW components. In more recent studies empl oyi ng hi gh resol ut i on 20 gel el ectrophoresi sand fl uorography, it was shown that in brain cortex and cerebellar neurons NHP and DNA-bind­ing proteins undergo substantial changes during development, the majority of which occurs in coincidence with the arrest of cell division and the beginning of differentiation (9). Our own studies (2) showed that the electrophoretic patterns of total NHP, extracted from nuclei of cerebral hemispheres of 10 and 30 day-old rats, reveal about 30 bands with MW ranging from 20 kD to 200 kD. Considerable changes occur at the two ages examined. Some bands in the high MW region are more evident at 30 than at 10 days. Similar studies performed on NHP extracted from nuclei of cerebellum of 10 and 30 day-old rats (3) resolved the NHP into 12-14 major bands and 4-8 minor ones, which were partially masked by the more heavily stained bands. A comparative analysis of NHP indicated age-specific differences, especially in the high MW regions. Certain components of NHP became more evident from 10 to 30 days of age, whereas other peaks disappeared or decreased as the brain developed. These variations may reflect changes in the template activity of chromatin during cerebellar cell differentiation. Moreover, they could mean that some constituents required for the differentiation process have fulfilled their functions and undergo structural changes or dis­appear, whereas new components appear and accumul ate duri ng the same period.

Studies focusing on neuronal and glial nuclei have suggested that qual itative and quantitative differences exist in the NHP patterns of these two nuclear types (9-12). Ople et al. (10) showed that when total brain nuclei were fractionated into large and small nuclei, the NHP of these two preparations displayed different densitometric profiles. The electrophoretic analysis of NHP extracted from neuronal, astroglial and

185

oligodendroglial nuclei (11) showed marked differences between neuronal and gl i a 1 nucl ei, whereas only a 1 imi ted degree of speci fi ci ty was observed among the NHP of astroglial and oligodendroglial nuclei. In particular, in adult animals, one polypeptide band of 10 kD (pI 8.5) was found in the NHP fraction of neuronal nuclei and was absent from the corresponding fractions of all other classes of glial nuclei. However, in 10 day-old rats, this polypeptide is present in all classes of nuclei. The content of NHP was higher in neuronal and astroglial nuclei than in oligodendroglial nuclei. Differences in the content of basic and acidic proteins between astrocytes and 01 igodendrocytes have been shown by histochemical studies (13).

In recent studies (14), we showed that age-specific changes occur in the gel electrophoretic patterns of NHP extracted from neuronal and glial nuclei. These changes might reflect differences in the template activity of neuronal and gl ial chromatin during cell differentiation. During postnatal development, the synthesis of NHP undergoes qualitative and quantitative fluctuations which occur in temporal association with the developmental shift to the short DNA repeat length (15). The major developmental change, which was apparent in neuronal nuclei from cere­bra 1 hemi spheres, was the synthes is at seven days after bi rth of an acidic NHP of 41 kD (pI 4.8) which was not apparent at one day after birth. This protein was not observed in glial nuclei, suggesting it is specific to neuronal nuclei, and appears in association with the attain­ment of the short nucleosomal DNA repeat length. Moreover, neuronal and glial nuclei show quantitative developmental changes in the synthesis of two basic chromatin-associated proteins (37 kD and 38 kD) and several high MW chromatin-associated acidic proteins. High mobility group proteins.

A group of spec i fi c NHP, des i gnated as the high mobil ity group (HMG) proteins, have recently come under intense investigation (for review, see ref. 16). They are loosely bound to chromatin, being dissociated from it with 0.35 M NaCl. The HMG have some properties in common with histones, having approximately 25% basic amino acids and are extractable with acids. However, they are present in much smaller quantities than histones, the total HMG proteins being about 1 to 5% the yield of histones, and contain a higher percentage of acidic amino

186

acids. Four major HMG proteins, designated as HMG 1, HMG 2, HMG 14 and HMG 17, have been descri bed. HMG 1 and HMG 2 protei ns are associ ated with the linker region of the nucleosome, and HMG 14 and HMG 17 with the nucleosomal core particle and preferentially with transcriptionally active chromatin (16).

All the four major HMG proteins have been identified in total brain nuclei. HMG 1, HMG 2 and HMG 17 were detected in neuronal and gl ia1 nuclei isolated from cerebral hemispheres of rabbit brain. However, although divergent chromatin structures are present in these two nuclear popul at ions, no di fference in the el ectrophoretic mobil iti es of the major HMG proteins, as analyzed on SDS-polyacrylamide gels, were found (17). Chromatin recombination experiments performed with HMG 14 and 17 demonstrated that these proteins are not involved in tissue-specific gene regulation, although they are bound to active chromatin (17). Post-translational modifications of chromatin-bound proteins.

Post-translational modifications of chromatin-bound proteins play an important role in the regulation of eukaryotic gene expression. Indeed, processes such as acetylation, methylation, phosphorylation and ADP-ribosylation of nuclear proteins may induce changes in DNA-protein interactions, which modify DNA conformation and affect the binding of enzymes and proteins involved in the regulation of specific gene expres­sion (7).

Since Allfrey et a1. (I8) first correlated changes of chromosomal proteins by acetylation and phosphorylation to changes in nuclear RNA synthesi s, many reports have impl i cated the modi ficat ions of nuc1 ear proteins in the control of chromatin transcription (8). All nuclear hi stones are subjected to postsynthet i c mod i fi cat ions, such as acety­lation, phosphorylation, methylation and ADP-ribosylation. The functional significance of such alterations has not been definitely established, but they should play an important role in the organization of chromatin during DNA replication and in the modulation of chromatin structure at times of gene activation for RNA synthesis (for review, see ref. 7). In brain, most of the data available on the post-translational changes of chromosomal proteins have mainly dealt with histones and NHP isolated from total brain regions.

Histone acetylation. The process of histone acetylation may

187

represent an enzymatic mechanism for modulating the interaction between histones and DNA. The modification of chromatin structure may, in turn, affect its template function. Histone acetylation has been correlated with increased transcriptional activity and, conversely, the deacetyla­t i on of hi stones has been correl ated to gene i nact i vat ion. Hi stone acetylation was often found to precede an increase in RNA synthesis (19-21). Acetylation of histones has a strong effect on the abil ity of isolated chromatin to support DNA-dependent RNA synthesis. Such modifications appear to alter the number of RNA initiation sites on chromat in and to facil i tate the movement of RNA polymerase along the chromatin fibers. Butyrate suppression of histone deacetylation leads to accumulation of multiacetylated forms of histones and increased sensitivity of the associated DNA sequences to DNase I, an enzyme that preferentially digests active regions of the chromatin. Histone acetylation involves mostly internal lysyl residues. Amino-terminal acetylation, in the form of acetyl serine, occurs in some histone frac­tions in the cytoplasm before the histones are transported to the nucl eus. Different hi stone mol ecul es are acetyl ated to a different extent.

Histone phosphorvlation. The process of histone phosphorylation is probab 1 y i nvo 1 ved in hormonal act ion, as well as in the changes of chromosome condensation wi th occur duri ng the cell cycl e (7). It has been observed that the incorporation of phosphate groups into histones renders them less effective as repressors of DNA transcription (8). Moreover, chromatin reconstitution experiments suggest an important role for hi stone phosphoryl at ion in the speci fi c regul at i on of gene trans­cription. Indeed, chromatin, reconstituted with phosphorylated histones, was a better template than chromatin reconstituted with unphosphorylated histones. Histone phosphorylation involves mostly different and highly specific serine and threonine residues, although the occurrence of 3-phosphohi st idi ne and phosphoryl-lysine in the H4 and HI fractions, respectively, has been reported. Histone phosphory­lation could be catalyzed by a cAMP-dependent histone kinase or by a less specific cAMP-independent kinase.

Hi stone methyl at ion. The process of hi stone methyl at i on affects the DNA-histone interactions and thereby may also be involved in the

188

regulation of gene expression (7). The methyl group of S-adenosyl­

methionine is transferred to arginine, histidine and lysine residues of different histone fractions which are converted to mono and dimethyl­arginine, 3-methylhistidine and mono, di and trimethyl-lysine deriva­tives, respectively. Histone methylation appears to be a relatively late event, with maximum methylation of the lysine residues occurring late in S phase or in G2, after the bulk of DNA and histones have been synthesized (7).

Histone ADP-ribosylation. Mono and poly(ADP)-ribosylation reactions constitute a general type of covalent modification of proteins that may play an important role in the regulation of chromatin function and in the metabolism of protein and nucleic acids. The reaction is catalyzed by a nuclear ADPribosyltransferase, sometimes referred to as poly(ADPR) polymerase, a chromatin-bound enzyme which transfers the ADPR moiety of NAD to acceptor proteins (histones, non-histone proteins and nuclear enzymes). The exact nature of the linkage has not been determined, although an ester linkage, involving the carboxyl group of either a glutamate or an aspartate residue, has been proposed, and the occurrence of a serine phosphate residue, associated with poly(ADPR) or an ester 1 inkage with an a-carboxyl group of the C-terminal lysine residue in HI histone, has been reported (22). The existence of poly(ADPR) and the presence of poly(ADPR) polymerase activity in the chromatin of all eukaryotic cells tested suggest an important role for ADP-ribosylation in different cellular processes. It has been proposed that this modification is involved in the regulation of DNA replication and repair, cell proliferation and differentiation, and in the mainte­nance of chromatin architecture (see also chapter by Mandel et al., this volume). In this chapter, we will report our studies on the changes in methylation, acetylation and phosphorylation of histones and NHP, which occur in different brain regions and in fractions of neuronal and glial cells during postnatal development.

RESULTS AND DISCUSSION Post-translational modifications of histones and NHP in neuronal and glial cells.

Very few studies have been performed on the post-translational changes of histones and NHP in neuronal and glial cells. Recently in

189

our laboratory, we have started to investigate the in vitro processes of acetylation, phosphorylation and methylation of histones and NHP in neurona 1 and gl i a 1 nucl ei i so 1 ated from rat cerebral hemi spheres at various times of postnatal development (14,23). The specific radio­activity of the total histones extracted from neuronal and glial nuclei, separated from the cerebral hemispheres of 1, 10 and 30 day-old rats, is shown in Fig. I. In both nuclear populations, the acetylation of total

c :! o a. 0> E

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Fig. I. Labeling of histone and non-histone chromosomal proteins (NHP) extracted from neuronal and glial nuclei isolated from cerebral hemi­spheres of 1,10 and 30 day-old rats. Isolated nuclei were incubated with [3H]acetyl-CoA and S-adenosyl [3H-methyl]methionine for 40 min and with [ 32 P]ATP for I min. Histones and NHP were extracted as previously described (13,14,24). The results are expressed as average ± SEM of 3 experiments performed in duplicate. Significance of the difference (Student's t-test) from the value of 1 day-old rats (a) and of 10 day­old rats (b), • p < 0.05; _ P < 0.01; & P < 0.001.

190

hi stones increases s ignifi cantly from 1 to 10 days and shows mi nor changes between 10 and 30 days, in agreement with previous studies (2). The level of incorporation is quite similar in both neuronal and glial nuclei at 1 and 10 days and slightly higher in neuronal than in glial nuclei at 30 days. The phosphorylation of total histones increases up to 30 days in both neuronal and glial nuclei and, at all the ages investigated, is higher in glial than in neuronal nuclei. The methyla­tion of neuronal nuclei increases from 1 to 10 days of age and decreases thereafter, whereas in glial nuclei it decreases significantly through­out the developmental period studied.

The electrophoretic analysis of the labeled histone fractions (14) showed that the post-transl at i ona 1 changes observed duri ng postnatal development mainly concern HI, H3 and H2B, whereas H2A and H4 show few changes duri ng postnatal development. Moreover, the argi ni ne-ri ch H3 was always more labeled than the other fractions and showed the major changes duri ng postnatal development, in agreement wi th previ ous data (2,3). These results indicate that the increased methylation of total histones observed soon after birth is related mainly to the methylation of neuronal histones, whereas the subsequent decrease (2) is attribu­table to changes in both nuclear populations.

With regard to the post-translational modifications of brain NHP, the only post-synthetic modification studied in detail is phosphoryla­tion (for a review, see ref. 24). It is known that a large number of enzymes responsible for phosphorylation and dephosphorylation of NHP at specific serine or threonine residues are present in mammalian cell nuclei (29). Several lines of evidences suggest that the phosphoryla­tion and dephosphorylation of these proteins is involved in gene regula­t ion: (a) phosphoryl ated NHP are highly heterogeneous and thei r phos­phorylation patterns are tissue specific; (b) changes in NHP phosphoryl­ation correlate with changes in chromatin structure and gene activity; (c) addition of phosphorylated NHP increases RNA synthesis ;n v;tro; (d) phosphorylated NHP bind specifically to DNA. Cyclic AMP has both stimulatory and inhibitory properties on NHP phosphorylation, depending on the enzyme fraction and substrate employed (24).

Relatively little is known about the effect of acetylation and methylat i on of NHP on chromatin structure and function. It has been

191

suggested that methyl at i on of NHP results in the neutra lizat i on of negative charges, allowing for the interaction of these proteins with the DNA-histone complex, thus regulating gene expression. Age-dependent changes in the methylation and acetylation of NHP, and their differen­tial modulation by various effectors, have been reported in rat cerebral cortex (25).

Our own electrophoretic studies showed that in rat cerebral hemi­spheres, the incorporation of the 14C-acetyl group into NHP appears to be generalized throughout the MW range, with small differences between 10 and 30 days (2). On the other hand, the incorporation of the [3H]­methyl group into NHP decreases during postnatal development throughout the MW range, except for a protein of approximately 118 kD which was

1 day

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Fig. 2. Gel electrophoretic patterns of NHP obtained from neuronal and glial nuclei isolated from cerebral hemispheres of 1, 10 and 30 day-old rats. Nuclei were incubated with [3H]acetyl-CoA for 40 min. NHP were extracted and fractionated as previously described (13,14,24). Markers of known MW are indicated by arrows: (a) ferritin, 220 kD; (b) phos­phorylase b, 94 kD; (c), bovine serum albumin, 67 kD; (d) ovalbumin, 43 kD; (e) trypsin inhibitor, 20 kD.

192

highly labeled at both ages. In rat cerebellum, the acetylation and methylation of NHP decrease from 10 to 30 days (3). Moreover, at both ages, a protein of approximately 110 kD was found to be intensely methy­lated.

Fi gure 1 shows the spec i fi c radi oact i vity of total NHP extracted from rat cerebral hemispheres at 1, 10 and 30 days. The acetylation and the phosphorylation of NHP increased up to 30 days in both nuclear popu­lations, the increase being more pronounced in neuronal as compared to gl ial nuclei from 1 to 10 days. The methylation of NHP is higher in neuronal than in glial nuclei at all the ages investigated. A decrease with age occurs in both nuclear populations, but is more drastic in the neuronal nuclei. These results are in agreement with those previously reported (2) and indicate that the modifications observed in total brain nuclei may be attributable to changes in both nuclear populations.

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193

nuclei show that the process of acetylation markedly increases up to 30 days throughout the MW range in both nuclear populations. The labeling is considerably higher, especially in the low MW range, in most NHP fractions from neuronal nuclei as compared to glial nuclei (Fig. 2).

The phosphorylation of the different NHP fractions (Fig. 3) also increases up to 30 days in both nuclear populations, the increase being

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Fig. 4. Gel electrophoretic patterns of NHP obtained from neuronal and glial nuclei isolated from cerebral hemispheres of 1, 10 and 30 day-old rats. Nucl ei were incubated with S-adenosyl [3 H-methyl]methi oni ne for 40 min. Other details as in Fig. 2.

194

more pronounced in the high MW proteins of neuronal nuclei while it is

spread throughout the low MW range ; n 91 i a 1 nuc1 ei. At 30 days, a highly phosphorylated protein of about 94 kD is present in both neuronal and glial nuclei.

The methylation of NHP decreases during postnatal development throughout the MW range (Fig. 4). The main difference between neuronal and gl i a 1 nuc1 ei cons i sts in the intense methyl at i on of protei ns of approximately 130 kD that are present in neuronal nuclei and virtually absent in glial nuclei. It may be concluded that the highly methylated proteins found in mixed brain nuclei (2) are of neuronal origin. Possibly, these proteins might be involved in the regulation of specific ce 11 funct ions.

CONCLUSION The results reported in the present study indicate that age-spe­

cific differences between neuronal and glial nuclei are present in the processes of acetyl at i on, phosphoryl ation and methylation of hi stones and NHP, especially during the first period of postnatal development. The observed modifications might reflect changes between the two nuclear populations in the activities of protein kinases, phosphatases, methy1-ases and acetyltransferases, as well as of chromatin-bound protease, although the latter activity may be minimal in rodent brain nuclei. The modifications might also be due to conformational changes of neuronal and glial chromatin, altering the availability of the sites for acety­lation, phosphorylation and methylation.

The differences in post-translational modifications of chromatin proteins in rat brain may be correlated with the different patterns of maturation and differentiation of neuronal and glial cells, with the divergent chromatin structures of neuronal and glial nuclei, as well as with the changes of RNA synthesis observed in neuronal and glial nuclei during postnatal development (26-29).

Recently, the importance of transcriptional enhancers, i.e. short DNA sequences that st imu1 ate transcri pt i on of 1 inked genes, have been emphasized. Tissue-specific cellular enhancers located upstream to the promoter region of the mammalian insulin and chymotrypsin genes have been i dent i fi ed (30). DNA methyl at i on has also been postu1 ated to be

195

involved in the regulation of gene expression (31). In brain cells, many expressed genes contain the so called identifier sequence (10), which was postulated to be involved in the specific regulation of these genes (32). However, although much progress has been achieved, under­standing the regulation of gene expression in specific cell types is far from complete.

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673-679, 1981. 18. Al 1 frey, V.G., Faulkner, R. and Mirsky, A. Proc. Natl. Acad. Sci.

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117-128, 1975. 20. Marushige, K. Proc. Natl. Acad. Sci. USA 73: 3973-3941, 1976. 21. Sarkander, H.J. and Kuoll-Kohler, E. FEBS Lett. 85: 301-304, 1978. 22. Ogata, N., Ueda, K., Kagamiyama, H. and Hayaishi, O. J. Biol. Chern.

255: 7616-7620, 1980. 23. Serra, I., Avola, R., Condorelli, D.F, Surrentino, S., Renis, M.,

Kamiyama, M., Hashim, G.A. and Giuffrida, A.M. J. Neurochem. (in press).

24. Kleinsmith, L.J. J. Cell Physiol. 85: 459-475, 1975.

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25. Supakar, P.C. and Kanungo, M.S. Biochem. Biophys. Res. Commun. 100: 73-78, 1981.

26. Serra, I., Avola, R., Vanella, A., Lombardo, B., Agodi, A. and Giuffrida, A.M. Neurochem. Res. ~: 1049-1061, 1984.

27. Giuffrida, A.M., Cox, D. and Mathias, A.P. J. Neurochem. 24: 749-755, 1975.

28. Giuffrida, A.M., Hamberger, A. and Serra, I. J. Neurosci. Res. Z: 203-215, 1976.

29. Giuffrida, A.M., Gadaleta, M.N., Serra, I., Renis, M., Geremia. E., Del Prete, G. and Saccone, C. Neurochem. Res. !: 37-52, 1979.

30. Walker, M.D., Edmunt, T., Boulet, A. and Rutter, W.J. Nature 306: 557-561, 1983.

31. Riggs, A.D. and Jones, P.A. Adv. Cancer Res. 40: 1-30, 1983. 32. Milner, R.I., Bloom, F.E., Lai, C., Lerner, R.A. and Sutcliffe, J.G.

Proc. Natl. Acad. Sci. USA 81: 713-717, 1984.

17 DNA CONTENT IN NEURONS G. BERNOCCHI AND E. SCHERINI Dipartimento di Biologia Animale, Centro di Studio per l'Istochimica del C.N.R., Universita di Pavia, Italy

ABSTRACT Microdensitometric and microfluorometric analyses on the nuclear

DNA content of Purkinje neurons demonstrated the presence of hyperdi­ploid values (H2c) in a small percentage of cells. Hyperdiploid nuclei appear or increase in number during development in the rat and in the eel, while their chromatin shows differing degrees of condensation. Comparative studies of two species differing in the degree of complexity of nerve circuits, i.e. the hedgehog and the frog, showed that during the active period of the annual cycle, some Purkinje neurons have H2c largely in association with more condensed chromatin. The number of H2c cells decreases during hibernation, in concomitance with further conden­sation of chromatin. Variations in chromatin condensation are mainly responsible for the presence of H2c, although a real increase in DNA content may also occur. The presence of H2c cells appears related to a high metabolic requirement, since H2c cells are more evident during the active period of the annual cycle.

INTRODUCTION There are different ideas in the 1 iterature supported by contro­

versial data as to whether DNA content can actually increase in post­mitotic neurons. In the last decade, this problem has been studied using different approaches and methods, mainly in neurons from brain and cerebellar cortices, in particular Purkinje neurons (2,5-8, 13-16,19,32, 33,39,41).

At present, there is no evidence for a surplus of DNA in cortical neurons (22,34) or for thei r abi 1 i ty to synthesi ze DNA in the post­mitotic period (34). The problem has been approached using biochemical, cytochemi cal and auto-radiographi c techni ques. However, some di screp­ancies exist in the data obtained using different reactions or fluores­cent dyes (fluorochromes).

198

Based on cytochemical findings, the coexistence of hyperdiploid

(H2c) and diploid (2c) nuclei in the Purkinje neuron population is now accepted (7,8,13,16,26,33,41), although the data are not yet conclusive and the meaning of the possible surplus in DNA content is still unclear.

Our studies have focused on the DNA content of Purkinje neurons in re 1 at i on to the degree of chromat in condensat ion in ontogenet i c and phylogenetic conditions that imply establishment of different levels of neuronal metabolism. We assumed that acquiring different types of loco­motor activity or becoming more active behaviorally is linked to changes in the cerebellum, a brain region involved in the regulation of move­ment. These variations can be reflected in changes in the properties of neuronal nucl ear DNA. Our approach has been excl us i vely cytochem­ical, since we wanted to establish whether these changes, if any, involve the entire neuronal population or are limited to only a few cells.

As the methodol ogi cal aspects of cytochemi cal studi es cannot be disregarded in the interpretation of the data, we shall first briefly di scuss the 1 imi ts of some of the cytochemi cal stai ns used for the detect i on of DNA and some of the errors inherent in the photometri c techniques. Critical aspects in the cytochemical demonstration of DNA.

The Feulgen reaction, which has been largely used both for micro­fluorometry and microdensitometry, is based on the detection by Schiff reagent of apurinic acid produced by HCl hydrolysis of both single- and double-stranded DNA. The amount of stained material (F-DNA) is the algebraic sum of apurinic acid production and DNA depolymerization, both of which are affected by the degree of chromatin compaction (27,37). As a consequence, the duration of the HCl hydrolysis is the most critical factor in the measurement of total DNA, since chromatins with di fferent condensation, such as those of granul e cell sand Purki nje cells in the cerebellum, react differently to the hydrolysis (8,11,39). The Feulgen reaction is useful for evaluating even small differences in the physical organization of chromatin within a given cell population. Information about the degree of chromatin compaction is given by the ratio between F-DNA values and the area of distribution of the stained material (F-DNA/area) (2,31).

199

The other stains we shall discuss require the use of the fluoro­chromes propidium iodide (PI) and Hoechst 33342 (HO 342). Both of them, if properly used, can give either qualitative (degree of chromatin condensation) or quantitative (DNA content) information. Propidium iodide intercalates within the double-stranded regions of DNA, RNA and DNA/RNA hybrids, thus requiring prel iminary RNA digestion in the DNA analyses. Its binding is not dependent on the content of GC or AT base pairs. When PI is used at low concentrations (O.5 ~g/ml), its reactiv­ity is greatly affected by the degree of chromatin compaction (17,28,36). For quantitative determinations, PI is used at relatively saturating concentrations (50 ~g/ml; PI 50) (28,36). Hoechst 33342 binds external­ly to DNA (but not to RNA) at neutral pH. Its capacity to bind depends chi efly upon the presence of AT base pai rs. When used at saturating concentrations (20 ~g/ml), it is suitable for quantitative DNA analysis and for the detection of double- and single-stranded DNA (23,24,29). At low ionic strength, dissolved in buffer containing 0.01 M NaCl, HO 342 stains both AT- and GC-DNA (25), although the latter is stained with 1 ess effi c i ency . Non -histone prote ins can hinder both the interact ion of fluorochromes with interphasic chromatin (1,17,38) and HCl hydrolysis. Their removal enhances the cytochemical reaction. RNA, directly or because of its association with proteins, can also limit the ability of chromatin to interact with various types of fluorochromes (17).

Finally, we will mention the errors of photometric techniques, mainly micro-densitometry, since these methods provided the first evi dence against the statement that "neurons have a 4c DNA content." Intranuclear, intracytoplasmic and extracellular compounds can cause non-specific light loss and induce spuriously high absorbance in conven­tional microdensitometry of Feulgen-stained material at 550 nm. Evalua­tion by the two-wavelength method (550 nm and 450 nm) (19) or after careful determination of the background setting, with the spot position close to the nucleus in cell-wall-place-cytoplasm (35) for tissue squashes or for isolated nuclei, decrease the frequency of high values of F-DNA, but H2c nuclei are still present (7).

Comparison of cells with different sizes and different dispersion of the chromophore can also be a source of error, such as glare, diffraction error and residual distributional error, leading to under-

200

estimation in small cells with condensed chromatin. Glare can be compensated for by electronic offset as recommended by Bedi and Goldstein (4), if lower than 3 to 4%. Diffraction error is reduced if measurements are taken with a closed field diaphragm. The residual distributional error is not a major problem with a scanning integrating microdensitometer and it cannot be the only error responsible for differences in DNA content. For example, in a comparison of two cell types, cerebellar granule cells and hepatocytes (the former with more condensed chromatin), hepatocytes yielded lower values, the opposite of what would be expected (Bernocchi et al., in preparation).

RESULTS AND DISCUSSION Developmental studies.

During the postnatal ontogenesis of the cerebellum, the most important change is the maturation of nerve circuits, which involves migration of cells, development of synapses and, especially, change in the metabo 1 i sm of neurons. The fi na 1 outcome of these events is the establishment of the cerebellar input in the control of locomotor activity. We have examined the appearance of cytochemically detectable vari at ions in the DNA content of devel opi ng Purki nje neurons in two species, the rat (mammals) and the eel (teleosts), differing in the degree of complexity of their cerebellar circuits (10,12). In both species, the chromatin of Purkinje neurons generally becomes less condensed duri ng maturation. The process of decondensat i on has been evaluated cytochemically by determining the F-DNA/area ratio, which decreases duri ng hi stogenes is, and in degree of i nterca 1 at i on of PI 0.5 which, on the contrary, increases.

In rat postnatal histogenesis, the number of Purkinje with H2c nuclei increases from postnatal day (PD) 7 (17% of the tion) to PD 12 (45%), as shown by the Feulgen method (Fig. 1).

neurons popula­In H2c

nuclei, chromatin appears generally decondensed (low F-DNA/area ratio) on PD 7, while on PD 12 some nuclei (22%) have condensed chromatin (high F-DNA/area ratio). In later developmental stages, on PD 21 and 30, most H2c nuclei show decondensed chromatin. The decondensation of the chromatin could be related to the enhanced RNA synthesis of mature neurons. With PI 50 staining (Fig. 2), 15 to 20% of the nuclei of

201

PO 7

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Fig. 1. Feulgen-DNA content and degree of chromatin condensation in rat cerebellar neurons during postnatal development. Upper graphs: distri­bution of DNA values (in arbitrary units) in Purkinje neurons (white columns) and granule cells (dashed columns). Bar indicates the mean value ( v) ± S.D. for 2c hepatocytes. Lower graphs: correlation between DNA values and degree of chromatin condensation (F-DNA/area ratio) in Purkinje neurons. Each point is the mean of three values. The quadrants on the left correspond to 2c nuclei with both decondensed and condensed chromatin, while those on the right to H2c nuclei.

Purkinje neurons are H2c on PD 12 and PD 21. The highest percentage is observed on PD 30. With HO 342 staining (Fig. 3), H2c Purkinje nuclei are present on PD 12, increasing to 10 to 15% of the population on PD 21 and PD 30.

Administration of [3H]thymidine during the period of appearance of H2c Purkinje nuclei indicated that DNA synthesis, though low, can occur in a few Purkinje neurons. Simultaneous treatment of the rats with cis­dichlorodiammine platinum, a cytostatic agent that interferes with DNA synthesis, suppresses the incorporation of [3H]thymidine. In addition,

101

PO 12

PO 30

PI- 0 N A

Fig. 2. Propidium iodide-DNA values in rat cerebellar neurons during postnatal development. PI-DNA values for Purkinje neurons (white columns) and granule cells (dashed columns). Columns below the 1 ine show the distribution of 2c and 4c hepatocytes on PO 30. (0.5),0.5 ].lg/ml PI; (50), 50 ].lg/ml PI.

microdensitometric data demonstrate the lack or the scarcity of H2c F­DNA in the Purkinje nuclei of the treated animals. Though these data seem to suggest a correl at i on between [3H]thymidi ne i ncorporat i on and the occurrence of H2c values in Purkinje cells, the nature of the extra­synthesized DNA has not been established yet and the possibility that the incorporation is due to enhanced turnover and/or repair mechanisms cannot be excluded.

Using a lower vertebrate such as the eel (Anguilla anguilla), endowed with a simpler cerebellar cytoarchitectonic structure, we have been able to study two phases of its life cycle. The period selected encompasses the transition from the trophic, sedentary phase (yellow

203

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Fig. 3. Hoechst 33342-DNA content in rat cerebellar neurons during postnatal development. HO 342-DNA values for Purkinje neurons (white columns) and granule cells (dashed columns). Columns below the line show the distribution of 2c and 4c hepatocytes on PD 30.

eel) to the reproductive, swimming phase (silver eel). During this transition, both anatomical and physiological changes take place (3,20). Some of these changes are linked to the developmental process and can be regarded as expression of the achievement of a more developed swimming capacity (elongated fins, more evident lateral 1 ine). In the cerebel­lum, changes in the gross morphology and cytoarchitectonics occur during the same transition.

Our data show that in the yellow eel, some Purkinje nuclei (22%) show H2c F-DNA, with different degrees of chromatin condensation (Fig. 4). In the silver eel, the frequencey of H2c F-DNA Purkinje nuclei, especially those with decondensed chromatin, increases to 33%. Using the PI 50 stain, H2c Purkinje nuclei represent 12% of the population in the yellow eel and 44% in the silver eel.

204

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z C I

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Fig. 4. Feulgen-DNA content and degree of chromatin condensation in cerebellar neurons during the transition from the sedentary (yellow eel; VE) to the swimming phase (silver eel; SE). Bar indicates mean value ( v ) ± S.D. for 2c erythrocytes. Other details as in Fig. 1.

On the basis of these results, it can be suggested that the appearance of H2c nuclei reflects the maturation of Purkinje neurons. In the rat, these nuclei appear at a critical stage in histogenesis, when numerous histological and physiological data indicate the occur­rence of important neuronal and extraneuronal events (for references, see 10). In the eel, the number of H2c Purkinje increases in number during the transition from the yellow eel to the silver eel, when morphological data suggest that further developmental processes are occurring in the cerebellum. The similarity of the results obtained with different cytochemical methods indicates, that at least in some of these nuclei, there is a real extra amount. of DNA. It is reasonable to assume that some decondensed H2c Purki nje nucl ei are probably synthe­sizing extra DNA and could become labeled after [3H]thymidine adminis­tration. On the other hand, some of the condensed H2c Purkinje nuclei might have already synthesized the additional DNA. Comparative studies.

The size of the cerebellum and its cytoarchitectonics (different numbers or types of neurons and synapses) are related to the functional

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Fig. 5. F-DNA values and degree of chromatin condensation in Purkinje neurons of active (Ac) and hibernating (Hi) hedgehogs. Other details as in Fig. 1.

complexity of the organ and to the degree of its involvement in loco­

motor control. Though the same basic circuit is present in all verte­

brates (30), the frog (Rana esculenta) , with a laminar cerebellum, has

primitive nerve circuits without basket cells and with few stellate

cells. On the other hand, in the hedgehog (Erinaceus europaeus) , a

mammal, nerve circuits are more complex. In the annual cycle of both

these species there is a period of activity and a period of hiberna­

tion. The latter is known to induce profound physiological changes in

the eNS (21).

Purkinje neurons of the two species differ in size, nucleo-cyto­

plasmic ratio and physical organization of the chromatin. For instance,

during activity only fine granular chromatin is observed in the hedge­

hog, while frog chromatin contains some clumps. These morphological

observations are confirmed by the values of the F-DNA/area ratios and by

the results of PI 0.5 staining. In both species, the degree of chroma-

206

tin condensation is generally lower during the active period. In the hedgehog, the Feulgen reaction shows that during activity

25% of the Purkinje neurons have H2c values which occur in nuclei with more condensed chromatin (Fig. 5). After PI 50 staining, only 2% of the neurons have H2c nuclei, while after treatment with HO 342, 15% of the neurons have H2c (Fig. 7). During hibernation, none of the Purkinje neurons show H2c with either stain.

In the frog (2), 60% of the Purkinje neurons show H2c-4c F-DNA values and more condensed chromatin during activity (Fig. 6). After staining with PI 50, the whole population has H2c-4c values, while after treatment with HO 342, only 40% have H2c values (Fig. 7). During hiber­nation, the frequency of H2c nuclei, as determined with the HO 342 stain, is drastically reduced (15%). Surprisingly, the number of H2c F­DNA cells increases to 70% of the total population. Perhaps this result is due to further condensation of the chromatin, which may affect the ratio between DNA depurination and depolymerization in favor of depuri­nation.

..

A •

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Fig. 6. Feulgen-DNA values and degree of chromatin condensation in Purkinje neurons of active (Ac) and hibernating (Hi) frogs. Other details as in Fig. 4.

207

The degree of complexity of nerve circuits does not appear to influence the cytochemical and functional properties of Purkinje nuclei. Indeed, in both species: (a) H2c nuclei generally show more condensed chromatin; (b) the possible increase in DNA content appears to involve at least part of this H2c population, mainly during the active period; (c) hibernation causes an increase in chromatin compaction, which may reflect a reduction in metabolic activity of the nerve cells. Nonetheless, there are differences between the two species. In the frog, in fact, a large percentage of Purkinje neurons maintain H2c-DNA values, irrespective of the period of activity or hibernation. This is noteworthy, since the greater degree of chromatin condensation which occurs during hibernation, should be expected to decrease the cyto-

~

u z ... :)

" ... II: II.

Frog

Hi

HO- DNA (a.u.)

Fig. 7. Hoechst 33342-DNA values in frog and hedgehog Purkinje neurons during activity (Ac) and hibernation (Hi). White columns, Purkinje neurons. Dashed col umns, granul e cell s. Bars, mean DNA val ues ( v ) ± S.D. for erythrocytes (frog) and 2c hepatocytes (hedgehog).

208

chemical response. These data suggest the need for other explanations, such as a true increase in DNA content or a di fferi ng re 1 at i onshi p between non-histone proteins and DNA.

CONCLUSION Our present knowledge about the problem of the "DNA content in

Purkinje neurons" can be summarized as follows: (a) different cyto­chemical stains indicate that H2c DNA is present in the Purkinje neurons of different vertebrate species. Their coexistence with 2c nuclei causes nuclear heterogeneity, which may correlate with different metabolic states within the population (6); (b) H2c are generally found in nuclei with condensed chromatin in all the species considered. During development, H2c nuclei with decondensed chromatin can also be observed; (c) an increase in DNA content might occur in some Purkinje nuclei. The amount of this "de novo" synthesized DNA is very small and may involve only a small percentage of the H2c nuclei. A high metabolic requi rement of the neuronal popul at i on appears to be rel ated to the presence of H2c nuclei, since H2c nuclei are detectable mainly during the active period of the annual cycle and in coincidence with the maturation of nerve cells.

The appearance of H2c Purkinje neurons seems to be a common factor in the ontogenesis of vertebrates, whatever their cerebellar cytoarchi­tectonics and complexity of nerve circuits. On the contrary, the complexity of nerve circuits achieved during phylogenesis seems to be inversely related with the percentage of H2c cells. These compara­tive findings suggest that cerebella with more complex nerve circuits, 1 ike those of mammals, requ i re fewer cells wi th increased DNA values. Two hypotheses can be put forward to explain the higher frequency of cytochemical H2c in lower vertebrates. The first hypothesis takes into account one of the 1 imi tat ions of the cytochemi cal approach, i. e. the DNA reactivity. For instance, qualitative and quantitative differences in nuclear non-histone proteins, known to be involved in gene regula­tion, might account for variable DNA-dye interaction. The second hypothes is states that there is a real extra amount of DNA ina 1 arge number of neurons. According to a fascinating speculation, these results might reflect an adaptative feature in a phylogenetically

209

primitive cerebellum whose size and structure does not allow for the presence of a large number of neurons. I f we take into account the theory of evolution, according to the "parcellation process" of Ebbesson (18), which correlates the migration of cells, the relative size of the regions and the number of neurons, in the brains of amphibians, the degree of parcell at i on must be very low and neurons must recei ve more input from afferent connections of heterogeneous origin. If this is the case, amphibian Purkinje neurons would require a remarkable amount of genetic information to respond to a great number of stimulations.

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Histochem. J. 13: 781-791, 1981. 2. Barni, S., Bernocchi, G. and Biggiogera, M. Bas. Appl. Histochem.

27: 129-140, 1983. 3. Barni, S., Bernocchi, G. and Gerzeli, G. Tissue & Cell II: 97-109,

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143-161, 1975. 6. Bernocchi, G. and Manfredi Romanini, M.G. Riv. Istoch. Norm. Pat.

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Lebedev, E.A. Histochemistry 59: 233-248, 1979. 17. Cowden, R.R. and Curtis, S.K. Histochemistry 72: 11-23, 1981. 18. Ebbesson, S.O.E. Behav. Brain Sci. I: 321-336, 1984. 19. Fukuda, M., Boehm, N. and Fujita, S. Progress Histochem. Cytochem.

11: 1-119, 1978. 20. Grasse, P.P. Traite de Zoologie, Masson, Paris, Vol. 13, 1958. 21. Heller, H.C. Am. Rev. Physiol. 41: 305-321, 1979. 22. Hobi, R., Studer, M., Ruch, F. and Kuenzle, C.C. Brain Res. 305:

209-219, 1984. 23. Kanter, P.M. and Schwartz, H.S. Mol. Pharmacol. 22: 145-151, 1982. 24. Lalande, M.E. and Miller, R.G. J. Histochem. Cytochem. 27: 394-402,

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18 ENZYMOLOGY OF DNA REPLICATION AND REPAIR IN BRAIN C.C. KUENZLE Institut fUr Pharmakologie und Biochemie, Universitat ZUrich-Irchel, ZUrich, Switzerland

ABSTRACT A number of enzymes involved in DNA replication have been

identified in brain. These include single-stranded DNA-binding proteins, topoisomerases I and II, DNA polymerase a., a protein that binds AP4A and might be classified as a DNA polymerase S accessory protein, RNase H, DNA polymerase S, DNA ligase, an endo- and an exo­nuclease of unknown function, DNA methyl transferase and poly(ADPR) synthase. In contrast, little is known about the enzymology of DNA repair in brain. The few enzymes identified comprise uracil-DNA glyco­sylase, a 3' to 5' exonuclease, possibly implicated in DNA repair, an endonuclease of unknown function, DNA polymerase B, DNA polymerase B (which in neurons is present only at immature stages), DNA 1 igase, poly(ADPR) synthase, and 06-alkylguanine-DNA alkyltransferase.

INTRODUCTION This article reviews current knowledge of the enzymology of DNA

replication and repair in the mammalian brain. I will follow the strategy of first presenting overall schemes of DNA replication and DNA repair, as they pertain to mammalian tissues and cells in general, and then focus on the enzymes that have been identified in the brain.

DNA REPLICATION Mechanism of DNA replication.

DNA replication is the process by which a cell duplicates its DNA in preparation of cell division (for full coverage of this topic, see ref. 1). Replication starts at predetermined sites scattered over the entire genome. The first proteins to bind to these origins of replica­tion are expected to be sequence-specific DNA-binding proteins, having the capacity to disrupt the hydrogen bonds involved in base pairing. Local strand separation is then propagated by the cooperative binding of

212

further helix-destabilizing proteins, all sharing the property of binding to single-stranded DNA.

The helix-destabilizing proteins are aided in their task of opening the double helix by helicases, which actively push the replication fork forward at the expense of ATP. This wedge-like action of the helicases would result in progressive overwinding of the DNA in front of the replication fork, were it not for the presence of topoisomerases. These enzymes release topological strain by cutting supercoiled DNA in either one strand (topoisomerase I) or both strands (topoisomerase II), followed by uncoiling and resealing of the cut strand(s).

In the meantime, a sufficient length of DNA will have been converted to the single-stranded form to accommodate the actual DNA synthetic machinery, the DNA polymerase a holoenzyme. This large multi enzyme complex occurs in at least two forms, termed the leading type and the lagging type DNA polymerase a holoenzyme. These names imply that the two daughter strands are synthesized at different rates by different enzyme forms. DNA polymerization proceeds only in the 5' to 3' direction. On the leading strand, DNA polymerase a can follow the advancing replication fork continuously, whereas on the lagging strand, longer stretches of single-stranded DNA must be exposed in a discontin­uous fashion before polymerization can begin.

The composite structures of the 1 eading and lagging strand DNA polymerase a holoenzymes are different. The leading type enzyme apparently contains fewer components. It consists of a DNA polymerase a domain plus an unspecified number of DNA polymerase a accessory proteins. The lagging strand DNA polymerase a holoenzyme is more complex in composition. It has at least one additional enzyme activity associated with it called a primase. This enzyme activity is probably contained in the same polypeptide as the polymerase. It synthesizes short pieces of RNA complementary to the parental DNA strand. These RNA primers are used by the DNA polymerase a to initiate the elongation reaction. DNA polymerase a accessory proteins probably increase the efficiency of the polymerization reaction. In order to clear the way for the advancing DNA polymerase a , RNase H removes the RNA primer from the previously synthesized stretch of DNA, called an Okazaki fragment. DNA polymerase a catalyzes DNA synthesis in a 5' to 3' direction, until

213

the newly-synthes i zed strand is separated from the precedi ng Okazaki fragment by a gap of approximately 15 nt. DNA polymerase CI. then detaches from the DNA template and is rep laced by DNA polymerase S , which fills in the missing nucleotides. The remaining interruption is sealed through a phosphodiester bond by DNA ligase.

DNA ligation concludes the sequence of steps leading to net DNA synthesis. However, the newly synthesized DNA is subject to modifica­tions. Replication by the DNA polymerases and their accessory proteins is not error-free. An editing system, consisting of repair endo/exo­nucleases can recognize sites of misincorporations, probably on the bas is of looped-out structures in the DNA. The defective pi ece is excised and replaced by the correct sequence (for further details, see section on DNA repair pathways).

Methylation of cytosine residues provides the repair enzymes, with information as to which of the two mismatched bases is the wrong one to be excised. Cytosine residues in the parental strand have previously been methylated in the 5 position by DNA methyl transferase. The newly­synthesized daughter strand does not contain methylated bases. Thus, methylation provides landmarks for the repair enzymes, allowing them to discriminate between parental and daughter strands.

DNA replication is controlled at different levels. Little is known about these processes but poly(ADP)-ribosylation of replication enzymes may be among the underlying mechanisms. It must, however, be kept in mind that the role of poly(ADP)-ribosylation in DNA replication is far from clear. The enzyme responsible for poly(ADP)-ribosylation is poly (ADPR) synthase. In this context, the report of a two-fold stimulation of DNA ligase by poly(ADP)-ribosylation is worth mentioning. In contrast, mammalian topoisomerase I is inhibited by this modification (see also chapter by Mandel et al. this volume). Rep 1 i cat i on enzymes i dent i fi ed .i n brain.

Helix-destabilizing and other single-stranded DNA-binding proteins. A survey of the nuclear proteins occurring in rat cerebral cortex and cerebellar neurons, as well as in cortex oligodendrocytes, has revealed several single-stranded DNA-binding proteins. However, it is not known whether, in addition to binding to single-stranded DNA, they are also able to destabilize the double helix as would be required for initiation

214

of DNA replication. The fact that most of these proteins begin to accumulate in the neurons when cell division stops during development, speaks against their involvement in DNA replication.

Helicase. No helicase has been identified in brain. Topoisomerases. Topoisomerase I has been assayed in the nuclei of

rat cerebral cortex neurons during differentiation (4). Enzyme activity per nucleus was identical in neuroblasts of 18 day-old fetuses and in differentiated neurons on postnatal days (PO) 14 and 60. In contrast, prel imi nary studi es by Duguet and HUbscher (unpubl i shed) have shown a rapid decline of topoisomerase I activity during late gestation, parallel to the fall in cell proliferation. This might indicate an involvement of topoisomerase I in DNA replication. In the same study, topoisomerase II remained constant from 19 days of gestation to PO 21.

DNA primase. Primase activity has not been investigated in brain. DNA polymerasea. DNA polymerase a has been identified in neuronal

perikarya from developing rat (5) and mouse (6) cerebral cortex. The enzyme has also been found in a glia-enriched fraction from mouse cerebral cortex (6). In neurons from both rat and mouse cerebral cortex, DNA polymerase a follows a developmental course closely paral­leling proliferative capacity. Its activity is high at prenatal stages, drops rapidly around birth, then further declines to negligible levels by PO 14.

DNA pol ymerase a accessory protei ns. Di adenosi ne tetraphosphate (Ap4A) is a dinucleotide that has been implicated in the triggering of DNA replication. A 57 kD subunit of calf thymus DNA polymerase a has been identified as the Ap4A-binding protein. Ap4A-binding has also been detected in rat neuronal perikarya, where its developmental profile accurately reflects the activity of DNA polymerase a (7). Whether the Ap4A-binding protein should be regarded as a subunit of DNA polymerase or as a DNA polymerase a accessory protein is, for the time being, a matter of definition.

RNase H. RNase H is detectable in the cerebellum and in the remalnlng pooled parts of the rat brain (8). The cerebellar activity increases up to about PO 6, then declines to approximately one third of the peak level. In the non-cerebellar regions, RNase H activity steadily falls from birth to adulthood. Both time courses are in good

215

agreement with the proliferative activity of the respective parts of the brain.

DNA polymerase B . DNA polymerase B occurs in neurons of the rat (5,9-11) and mouse (6) cerebral cortex, as well as in mouse cerebral cortex glia (6). In neuronal nuclei from adult rats, DNA polymerase B is the predominant (99.2%) DNA polymerase, the remaining activity (0.8%) being contributed by DNA polymerase y (10-12). The activity of the neuronal enzyme declines only slightly during perinatal development (5,6,9). DNA polymerase B has been isolated from neuronal perikarya of the rat cerebral cortex (11). The molecular and functional character­istics of this enzyme are those of a typical mammalian DNA polymerase B.

DNA ligase. DNA ligase is present in neuronal and glial nuclei, isolated from the cerebral cortex of adult guinea pigs (13-15). Its activity is approximately II-fold higher in neurons than in glia (14). In the rat cerebellum, ligase activity increases until PO 6, then falls to very low levels in adult animals (16). In contrast, in pooled'non­cerebellar regions of the brain, the activity remains essentially constant during postnatal development.

DNA repair endo/exonucleases. An endo- (17) and an exonuclease (18,19) have been isolated from brain, but their roles in DNA repair, if any, have not been clarified (for further details, see section on repair enzymes).

DNA methyl transferase. Neuronal perikarya isolated from rat cerebral cortex harbor a DNA methyltransferase of subunit MW approx­imately 50 kD (20). Mature neurons obtained on PO 28 appear to contain at least twice the amount of enzyme than do neuroblasts prepared on the last day of gestation.

Poly(ADPRl synthase. The existence of poly(ADPR) synthase in bovine brain has been reported (21). Comparable activities were found in neuronal and glial nuclei from the cerebral cortex and the white matter, respectively. In a preliminary development study, Hubscher and Kuenzle (unpublished) have shown the presence of poly(ADPR) synthase in neuronal perikarya from the rat cerebral cortex. The activity was detectable at all developmental stages investigated, namely from day 19 of gestation to PO 21. A peak between PO 4 and 7 made an association with DNA replication unlikely. More information on poly(ADPR) synthase

216

in brain can be found in the chapter by Mandel.

DNA REPAIR DNA repair pathways.

DNA repair is a cell's response to DNA damage and results in restoration of the DNA's functional integrity (for full coverage of this topic, see ref. 22). Damage can be inflicted on DNA by various agents, causing multiple types of lesions. Repair pathways can be divided into six groups, depending on the type of lesion but not necessarily on the causative agent. These pathways are characterized by the primary repair mechanism, namely a) ring closure in the case of ring-opened purines, b) strand ligation in the case of simple breaks, c) base removal in the case of converted, damaged or N- and 02-alkylated bases, d) transfer of alkyl group from 0 6_ and 04-alkylated bases to a cysteinyl residue of the repair enzyme, e) strand incision at sites of hel ix distortions caused by bulky substituents and cross-links, and (f) strand incision/ excision at sites of looped-out DNA regions caused by mismatched bases.

Ri ng closure. Ionizing radi at ions cause damage to bases among other lesions. One type of damage that affects purines consists in breakage of the imidazole ring between Cs and N9. An enzyme which restores the ruptured bond in imidazole ring opened adenine and guanine residues has been described very recently (23). It acts by simple ring closure and, therefore, has been termed purine imidazole ring cyclase. It has been isolated from bacteria, but an analogous activity has also been found in cultured mammalian cells.

Strand ligation. Interruptions of the sugar-phosphate backbone in ei ther one or both strands of DNA are caused by i oni zi ng radi at ions, oxygen radicals, chemical mutagens (e.g. bleomycin) and endonucleases present ina 11 tissues. Li gat i on is effected by DNA 1 i gase at the expense of ATP. Two types of DNA ligase have been described, with some indication that type I is involved in DNA replication and type II in DNA repair. Poly(ADP)-ribosylation, catalyzed by poly(ADPR) synthase, is known to playa role in DNA repair. It has been reported that DNA 1 igase II is stimulated two-fold by poly(ADP)-ribosylation (24a), but other targets of poly(ADPR) synthase must also exits.

Base removal. Base removal occurs with different types of damaging

217

agents and different types of lesions. The simplest form of base removal is spontaneous hydrolysis of the N-glycosyl bond, linking a puri ne to its deoxyri bose moi ety. Actua 11 y, thi s event shoul d not be listed here since, in this case, the loss of the base constitutes the primary damage and not the repair mechanism. However, the resulting apurinic site is repaired by a mechanism common to the repair of all apuri n i c sites {see below}. Spontaneous depuri nat i on occurs at a rate of 14,000 events/cell/day. This implies that within the life-time of a human neuron approximately 300 million bases are lost and must be replaced.

Spontaneous and chemical deamination of cytosine and adenine residues occurs at a rate of 200 deaminations/cell/day. Cytosine is converted into uracil and adenine into hypoxanthine. Both are premuta­genic lesions, since upon replication uracil pairs with adenine, replacing the normal C-G pair, and hypoxanthine pairs with cytosine, replacing the normal A-T pair. Specific DNA glycosylases remove the converted bases by scission of the N-glycosyl bond. Uracil-DNA glycosy­lases and hypoxanthine-DNA glycosylases have been isolated from various tissues. The resulting apurinic or apyrimidinic (AP) site is recognized by two types of AP endonuc 1 eases. Type I cl eaves the phosphodi ester bond 3' to the apurinic site, whereas type II cleaves on the 5' side. 3' to 5' and 5' to 3' exonucleases then excise the apurinic site together with a few adjacent nucleotides to produce a single-strand gap. This relatively small gap is filled, predominantly, by DNA polymerase S and to a lesser extent by DNA polymerase ~. The relative contribution of these two enzymes depends on gap size, type of damaging agent, and dose. Following completion of repair repl ication by DNA polymerase S or ~ , ligation is effected by DNA ligase.

An alternative mechanism for the repair of apurinic sites has been proposed (24b). An enzyme has been i so 1 ated, termed puri ne i nsertase, which seems to reinsert purines, but not pyrimidines, into DNA contain­ing apurinic sites. Insertion of purine bases is apparently template­specific with guanine but not adenine, being inserted into depurinated poly(dG-dC), and adenine but not guanine being inserted into poly(dA­dT). However, several arguments make the occurrence of a purine insertase very questionable.

218

Ionizing radiation and UV light cause, among other lesions, damage

to bases. Many types of base damage have been recognized, but some of

the more common features are saturation and di hydroxyl at ion of bases,

hydroxymethyl substitutions and ring cleavages. DNA glycosylases,

specific for a particular type of lesion, remove the damaged base. The

resulting apurinic site is repaired as above.

Al kyl at i ng agents attack bases at various pos i ti ons.

pathways operate depending on the site of alkylation.

Two repair

N-alkylated

purines and 02- alkylated pyrimidines are eliminated by fission of the N­

glycosyl bond, either by spontaneous depurination or by specific DNA

glycosylases. Apurinic sites are generated in all instances and are

repaired by the common pathways described above. In contrast, 06

-alkylguanine and 04-alkylthymine are repaired by abstraction of the

alkyl group as described in the following paragraph.

Transfer of alkyl group to cysteinyl residue of repair enzyme.

This pathway is responsible for the repair of 06-alkylguanine and O!l­

alkylthymine. These two lesions have been implicated in the carcino­

genicity of alkylating agents (24,25). The repair of 06- alkylguanine

and 04- alkylthymine occurs by direct abstraction of the alkYl group from

the modified base with transfer to a cysteinyl residue of the repair

enzyme. Thus, the correct structure of the DNA is restored in a single

step at the cost of permanent inactivation of the enzyme. The enzyme

responsible for OLalkylguanine repair is 06-alkYlguanine-DNA alkyl­

transferase. It is likely that the same enzyme also repairs 04-alkyl­

thymine.

Strand incision at sites of hel ix distortions. Hel ix distortions

are caused by DNA-reactive bul kY carcinogens (e.g. N-acetoxY-2-acetyl­

ami nofl uorene and substituted benzpyrenes), DNA-reactive cross-l inking

agents (e.g. mitomycin e and psoralen plus UV light) and UV light

alone. Both the bul kY carci nogens and the cross-l i nki ng agents, upon

reaction with the DNA, become covalently attached to the bases. UV

light causes intrastrand cross-links between adjacent pyrimidines. The

best known photoreaction product is a cyclobutane thymine dimer but, in

addition, Te and ee sequences can also form what is called a (6-4)­

photoproduct. These lesions are acted upon by repair endonucleases, which

219

recognize backbone deformation but otherwise are not very specific. Until recently, it was thought that these enzymes initiated repair by producing a single incision on either side of the damaged region, followed by excision by a 3' to 5' or 5' to 3' exonuclease, gap filling by DNA polymerase Banda., and ligation by DNA ligase. Recent progress with bacterial systems have, however, shown that the repair endonuc 1 ease act i ng on UV damage in E. co 1 i, termed UVRABC exc is ion nuclease, cuts a strand on both sides of the damaged region (26). This two-site incision is then followed by either exonucleolytic removal of the intervening, damaged oligonucleotide or by displacement through the act i on of a he li case (27). The further steps of repair woul d then be gap filling and ligation as outlined above. It is not known whether the two-cut mechanism also operates in mammalian cells, but similarities between the UVRABC system of bacteri a and analogous rep a i r systems in mammals make this a most likely assumption.

Strand incision/excision at sites of looped-out DNA regions. DNA replication by DNA polymerases and their accessory proteins is associ­ated with an error rate of 10-7 to 10-8 (see section on DNA repl ication). Mispairing of bases results in looping out of corres­ponding DNA regions. The mechanism by which misincorporations are corrected is not clear in mammalian cells. One possibility is that repair endo- and exonucleases initiate an incision/excision mechanism similar to that acting on helix distortions. An alternative possibility is that mammalian replicative DNA polymerases are accompanied by a proof-reading 3' to 5' exonuclease similar to that observed with DNA polymerase III, the replicative enzyme of E. coli. In fact, it has been shown that certain forms of DNA polymerases a. are associated with 3' to 5' exonuclease activity. Maybe, in DNA replication, both of these mechanisms operate in concert to achieve the highest possible level of fidelity. Repair enzymes identified in brain.

In general, the brain repairs DNA damage more slowly than other tissues (28-30). This indicates that levels of repair enzymes are generally low in the brain.

Uracil-DNA gl ycosyl ase. Thi s act i vi ty has been ident ifi ed in the brains of five human fetuses at 18-20 wk of gestation (31). On average,

220

enzyme levels were comparable (42 to 115%) with those of eight other fetal tissues which were grouped in the order kidney> liver> stomach>

colon> brain> spleen> pancreas> small intestine> lung. 3' to 5' exonuclease. An exonuclease with single-stranded DNA

specificity has been isolated from the whole brain of adult rats (18, 19). It is local ized pre-dominantly in the nuclei of neurons. Enzyme activities were minimal in preparations enriched in gl ial nuclei and were absent in mitochondrial and cytoso1ic fractions. The enzyme has a MW of 60 kD. The rate of hydrolysis is approximately the same for single-stranded DNA from rat brain, calf thymus and E. coli, single­stranded depurinated DNA, synthetic poly(dA), as well as extensively nicked double-stranded DNA. The enzyme does not attack RNA and double­stranded native, depurinated or ultraviolet-irradiated DNA. Nucleoside-5'-monophosphates are liberated during its action. The terminus preferentially attacked by the enzyme has not been defined precisely, but properties reminiscent of mammalian DNase III suggest that the enzyme might be a 3' to 5' exonuclease. The fact that it acts on depurinated single-stranded DNA makes it a likely candidate for a repair enzyme, functioning in the excision of apurinic sites.

An endonuclease releasing oligonucleotides with 5'-phosphate ends from single-stranded DNA has been isolated from lamb brain (17). Its action on double-stranded DNA was minimal. However, its role in DNA repair is not clear. For further information on brain DNases, see chapter by Subba Rao.

DNA polymerase a and B. Both types of enzyme (including DNA polymerase y, ref. 12) have been identified in the brain (5,6,9-11). DNA polymerase a is present in neurons only in early developmental stages (up to PO 14 in the cerebral cortex of rats and mi ce) and, therefore, can be involved in neuronal DNA repair only in early develop­ment. In contrast, neuronal DNA polymerase B remains at an almost constant level during brain maturation. Since it accounts for 99.2% of the total DNA polymerase activity in the neuronal nuclei of adult rat cerebral cortex, it can be regarded virtually as the exclusive DNA poly­merase functioning in DNA repair in neurons of the adult brain (10). For a fuller discussion of DNA po1ymerases a and B in brain, see section on replication enzymes.

221

DNA ligase. DNA ligase in brain has been discussed in the section on replication enzymes.

Poly(ADPR) synthase. Poly(ADPR) synthase in brain has been discussed in the section on replication enzymes.

06-alkylguanine-DNA alkyltransferase. It has been known for some time that 06-alkylguanine is removed from the rat brain at a rate much slower than it is from kidney, lung and liver (24,32,33). However, the enzyme responsible for this repair, 06- alkylguanine-DNA alkyltransferase has been identified in the brains of rats (34) and humans (29,31,34) only very recently. Enzyme activities in the adult rat and human brain were low, compared with other tissues. Within both species, brain levels were only one-tenth that of 1 ;ver, with colon, oesophagus and lung, ranging inter-mediately (29). In contrast, organ differences were less pronounced in human fetal tissues. Transferase activities were 30 to 70% those of eight other tissues, which ranged in the order stomach> lung> pancreas> colon> small intestine = kidney> liver> spleen> brain (31). Similarly, the activity in normal adult human brain was comparable to that in several human brain tumors (34). An interspecies comparison revealed that organ transferase activities were lower by a factor of 2 to 12 in rats as compared to humans (29).

The apparent MW of the human brain alkyltransferase is approx­imately 22 kD (34) in agreement with that found for other human (35,36) and rodent (37) tissues. It has also been shown that the enzyme acts by transferring the alkyl group stoichiometrically to one of its cysteinyl res idues, thereby becomi ng i nact i vated. The report that the activity present in rat brain does not repair 04- alkythymine (33) is at variance with the view that 06- alkylguanine and 04-alkylthymine are repaired by the same enzyme (38).

ACKNOWLEDGEMENTS I am grateful for Drs. F.R. Althaus, U. HUbscher, P. Kleihues and

G. Morgenegg for help in preparing this manuscript. This work was supported by the Swiss National Science Foundation, grant 3.391-0.83.

REFERENCES 1. Kornberg, A. DNA Replication. W.H. Freeman & Co., San Francisco,

1980, 1982 supplement.

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2. Heizmann, C.W., Arnold, E.M. and Kuenzle, C.C. J. Biol. Chern. 255: 11504-11511, 1980.

3. Heizmann, C.W., Arnold, E.M. and Kuenzle, C.C. Eur. J. Biochem. 127: 57-61, 1982.

4. Ivanov, V.A. and Melnikov, A.A. FEBS Lett. 177: 300-304, 1984. 5. HUbscher, U., Kuenzle, C.C. and Spadari, S. Nucleic Acids Res. !:

2917-2929, 1977. 6. Shrivastaw, K.P., Philippe, M. and Chevaillier, P. J. Neurosci.

Res. 2: 1-10, 1983. 7. Grummt, F., Waltl, G., Jantzen, H.-M., Hamprecht, K., HUbscher, U.

and Kuenzle, C. Proc. Natl. Acad. Sci. USA 76: 6081-6085, 1979. 8. Sawai, Y., Sawaski, Y. and Tsukada, K. Life Sci. Z!: 1351-1356,

1977. 9. HUbscher, U., Kuenzle, C.C., Limacher, W., Scherrer, P. and Spadari,

S. Cold Spring Harbor Symp. Quant. 43: 625-629, 1978. 10. HUbscher, U., Kuenzle, C.C. and Spadari, S. Proc. Natl. Acad. Sci.

USA 76: 2316-2320, 1979. 11. Waser, J., HUbscher, U., Kuenzle, C.C. and Spadari, S. Eur. J.

Biochem. 97: 361-368, 1979. 12. HUbscher, U., Kuenzle, C.C. and Spadari, S. Eur. J. Biochem. 81:

249-258, 1977. 13. Inoue, N. and Kato, T. Proc. Jap. Acad. 52: 461-464, 1976. 14. Inoue, N. and Kato, T. J. Neurochem. 34: 1574-1583, 1980. 15. Inoue, N., Ono, T. and Kato, T. Biochem. J. 180: 471-480, 1979. 16. Nakaya, N., Sawasaki, Y., Teraoka, H., Nakajima, H. and Tsukada, K.

J. Biochem. 81: 1575-1577, 1977. 17. Healy, J.W., Stollar, D., Simon, M.I. and Levine, L. Arch. Biochem.

Biophys. 103: 461-468, 1963. 18. Ivanov, V.A., Terpilovskaya, O.N., Tretyak, T.M. and Smirnova, G.N.

Biochemistry (engl. transl. Biokhimiya) 47: 337-343, 1982. 19. Ivanov, V.A., Gaziev, A.I. and Tretyak, T.M. Eur. J. Biochem. 137:

517-522, 1983. 20. HUbscher, U., Pedrali-Noy, G., Knust-Kron, B., Doerfler, W. and

Spadari, S. Anal. Biochem., submitted. 21. Bilen, J., Ittel, M.E., Niedergang, C., Okazaki, H. and Mandel, P.

Neurochem. Res. 2: 1253-1263, 1981. 22. Friedberg, E.C. DNA repair. W.H. Freeman & Co., San Francisco,

1985. 23. Chetsanga, C.J. and Grigorian, C. Proc. Natl. Acad. Sci. USA 82:

633-637, 1985. 24. Kleihues, P., Schmerold, I. and Wiestler, O. In: Developments in

the Science and Practice of Toxicology (Eds. A.W. Hayes, R.C. Schnell and T.S. Miya), Elsevier, Amsterdam, 1983, pp. 255-264.

24a.Creissen, D. and Shall, S. Nature 296: 271-272, 1982. 24b.Deutsch, W.A. and Lim, S. Proc. Natl. Acad. Sci. USA 76: 141-144,

1979. 25. Kleihues, P. and Rajewsky, M.F. Prog. Exp. Tumor Res. 27: 1-16,

1984. 26. Sancar, A. and Rupp, W.D. Cell 33: 249-260, 1983. 27. Kumura, K., Sekiguchi, M., Steinum, A.-l. and Seeberg, E. Nucleic

Acids Res. ~: 1483-1492, 1985. 28. Gibson-D'Ambrosio, R.E., Leong, Y. and D'Ambrosio, S.M. Cancer Res.

43: 5846-5850, 1983.

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29. Grafstrom, R.C., Pegg, A.E., Trump, B.F. and Harris, C.C. Cancer Res. 44: 2855-2857, 1984.

30. Wheeler, K.T. and Wierowski, J.V. Radiat. Environ. Biophys. 22: 3-19, 1983.

31. Krokan, H., Haugen, A., Myrnes, B. and Guddal, P.H. Carcinogenesis !: 1559-1564, 1983.

32. Rajewsky, M.F., MUller, R., Adamkiewicz, J. and Drosdziok, W. In: Fundamental Mechanisms and Environmental Effects (Eds. B. Pullman, P.O.P. Ts'o and H. Gelboin), Reidel Publ. Co., 1980, pp. 207-218.

33. Singer, B., Spengler, S. and Bodell, W.J. Carcinogenesis Z: 1069-1073, 1981.

34. Wiestler, 0., Kleihues, P. and Pegg, A.E. Carcinogenesis~: 121-124, 1984.

35. Myrnes, B., Giercksky, K.E. and Krokan, H. J. Cell Biochem. 20: 381-392, 1982.

36. Harris, A.L., Karran, P. and Lindahl, T. Cancer Res. 43: 3247-3252, 1983.

37. Pegg, A.E., Wiest, L., Foote, R.S., Mitra, S. and Perry, W. J. Biol. Chern. 258: 2327-2333, 1983.

38. Becker, R.A. and Montesano, R. Carcinogenesis 2: 313-317, 1985.

19 BRAIN DNases AND THEIR FUNCTIONAL IMPORTANCE K. SUBBA RAO School of Life Sciences, University of Hyderabad, AP, India

ABSTRACT The available information on the deoxyribonuclease (DNase)

activities in various regions and cell types of developing and aging brain is reviewed. Two major endo-deoxyribonucleases, one with acidic pH optimum and the other with alkal ine pH optimum, have been demon­strated. An exodeoxyribonuclease, specific for single-stranded DNA, is a 1 so present in rat brain. Modul at ions in the act i vi ties of these DNases, with respect to age and cellular replicative schedules, suggest a role for acid DNase in DNA replication. On the other hand, alkaline DNase could be involved in DNA repair.

I NTRODUCTI ON One of the challenges a living cell has to face is the attempt by

several agents to alter or damage its genetic material. This damage is induced by a large number of agents like UV, X-rays, and an array of alkylating agents and bulky ring compounds. In addition, DNA is attacked by free radicals produced within the cell. Spontaneous as well as induced depurination is also a major source of DNA damage. In response to these alterations, living systems appear to have developed compl icated mechanisms to affect DNA repair. For example, DNA repair may be achieved by nucleotide excision, base excision, recombination, etc. (1,2). The enzymology of these repair processes is reasonably well understood in lower organisms. In mammalian cells, however, a clear picture of these mechanisms has not yet emerged (see chapter by Kuenzle, this volume). It is of course predictable that several types of DNases, both exo- and endo- in their catalytic nature, must be playing important roles.

In a post-mitotic tissue like brain, DNA damage and repair assume special significance. In the non-dividing nerve cell, any damage, if unrepaired, would persist throughout the life span of the organism and cause several met abo 1 i c errors wh i ch may 1 ead to senescence and cell

225

death. In spite of these possibilities, brain tissue, for some reason, has been largely ignored as far as DNase studies are concerned). It is the purpose of thi s art i cl e to revi ew the work done to date on brain DNases, including that from the author's laboratory, and also to suggest some lines in which further research could proceed in order to better understand their biological role in brain DNA metabolism.

RESULTS Probably the first demonstration of the presence of two brain

DNases, the acid and the alkaline DNase, has come from the laboratory of Sung (3). Two DNases were isolated from rat brain and separated by ammonium sulphate fractionation. One of the enzymes acts optimally at pH 5.0, hydrolyzing preferentially native DNA. The other DNase has its optimal pH between 7.4 and 8.9, and acts preferentially on heat-de­natured DNA. The products of the acid DNase were reported to be oligo­nucleotides terminating in 3'-phosphate while the alkaline DNase released 5'-phosphate oligonucleotides. The latter enzyme was thought to be s imil ar to a phosphodi esterase i sol ated earl i er from 1 amb brain by Healy et al. (4). Both of these enzymes appeared to be localized in the mitochondrial/microsomal fraction and to be endonucleases, as neither mononucleotides nor nucleosides were found even after 25% of the DNA added as substrate was hydrolyzed to acid soluble nucleotides. The possible role of these DNases, however, remained obscure.

The presence of an acid DNase in human cerebrum and cerebellum was demonstrated by this author (5). The highest activity was noticed during the early stages of embryonic development. With the advancement of gestation, the activity decreased. In the cerebellum, however, the activity increased in late gestation, and maximum specific activity was found at term (Table 1). This temporal pattern of activity correlated well with the rates of DNA synthesis in these regions of the developing human foetal brain (6).

The observations made on human brain prompted us to continue our investigation in a systematic manner using other easily obtainable spe­cies. In chick brain, two prominent activities, comparable to the acid

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TABLE 1. Acid DNase activity in foetal human brain.

Gestational Age Cerebrum

9 - 20 wk 6.4 ± 1.08 (8)

24 - 32 wk 4.6 ± 0.57 (3)

Full term 3.4 ± 0.88 (6)

Cerebellum

5.9 ± 1.27 (3)

10.1 ± 3.12 (6)

Activity expressed as ~g acid soluble deoxyribose-P released/mg protein/ 2 hr. In parenthesis, number of analyses. Data recalculated from ref.5.

and alkaline DNases described by Sung for rat brain, have been observed (7). Both acid and al kal ine DNases exhibited maximum activity during embryonic life, at a time of rapid cellular proliferation. During the post-hatching period, acid DNase showed a marked and abrupt decline. This low activity remained throughout the 1 ife span, thus showing no correlation to the increase in brain DNA content observed during adult­hood in these species. On the other hand, alkaline DNase continued to show high activity throughout the post-hatching period. The

TABLE 2. DNA content and DNase activities in developing and old chick brain.

Age (days)

16 - (E)

1 - (PH)

10 - (PH)

730 - (PH)

DNA content (~g/g tissue wt)

600

800

900

1500

Acid Alkaline DNase DNase

14.5 18.8

11.0 21.3

8.5 19.4

2.0 9.4

Enzyme activities expressed as in Table 1. E, embryonic life; PH, post­hatching. Values are averages of 5 to 10 experiments. Data recalcu­lated from ref. 7.

227

TABLE 3. DNase act i vi ties in the white and gray matter of chick brain at different ages.

White Matter Gray Matter

Age Total Activity Total Activity Enzyme (days) Activity mg/DNA Activity mg/DNA

Acid 13 68 ± 13 431 ± 84 168 ± 15 306 ± 31

DNase 130 246 ± 60 439 ± 79 412 ± 76 354 ± 67

422 180 ± 22* 235 ± 34* 396 ± 58 200 ± 42*

Alkaline 13 297 ± 19 1896 ± 205 991 ± 111 1767 ± 131

DNase 130 1066 ± 169 1839 ± 139 2347 ± 356 2115 ± 261

422 1540 ± 26 2011 ± 148+ 3015 ± 208* 1511 ± 183

Activity expressed as in Table 1. All values are averages ± S.D. from si x edperiments. *, p < 0.005 and +, p < 0.05 in compari son to the corresponding value at 130 days. Data taken from ref. 9.

activity, although decreased in old brain (2 yr-old), still remained at a significant level (Table 2). A similar pattern of change in DNA and DNases was also observed in the cerebellar region of chick brain (8).

A subsequent study (9) confirmed that the age-related decrease in the specific activity of acid DNase was more pronounced compared to alkaline DNase (Table 3). A marked reduction in total acid DNase beyond the age of 130 days was also observed. On the other hand , total alkaline DNase continued to increase with age in both white and gray matter. Furthermore, its activity, when expressed per mg DNA (a measure of activity in each cell), also increased in the white matter of the aging brain.

The above results suggested that acid DNase might be restricted to cells proliferating during early embryonic stages (neurons ?). By contrast, alkaline DNase might playa continuing role throughout the life span of the organism and be involved in the activities of the glial cells formed at later stages. This postulate, however, was proved to be

228

TABLE 4. DNase activities in different cell types of chick brain.

Neurons Astrocytes Oligodendrocytes

Age Acid Alkaline Acid Alkaline Acid Alkaline (days) DNase DNase DNase DNase DNase DNase

16(E) 37.I±4.6 32.3±3.4 28.0±2.1 31.3± 2.1 35.3±3.0 2.32±O.2

IO(PH) 8.6±1.0 26.6±3.2 7.4±O.8 24.0± 2.3 42.3±5.S 4.70±O.3

540(PH) 12.3±1.2 5S.9±3.5 12.5±I.0 55.0±IO.2 IO.5±l.I 1.6S±O.IS

Values are averages of 4 to 6 experiments. Other details as in Table 2.

incorrect by our recent work with fractions enriched in neurons, astroglial, and 01 igodendroglial cells isolated from chick brain of different ages. Our analyses concerned the DNA content and the activi­ties of DNases and DNA polymerase. Both acid and alkaline DNases were found in all the three cell types at all ages, although alkaline DNase was minimal in 01 igodendrogl ia (Table 4). The data revealed an inter­esting phenomenon. Acid DNase shows the highest activity in all brain cell types at a time when these cells are supposed to undergo replica­tion (embryonic day 16 for neurons and astrocytes, and postnatal day 10 for oligodendrocytes). The activity exhibits a precipitous decline immediately after the replicative period. On the other hand, alkaline DNase not only shows high activity during the embryonic period, but this activity actually increases with age, reaching a maximum in old age both in neurons and astrocytes. These results confirm the intimate associa­tion of acid DNase with DNA replication (cell-replication). On the other hand, alkaline DNase appears to be involved in a continuous process which assumes greater importance in old age, probably DNA repair.

We have extended these studies to yet another species, the rat, in order to establ ish the rol e of DNases in mammal i an brain. In these experiments we determined the content of DNA and the activities of acid and alkaline DNases in cerebral white and gray matter at different ages of the rat (IO). DNA polymerase activities were also measured at various stages of the life span (II). The white matter, as opposed to the gray matter, exhibited continuous growth throughout the period

229

studied (from 1 to 750 days). By various biochemical parameters, including DNA content, it was clear that, between 225 and 750 days, there was a second round of DNA accumulation. Acid and alkaline DNases showed a trend which is reminiscent of that observed in chick brain. Thus, the specific activities of both enzymes were high during the early stages of development. A marked decl ine in the specific activity of aci d DNase occurred from the 15th day onwards. However, the act i vi ty increased again between 225 and 750 days in concomi tance to the DNA increase and to a second peak of DNA polymerase activity (Fig. 1).

Alkaline DNase also exhibited an initial, reasonably high specific activity and continued to increase with age up to 60 days. The activity decreased at 225 days but rai sed again by 750 days. In fact, the specific activity was highest in 750 day-old rat brain. A similar pattern of changes also occurred in the cerebellum. Thus, a correlation between early cell proliferation and acid DNase activity can be envisaged (12).

A physiologically important role for brain DNases is suggested by another set of experi ments carri ed out in our 1 aboratory. In these studies, we examined the effect of early postnatal undernutrition and

c ~l " -J 8.0 ,

~ " , I, ,

~ 250 Of 4.0 I ,

I , , '" o " I , I .J:: L .. I N 0. .. I 6D 'C-c- 120 :;:: 1.20

! E ~ 'it-

~ ~

::E 0.

.... « c-'0

z 4D-$. 80 0 0.8 .. '" ";

Of E « "0 z E 0 0 .Il

40 0.4 _ DNA

2.0 0> Q, - DNA POL

:L

_ At DNa ...

6---t\ ALK DNaSG

16 0 7 15 30 225 360 540 750 PrenataL Postnatal

AGE IN DAYS-

Fig. 1. DNA content, DNA polymerase, and DNase activities the in white matter of rat brain at various ages. Data taken from ref. 10 and 11.

230

subsequent rehabilitation on DNA content, and on the activities of acid

and alkaline DNases in white and gray matter of rat brain. An interesting result of these studies was that the specific activities of both DNases were unaffected by weanling undernutrition in both white and gray matter. Moreover, during rehabilitation, the total activities of these enzymes staged a remarkable recovery to greater than normal levels. These results indicate that these proteins are made preferen­tially during rehabilitation (13).

Chanda et al. (14) also independently observed that in rat cere­bellum, the activity of acid DNase steadily decreases with maturation (units/g tissue wt), whereas alkaline DNase steadily increases with age. These findings are in agreement with the results obtained in our laboratory.

DISCUSSION After havi ng enumerated the vari ous studi es concerned wi th brain

DNases, it is worthwhile to have a fresh look at the overall results as these might give clues as to the true function of these enzymes. It is clear that all the data indicate, although indirectly, that acid DNase has something to do with cell replication and, therefore, DNA replica­tion. Indeed, a close correlation between acid DNase activity and replicative capacity of a given tissue had been reported by Allfrey and Mirsky (15) more than 30 years ago. However, for some reason, 1 ittle attention was paid to this finding. Some major objections to assigning a function for acid DNase, other than the degradatory role, are: (a) its pH optimum (around 5.0); (b) its intracellular localiza­tion (lysosomal); (c) the nature of its reaction products (oligonucleo­tides with 3'-phosphate rather than 3'-OH). However, evidence has already accumulated that this enzyme is active even at pH 7.0, given appropriate conditions of low ionic strength and divalent cations (16). In our laboratory, we have found that partially purified acid DNase from rat brain attacks both native as well as UV-irradiated DNA with almost equal efficiency at pH 5.0 (unpublished observations). Furthermore, the acid DNase activity is present in the nuclear fraction of calf thymus (17), as well as in isolated neuronal, astroglial and oligodendroglial cells from rat brain (18). Additional work seems to be necessary

231

regarding the nature of the released products. A role for at least two endo-DNases in the DNA replication of

bacteriophages has been well documented (19). These nucleases are the ~ 174 gene-A protein and the fd gene-2 protein. Both of these nucleases are shown to produce site-specific nicks essential for the initiation of DNA replication in these phages. We are inclined to speculate that the acid DNase may be playing a similar role in brain DNA replication. It is also possible that this enzyme is responsible for post-replication recombi-national DNA repair, as it occurs in E. coli (20) and HeLa cells (21).

The results on the DNase activities of isolated brain cell types (Table 4) indicate an important role for alkaline DNase in a process that is operative through-out the life cycle of the organism and is probably prevalent in the later stages of the life span. It is logical to assume that this process may be DNA repair. Several types of exo-, endo-, and site-specific nucleases, both in lower organisms and in mammalian systems, have been implicated in DNA repair. Recently, this subject has been elegantly reviewed (2,22). It is beyond the scope of this article to summarize the vast amount of available information. Suffice it to say that the nucleotide and base excision repair mechanisms in lower organisms are reasonably well understood. By contrast, information about mammalian systems is scanty, although some exonucleases and endonucleases capable of acting at pyrimidine dimers and apurinic sites have been described. It is pertinent to mention the recent isolation from rat brain of a neuronal exodeoxynuclease, which can hydrolyze homologous, heterologous, synthetic, and depurinated substrates at the same rate, liberating nucleoside-S'-monophosphates (23). However, the enzyme fails to attack UV-irradiated polydeoxyribo­nucleotides. The enzyme was named DNase B III and is considered to be a repair enzyme (for further information on brain DNA repair mechanisms, see Kuenzle's chapter).

In conclusion, it appears more than likely that the brain alkaline DNase is participating in DNA repair. All of its known properties are suited for thi s purpose. Very recent experiments from our 1 aboratory have shown that a partially purified alkaline DNase preparation from rat brain also attacks UV-irradiated and depurinated DNA. Further work

232

wi th extensi vely puri fi ed brai n DNases and construction of in vitro systems for DNA replication and repair, should pinpoint the exact mechanisms through which brain DNases participate in these processes.

ACKNOWLEDGEMENTS Drs. K.P. Shrivastaw, K.V.S. Rao and B. Usha Rani have contributed

to various phases of the work carried out in my laboratory. This work was supported by the Indian Council of Medical Research and the University Grants Commission, New Delhi, India.

REFERENCES 1. Friedberg, E.C., Anderson, C.T.M., Bonura, 1., Cone, R., Radany,

E.H. and Reynolds, R.J. Prog. Nucleic Acid Res. Mol. Biol. 26: 197-215, 1981.

2. Linn, S. In: Nucleases (Eds. S.M. Linn and R.J. Roberts), Cold Spring Harbor Laboratory Publications, New York, 1982, pp. 59-83.

3. Sung, S.C. J. Neurochem. 12: 477-481, 1968. 4. Healy, J.W., Stollar, D., Simon, M.I. and Levine, L. Arch. Biochem.

Biophys. 103: 461-468, 1963. 5. Subba Rao, K. Life Sci. 1Z: 89-96, 1973. 6. Dobbing, J. In: Chemistry and Brain Development (Eds. R. Paoletti

and A.N. Davison), Plenum Press, New York, 1971, pp. 399-412. 7. Shrivastaw, K.P. and Subba Rao, K. J. Neurochem. 25: 861-865, 1975. 8. Subba Rao, K. and Shrivastaw, K.P. J. Neurochem. 27: 1205-1210,

1976. 9. Subba Rao, K. and Shrivastaw, K.P. J. Biosci. 1: 69-74, 1979. 10. Subba Rao, K.V. and Subba Rao, K. Mech. Ageing Dev. 18: 225-238,

1982. 11. Subba Rao, K.V. and Subba Rao, K. Biochem. Intl. 2: 391-397, 1984. 12. Subba Rao, K.V. and Subba Rao, K. J. Biosci. !: 139-144, 1982. 13. Subba Rao, K.V. and Subba Rao, K. J. Neurosci. Res. I: 279-287,

1982. 14. Chanda, R., Woodward, D.J. and Griffin, W.S.T. J. Neurochem. 24:

723-727, 1975. 15. Al 1 frey, V.G. and Mirsky, A.E. J. Gen. Physiol. 36: 227-241, 1952. 16. Slor, H. and Lev, J. J. Biol. Chem. 247: 2926-2930, 1972. 17. Slor, H. and Lev, J. Biochem. J. 123: 993-995, 1971. 18. Stambolova, M.A., Cox, D. and Mathias, A.P. Biochem. J. 136:

685-695, 1973. 19. Brown, D.R., Hurwitz, J., Rei nberg , D. and Zipursky, S.L. In:

Nucleases (Eds. S.M. Linn and R.J. Roberts), Cold Spring Harbor Laboratory Publications, New York, 1982, pp. 187-209.

20. Ganesan, A.K. J. Mol. Biol. 87: 103-119, 1974. 21. Meneghini, R. and Hanawalt, P.C. In: Molecular Mechanisms for

Repair of DNA, Part B (Eds. R. Hanawalt and R.B. Setlow) , Plenum Press, New York, 1975, pp. 639-642.

22. Lindahl, T. Ann. Rev. Biochem. 51: 61-87, 1982. 23. Ivanov, V.A., Gaziev, A.I. and Tretyak, T.M. Europ. J. Biochem.

137: 517-522, 1983.

20 POLYADP-RIBOSE POLYMERASE AND ADP-RIBOSYLATION REACTION P. MANDEL, C. NIEDERGANG, M.E. ITTEL, H. THOMASSIN and A. MASMOUDI Centre de Neurochimie du CNRS, Strasbourg, France

ABSTRACT Twenty years ago, polyADP-ribose, poly(ADPR), and poly(ADPR)

polymerase, poly(ADPR)-P, were discovered in liver nuclei. A growing body of evidence suggests that poly(ADPR)-P, which appears to also be a poly(ADPR) transferase, is ubiquitously distributed in eukaryotic nuclei and involved in DNA replication, transcription and repair. Poly(ADPR) or mono (ADPR) transferase react ion is present i n several subce 11 ul ar fractions, including mRNP particles. Poly(ADPR)-P activity is very high in neurons. Rather lower activity exists in normal and transformed astrocytes. Nicotinamide (Nic) or 3-aminobenzamide (Nic analogue), known poly(ADPR) -P i nhi bi tors, decrease prol iferat i on of normal astro­cytes and C6 gl ioma cell s, as reported for other prol iferating cell types.

INTRODUCTION In 1962, we observed that in rats, after ablation of a kidney,

ni cot i namide (Ni c) admini strat ion blocked the compensatory hypertrophy and hyperplasia of the remaining kidney. Moreover, inhibition of the RNA, DNA and protein increase caused the appearance of a polyadenyl ic compound in the nuclei (l). Since Nic injection produced an increase of nicotinamide adenine dinucleotide (NAD) , we tested whether nicotinamide mononucleotide (NMN), which is transformed into NAD in nuclear extracts, would also induce a synthesis of the polyadenylic compound. Actually it appeared that in extracts of 1 i ver nucl ei, NMN or NAD enhanced the incorporation of [ 14 C]ATP into the polyadenyl ic acid polymer at least one thousand-fold (2). From enzymatic degradation and methylation experiments, we could conclude that the polymer synthesized from NAD by poly(ADPR) polymerase-transferase, poly(ADPR}-PT, was poly(ADPR} (3) (Fig.I). The synthesis of the polymer appeared to be DNA-dependent. The structure of the polymer was confirmed one year later by Hayaishi et al. (4) and by Sugimura et al. (5).

134

Ada Ade ,.----"---'. , I , Ribi Rib - Rib Rib-

, . I I I p~p p-p

I _4

I • Ade : Ade Ade I i I I

-Rib, Rib-Rib: Rib-Rib Rib-I il I I I I P4-P p-;..p p-p . , , ~

Fig. 1. The structure of poly(ADPR). Ade, adenine; R, ribose; P, phosphate. The P-P bond is hydrolyzed by phosphodiesterases; the Rib­Rib, bond is hydrolysed by glycohydrolases.

The fundamental step in understanding the role of poly(ADPR)-PT and ADP-ribosylation reaction has been the demonstration of the transfer of ADPR to proteins in nuclei (6,7), in plasma membrane (8,9), in mitochon­dria of rat liver (9,10,11), testis (12) and brain (13), as well as in cytoplasmic ribosomes (14) and in mRNA particles (IS). It appears that the enzyme which produces poly{ADPR) is also a poly{ADPR) transferase, poly{ADPR)-T, which is responsible for the transfer of the ADPR moiety of NAD to proteins and/or to a poly{ADPR) chain. Since its discovery (2,3), the involvement of the ADP-ribosyl transferase reaction in cell proliferation and differentiation and in hormone action has been extensively investigated (for a review, see 16-19).

ADP-RIBOSYLATION REACTION Enzymology. In 1976, we developed a procedure for the purification of the nuclear poly{ADPR)-PT from bovine thymus yielding a fraction with less than 1% contamination by other proteins. Until the next to the last purification step, the enzyme contained about 10% DNA on a weight

235 basis and its activity was DNA-independent. After removing the DNA fraction (which we called sDNA), the enzyme became DNA-dependent. The activity of the enzyme preparation was restored by addition of calf thymus DNA or sDNA. The saturating concentration of calf thymus DNA was one hundred times higher than that of sDNA. The apparent Km for NAD of the DNA-dependent poly(ADPR)-PT was shown to vary with the DNA concen­tration. A minimum value of Km was obtained when the amount of sDNA was 10% that of the enzyme. The ratio of the apparent Km for sDNA to enzyme concentrat i on was constant at all enzyme concentrations studi ed. The minimum length of sDNA required for maximal activation of the enzyme was 16 bp, while for calf thymus DNA this number appeared to be 640 bp (20,21). These results suggested that the activation of the enzyme required the formation of a complex between the protein and a specific DNA segment. This complex was probably preserved in the DNA-independent enzyme preparation.

In mammalian cells, the nuclear enzyme is found tightly bound to chromatin. Recently, it has been suggested that poly(ADPR)-PT activity is local ized in nucl eosome 1 i nker regions (22) and between adjacent solenoidal helices (23). The interaction between purified calf thymus poly(ADPR)-PT and its activating copurified sDNA was investigated by electron microscopy. It was shown that the enzyme-DNA complex possesses a nuclesome-like structure. The enzyme-bound sDNA is enriched in single-stranded regions and branched structures, presumably replication forks (24).

Using the protein blotting technique and polyclonal antibodies, we found a MW of 130 KD for the nuclear poly(ADPR)-P of different tissues of beef, rat, chicken and pig (25). This finding suggests that the overall structure/function relationship of the enzyme has been well preserved during evolution. This is analogous to the evolutionary behavior of histones and other nuclear enzymes, such as RNA polymerase and or. and B DNA polymerases. Nevertheless, no immunoprecipitation 1 ine was observed with poly(ADPR)-P from rabbit or rat brain or from liver nuclei treated with an anti calf thymus poly(ADPR)-PT antibody. This result demonstrates the existence of species-specific antigenic deter­minants on poly(ADPR)-P molecules (26). The content of poly(ADPR)-PT per mg DNA was evaluated by the micro complement fixation method. The

236

highest value was obtained in chick neurons (0.50 ~g), followed by calf thymus nuclei (0.16 ~g), whereas the lowest value was found in rat liver nuclei (0.001 ~g) (25). Using the micro complement fixation method, it was also demonstrated that the enzyme-bound DNA must occupy a specific site on the poly(ADPR)-PT molecule (25). It seems likely that the ADPR-ribo-sylation site of the enzyme differs from the antigenic sites, at least with regard to those expressed in our antibodies.

Since our description that the poly(ADPR) structure contains a 1"2' -ribose phosphate backbone (2), the occurrence of a branched structure 2' -[1"-ribosyl-2"(or 3") (I'" -ribose)] was demonstrated by physicochemical methods (32) and visualized by electron microscopy (24). The frequency of branching was estimated as one per 30 to 50 ADPR residues (33,34). A double-stranded structure of poly(ADPR) (35) and even a helical conformation of long chain poly(ADPR) was suggested (36).

When incubated with NAD, the poly(ADPR)-PT undergoes an ADP-ribosy­lation (37-39). The auto ADP-ribosylated polymerase, as well as the branched poly(ADPR), was vi sua 1 i zed by dark fi el d el ectron mi croscopy (24). During poly(ADPR) formation, the enzyme molecule appeared to be located on sDNA. The release of the "enzyme-bound product" from sDNA reduced the enzyme activity. It may be argued that the detachment of

TABLE 1. Poly(ADPR)-PT activity of neurons and glial cells in culture.

Cell Type nmol! Differentiation Proliferation mg prot.*

Neurons (chick) 0.475 +++ 0

C6 glioma 0.512 ++ +++

Rat astroblasts primary culture 0.470 ++ ++

spontaneously transformed 0.150 + ++

Chick neurons were cultured according to Pettmann et al. (27), C6 cells were cultured according to Benda (28), rat astroblasts and spontaneously transformed cells according to Booher (29) and Mersel (30). Enzymatic activity was measured in permeabilized cells by the incorporation of the ADPR poiety of [JH]NAD into acid-insoluble material according to Bilen et al. (31). *, 5 min, 25°.

237

the polyADP-ribosylated enzyme from DNA may be a part of an auto-regula­tory mechanism controlling the enzymatic activity.

Three types of enzymes insure poly(ADPR) hydrolysis: (1) glycohy­drol ases that spl it the ri bose-ri bose and ri bose-ri bose-ri bose bonds (40); (2) phosphodiesterases (with pyrophosphatase activity), like those present in snake venom, rat liver and tobacco cells, which hydrolyze the pyrophosphate bond of poly(ADPR) (40) (Fig.I); and (3) ADPR hydrolase which cleaves the residual monomeric unit after excision of the ADPR units. No glycosidase has been found to hydrolyze the bond between ribose and adenine, nor a phosphodiesterase which hydrolyzes the bond between ribose and phosphate. The mode of hydrolysis by the calf thymus glycohydrolase is exoglycosidic. No enzyme that hydrolyzes poly(ADPR) endoglycosidically has been reported. After partial hydrolysis of poly(ADPR) with poly(ADPR) glycohydrolase, the residual fragment may still be ADP-ribosylated by poly(ADPR)-PT with chain elongation. Considering the Km values of poly(ADPR) glycohydrolase and rat 1 iver phosphodi esterase, it is concei vabl e that glycohydrol ase pl ays a major role in the hydrolysis of poly(ADPR). Thus, three kinds of enzymes yielding two kinds of products participate in poly(ADPR) hydrolysis. Poly{ADPR) glycohydrolase and ADP-ribosyl hydrolase produce ADPR, while the product of the phosphodi esterase action is phosphori bosyl-AMP or iso-ADPR (Ade(P)-Rib-P) (3,40). Although snake venom phosphodiesterase is generally an exonuclease attacking the 3'OH, it hydrolyzes poly(ADPR) endonucleolytically. Extra nuclear poly and oligo ADP-ribosylation.

Poly(ADPR)-T activity is present in the ribosomal/microsomal fraction (14), as well as in mitochondria of rat liver (IO,Il), testis (12) and brain (13) (Table 2). Apparently, the ADP-ribosylation activity is strikingly higher in brain mitochondria as compared to liver (Table 2). The enzyme found by Kun in rat liver mitochondria differs apparently from the nuclear enzyme in its insensitivity to thymidine and DNA (II). Evidence was also obtained in favor of a regulatory role of ADP-ribosylation in mitochondrial DNA replication (II). Moreover, ADP­ri bosyl at i on by mi tochondri al glycohydrol ase was suggested by Hil z et al. (43).

We have demonstrated that poly(ADPR)-PT activity is also associated

238

TABLE 2. ADP-ribosylation activity in mitochondria.

Mitochondria

Synaptic

Non-synaptic

Liver

Details as in Table 1.

pmoles/mg protein hr

195

230

117

with free mRNA particles in liver and in plasmocytoma (15,44). In the latter, the enzyme activity was not stimulated by added DNA or histone HI, and was not inactivated by DNase digestion. The poly(ADPR)-PT activity of mRNP particles represented 34% of the total cellular activity, while the DNA content was less than 4% of the total cellular DNA. As a result, the poly(ADPR)-PT specific activity per mg DNA was about 75-fold higher in free mRNP than in the nuclei. Several ADP-ribo­syl ated protei ns were detected by 1 i thi urn dodecyl sulfate gel e 1 ectro­phoresis after incubation of mRNP particles with NAD (15,44).

ADP-RIBOSYLATION OF PROTEINS In addi ti on to phosphoryl at i on, acetyl at i on and methyl at ion

reactions, the ADP-ribosylation gives rise to a biologically important post-translational modification of proteins.

Two types of protein ADP-ribosylation may be considered. Mono ADP-ribosylation, when a single ADPR is transferred from NAD to protein, and polyADP-ribosylation, when the first transfer is followed by poly­(ADPR) formation. A number of nuclear proteins have been identified as acceptors for polyADP-ri bosyl at i on. Of parti cul ar rel evance are histones (especially HI), the high mobility group proteins, nucleosomal core histones (16,45-48) and several enzymes involved in DNA and RNA metabolism, such as topoisomerase I (49), magnesium-dependent (50) and magnesium/calcium-dependent endonuclease (51), RNases (52), DNA poly­merase ex and S , terminal deoxynucleotidyl transferase and DNase 1 igase II (54), as well as the SV40 T antigen (16,53).

239 Several amino acids may serve as ADPR acceptors including glutamic

acid, aspartic acid, arginine, diphtamide and probably others (18, 19, 55). It seems that ADPR transfer may be controlled, in part, by the accessibility of a specific amino acid chain present in potential acceptor proteins, and that a given ADP-ribosyl transferase can use only one amino acid as acceptor. In rat liver histones, glutamic acid or lysine are the sites for polyADP-ribosylation (56,57). The 1 inkages involved in the covalent attachement of poly(ADPR) chains in nuclear protei ns are ester bonds between the termi na 1 ri bose and the carboxyl group of glutamic acid or of a terminal lysine residue. However, a small fraction of ADPR may be attached to proteins through linkages not susceptible to neutral NH20H and/or dilute alkali (58). Rat liver extracts also possess an ADP-ribosyl transferase activity which utilizes arginine, or other guanidino compounds as ADPR acceptors. The existence of ADPR conjugates with phosphoserine has also been considered (59).

Choleragen and E. coli heat-labile enterotoxin catalyse the ADP-ri bosyl ati on of an argi ni ne ina regul atory moi ety of adenyl ate cyclase (60,62). Diphteria toxin and Pseudomonas exotoxin A ADP-ribosy­late diphtamide, a modified histidine residue, in elongation factor II (63,64). In turkey erythrocytes, an ADP-ri bosyl transferase has been identified which is catalytically similar to choleragen and E. coli heat-labile enterotoxin (8).

FUNCTIONAL ASPECTS OF ADP-RIBOSYLATION ADPR in DNA repair.

All DNA damaging agents lower cellular NAD and promote biosynthesis of poly(ADPR}. Nuclear poly(ADPR)-PT is activated by both single and double-stranded breaks in the associated DNA. After chemical or radia­tion damage, inhibitors of poly{ADPR}-PT prevent the drop in cellular NAD as well the DNA excision/repair process. Similarly, nutritional deprivation of nicotinamide lowers NAD level and blocks DNA excision and repair. Poly{ADPR}-PT inhibitors and nicotinamide deprivation synergis­tically increase the lethality of a great variety of DNA damaging agents, such as alkylating agents and radiations. It is expected that the enhanced toxicity of some alkylating agents brought about by poly(ADPR}-PT inhibitors, may be of cl inical importance. The inter-

240

ference of poly(ADPR)-PT inhibitors with DNA repair is also of impor­tance in carcinogenesis. Poly(ADPR)-PT inhibitors, when present in the body, may inhibit efficient DNA repair with increased risk of carcino­genesis. As to the molecular mechanism underlying the involvement of poly(ADPR)-PT in DNA repair, it seems likely that it modulates DNA ligation. It is noteworthy that nuclear poly(ADPR)-PT activity is stimulated by breaks in the DNA strand and is required for the efficient rejoining of these breaks. For review, see 16-19,55,59,65. PolyCADPR) and cell proliferation.

There is evidence to suggest that ADP-ribosylation may be an important post-translation event during cell proliferation and DNA replication (for review, see 15-18,67,68). Poly(ADPR)-P activity is high in nuclei during the G1 phase and is low during the S phase. This suggests that ADP-ribosylation may be involved in the initiation of DNA synthesis. Moreover, in fast dividing cells NAD is low (69) while poly­(ADPR)-PT activity is high. This contrasting behavior suggests an increase of pol y(ADPR) synthes is whi 1 e the precursor NAD decreases. Inhibition of poly(ADPR)-PT by Nic treatment results in the prevention of the NAD decrease and in the inhibition of cell proliferation.

It was shown in our laboratory that phytohemoagglutinin (PHA)­treated human lymphocytes undergo an active prol i ferat i ve response in parallel to the observed increase in poly(ADPR)-PT activity measured in permeabil ized cell s (70). When nicotinamide or 3-aminobenzamide was added at the onset of the PHA treatment, a marked inhibition of the PHA­induced DNA synthesis, cell proliferation and poly(ADPR)-PT activity increase was observed. When the inhibitors of poly(ADPR)-PT were added at a later stage (48 hr), no inhibition of the PHA-stimulated DNA synthesis and cell proliferation was observed (71). The involvement of ADP-ribosylation during the initiation of DNA synthesis was thus suggested.

Another interesting approach is offered by the fact that diadeno­sine 5'5"P1P4tetraphosphate (Ap4A), which increases in cells experienc­ing division, can be ADP-rybosylated. This reaction may be involved in the conrol of DNA replication (72).

Several other observations are suggestive of a modulation of cell proliferation through regulation of ADP-ribosylation. In this respect,

241

it is noteworthy that activation of DNA polymerase activity follows ADP­ri bosyl at i on of chromat i n protei ns, as it was shown in our 1 aboratory (47) .

A useful model to study the relationship of ADP-ribosylation to DNA synthesis is represented by transformed or tumor cells. We have shown that the content of pyrimidine nucleotides in tumors and tissues under­going rapid proliferation is low (69). Moreover, the poly(ADPR)-PT activity of leukemic and hepatoma cells is significantly higher than in normal lymphocytes and in liver cell nuclei, respectively (67,73,74).

It was also shown in our laboratory that the poly(ADPR)-PT activity measured in permeabilized cells in culture is higher in glioma cells than in normal astrocytes (Table 1). The proliferation of both cell types is blocked by inhibitors of ADP-ribosylation, suggesting a possible application of poly(ADPR)-PT modulators in the chemotherapy of tumor cells, including the most malignant glioma cells (Fig. 2 and 3).

co I

o

x :I: <II i5 iX .... UJ

!!:: <II ..J ..J UJ o

10

8

6

4

2

I. , , , ! , , J I

'5 7 9 11 13 15 17 19 21

DAYS

Fig. 2. Effect of nicotinamide (5 mM,'" and 15 mM, _ ) amd of 3-amino­benzamide (5 mM, • and 10 mM, *) on astroblasts proliferation in primary culture. Controls, x. Astroblasts of newborn rats were cultured accor­ding to Booher (29). Nicotinamide and 3-aminobenzamide were present unt il day 13.

242

ADP-ribosylation and cellular differentiation. Various reports suggest that poly(ADPR)-PT is involved in cell

differentiation and that this enzymatic activity may be correlated with differential gene expression during development (15-18). Changes in the activity of poly(ADPR)-PT occur during the normal differentiation of cardiac muscle cells (75), intestinal epithelial cells (76), embryonic chick muscle cells in vitro (77), embryonic cells of Xenopus laevis (78) and in the slime mold Dictyostelium discoideum (79). Similar changes of poly(ADPR) synthesis and of polymer levels were observed in the chemically induced differentiation of Friend erythroleukemia cells (80), in the hormonally promoted differentiation of quail oviduct goblet cells (81) and in the early phase of mesenchimal differentiation (16,82-84). It is likely that the mechanisms mediating the action of ADP-ribosyla­tion on cell differentiation are mainly alterations of nuclear chromatin

6 x

5

to I

0 4 )(

:I: VI a 3

ii: .... UJ Q.. 2 -VI ...J ...J UJ U

5 7 9 11

DAYS

Fig. 3. Effect of nicotinamide (5 mM,. and 15 mM, _ ) and of 3-amino­benzamide (5 mM, .) on C6 glioma cell proliferation. Controls, x. C6 cells were cultured according to Benda (28). Nicotinamide and 3-amino­benzamide were present until day 8.

243

DNA strand breaks, increased avai 1 abil i ty of ADP-ri bosyl at i On acceptor enzymes or other events may also be i nvo 1 ved. One may specul ate that local AOP-ribosylation may leave the chromatin in a permanent state in which certain regions are differentially accessible for transcription. If this view is correct, one may expect that the quantitation of poly­(AOPR)-PT during development may be a probe for structural alterations of the chromatin during development.

CONCLUSION Mono and polyADP-ri bosyl at i on can be def) ned as a postsynthet i c

modification of proteins by the covalent attachment of AOPR from NAO. For elucidation of the role played by ADP-ribosylation, the identifica­tion of the acceptor protein(s) is of paramoumt importance. An increasing body of evidence implicates AOP-ribosylation of nuclear proteins in DNA duplication and repair, protein synthesis and cell differentiation. The discovery of an extranuclear ADP-ribosylation of proteins has stimulated investigations of this reaction in cytoplasmic components and in membrane fractions. The activation of membrane adenyl ate cyclase by AOP-ribosylation of the associated GTP-binding protein suggests its involvement in signal transmission. The use of poly(AOPR)-PT inhibitors has prompted further investigations of potential interest for cancer therapy. It is noteworthy that until now, the highest poly(AOPR)-PT activity has been observed in neurons. Since this enzyme activity cannot be solely associated to DNA duplication or repa i r, a major role in other cell functions cannot be rul ed out. Finally AOP-ribosylation activity appears to be higher in brain mito­chondria as compared to liver. Since oxygen consumption is highest in brain sl ices as compared to several other organs (85), one may wonder whether the mi tochondri a 1 poly (AOPR) -PT activity may correl ate to the basic mitochondrial function of energy production.

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21

DNA SYNTHESIS AND CELL NUMBER HOMEOSTASIS IN THE BRAIN y

V. MARES

Institute of Physiology, Czechoslovak Academy of Sciences, Prague,

Czechoslovakia

ABSTRACT

The distribution of DNA synthesizing and/or dividing non-neuronal

cells is unequal within the brain and meningeal envelopes of mice at

different ages. In addition to large scale regional differences, a

focal occurrence of labeled cells (LC) has been demonstrated in the

subependymal layer and in the brain parenchyma; the latter is attributed

to the presence of "stem" cells or their transiently activated dormant

forms. The number of LC decreases during adulthood, but not equally in

all brain regions. In the periventricular subependymal layer of the

forebrain, the decrease is relatively less profound than in the main

mass of the brain. A certain number of cells synthesizing DNA is still

present in all regions of the mouse brain till early senescence. A

SUbstantial part of the newly-formed cells, or their DNA, undergoes

disintegration, soon after their origin or synthesis.

INTRODUCTION

Data on cell number, pre-mitotic DNA synthesis, and cell division

in the adult brain are still fragmentary (for reviews, see ref. 1-3).

Cell division, as indicated by pre-mitotic synthesis of DNA, has been

shown in all types of non-neuronal cells (N-N cells), including glial

and endothelial cells, and the cells of brain appendices. The present

paper deals with the pre-mitotic synthesis of DNA in N-N cells of the

mouse and rat brain in terms of the distribution of dividing cells,

their number at different ages, and also with the impact of cell multi­

plication on cell number homeostasis in the differentiated brain. The

aim was to show that this process is not as low and steady as is usually

considered, and to provide morphological background for the biochemical

studies of non-replicative brain DNA synthesis.

248

RESULTS AND DISCUSSION Appearance and distribution of DNA synthesizing cells in mouse brain.

Mice 1 to 16 month-old received an intraperitoneal injection of 5 uCi r3H]thymidine (3HTdR)/g body wt and were killed after 2 hr. Fifty percent of the animals survived only till the age of 16 mo and displayed severe symptoms of senil e atrophy. The brai ns were routinely fi xed, embedded and autoradiographed (4,5).

Distinct incorporation of 3HTdR takes place mainly in N-N cells (1,2,4-11). Although further cytological classification of these cells is difficult in paraffin embedded material (necessary for quantitative studies, see below), it is clear that most of the labeled cells (LC) are glial cells of different degrees of maturity, immature cells of the subependymal layer of the forebrain, endothelial cells, and cells accom­panying brain vessels. In all the age groups, there are, however, some cells of ambiguous appearance. Cytological classification of LC was given in previous studies (6-9). However, many of the LC have probably not yet been definitely classified, and the nature of the LC population is, thus not fully known. In our material, LC range from 3 to 10 um in diameter. Cells with relatively small nuclei prevail over those with larger nuclei in the forebrain gray matter of adolescent animals. The prevalence of smaller LC is even more apparent in the callosal white matter. Typical large nerve cell nuclei were practically without labeling except for a few cases which require further verifica­tion. There are, however, a few good examples of microneuron-like labeled nuclei in the hippocampus and olfactory bulbs, especially in animals which have received 3HTdR repeatedly. Labeled cells also occur in brain accessory structures, such as the leptomeninges and the choroid plexus (4,5).

Labeled cells are not distributed equally within the mouse brain (1,4, and Fig. 1). They are most frequent in the persisting germinative subependymal layer around the lateral ventricles of the forebrain, reachi ng thei r anteri or and posteri or extensi ons into the olfactory bul bs and the hi ppocampus (IS to 20%). In the white matter of 1 arge commissures of young adult mice (I to 2 month-old), LC are relatively more frequent (I to 2%) than in the surrounding gray matter (0.05 to 0.1%). In the olfactory bulb and in the hippocampus, LC are more

249

numerous than in other parts of the brai n (0.2 to 0.3%); the lowest number of LC occurs regularly in the cerebellum « 0.01%). The same pattern of distribution of LC is present in 2 month-old animals, which received 6 doses of 3HTdR within 48 hr and were killed 3 hr later (4). The incidence of LC is somewhat higher in the frontal, occipital and pyriform parts of the cerebral cortex (Fig. 1). An unequal distribution of LC has also been revealed in the leptomeningeal envelopes, where LC are more numerous around the rostral part of the brain than more caudally, especially over the cerebellum (Fig. 1). A microfocus of LC can, however, also appear at other places, for example around the large blood vessel in the roof of the third ventricle and its branches penetrating brain parenchyma (Fig. 1).

Labe 1 ed cell s often occur in groups of different sizes. Th is is most evident in the subependymal layer of the forebrain, but also in other regi ons where groups of three to fi ve cell s, often beari ng a different number of silver grains, are present.

211101'11w

Fig. 1. Distribution of labeled cells in the brain of 2 and 16 month­old mice after injection of 3HTdR (5 ~Ci/g bogy wtj 2 hr pulse). Each black dot indicates the presence of one LC; an open circle corresponds to ten LC. Six sections prepared from three animals were superimposed for each age group.

250

Age changes in the number and distribution of labeled cells. Dalton et al. (8) reported an age-dependent decrease in LC in the

mouse brain. A significant difference, however, appeared only between animals 23 and 400 day-old. Similar studies performed later (1,4) showed that in some regions, for example in the forebrain gray and white matter, the decrease in labeling index (LI, number of LCjtotal number of cell s x 100) is continuous duri ng adol escence and adulthood. In the cerebra 1 cortex, the decrease is arrested or cons i derab 1 y slowed down after 12 mo, but progresses further, for instance, in the corpus callosum of the hippocampus. The age-dependent decrease in LI stops even earlier (at about 6 mo) in the population of germinative subepen­dymal cells. This is accompanied by a slight reduction in the total cell number in the layer (by about 30%; ref. 5). Evaluation of different types of labeled N-N cells revealed that the age decrease occurs mostly in the glial cells, while endothelial cells and other N-N cells continue to incorporate 3HTdR in some regions (e.g. in the cerebra 1 cortex) at the same rate or perhaps even at an i ncreas i ng rate. An extension of the study to other brain regions (Fig. 2), performed in terms of total number of LC instead of LI values (which are difficult to determine in regions with densely packed cells), showed that the age decrease is a general phenomenon in mouse brain. The decrease is smaller and terminates earlier in the germinative subepen­dymal layer than in the other brain regions. In some parts of the brain, there is no further decrease after about 12 mo (e.g. cerebral cortex, basal ganglia, brain stem), while in others the decrease continues further, for instance, in the corpus callosum, cerebellum, and hippocampus (Fig. 2). In the brain meninges the number of LC does not decrease until the 16th mo.

The age changes in the number of LC most likely reflect the decrease in the growth fraction (GF) of cell populations. The GF remains, thus, relatively high in the subependymal layer until early senescence, while it almost disappears in the brain parenchyma. In brain parenchyma, the possibility of a cell division, activated by heal i ng or other compensatory processes, shoul d be di sti ngui shed from that associated with the original, persisting pool of dividing cells.

.. o • I

i ~ .. o

III

251

RG£ CHANGES IN 3HTdR LABELLING IN DIFFERENT REGIONS Of' THE I10USE BRAIN

----~m~,g~!~ ~~n:. diencephalon ig"J'1Mgrlr' .. ••• encepbal.oD baoal _&1111

cerebel.lua h1ppoo..,... corplW callona

Ie 12 14 16 IS Age [lftOnthal

Fig. 2. Total number of LC in different regions of mouse brain after injection of 3HTdR (5 llCi/g body wt; 2 hr) in animals of different ages.

It is noteworthy that in the subependymal layer, the incidence of LC is not random but varies greatly between groups of cells (Fig. 3).

In addition, a slight shift in the modes of the frequency distribution curves towards a lower LI appears between postnatal mo 1 and 16 (Fig. 3; ref. 5). The differences in LI are likely to be due mainly to varia­tions in the GF. If cell cycle parameters (11) are taken into consider­ation, the groups with LI values of 40 to 50% should contain only dividing cells, while in those with lower LI, a reasonable number of cells should be non-cycling. There is also a slight tendency for higher LI to occur in smaller nests and vice versa (5). The biological basis for the local variations in LI in the subependymal layer and possibly in other brain regions cannot, as yet, be reasonably explained. It is, however, very suggestive that the activity of the nest-forming "stem" cells may be undergoing transient modulations. The reasons and mechanisms governing this process are not known, especially outside the subependymal layer of the forebrain. Proliferative microfoci might originate from germinative cells released from the periventricular layer or by spontaneous reactivation of cell division in dormant stem cells, or even in some predisposed, more differentiated cells. Elucidation of this phenomenon should provide new insight into compensatory and healing processes as well as the origin of brain tumors.

252

2 months

12-16mcrths

1J 15 20 25 XI :J) l.() 45 50 55 6) L.l.%

1- 2 months

0--0 12-15 mDI"IIw

o 5 'D 5 20 25 :JJ 3S 40 45. fLI 55 60 loL%

Fig. 3. Frequency distribution of LC in "nests" of dividing cells of the subependymal layer (ordinates), in relation to different labeling index (abscissa) in mice of different ages.

Life-span of newly formed cells and their impact on brain cell kinetics. Glial cell division may lead to a small but long-lasting increase

in the cell population (12) or it may reflect a process of cell renewal

(1,4,6). It has been calculated that the observed rate of cell divi­

sion, however low, may lead to an increase of the cell population in

mouse brain hemispheres of about 50% within 1 to 12 mo of postnatal life

(1). However, a comparable increase in the total brain DNA content was

not found (13) and ce 11 d i vi s i on was, thus supposed to be balanced by

cell death. The process of cell renewal need not concern all cells of

the glial population equally, i.e. it does not necessarily entail a

linear addition of new cells from the proliferating pool and a process

of cell loss limited to the static compartment of the population. A

closer relationship between newly-formed and dying cells has already

been suggested for the popul at i on of immature subependyma 1 cell s of

mouse forebrain (8) and, recently, also in the dividing N-N cells of

253

brain parenchyma (2,13,14). Examination of the retention of newly­synthesized (3H]DNA at longer post-injection (pi) intervals has shown (12,14) that the amount of [3H]DNA decreases substantially in all the brain regions by 72 hr, except for the olfactory bulbs, and continues to diminish, such that only about 20% of the initially labeled DNA is present 21 days pi. In the olfactory bulbs, an increase of 55% occurred towards pi day 7; this was followed by a decrease similar to that of the other regions. The same observations were made after different doses of 3HTdR (0.5 to 1.0 pCi/g body wt). A parallel autoradiographic examina­t i on of the cerebral cortex revealed a s 1 i ght increase in the tot a 1 number of LC towards pi days 7 and 21, accompanied by a decrease in the intensity of labeling of cell nuclei and a greater range of grain

density counts in the labeled population. The histological determina­tion of cell death, performed in the hippocampus, has not revealed significant differences in the number of pyknotic cells (1 to 1.5/sec­tion) between non-injected and 3HTdR injected animal s at all pi inter­vals, except for 8 hr, when a transient increase (2.5 pyknoses/section) occurred (13,14). Some pyknotic cells were labeled and their LI ranged from 12 to 20%, with the exception of 8 hr pi interval, when a transient increase appeared (14). The loss of [3H]DNA can be attributed either to death of some newly-formed cells or to molecular turnover of DNA.

Data on [3H]DNA loss in the brain of adult rats (16) and mice (15), very similar to ours, were reported earlier and attributed to radiotox­icity (16) or turnover of DNA, especially in post-mitotic cells. The latter alternative was suggested because the rate of [3H]DNA disappear­ance was the same in samples "rich and poor" in dividing cells observed in a parallel study of 1 day-old mice. The influence of radiotoxicity was excluded as the same findings were obtained after different doses of

HTdR. It has also been shown (1,2,7,11) that dividing cells are present in all regions of the brain, even in adults, and, furthermore, that there is a different mode of proliferation and physiological cell death in the immature and differentiated brain (2). The relatively close topographic correlations between the values of DNA synthesis measured bi ochemi cally and autoradi ographi cally (14), the presence of labeled mitoses and the autoradiographic results on cell kinetics (2) are all more in favor of pre-mitotic rather than non-replicative

254

synthesis of DNA shown in our auto-radiograms. The loss of [3H]DNA can, thus, be attributed mainly to cells of the dividing pool. This fits in well with the autoradiographic estimates (2) indicating that 17% of the newly-formed daughter cells undergo pyknosis in the adult mouse brain after each cell cycle. Analogous values of labeled pyknoses (12 to 20%) were found in our utoradiograms at most pi intervals. The loss of [ H] DNA, thus, seems to be a physiological phenomenon, reflecting redundancy in cell division in a small pool of dividing cells present in the mouse brain parenchyma.

Cell overproduction, followed by cell death, is relatively common at early stages of brain development (17), but contrasts with the low rate of cell proliferation in the adult brain. The significance of this process as well as its regulatory mechanisms are, as yet, unknown. An equilibrium between the rate of cell formation and cell death should, however, be quite essential for the maintenance of the population homeostasis of the brain. Its disturbance may lead to some pathological states as, for instance, gliosis or brain tumors. The danger of tumors is enhanced by the poss i bil i ty of "propagation and fixation" of unre­paired, occasional damage to DNA in repeatedly dividing cells.

The influence of molecular renewal of DNA on the disappearance of [3H]DNA can-not, however, be completely excluded. A recent study of bulk isolated brain cells (18) considers DNA turnover in brain cells, especially in neurons, to be the main reason for the [3H]DNA disappear­ance from the brain of rats injected with 3HTdR intracranially.

Finally, it should be pointed out that the number of LC reflects, to a certain extent, the functional load of the brain. It has been shown that in 11 wk-old rats kept in darkness since birth, the incidence of LC was significantly lower in the lateral geniculate body, a primary visual centre and, also, in the deep layers of the visual cortex (19). In addition, an unexpected deficit in the number of mast cells appeared in the brain of animals raised in the dark (19).

REFERENCES 1. Mar~s, V., Lodin, Z. and Jilek, M. J. Compo Neurol. lQl: 471-482,

1975. 2. Korr, H. Adv. Anat. Embryol. Cell Biol. 61: 1-72, 1980. 3. Reznikov, K. Yu. Proc. Acad. Sci. USSR 181: 467-469, 1968. 4. Mares, V. and Lodin, Z. Brain Res. 76: 557-561, 1974.

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5. Mare~, V., Fulop, Z., Gerstejn, L.M. and Narovec, T. Physiol. Bohemoslov. (in press).

6. Mare~, V., Lodin, Z., Treska, J. and Faltin, J. In: Metabolism of Nucleic Acids and Proteins and The Function of the Neuron (Eds. Z. Lodin and S. Rose), Excerpta Med. Fdn., 1968, pp. 62-72.

7. Paterson, J.A., Privat, A., Ling, A.E. and Leblond, C.P. J. Compo Neurol. 149: 83-102, 1973.

8. Smar~, I. J. Compo Neurol. ll§: 325-347, 1961. 9. Mares, V. Acta Histochem. 53: 70-76, 1974. 10. Dalton, M.M., Hommes, O.R. and Leblond, C.P. J. Compo Neurol. 134:

397-399, 1968. 11. Lewis, P.O. Exp. Neurol. 20: 208-214, 1968. 12. Brizzee, K.R., Sherwood, N. and Timiras, P.S. J. Gerontol. 23:

289-~97, 1?68. . 13. Mares, V., Langmeler, M. and Lisy, V. Physiol. Bohemoslov. 26:

481-487, 1977. 14. Mare~, V. and Lisy, V. Physiol. Bohemoslov. 32: 385-392, 1983. 15. Kimberlin, R.H., Shirt, D.B. and Collis, S.C. J. Neurochem. 23:

241-248, 1974. 16. Merits, I. and Cains, J. Biochim. Biophys. Acta lZ!: 315-321, 1969. 17. Hyndman, A.G. and Zamenhof, S. Develop. Neurosciences 1: 216-225,

1978. 18. Perrone Capano, C., D'Onofrio, G.D. and Giuditta, A. J. Neurochem.

38: ~2-56, 1982. 19. Mares, V., Bruckner, G., Narovec, T. and Biesold, D. Life Sci. Zl:

727-732, 1977.

BRAIN DNA CHANGES DURING LEARNING S. REINIS

22

Department of Psychology, University of Waterloo, Ontario, Canada

ABSTRACT In this paper, evidence is considered which implicates neuronal DNA

in the formation of a permanent memory trace. It is postulated that one of the early changes necessary for permanent morphological and biochem­ical adjustments of neurons, which are ultimately responsible for the formation of the memory trace, may concern the structure of DNA.

INTRODUCTION DNA is a carrier of most of the genetic information necessary for

normal cellular development and functioning. As a result, DNA is one of the prime regulatory factors within body cells. Since the neurons in the adult CNS usually do not divide, brain DNA was considered to be very stable. Koenig (1), however, reported that there is a slow turnover of brain DNA. These findings were confirmed several times (2-6). Unambig­uous proof of DNA synthetic activity persisting into adulthood has been presented by Kuenzle et al. (7). Slow, protracted DNA synthesis in neurons is also accompanied by DNA loss. However, this "unstable" DNA represents just a portion of total neuronal DNA (6).

The turnover of DNA in adult neurons requires the activity of several enzymes whose presence in the brain has been documented (see chapters by Kuenzle and Subba Rao). Activity of these enzymes may possibly lead to an increase in the total amount of DNA in the neuronal nucleus, although this problem has not been solved with certainty (see chapter by Bernocchi and Scherini).

The importance of these DNA changes is probably related to increased metabol i c needs associ ated with the functional act i vat i on of neurons. In a series of experiments which lasted several years, we made an attempt to relate these changes to learning, as well as to the recovery from brain injury.

257

RESULTS Effects of purine and pYrimidine derivatives on learning.

In our early studies on the effects of purine and pyrimidine derivatives on learning, we used 2,6-diaminopurine, 6-mercaptopurine,

6-thioguanine, 5-iodouracil, and 5-bromouracil (8-10). The intracranial injection of these substances, at least 2 hr before the learning session (passive avoidance or V-maze) in strain A mice, caused a defect of performance of animals tested from 24 hr to 1 wk after training. The effect was still present when the drugs were injected 1 hr after training (with the exception of 6-mercaptopurine), but not 2 to 24 hr after the training session.

These five compounds are incorporated into RNA and, therefore, may disturb the process of translation into proteins. However, at the same time, they also act as mutagens. They may be incorporated into DNA, and may either change base-pairing properties, or be excised and induce DNA repair processes (11). Most of these compounds may also enter other metabolic processes in the cell, and act as inhibitors of protein synthesis (12). 6-mercaptopurine inhibits metapyrophosphate amino­transferase and, thus blocks de novo purine synthesis. It may also inhibit purine oxidases (13). Generally, 2-mercaptopurine follows closely the known pathways the anabolism and catabolism of the purine bases. Therefore, the effects of these compounds and of other purine and pyrimidine derivatives cannot unequivocally demonstrate which metabolic process underlies learning and long-term fixation of the memory trace.

For this reason, in later studies, we used a more specific antimetabolite which interferes with DNA replication. 5-iodo-2'-deoxy­uridine (IUdR) is a compound which, with reasonable specificity, is incorporated into DNA instead of thymidine, but is transcribed as if it were cytosine (14). The incorporation is permanent. The IUdR-substi­tuted DNA is unable to act as template for the synthesis of the normal enzymes necessary for cell function, although some enzyme molecules are produced (15). IUdR may also induce alterations of normal development, as shown in Drosophila (16). Although a portion of IUdR is deiodinated before its incorporation into DNA (17), most of the compound is incor­porated as such into the DNA of mouse tissues in vivo (18). In many

258

respects, its effects are identical to those of 5-bromo-2' -deoxyur­idine. Thus, if DNA changes are involved in the biochemical process necessary for the storage of memory, the DNA alterations induced by incorporation of this antimetabolite will cause a defect in the memory trace. Our experiments demonstrated that IUdR, if administered immediately before passive avoidance training, blocked the expression of the memory trace of mice tested up to 1 wk after the initial training session (19). The injection of IUdR, 24 hr before training or 2 hr after training, did not change the performance of the mice in the testing trials. In order to confirm that the retention deficits were due to the incorporation of IUdR into DNA in competition with thymidine, rather than to general or unspec i fi c effects, we exami ned the effects induced by the simultaneous adminis-tration of thymidine and IUdR. It has been shown, both in vitro and in vivo, that increasing concentra­tions of thymidine reduce the incorporation of labeled IUdR into DNA (20,21). The rate of thymidine incorporation is about five times faster than that of IUdR (18). The competition between IUdR and thymidine was used to demonstrate the specificity of the effect of IUdR on retention. By injecting increasing doses of thymidine together with IUdR, we expected to suppress the incorporation of IUdR into DNA, as well as its effect on learning. Our results showed that thymidine is able to suppress the negative effect of IUdR on retention of the passive avoid­ance task. It should be added, however, that high doses of unlabeled thymidine (10 ).11 of a 0.1 M solution, intracranially) interfere with the performance of mice in the testing trial of passive avoidance training. The injection of the same amount of uracil was without effect (Table 1). This finding may be related to the inhibition of DNA synthesis induced by very high does of thymidine (22).

The effects of various purine and pyrimidine derivatives on Y maze and passive avoidance learning in mice might, therefore, indicate the existence of some DNA changes probably necessary for the fixation of the memory trace. For this reason, we decided to study these DNA changes directly, by biochemical and autoradiographic methods. Biochemical studies of DNA changes during learning.

In a paper published in 1972 (23), we described that passive avoidance training in a step-through apparatus induced an increased in-

259

TABLE 1. Effect of intracranial injection of unlabeled thymidine (T) or unlabeled uracil (U) on passive avoidance learning.

Interval between injection and acquisition

tri al

24 hr before 2 hr before 5 min after 1 hr after

24 hr after

Interval between acquisition and testing trial

24 hr T U

12 11 10 12 8 12

12 10 12 11

48 hr T U

12 11 11 11 3 11

11 12 11 12

72 hr T U

10 11 12 12

1* 12 10 11 12 12

1 wk T U

11 12 12 10 0* 11

12 10 11 12

Values refer to number of mice not entering the dark space in the testing trial. Thymidine and uracil injected in volumes of 10 pl (0.1 M solution). *, p < 0.05, X2 test.

corporation of radioactive thymidine into brain DNA. The increase was significant in comparison with untrained animals, or with animals exposed to foot shocks without the opportunity for escape. Two indepen­dent sets of experiments were performed. One study involved the intra­peritoneal injection of 2 pCi [3H-6]thymidine/g body wt in order to avoid brain damage, while in a second investigation, 1.5 pCi [3H-6]­thymidine was injected intracranially to avoid the blood-brain barrier. In both studies, the mice were divided into three groups: quiet controls (NS), shocked controls without the possibility of escape (S), and experimental passive avoidance (PA) animals.

In another experiment, the labeled thymidine was injected intraper­itoneally and the S group was replaced by an escape (E) group. The animals were placed in the dark compartment of the passive avoidance apparatus and treated with foot shocks for at least 1 sec. They were, however, allowed to escape into the illuminated compartment of the step­through apparatus. In all these experiments, DNA was extracted from mouse brains by the method of Kirby (24) and purified by proteinase and RNase digest i on. The concentrat i on of DNA in the fi na 1 sample was determined by optical density reading at 260 nm. Radioactivity was

260

TABLE 2. Specific activity of purified brain DNA extracted from the brains of mice sacrificed at different time intervals following passive avoidance training.

Time Period

25 min

24 hr

1 wk

2 wk

Non-Shocked Controls

273 ± 68

547 ± 122

344 ± 96

292 ± 167

Shocked Controls

430 ± 118*

653 ± 152

346 ± 90

327 ± 139

Passive Avoidance

566 ± 179**

983 ± 186***

294 ± 99

271 ± 136

Results (cpm/mg DNA) are average ± S.D.; *, p < 0.02 and **, p < 0.01, compared with non-shocked controls. ***, p < 0.01, compared with non­shocked and shocked controls (Wilcoxon rank sum test, two-tailed).

measured by liquid scintillating counting (for details, see refs. 23, 25).

The data presented in Table 2 indicate that after intraperitoneal injection of 2 ~Ci tritiated thymidine/g body wt, the specific activity of purified DNA, was significantly higher in the PA group than in the NS group, from 25 min to 24 hr following the training session. In addi­tion, the S group incorporated more label into DNA than the NS group, the difference being statistically significant 25 min after the behav­ioral event. Following the intracranial injection of labeled thymidine, similar differences were observed.

When the shocked animals were allowed to escape, the PA group still incorporated significantly more label than the NS or E group. The E group incorporated slightly more radiolabel than the NS group, but the difference was not significant (see Table 3).

The pyrimidine bases, including tritiated thymidine, may be degraded in brain soon after their injection. The principal product of this metabolism is ~-alanine, which may then be degraded further. Also, the metabolic products of thymidine degradation may be incorporated into other macromolecules, such as RNA and various protein species. For this reason, we considered it necessary to purify the extracted DNA with

261

TABLE 3. Specific activity of purified brain DNA, extracted from the brains of mice sacrificed 24 hr following escape training and passive avoidance training.

Group

Non-shocked controls

Escape controls

Passive Avoidance

N

6

7

7

Mean + S.D. Specific Aciticity

(cpm/mg DNA)

234 ± 50

266 ± 25

429 ± 200*

Values given as the mean ± S.D. *, P < 0.05, compared with NS. Adapted from ref. 25.

RNase and proteinase. When this step was omitted, the resulting "DNA" extracts did not show any radioactivity increase induced by learning.

In this experiment, reported originally in 1974 in the doctoral dissertation of R.W. Lamble (25), DNA was extracted by the method of Mori et al. (26). The brains were homogenized in cold 0.25 M sucrose, 3 mM CaC12 solution and centrifuged. The pellet was washed twice with 5% TCA, chloroform/methanol, hr at 37° to degrade RNA. tion at 85° for 20 min in

ethanol and ether, and incubated in KOH for 2 DNA was hydrolyzed from the pellet by incuba-

1 N PCA. The solution was centrifuged and the supernatant was analyzed for DNA at 260 nm. The highest radioactivity was found in the "DNA" extract from the brains of S animals. In the PA group, the relative radio-activity was actually slightly lower than in the other experimental groups. "DNA" extracts were, however, markedly contaminated by protein and RNA, as determined by the method of Lowry et al. (27), and by the orcinol test (28), respectively. These results i nd i cate that to determi ne the 1 eve 1 of thymi dine i ncorporat ion into brain DNA, the samples have to be carefully purified.

Radioactive thymidine itself, following its incorporation into DNA, may cause disruption of the labeled DNA molecules, due to radiation damage (21). DNA strands may be broken at a rate of 2.1 breaks/disinte­gration (29), and the appearance of fragmented chromosomes has also been reported by other authors. We took advantage of this phenomenon to

161

TABLE 4. Effect of different doses of [3H]labeled thymidine on retention of passive avoidance learning in C57BL/6j mice.

Thymidine (].lCi)

o 3

10

50

Latency in the testing trial

(sec)

262.6 ± 51.4

271.1 ± 68.8

134.4 ± 44.1

88.2 ± 71.8

Results expressed as mean ± S.D. [3H]thymidine was dissolved in 10 1 phosphate buffer and injected 5 min after the training session. Animals tested 72 hr later.

obtain indirect evidence of the involvement of brain DNA in learning. In our experiment, we injected increasing doses of [3H]thymidine (1 to 50 ].lC, intracranially) to C57BL/6j mice before passive avoidance training. The results shown in Table 4 indicate that the degree of retention decreases following injection of higher doses of labeled thymidine. Presumably, the radiochemical damage inflicted to brain DNA interferes with the performance of the trained animals in the testing tri a-l . Autoradiography of DNA changes during learning.

An important quest i on concerns the 1 ocat i on of the DNA changes related to long-term retention of the memory trace. An autoradiographic study (30) indicated that after [3H]thymidine incorporation into brain DNA, not only glial cell nuclei but also many neurons were labeled in the cerebral cortex. These neurons do not form any anatomically defined group, but are scattered throughout the whole cerebral cortex, with maximal labeling in the parietal area. They are also present in handled and yoked controls, but their number is lower. In the cerebral cortex, they are localized exclusively in the second and sixth layers. We also found them in many subcortical areas from olfactory bulbs to the medulla.

263

Fig. 1. Autoradiography of layer 2 of mouse parietal cortex, showing incorporation of radioactive thymidine into neuronal nuclei. The animal was sacrificed 24 hr after the injection.

We counted these labeled cells in the parietal cortex of NS, S, and PA mice and found that their numbers were significantly higher in the PA group than in the other two groups. The detailed data pertaining to this experiment have been reported (30).

The labeled neurons of all experimental groups often showed a nucleolar localization of the label, or a localization in the nucleolus combined with one or two more areas of the cell nucleus. This observa­tion is very similar to the figures published by Dropp and Sodetz (31),

who reported the incorporation of [3H]thymidine into the neuronal nuclei of the superi or cervi ca 1 and coe 1 i ac gangl i a of adult rats, fo 11 owi ng their long-lasting stimulation. Wintzerith et al. (32) also found that following injection of radiolabeled thymidine, about 50% of the incor­porated label was localized in neuronal nucleoli, the rest being distributed in clumps throughout the neuronal nucleus.

264

DISCUSSION Thus, we were able to provide three lines of evidence - pharmaco­

logical, biochemical, and autoradiographic - showing that some changes may take place in the DNA of cortical neurons during learning, and that these changes are probably necessary for the permanent fixation of the memory trace.

An open question still remains as to the nature of the DNA changes involved in the process of learning. One possibility is repair DNA synthesis, as proposed by Vilenchik and Tretjak (33). These workers found that hydroxyurea, which blocks replicative DNA synthesis but not DNA repair, is unable to block unscheduled DNA synthesis in the brain. In a group of unpublished experiments, we found that intracranial injection of hydroxyurea does not influence the acquisition of passive avoidance in mice, although several other antimetabolites do. However, all this evidence is indirect. It suggests that replicative DNA synthesis is not involved in the process of neuronal plasticity, but it does not show which other DNA modifying system is involved.

The second possibility concerns the activity of reverse transcrip­tase, as proposed by Sal gani k et al. (34). These authors cl aim that RNA-dependent DNA polymerase is more active in the hippocampus of fast­learning Wistar rats than in slow learners. The acceptability of this proposal depends, however, on the unequivocal proof that reverse transcriptase really exists in mammalian brain. The amplification of genes descri bed, for example, by Montgomery et a 1. (35) inhuman neuroblastoma cells, may be accomplished by DNA-dependent DNA poly­merase. Such ampl ifi cat i on of genes may alter phenotypi c expressi on when increased amounts of specific proteins are required, and may be eventually followed by excision of the newly-formed genes.

The third possibil ity to be considered regards the repl ication of the genome involving all segments of DNA. Kuenzle et al. (7) favored the existence of such replication, since they did not find a preferen­t i a 1 enri chment of defi ned portions of the genome duri ng 1 abe 1 i ng of brain DNA.

An important observation, related to this problem, may be the observation made by Giuditta et al. (36), who found that the amount of DNA labeling in rat brain may actually decrease following electro-

265

shock treatment. This shows that there are two possible directions of DNA changes - toward an increase and/or toward a decrease in the rate of synthesi sand/or degradation. Gi udi tta and hi s group also showed the newly-labeled DNA disappears from rat cerebral cortex relatively quickly (4). The metabolic processes involved in the synthesis of extra DNA in neurons are, therefore, probably not identical with the synthetic processes responsi bl e for dupl i cat i on of the genome duri ng neurobl ast division, leading eventually to permanent labeling of neuronal DNA.

Another important problem is how these DNA alterations are related to the many metabolic changes which, in the past three decades, were described in the CNS as attributes of learning and memory. Changes in the levels of RNA, pre- and post-translational modification of proteins, alterations in the second messenger systems, levels of various neuro­transmi tters, sensit i vi ty of their receptors, receptor synthes is and degradation, methylation and other changes of various membrane lipids, as we 11 as changes in energy metabo 1 ism, have a 11 been imp 1 i cated as components of the bi ochemi cal systems affecting neuronal exci tabil i ty. Data accumulated in the literature indicate that the response of the nerve cell to an altered input is extremely complex, involving many, and perhaps all cellular metabolic systems (37). On a conceptual level, we may assume that specific memories are stored in complex neuronal systems (assemblies), involving large numbers of nerve cells (38). Within such systems, each individual nerve cell receives information in the form of impulses from large numbers of other neurons. Many nerve cells may respond to an increased or decreased input by a permanent change in excitability. Further, these shifts of neuronal excitability may depend on the simultaneous functioning of many cellular metabolic systems. The changes in neuronal excitability dispersed over large areas of the brain would result, therefore, in a number of alterations of metabolic processes, which may vary, both quantitatively and qual itatively, in each nerve cell involved. Thus, the DNA changes observed here are just one component of these systems. However, DNA alterations may represent the critical factor which controls many other metabolic changes, leading to the modified neuronal excitabil ity necessary for long-term memory storage. From these consid-erations, we may conclude that DNA changes in individual neurons forming such neuronal assemblies may be extremely

266

varied, involving an increase or decrease in the rate of DNA synthesis, as well as various parts of the genome. However, until now, the bio­chemical analysis of brain DNA has concerned only the average DNA state of large numbers of both neurons and glia cells.

The role of 1 abe 1 ed neurons in the formation and maintenance of the memory trace is still uncertain. The autoradiographic data presented in this paper indicate that, at least in the cerebral cortex, only a small portion of the neuronal nuclei is labeled. What is the role of these neurons in the activity of large neuronal assemblies? Do they act as command neurons, or as neurons controlling input or output into neuronal systems responsible for certain logical operations? This question is actually related to one of the ultimate problems in neuro­sCience, which concerns the participation of individual neurons in large cell assemblies and their mutual interactions and cooperation. To date, very little work has been done to approach this problem.

REFERENCES 1. Koenig, H. J. Biophys. Biochem. Cytol !: 785-792, 1958. 2. Inoue, N., Suzuki, O. and Kato, T. J. Neurochem. 27: 113-119, 1976. 3. Cameron, I.L., Pool, M.R.H. and Hoage, T.R. Cell Tissue Kinet. 12:

445-451, 1979. 4. Perrone Capano, C., D'Onofrio, G. and Giuditta, A. J. Neurochem.

38: 52-56, 1982. 5. Kaplan, M.S. and Bell, D.H. Exp. Brain Res. 52: 1-5, 1983. 6. Kimberlin, R.H., Shirt, D.B. and Collis, S.C. J. Neurochem. 23:

241-248, 1974. 7. Kuenzle, C.C., Bregnard, A., Hubscher, V. and Ruch, F. Exp. Cell

Res. ~: 151-160, 1978. 8. Reinis, S. J. Biol. Psychol. 12: 45-48, 1970. 9. Reinis, S. Ghana Med. J. g: 255-259, 1970. 10. Reinis, S. Psychopharmacologia Jg: 34-39, 1971. 11. Pietrzykowska, I. Mutation Res. Jg: 1-11, 1973. 12. Smith, J.L. and Forbes, I.J. Austr. J. Exp. Biol. Med. Sci. 48:

267-274, 1970. 13. Chalmers, A.H., Knight, P.R. and Atkinson, M.R. Austr. J. EXp.

Biol. Med. Sci. 47: 263-270, 1969. 14. Prusoff, W.H. Biochim. Biophys. Acta 39: 327-341, 1959. 15. Aamodt, L. and Goz, B. Biochem. Pharmacol. 19: 2400-2403, 1970. 16. Rizki, R.M. and Rizki, T.M. Experientia 28: 324-325, 1972. 17. Commerford, S.L. and Joel, D.O. Biochem. Biophys. Res. Commun. 86:

112-118, 1979. 18. Myers, O.K. and Feinendegen, L.E. Can. J. Physiol. Pharmacol. 53:

1014-1026, 1975. 19. Reinis, S., Abbott, J. and Clarke, J.J. Physiol. Chern. Phys. !:

440-448, 1972.

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20. Hampton, E.G., Rich, M.A. and Eidenhoff, M.L. J. Biol. Chern. 235: 3562-3567, 1960.

21. Hofer, K.G. and Hughes, W.L. Radiat. Res. 47: 94-109, 1971. 22. Cooper, R.A., Perry, S. and Breitman, T.R. Cancer Res. 26:

2267-2272, 1966. 23. Reinis, S. and Lamble, R.W. Physiol. Chern. Phys. !: 335-338, 1972. 24. Kirby, K.S. Biochem. J. 66: 495-504, 1957. 25. Lamble, R.W. Doctoral Dissertation, York University, Toronto, 1974. 26. Mori, K., Yamagami, S. and Kawakita, Y. J. Neurochem. lZ: 835-843,

1970. 27. Lowry, O.H., Rosebrough, N.H., Farr, A.L. and Randal, R.J. J. Biol.

Chern. 193: 265-275, 1951. 28. Mejbaum, W. Zschr. Physiol. Chern. 258: 117-120, 1939. 29. Cleaver, J.E., Thomas, G.H. and Burki, H.J. Science 177: 996-998,

1972. 30. Reinis, S. Physiol. Chern. Phys. !: 391-397, 1972. 31. Dropp, J.J. and Sodetz, F.J. Brain Res. 33: 419-430, 1971. 32. Wintzerith, M., Wittendorp, E., Rechenmann, R.V. and Mandel, P. J.

Neurosci. Res. ~: 217-230, 1977. 33. Vilenchik, M.M. and Tretjak, T.M. J. Neurochem. 29: 1159-1161,

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23

ROLE OF DNA IN BRAIN INFORMATION PROCESSING A. GIUDITTA, M.V. AMBROSINI 1 , F. MORELLI, C. PERRONE CAPANO, T. MENNA, C. BUONO, C. LAMBERTI, A. CERBONE 2 AND A. SADILE2 International Institute of Genetics and Biophysics and Department of General and Environmental Physiology, Naples, Italy; lInstitute of General Biology, University of Perugia, Italy; 2 Institute of Human Physiology and Medical Physics, Naples,Italy

ABSTRACT In the adult rat, brain DNA is in a state of turnover, which also

involves a neuronal perikaryal fraction. Brain DNA synthesis is modul ated by 1 earni ng in upward or downward shi fts, dependi ng on the nature of the learning task. The concentration of radioactive brain DNA synthesized during training is inversely dependent on the amount of post-training paradoxical sleep, but only in non-learning rats. These results are taken as evi dence that brain DNA is synthes i zed duri ng learning in relation to the process of information storage, and may eventually be degraded in relation to the removal of that information. The latter process requires paradoxical sleep, i nformat i on proves to be wi thout adaptive val ue.

and occurs if the It is proposed that

transpos i t ion mechan isms underly the observed brai n DNA turnover and the corresponding events of information processing. The data also support the sequential hypothesis of sleep function, which holds that the information gathered by brain during wakefulness is processed during sleep in two major steps occurring sequentially during synchronized and pardoxical sleep.

I NTRODUCTl ON During the last 25 years, a great deal of information has accumu­

lated on the molecular aspects of learning. Most studies have concerned the acquisition step of long-term memory, while little attention has been paid to the moleuclar basis of the following steps in information processi ng. Some of the key steps of thi s further el aborat i on of the memory trace occur during sleep. It is known that the amount of para­doxical sleep (PS) increases after the acquisition of complex learning tasks, and that selective deprivation of PS in the immediate post-train-

269

ing period impair the recovery of memories (3,4). The neurochemical study of sleep is, however, still in its infancy and little biochemical data is available with regard to post-training sleep (5,6). We have started to investigate this neglected area of research, which holds promise of yielding important clues regarding the nature of molecular information processing in brain.

Our approach is based on a novel way of considering sleep and its relationship with wakefulness (7). Most hypostheses on the function of sleep attribute distinct roles to synchronized sleep (SS) and PS, as if they were independent (5,7). On the other hand, we believe that PS and SS are strictly related to each other and to the previous period of wakefulness (W). According to our "sequential" hypothesis (7), the information acquired by the brain during W is processed during SS to reach an intermediate stage, and eventually during PS, to reach a final stage. The hypothesis may be tested at the molecular level on the assumption that changes in brain state bring about corresponding biochemical changes. It is thus implied that the biochemical modifications of the brain occuring during the acquisition step, should be expected to undergo further change in concomitance with the processing of the acquired information. A standard method used to identify the molecular correlates of the acquisition process in brain relies on the injection of suitable radioactive precursors, and on the determination of the labeled brain products synthesized during the learning session. A similar experimental design may be used to assess the post-training fate of molecules synthesized during the training period. The amount and the types of sleep occurring in the post-train­ing period can be determined by EEG recording.

RESULTS DNA synthesis in the adult brain.

In principle, the above experimental paradigm can be applied to the study of the fate of different classes of molecules presumably involved in brain information processing. Initially, we chose to investigate the role played by DNA in brain information processing, a rather unusual choice. Of course, we had reasons for doing so. Our interest in brain DNA goes back several years, and was initially aroused by the observa-

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270

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tion that the cellular DNA content of the subesophangeal region of octupus brain progressively increases with age (Fig.I) (8, see also 9 and 10). Since then, we considered the idea that brain DNA might be directly involved in brain function. Pelc's work on metabolic DNA (11) prompted a search for a similar kind of DNA turnover in the mammal ian brain. We found that following intracranial injection of [3H-methyl] thymidine into adult rats, brain DNA became maximally labeled within 4 to 5 hr, and that approximately half the radioactive DNA disappeared in the following 10 to 15 hr. A similar DNA turnover occurred in a fraction of neuronal perikarya purified from the cerebral cortex, but no loss of labeled DNA was observed in liver (12). Previous data, showing the occurrence of a slower process of turnover of brain DNA, were explained in terms of repair reactions enhanced by the radiation damage caused by the incorporation of the radioactive precursor (13). This interpretation, however, did not account for a) the initial process of [3H]thymidine incorporation, b) the lack of DNA turnover in liver, and c) the low rate of DNA turnover observed in deve 1 opi ng mouse bra in, despite its marked rate of incorporation (14). In our view, the loss of a substantial portion of radioactive DNA was 1 ikely to reflect a true process of turnover. Influence of learning on brain DNA synthesis.

To support our interpretation and to investigate the significance

271

of DNA turnover in brain function, we decided to study the effect of various manipulations of brain activity on brain DNA synthesis, includ­ing electroconvulsive shock (15) and, in later studies, several learning paradigms. We thought that the demonstration that brain DNA synthesis was affected by behavioral variables would argue against the repair hypothesis, since DNA repair reactions could hardly be modulated by learning or other types of activity. We were quite intrigued, at that time, to discover that brain DNA synthesis had already been shown to be influenced by learning. Indeed, in mice trained in a passive avoidance apparatus, the synthesis of brain DNA was reported to increase signifi­cantly in comparison with control groups (16,17; for a review, see 18). To confirm the validity of these results and extend them to a different animal species and other types of training, we started a series of investigations using rats as experimental animals. Our results can be summarized as follows: 1) When female Wistar rats were trained in a two-way active avoidance apparatus, the concentration of radioactive cortical DNA synthesized during the training session from [3H-methyl ]thymidine was higher in learning animals than in paired control rats kept in their home cage in the same experimental room. An increase in DNA labeling was also present in animals which failed to reach the learning criterium and in their paired control mates. A comparable pattern of changes occurred in the radioactivity of the PeA-soluble fraction. No effect was present in liver. However, when the concentration of brain cortex radioactive DNA (or the percent incorporation) was plotted as a function of the total number of avoidances scored, a linear inverse relationship emerged, both in the learning group and in the non-learning group, but the regression line obtained with learning rats was significantly different from that of non-l earners rats. In part i cul ar, 1 earni ng rats showed consi stently higher values at all levels of avoidances. Since the number of avoid­ances are complementary to the number of footshocks received and, as such, may be taken as an inverse measure of the amount of stress, we interpreted these results to indicate that the incorporation of [3H]thy­midine into brain cortex DNA was specifically enhanced by learning, in addition to its dependence on the level of stress. The observation that control rats paired to non-learning animals had higher incorporation

272

values than control rats paired to learning animals, appeared to be in accord with a facilitatory role of stress on brain DNA synthesis, since

the former controls had presumably received more stressful signals from their paired mates who were unable to avoid footshock (19). An increased synthesis of brain DNA during active avoidance learning, has also been reported by Ashapkin et al. (20). 2) In female Sprague-Dawley rats trained to retrieve food pellets with their non-preferred paw (reversed handedness training; an appetitive form of learning), the percent incorporation of radioactive thymidine into DNA was lower than in quite controls in several brain regions, including hippocampus (Fig. 2A), entorhinal cortex, visual cortex and the remaining brain (mostly other cortical areas and brain stem). No effect occurred in the sensory-motor cortex. In the group of act i ve controls which were exposed to the same behavioral situation, but left free to use their preferred paw, a significant decrease was present only in the entorhinal cortex. A marked decrement in DNA specific activity was observed in neuronal perikaryal fractions isolated from the hippocampus (Fig. 2B) and the remaining brain of learning rats. In active control rats, a significant decrease occurred in the hippocampus only (21). These results were unexpected, since they indicated that the concentration of newly-synthesized brain DNA can be modulated downwards by learning an appetitive task, while it is modulated upwards by avoid­ance learning. In the same reverse handedness experiment, we used DNA renaturation experiments to show that radioactive DNA isolated from the brain of control animals was present in all kinetic fractions (from Cot 2 to Cot 50,000). Its pattern of distribution, however, differed significantly from that of total genomic DNA. Significant differences were also found in the extent of reannealing of radioactive DNA at various Cot values in the comparison of learning animals with active and passive control rats (21). 3) The condition of behavioral habituation (exposure to a square enclosure, a form of non-associative learning) also resulted in decrements in the concentration of radioactive DNA in a limited number of brain regions. These effects only occurred in an inbred strain of Sprague-Dawley rats, i.e. the Naples Low Excitability strain, which was originally selected on the basis of its response to the behavioral

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273

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Fig. 2. A. Values of percent [3H]thymidine incorporation in the hippocampus of passive control rats (PC), active control rats (AC) and rats learning the reverse handedness task (L). B. DNA specific activities determined in the neuronal perikaryal fraction prepared from the hippocampus of the same groups of rats.

habituation test. No significant changes were found in the complemen­tary inbred strain, the Naples High Excitabil ity strain, nor in the parent stock (Lamberti et al., in preparation).

The above results supported the view that learning modulates the rate of brain DNA synthesis in a rather complex way, inducing upward or downward shifts, presumably depending on the nature of the learning task and on the amount of stress. This conclusion is in contrast with two widely held opinions according to which 1) brain DNA synthesis reflects only replicative or repair events, and 2) the role of brain DNA in learning is 1 imited to its participation in transcription per se. With regard to the first point, we have already mentioned the main reasons that 1 ead us to bel i eve that brain DNA undergoes a cont i nuous process of turnover. As to the second opinion, it is important to point out that several biological features of eukaryotic DNA are well known,

274

J. TRAINING REST t - - - -

I I

1 2 3 4 5 6 7 HOURS

Fig. 3. Experimental paradigm used to test the sequential hypothesis of sleep function. L intraventricular injection of [3H]thymidine; t, death. The filled boxes indicate the training sessions.

which demonstrate the great functional flexibility of the genome. These i ncl ude phenomena such as ampl i fi cat i on, transposi t ion, reverse trans­cription, genomic rearrangement, etc. (22). It is not unreasonable to expect that these or comparable features mi ght be occuri ng in brain, as required by the molecular mechanisms underlying brain information processing. Brain information processing and brain DNA turnover.

Following this line of thought, we performed experiments aimed at verification of the sequential hypothesis of sleep function, using DNA as test molecule. Our experimental design was as follows (Fig. 3)

(23). Rats were injected with [3H-methyl]thymidine intraventricularly and 30 min later exposed to an active avoidance session lasting four hours, or left in their home cages. At the end of this period, they were transferred to an EEG recording chamber to which they had been acclimatized, and left free to sleep for three additional hours before sacrifice. EEG activity was continuously monitored during the rest period.

On the basis of the total number of avoidances, the population of trained rats segregated into two quite distinct subpopulations of 1 earned and non-l earned animal s. When the bi ochemi cal parameters were related to the behavioral and sleep variables, it was found that in the major brain regions (cerebral hemispheres, cerebellum, hippocampus, brain stem), but not in liver, the concentration of radioactive DNA of non-learning animals was inversely related to the amount of PS recorded in the post-training rest period (Fig. 4). No effect was observed in learning or control animals. Since PS was largely confined to the last two hr of the whole experimental period (lasting 7.S hr), we interpreted the correlation as reflecting a modulation of DNA degradation, rather

275

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4

>-1---'" >' - a 3 b ~ « x u « LL z U

0 2

UJ 0)

a.. ~ tI) E « a. Z u 0 1

L NL L NL L NL L NL

Fig. 4. Effect of post-training PS on the specific activity of DNA from cerebral cortex (A), hippocampus (B), cerebellum (C) and brain stem (D). L, learning rats; NL, non-learning rats. Open bars, rats with less than 660 sec PS; hatched bars, rats with more than 660 sec PS (23).

than of its synthesis. Brain DNA labeling was, in fact, presumed to be of minor significance 5.5 hr after the injection of the radioactive precursor. This assumption remains to be proven. Furthermore, since the concentration of radioactive DNA was inversely related to the amount of PS in non-learning animals only, we hypothesized that the degradation of brain DNA synthesized during the training period was associated with the removal of information devoid of adaptative value (23). The latter process appeared to require the occurrence of high amounts of PS, as suggested by a recent hypothesis (24) and as previously postulated by Dewan(25). Our results on the fate of radioactive brain DNA in non­learning rats support these views. At the same time, the lack of a rel ationship between radioactive brain DNA and PS among learning rats may indicate that in these animals, brain DNA synthesized during training is related to information of adaptive value, which may become

276

further val idated during PS. As a result, radioactive brain DNA is retained rather than degraded, as it occurs in the group of non-learning animals. Much additional work is needed to establish the validity of this interpretation.

These results also lend support to the sequential hypothesis of sleep function (7), inasmuch as the PS-modulation of a brain biochemical parameter was dependent on the nature of the precedi ng waki ng experi­ence. The data, however, did not reveal strong correlations between the biochemical parameters and SS, at variance with an additional prediction of the sequential hypothesis. One possible explanation of this negative finding concerns the method of sleep analysis used, which could only detect modifications in the duration of the sleep states. On the other hand, SS is also known to be capable of intensity-modulation, a dimen­sion which requires a power spectral analysis of EEG patterns.

DISCUSSION The results presented in this paper and additional data from the

literature (18, 26, see chapter by Reinis) contribute to the conclusion that brain DNA turnover is modulated by learning and sleep. The acqui­sition of new information by the brain is associated with an increase or decrease in the concentration of newly-synthesized brain DNA, presumably depending on factors related to the nature of the learning task, such as the type of reward and the amount of stress. Presumably, it is the rate of DNA synthesis that is modulated in the acquisition step. On the other hand, in the further processing which occurs during post-training sleep, it is the rate of DNA degradation which appears modulated. In our view, newly-synthesized DNA represents one of the key molecular counterparts contributing to the brain record of past experience. If

experience does not lead to a meaningful pattern of adaptative response, the acquired record and its molecular counterparts are deleted. We have used these views to interpret our data on the behavior of radioactive brain DNA following shuttle-box training and post-training sleep. Additional work is necessary to put this interpretation on a sounder basis.

While not definitely proven by the experimental data, the sequen­tial hypothesis of sleep function finds good support in the observed

277

strict dependence of a brain biochemical parameter (the concentration of DNA synthesized during the training session) on the nature of the previous waking experience and on the duration of post-training PS. The merits of the hypothesis lay in its simplicity and in the relative ease with which it may be amenable to experimental test.

What is the role then of DNA turnover in brain information proces­sing? Better, what are the presumed mechanisms that might account for the very existence of a brain DNA turnover, and for its involvment in neural information processing? Unfortunately we know very little regarding the molecular nature of newly-synthesized DNA, besides its heterogeneous distribution among DNA fractions with high, moderate and low degrees of repetitiveness. We also know that experience modifies this pattern of distribution which, in turn, is not equivalent to that of bulk DNA. Perhaps, this little information is sufficient to exclude amplification events as a major cause of the observed brain DNA synthe­sis. Other authors have suggested alternative explanations, such as transcription-modulated DNA repair (20) and reverse transcription (26).

On the basis of the behavior of newly-synthesized brain DNA in different learning conditions and in post-training sleep, we have favoured a transposition hypothesis (27). Suitable variants of this mechanism (22) are sufficiently flexible and plastic to correlate well, at the molecular level, with processes such as information storage and decay. The basic underlaying concept envisages that by transposing certain DNA segments (containing controlling and constitutive sequences) to other regions of the genome, the transcription capacity of these regions would become markedly altered. Conversely, by deleting or transposing away the same segments from their new location, the original state of that genomic region would be restored, thereby erasing at the molecular level whatever record was originally laid down. The initial transposition event and the subsequent erasing process would require DNA synthes is and degradation, respectively. The more recent concept of retrotransposition (28), which we find even more intriguing, would have the additional merit of also accomodating the hypothesis of reverse transcription (26).

178

ACKNOWLEDGEMENTS We thank J.-l. Knox for assistance in the preparation of the

manuscript. Financial support for this work was provided by the Ministero della Pubb1ica Istruzione, the Progetto Fina1izzato di Medicina Preventiva e Riabi1itativa, CNR, the Universita de Perugia and the Universita de Napoli.

REFERENCES 1. Komitiani, P.A., A1eksidze, N.G. and L1ein, E.E. Progr. Neurobio1.

18: 181-229, 1982. 2. Dunn, A. In: Molecular Approaches to Neurobiology (Ed. I.R.Brown),

Academic Press, New York, 1982, pp. 317-340. 3. McGrath, M.H. and Cohen, O.B. Physio1. Bull. 85: 24-57, 1978. 4. Perlman, C.A. Neurosci. Biobehav. Rev. ~: 57-68, 1979. 5. Giuditta, A., Perrone Capano, C. and Grassi Zucconi, G. In:

Handbook of Neurochemistry, 2nd edition (Ed. A. Lajtha), Plenum Press, New York, Vol. 8, 1984, pp. 443-476.

6. Karnovsky, M.l. and Reich, P. In: Advances in Neurochemistry (Eds. B.W. Agranoff and M.H. Aprison) , Plenum Press, New York, Vol. 2, 1977, pp. 213-275.

7. Giuditta, A. In: Sleep '84 (Eds. W.P. Koe11a, E. RUther and H. Schulz), Fischer Verlag, Stuttgart (in press).

8. Giuditta, A., Libonati, M., Packard, A. and Prozzo, N. Brain Res. 25: 55-62, 1971.

9. De Marianis, B. and Giuditta, A. Brain Res. l2!: 134-136, 1978. 10. De Marianis, B., 01mo, E. and Giuditta, A. J. Compo Neuro1. 186:

293-300, 1979. 11. Pe1c, S.R. Intern. Rev. Cyto1. 32: 327-355, 1972. 12. Perrone Capano, C., D'Onsfrio, G. and Giuditta, A. J. Neurochem.

38: 52-56, 1982. 13. Merits, I. and Cain, J. Biochim. Biophys. Acta ~: 327-338, 1970. 14. Kimberlin, R.H., Shirt, D.B. and Collis, S.C. J. Neurochem. 23:

241-248, 1979. 15. Giuditta, A., Abrescia, P. and Rutigliano, B. J. Neurochem. 31:

983-987, 1978. 16. Reinis, S. and Lamb1e, R.W. Physio1. Chem. Phys. !: 335-338, 1972. 17. Reinis, S. Physio1. Chem. Phys. !: 391-397, 1972. 18. Giuditta, A. In: Handbook of Neurochemistry, 2nd edition (Ed.

A. Lajtha), Plenum Press, New York, Vol. 5, 1983, pp. 251-276. 19. Scaroni, R., Ambrosini, M.V. Principato, G.B., Federici, F.,

Ambrosi, G. and Giuditta, A. Physiol. Behav. 30: 577-582, 1983. 20. Ashapkin, V.V., Romanov, G.A., Tushma10va, N.A. and Vanyishin, B.F.

Biokhimiia 48: 355-362, 1983. 21. Giuditta, A., Perrone Capano, C., D'Onofrio, G., Toniatti, C.,

Menna, T. and Hyden H., submitted. 22. Lewin, B. Genes, J. Wiley & Sons, New York, 1983. 23. Giuditta, A., Ambrosini, M.V., Scaroni, R., Chiuru1la, C. and

Sadile, A. Physiol. Behav. 34: 769-778, 1985. 24. Crick, F. and Mitchison, G. Nature 304: 111-114, 1983.

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25. Dewan, E.M. In: Sleep and Dreaming (Ed. E. Hartman), Little Brown, Boston, 1970, pp. 295-307.

26. Salganik, R.I., Parvez, H., Tomson, V.P. and Shumskaya, I.A. Neurosci. Lett. 36: 317-322, 1983.

27. Giuditta, A., Perrone Capano, C. and Ambrosini, M.V. In: Brain Plasticity, Learning and Memory (Eds. B.E. Will, P. Schmitt and J.C. Dalrymple-Alford), Plenum Press, New York, 1985.

28. Baltimore, D. Cell 40: 481-482, 1985.

24 RNA AND LEARNING , H. HYDEN Institute of Neurobiology, Faculty of Medicine, G8teborg, Sweden

ABSTRACT This review discusses evidence that specific RNA species are

produced in neurons and glia during learning. Using microchemical techniques, we have shown that an increased RNA and protein synthesis is correlated with learning a new task in rats and rhesus monkeys. Learning produces a rapid response in the synthesis of soluble proteins, which begins in the hippocampus and entorhinal cells and spreads to cort ica 1 areas. Eventually, thi s RNA/protei n act i vat i on and the synthesis of neuronal membrane proteins may lead to a synapse and membrane differentiation, modifying neural function. In hippocampal CA3 pyramidal cells, in comparison to active controls, there is an overall increase in RNA synthesis and the production of 25S RNA and of 8-9S and 16-18S poly(A)+mRNA. Thus, learning a new behavior initiates a process of gene activation (Hyden and Cupello, 1981-1983). Previous studies gave the same concl usi on, and demonstrated an RNA increase and base ratio changes towards mRNA values in 5th layer cortical neurons and glia in rats and monkeys subjected to instrumental 1 earni ng. Nonspeci fi c stimulation and stress gave no effect. The astrocytic S-100 protein complex in its Ca++-form increases within miDutes and is inserted into synapse 'and plasma membranes. Ca++per se increases in the hippocampus. This produces a functional differentiation: S-100 induces a 20 to 25% increase in the transport of GABA across GABAergic synapses and a 40% increment in the 36Cl- passage into the post-synaptic neuron. In this way, the process of GABA inactivation is accelerated and the restoration of the neurons excitatory state becomes more effective. This effect can, thus, be traced back to a glial protein, the S-100. Antiserum to S-100 injected into the hippocampus of training animals produces amnesia and temporally blocks further learning. The prompt response of the neuron-glia unit persists in old age. Similar RNA and S-100 effects have been demonstrated by V. Shashua in goldfish brain. A strictly localized RNA-specific response occurred in monkey cortical neurons

281

during learning. Hippocampal and entorhinal cells seem the first to be biochemically activated when something new is learned.

The turning on (or off) of gene sets in brain cells, when the system performs new functions, is in 1 i ne with most general systems act i vity. How cognit i ve st imul i from the outer or inner envi ronment can impinge on brain cell nuclei altering gene expression will probably remain unsolved for a long time.

INTRODUCTION This discussion deals with specific RNA species in neurons and

glial cells produced during behavioral tests, the concomitant synthesis of specific proteins, and what influence they may have on learning processes. The evidence is taken from recent as well as previous data in the field. My theme is that learning is a life-long differentiation, molecular and functional, involving neuronal plasma membrane, synapses, and glia. The rationale for the discussion is that one cannot discuss brain RNA only, but is bound to consider the translation products resulting from cognitive stimuli and, even if information is incomplete, the question of what effects such proteins may have on neural function.

It is clear, furthermore, that reductionism cannot be carried out to its extreme. Detailed knowledge of RNA molecules cannot give a full explanation of learning processes, and of the storage and retrieval of i nformat i on in bra in wi th all its enormous complexity. There is no foundation for the view that RNA could store information in its inner structure as in a magnetic tape, a point which I have stressed for many years in publication and elsewhere.

In view of the high complexity of the brain, the marked hetero­geneity of its cell population and their differential functional roles, it is not surprising that gene expression in brain cells is approxi­mately five times more diversified than in other somatic cells. It is important to keep in mind that there is an intimate relationship, bio­chemically and functionally, between the glia cells and the neuron they surround. This is also expressed during learning at the gene level, as examples will show.

Brain cells contain RNA of high sequence complexity. Forty-five percent of the single-copy DNA is transcribed in a young adult rodent.

282

TABLE 1. Content and base composition of RNA in pyramidal cells of the hippocampal CA3 region in rats of different ages.

RNA Base Composition Content

Age N (pg/cell ) A G C U G+C/A+U

Foetal 4 19± 3 20.9±0.8 26.2±0.5 30.8±1.2 22.1±0.5 1.33±0.06

Newborn 4 24± 1 21.0±0.7 27.6±0.8 31.0±1.2 20.8±0.3 1.41±0.04

Adult 4 1l0±10 17.7±0.8 24.5±2.0 37.8±2.0 20.0±0.5 1.66±0.04

Old 2 53± 1 14.9±1. 7 29.0±1.6 36.9±0.2 19.3±0.3 1. 95±0.13

Results are mean values ± SEM; A, adenine; G, guanine; C, cytosine; U, uracil ; N, number of animals (from ref. 3).

The polysomal RNA complexity of the mouse is equivalent to about 100,000 different sequences. This complexity is divided equally in poly(A)+ and poly(A)-RNA. About 65% of the poly(A)+mRNA seems to be brain-specific. Gene activation, with specification of mRNA, occurs postnatally and approximately 20% of the poly(A)+ RNAs are not present at birth. In contrast, the majority of nonadenylated RNAs are expressed after birth, requiring 30 days to reach adult levels (1,2) (see chapters by Kaplan et al., and Hahn et al.). For this discussion, it is important to keep such data in mind. Particularly, that brain-specific mRNAs are now rapidly being isolated and specific genes and their expression to a defined protein are now being described (2). Evidence has been presented that "identifier" species of RNA are transcribed in brain and are thought to playa regulatory role in the expression of brain-specif­ic genes. This problem is still under productive discussion.

After this brief introduction, some basic data on neuronal and glial RNA will serve to introduce examples of the synthesis of these RNAs during learning. In two cases, a protein produced during the learning session will be demonstrated to have specific effects on the neuronal mechanisms involved in learning.

283

RESULTS Neuronal RNA and its changes with age.

Postnata lly, nerve cell tot a 1 RNA increases to adult 1 eve 1 s. It

may then vary 10 to 50%, depending on neural activity. In early experi­ments, the RNA content of pyramidal neurons of the hippocampal CA3 region was measured at different ages. The base composition of the RNA was determi ned after hydro lysi sand e 1 ectrophoret i c separat i on of the products on cellulose threads 1 ~m wide (4,5).

In old age, there is a decrease of total RNA, mainly of the rRNA type, which prevails in large neurons (Tables 1 and 2). In most of the cortical mammalian cells, however, the nuclear RNA may dominate because of the scanty cytoplasm and relatively large nucleus. In the rat, the processing of rRNA (18S and 28S) and nuclear RNA is slower at 1 mo than at 10 mo of age (7). The proportion of poly(A)+RNA increases with age and is maximal in synaptosomal RNA (7).

On the basis of quantitative and base ratio RNA analyses, Hyden and Lange (8,9) suggested that gl i al RNA was transported to nerve cell perikarya. In the giant squid axon, there is evidence for a local synthesis ofaxoplasmic RNA, presumably synthesized in peri axonal glia (10). Giuditta has shown that mRNA species are present in the giant axon (see chapter by Giuditta et al.).

TABLE 2. Total RNA content in human motorneurons.

Age (yr)

0 - 20

21 - 40

41 - 60

61 - 80

over 80

RNA content (pg/cell )

402 ± 28

553 ±38

640 ± 55

504 ± 31

420 ± 30

C5 motorneurons (nucleus ventralis lateral is) were obtained from healthy persons, deceased in traffic accidents (from ref. 6).

284

Handedness reversal learning.

Three month-old Sprague-Dawley rats were trained to reverse handed­

ness in retrieving food pills, using the non-preferred paw. Rats are

45% left-handed, 40% right-handed, and 15% ambidextrous, when assayed in

compl icated motor tasks. They were trained in 25 min sessions, (two

sessions/day) for four days. [14C]orotic acid or [3H]uridine were

injected intraventricularly before the last session and a 1 hr pulse was

allowed (13). The CA3 pyramidal nerve cells of the hippocampus were

rapidly dissected out with minimal glial contamination. After RNA

extraction, poly(dT}cellulose RNA binding and separation on 0.7% agarose

- 1.7% acrylamide gels of 1 mm diameter, we found these RNA changes in

comparison with active controls: (a) an overall increase of neuronal

RNA synthesis; (b) a stimulation of 8 to 9S and 16 to 17S poly(A}+ RNA

and 30S hnRNA (Fig. 1 and 2).

4S 8-95 ~~s 1BS 285

! !

1.0

NUMBER or fRACT J ONS

" Average iractional distribution of radioacrivity

Fig. 1. Electrophoretic separation of newly-synthesized RNA in the CA3 region of rat hippocampus of trained and control animals (average of eight separate runs). -----, control; -----, trained (from ref. II).

285

CONTROLS 188 288

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20 15 NUMBER OF FRACTIONS

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18. 288

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5 NUMBER OF FRACTIONS

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Fig. 2. Electrophoretic fractionation of newly-synthesized poly(AtRNA of the CA3 region of rat hippocampus. The arrows mark the positions of 18S and 28S RNA added as optical density markers. --, optical density; -----, radioactivity (from ref. 11).

Zomzely-Neurath et al. (12) found that 8S and 16S RNA represent mRNA in the rat brain. Our conclusion was that in learning this new behavior, a gene activation occurred in the CA3 pyramidal nerve cells of the hippo­campus with the appearance of specific RNA species.

In several other learning tests, pyramidal nerve cells from CA3, CA4, and CAl have been used for RNA and protein analysis. In the following discussion, we will consider only examples taken from instru­mental 1 earni ng experiments. In mammals, the hi ppocampus must function correctly bil atera lly for 1 earni ng compl i cated tasks. The hi ppocampus is a regulating center for feeding, rage and fear, and enables the rat to construct and remember a 3-dimensional environmental map; the CA3 neurons form an "integrating center" of the involved neural network.

This conclusion was also drawn from RNA analyses made in the same learning test. Pyramidal nerve cells from the 5th layer of rat cortex,

286

were analyzed with respect to base ratios and amount of RNA (13). The nerve cells were sampled from a cortical area close to the bregma. If this area is destroyed, reversal learning is no longer possible. It should be noted that in this experiment, the pyramidal nerve cells of the corresponding contralateral area of the same animal were used as controls. As additional controls, we used caged rats of the same age, rats performing the same task with the preferred paw, and rats subjected to stress. In these controls, a slight RNA increase was found, but no base ratio changes were observed. In learning rats, the RNA content increased 20% and the ratio G + CIA + U decreased from 1. 72 to 1. 51, approaching mRNA values (Table 3).

A quotation from the conclusion of this paper (13) reads, "an acute learning situation with no precedence in the animal's life acts as a gene st imul at i on, resulting ina production of RNA wi th speci fi c base ratios in the neurons immediately involved." Glial and neuronal RNA during vestibular learning.

Similar RNA changes were observed in another rat learning paradigm (14). Rats learned to balance on aIm long, 1.5 mm diameter steel wire, strung from the floor of the cage to a platform containing food

TABLE 3. Changes in the RNA base composition of cortical neurons from the control (left) side and from the learning (right) side of rats performing the reverse handedness task.

Adenine

Guanine

Cytosine

Uracil

A + C C + U

G + C A + U

From ref. 13.

Controls Learning

18.4 ± 0.48 20.1 ± 0.11

26.5 ± 0.64 28.7 ± 0.90

36.8 ± 0.97 31.5 ± 0.75

18.3 ± 0.48 19.6 ± 0.56

0.81 ± 0.027 0.95 ± 0.035

1.72 ± 0.054 1.51 ± 0.026

% Change

9.2

8.3

-14.4

7.1

17.3

-12.2

p

0.02

0.01

0.01

0.05

0.01

0.02

287

pellets. The Deiters' nerve cells, in the lateral vestibular nucleus, are an important station of the vestibulo-spinal pathway and are directly involved in the vestibular control in this learning task. These neurons were analyzed with respect to RNA content and base ratios. The large size of these nerve cells required a special analytical scheme. They have a cell body volume of approximately 40,000 ~m3, and a total RNA content of about 700 pg, the base composition of

which is dominated by G and C. The nucleus, on the other hand, is small and contains 30 pg RNA. In order to analyze nuclear RNA, Deiters' nerve cells were isolated from glia and the nucleus of each nerve cell was removed, using a de Fonbrune micromanipulator. Twenty nuclei were sampled for each analysis. The adenine/uracil ratio was changed from 1.06 to 1.35 in the nuclear RNA from learning animal s (Table 4). No such alteration of nuclear RNA occurred in the controls subjected to passive (horizontal and vertical) rotation, or in cells of the reticular formation. This RNA study was extended to glial cells surrounding the Deiters' nerve cells (15). The glial sample corresponded in dry wt to that of the nerve cell they surrounded, as determined by X-ray micro­radiography at 8 to 12 X and based on the mass absorption coefficients of C, N, and 0 (16,17). The glial cell samples of the trained animals showed similar base ratio alterations as did the Deiters neurons (Table 4). In both cases, the RNA composition changed towards that character-

TABLE 4. Changes in RNA base composition in nuclei of Deiters neurons and in their surrounding glia during learning.

Neuronal Nuclei Glia Cells Controls Learning Controls Learning

Adenine 21. 4±0. 44 24.1±0.39*** 25.3±0.16 28.3±0.45***

Guanine 26.2±0.45 26.7±0.87 29.0±0.24 28.8±0.31

Cytosine 31.9±0.77 31.0±0.95 26.5±0.43 24.3±0.36**

Uracil 20.5±1.01 18.2±1.11* 19.2±0.27 18.6±0.21

* , P < 0.05; **, P < 0.01; ***, p < 0.001. From ref. 14,15.

288

istic of mRNA. On the basis of the type of change in neuronal nuclear RNA, an increase in protein synthesis could be expected to occur during the learning session. A similar effect could also be expected for glial proteins. Training induced synthesis of a specific glial protein. Its effect on neuronal membranes in RNA responding loci.

In brain, the acidic protein $-100, originally described by Moore (18), is a product of glial cells (19), as shown by antiserum precipita­tion and immunofluorescence. Most $-100 is soluble. $hashoua et al. (20) have shown a release of $-100 in the extracellular space. $-100 has an affi ni ty for Ca ++, undergoes i on- induced conformat i ona 1 changes, and can be inserted into neuronal membranes. $-100 has been demons­trated in synapses, and in plasma and nuclear membranes, although it has not been proven that neurons produce $-100. Poletaev et al. (21) have presented evidence supporting a transfer of the glial $-100 to neurons.

During instrumental and maze learning, the synthesis of $-100 increases in the hippocampus (22,23), and the membrane-bound $-100 also increases. Monospecific antiserum against $-100, injected intraventri­cularly, produces amnesia of a newly learned task (21-24). It should be noted that Ca ++ becomes concentrated in the hippocampus early during learning, at variance with Na+, K+, and H20 (25). This is an inter­esting example of correlation in time of several learning-associated events, such as synthes is of neuronal and gl i a 1 mRNAs, increase in the concentration of Ca++, and appearance of the glial $-100 in synapses and neuronal membranes.

The question for 20 years has been: What does the $-100 protein do? Two effects of $-100 have now been demonstrated with respect to its neuronal function which seem important for the acquisition of a new behavior. They concern the GABA intake into the postsynaptic neuron (26,27) and the transport of 36 Cl 1 nto the same neuron (28). We analyzed these effects on Deiters neurons which have receptors for GABA (29). Isolated plasma membranes were made from Deiters neurons by rapid free-hand dissection. Figure 3 shows a Deiters nerve cell photographed with an interference contrast microscope. Note the density of the spine-like synaptic knobs. These can be easily removed and the post­synaptic areas exposed. The cell is opened by micro-knives and a very thin outer cell layer containing the plasma membrane is prepared. The

289

Fig. 3. Deiters neurons photographed by interference microscopy. Spine-like synaptic knobs (left panel) may be removed by free-hand dissection (right panel).

plasma membrane is highly polarized with respect to GABA. The membrane is rapidly and tightly placed over a hole in a partition between two compartments of a micro-chamber (Fig. 4). The kinetics of [3H]GABA and 36Cl-transport are measured in the two directions. The membrane (except for controls) is treated with 5-100 and Ca++ added un its outer side. The effect of 5-100 consisted of a 40% increase of the 36Cl-transport in 20 sec and a 25% increase of GABA uptake and transport across the membrane. Since the synthesis of glial 5-100 increased in the hippo­campus during learning, as did mRNA, these results may point to a two­cell collaboration at the molecular level, leading to a more rapid extinction of the GABA inhibitory effect and restoring the neuron's excitatory state more effectively (29).

290

75 j.JI M 1

0.2 mM GABA

membrane Au 2.0 mM GABA

7.5 pI M 2

Fig. 4. Schematic representation of the microchamber used to measure GABA transport across Deiters plasma membrane (bottom).

The regions with membrane-bound S-100 on the neuronal surface, including post-synaptic areas, are irregular and increase in size from early postnatal period into adulthood (30-32). A chemical differentia­tion of synapses and plasma membranes occurs as a function of training and learning. Inactivity and isolation prevents these important processes. Relearning of swimming in fish: mRNA and a glial protein.

The second example showing the involvement of RNA and protein in learning, comes from a series of studies by Shashoua (33,34) on RNA synthes is in gol dfi sh, and on the production of another gl i a 1 protei n called ependymin. A float was sutured ventrally on the goldfish, at the base of the pectoral fins, forcing the animals upside down. In 4

291

hr, they learned to adjust and swim normally. Labeled orotic acid was injected intracerebrally. The RNA synthesized during this learning tri a 1 had a base compos it i on approach i ng that of mRNA wi th 20 to 80% higher uridine to cytidine ratio, compared to controls for motor activity, stress, and convulsions. Changes in the precursor pools were excluded. Shashoua used a double-labeling technique and found an increase of 13S and 32S RNA in learned fish, and an increased uridine incorporation.

As in the rat brain studies, the synthesis of such presumptive mRNA could be expected to lead to an increased protein synthesis. Indeed, three acidic proteins were found, which were produced by glial-like cells. It was later shown that a single protein, named ependymin, is released from the glia into the extracellular space. This protein requires Ca ++ to remain soluble. A monospecific antiserum against ependymi n produced amnesi a of the acqui red task. Li ke S-100 protei n, ependymin is present in the brain from man to fish. Shashoua suggests that specific firing during learning can give rise to a local Ca++ dep 1 et i on at the cell surface, a format i on of ependymi n fi bers, and eventually formation of new synapses.

DISCUSSION How do cognitive stimuli act? A suggestion.

In the initial stages of training, we found a prompt Ca++ increase in the CA3 region of the hippocampus. Soluble S-100 increased extracel­lularly, as well as membrane-bound S-100 and Ca++, whose confluence in irregular areas increased with training and time. On the basis of these observations, I propose that S-100-differentiated membrane areas, formed over mill ions of neurons during learning, may have fixed charges of coherent energy 1 evel s accordi ng to the observations and hypothesi s of Adey (35). Adey has shown that weak electro-magnetic fields (mW/cm, with a gradient of 0.01 V/cm) can produce a release of Ca++ in brain cortex, if modulated 3 to 40 Hz for defined window effects. Small membrane patches may initiate intracellular events by amplifying specific energy waves across the membrane. The glial S-100 is released into the extracellular space and is incorporated into the nerve cell surface, and also (and this is a main point) in the nuclear membrane.

292

Weak cognitive electro-magnetic fields, initially amplified at neuronal membran S-100/Ca++ patches, could be propagated via the intra­

cell ul ar net of Ca++ -dependent mi crofil aments (36,37) to the nuclear membrane S-100, and amplified there a second time to initiate gene expression by defined solitons (38).

REFERENCES 1. Chandhari, N. and Hahn, W.E. Science 220: 924-928, 1983. 2. Sutcliffe, J.G., Milner, R.J., Gottesfeld, J.M. and Reynolds, W.

Science 225: 1308-1315, 1984. 3. Ringborg, U. Brain Res. l: 296-298, 1966. 4. EdstrBm, J.-E. Biochim. Biophys. Acta 12: 361-386, 1953. 5. EdstrBm, J. -E. and Hyden, H. Nature 174: 128, 1954. 6. Hyden, H. In: The Neurosciences (Eds~.C. Quarton, T. Melnechuk

and F.O. Schmitt), The Rockefeller University Press, New York, 1967, pp. 248-266.

7. Serra, r., Cupello, A., Gadaleta, M.N., Viola, M., Ragonese, P. and Giuffrida, A.-M. Neurochem. Res. 8: 433-447, 1983.

8. Hyden, H. Curro Mod. Biol. 2: 57-60, 1968. 9. Hyden, H. and Lange, P.W. Naturwiss. 53: 64-70, 1966. 10. Cutillo, V., Montagnese, P., Gremo, F., Casola, L. and Giuditta, A.

Neurochem. Res. 8: 1621-1634, 1983. 11. Cupello, A. and HyMn, H. Exp. Brain Res. 31: 143-152, 1978. 12. ZomzelY-Neurath, C., Roberts, S. and Peache, S. Proc. Natl. Acad.

Sci. USA 67: 644-651, 1970. 13. Hyden, H.-and Egyhazi, E. Proc. Natl. Acad. Sci. USA 52: 1030-1035,

1964. 14. Hyden, H. and Egyhazi, E. Proc. Natl. Acad. Sci. USA 48: 1366-1373,

1962. 15. Hyden, H. and Egyhazi, E. Proc. Natl. Acad. Sci. USA 49: 618-624,

1963. 16. EngstrBm, A. and LindstrBm, B. Biochim. Biophys. Acta !: 351-362,

1950. 17. Brattg~rd, S.-D. and Hyden, H. Acta Radiol., Suppl. 94, 1-48, 1952. 18. Moo~e, B.W. Biochem. Biophys. Res. Commun. 19: 739-744, 1965. 19. Hyden, H. and McEwen, B.S. Proc. Natl. Acad. Sci. USA 55: 354-358,

1966. 20. Shashoua, V.E., Hesse, G.W. and Moore, B. J. Neurochem. 42:

1536-1541, 1984. 21. Poletaev, A.B., Miani, N., Michetti, F. and Donato, R. Biochemistry

SSSR 48: 1565-1568, 1983. 22. Hyden~H. and Lange, P.W. Proc. Natl. Acad. Sci. USA 67: 1959-1966,

1970. 23. Zomzely-Neurath, C. and Keller, A. In: Mechanisms, Regulation and

Special Functions of Protein Synthesis in Brain (Eds. S. Roberts, A. Lajtha and W.H. Gispen), Elsevier, Amsterdam, 1977, pp. 279-298.

24. Karpiak, S.E., Serokosz, M. and Rapport, M.M. Brain Res. 102: 313-321, 1976.

25. Haljamae, H. and Lange, P.W. Brain Res. 38: 131-142, 1972. 26. Hyden, H., Lange, P.W. and Larsson, S. J. Neurol. Sci. 45: 303-316,

1980.

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27. Hyden, H. and Lange, P.W. Cell. Molecul. Neurobiol. 1: 313-317, 1981.

28. Cupello, A., Hyd~n, H. and Palm, A. Neurochem. Res. (in press). 29. Hyden, H., Cupello, A. and Palm, A. Brain Res. 294: 37-45, 1984. 30. Hyd~n, H. and'R6nnback, L. Neurobiol. a: 291-302, 1975. 31. Hyd~n, H. and R6nnback, L. Brain Res. 100: 615-628, 1975. 32. Hyden, H. and R6nnback, L. J. Neurobiol. ~: 489-492, 1978. 33. Shashoua, V.E. Proc. Natl. Acad. Sci. USA 74: 1743-1747, 1977. 34. Shashoua, V.E. In: Advances in Cell Neurobiology (Eds. S. Fedoroff

and L. Hertz), Academic Press, New York, Vol. 3, 1982, pp. 97-141. 35. Adey, R.W. In: Biological Effects and Dosimetry of Nonionizing

Radiation (Eds. M. Grandolfo, S.M. Michaelson and A. Rindi), Plenum Press, 1983, pp. 359-391.

36. Han~son, H.A. and Hyden, H. Neurobiol. 4: 364-375, 1974. 37. Hyden, H. Proc. Natl. Acad. Sci. USA Zl: 2965-2968, 1974. 38. Davydoo, A.S. Physica Scripta 20: 387-394, 1979.

25 MOLECULAR GENETICS OF THE NERVE CELL ADHESION MOLECULE N-CAM: EVIDENCE FOR MULTIPLE, DEVELOPMENTALLY REGULATED, mRNA SPECIES G. GENNARINI, M.R. HIRSCH, M. HIRN AND C. GORIDIS Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Marseille, France

The nerve cell adhesion molecule (N-CAM) is a surface glycoprotein and the most extensively characterized cell adhesion molecule of the nervous system (1,2). It is composed of a triplet of transmembrane oriented, glycosylated peptides. Developmentally regulated modifica­tions in its sugar content have been described. The embryonic, highly sialylated form, migrates in polyacrylamide gel electrophoresis as a diffuse band in the MW range of 200 to 250 kD. In adult animals, on the contrary, a triplet of 180, 140 and 120 kD polypeptides is found (3). These biochemical changes are related to corresponding modifications of the functional properties: adult N-CAM displays an adhesive behavior which is not shared by embryonic N-CAM (4).

A large number of studies focused on the structural properties of N-CAM (5-7), as well as on the possible relation to other adhesion molecules (8,9). We approached these problems by molecular genetics studies (10). An N-CAM-specific cDNA clone was isolated by antibody screening of a mouse neuroblastoma library prepared in the expression vector Agt11. The sequence was subcloned in the plasmid pBR328 and ut il i zed to probe Southern blots of mouse genomi c DNA restri ction fragments, as well as Northern blots of poly(AtmRNA prepared from adult and developing mouse forebrain.

One to two N-CAM related sequences were detected in genomic blots. These results, taken together with restriction fragment mapping of cosmid clones of genomic DNA, strongly indicate that a single N-CAM gene exists in the mouse genome. On the other hand, the probe hybridized to 5 different mRNA species ranging between 2.9 and 7.4 kb in length. All of them were specifically expressed in the brain and developmentally regulated. In young animals, the two higher MW mRNAs (7.4 and 6.7 kb) were predominant, while adult mouse brain mainly contained the 5.2 and 2.9 kb species. A 4.3 kb mRNA was relatively more abundant in embryonic and perinatal brain. The derivation of multiple, developmentally

295

regulated mRNA chains from a 1 imited number of genes (probably only one), would involve differential splicing of precursor nuclear transcripts or could require the utilization of different initiation or polyadenylation sites.

Irrespective of the exact mechanism of N-CAM mRNA formation, it appears that different messengers code for the N-CAM polypeptides and that the reported developmental and regional heterogeneity of the molecule is controlled, at least in part, at the transcriptional level.

REFERENCES 1. Goridis, C., Oeagostini-Bazin, H., Hirn, M., Hirsch, M.R., Rougon,

G., Sadoul, R., Langley, O.K., Gombos, G. and Finne, J. Cold Spring Harbor Symp. Quant. 48: 527-538, 1983.

2. Edelman, G.M., Hoffman, S., Chuong, C.M., Thiery, J.P., Brackenbury, R., Gallin, W.J., Grumet, M., Greenberg, M.E., Hemperly, J.J., Cohen, C. and Cunningham, B.A. Cold Spring Harbor Symp. Quant. 48: 515-526, 1983.

3. Rougon, G., Deagostini-Bazin, H., Hirn, M. and Goridis, C. EMBO J. 1: 1239-1244, 1982.

4. Sadoul, R., Hirn, M., Deagostini-Bazin, H., Rougon, G. and Goridis, C. Nature 304: 347-349, 1983.

5. Hoffman, S., Sorkin, B.C., White, P.C., Brackenbury, R., Mai 1 hammer, R., Rutishauser, V., Cunningham, B.A. and Edelman, G.M. J. Biol. Chem. 257: 7720-7729, 1982.

6. Gennarini, G., Rougon, G., Deagostini-Bazin, H., Hirn, M. and Goridis, C. Eur. J. Biochem. 142: 57-64, 1984.

7. Gennarini, G., Hirn, M., Deagostini-Bazin, H. and Goridis, C. Eur. J. Biochem. 142: 65-73, 1984.

8. Grumet, M., Hoffman, S. and Edelman, G.M. Proc. Natl. Acad. Sci. USA 81: 267-271, 1984.

9. Kruse, J., Mail hammer, R., Wernecke, H., Faissner, A., Sommer, I., Goridis, C. and Schachner, M. Nature 311: 153-155, 1984.

10. Goridis, C., Hirn, M., Santoni, M-J., Gennarini, G., Deagostini­Bazin, H., Jordan, B.R., Keifer, M. and Steinmetz, M. EMBO J. !: 631-635, 1985.

26

THE CHARACTERIZATION OF cDNAs ENCODING BRAIN-SPECIFIC AND UBIQUITOUS mRNA BY HYBRID-SELECTED TRANSLATION C. HALL, T. LEUNG AND L. LIM Department of Neurochemistry, Institute of Neurology, London, UK

Many membrane and secretory proteins are synthesized by membrane­bound polysomes and may undergo post-translational modification, such as glycosylation in the rough endoplasmic reticulum and/or in the Golgi apparatus. In the brain, membrane proteins may playa key role during development, when cellular contacts are established and membrane glyco­proteins (such as the acetylcholine receptor) are initially involved in synaptic transmission. In addition to neurotransmitter receptors, other proteins of synaptic plasma membranes have been identified. These include cytoskeletal proteins, e.g. tubulin and actin (1), spectrin (2,3), 68 kD microtubule-associated protein (68K MAP) (4-6) and the enzymes Ca++/calmodulin-dependent protein kinase (PSD-50) (7), creatine kinase, neuron-specific enolase and pyruvate kinase (5). In the brain, these 1 atter enzymes are synthesized by both free and membrane bound polysomes (5,9). Other translation products are exclusive to membrane­bound polysomal mRNA (9-11) and may encode as yet unidentified synaptic membrane proteins. Several of the products of abundant mRNAs specifi­cally associated to membrane-bound polysomes are also developmentally regulated (10). To investigate these mRNAs further, cDNA copies of membrane-bound polysomal mRNA were cloned in E. co7i.

Poly(A)+RNA, isolated from rat brain membrane-bound polysomes, was size-fractionated on sucrose density gradients to enrich for mRNAs encoding developmentally regulated polypeptides (20 to 50 kD). Double­stranded (dC)-tailed cDNAs, synthesized from pooled mRNA fractions (10 to 18S approximately), were annealed to Pstl-cut, (dG)-tailed plasmid pAT153 and used to transform E. co7i RRI.

cDNA clones were grown on duplicate nitrocellulose filters, and those containing sequences exclusive to membrane-bound polysomal mRNA, were identified by differential colony hybridization (12). [32P]labeled cDNA copies of either free or membrane-bound polysomal RNA were used as hybridization probes. Approximately 27% of the hybridizing colonies

297

were specific to the membrane-bound polysomal fraction, including clones

M444 and M1622. Others hybridized with equal intensity to both probes

(these would probably include cDNAs for tubulin and actin) and some

clones (e.g. FM564 and FM1636) apparently contained sequences which,

a lthough present in membrane-bound po lysoma 1 mRNA, are more abundant

in free polysomal mRNA. Plasmid DNA from selected clones was purified

and cDNA inserts excised with Pstl. The restriction digests analyzed on

ethidium bromide-stained agarose gels indicated that the inserts ranged

from 200 to 1200 bp in size.

Membrane-bound polysomal mRNA was hybridized to selected nitrocel­

lulose-bound cDNAs. After stringent washing (6), hybridized mRNA was

eluted, translated in vitro and the products analyzed on 2D-gels. In

control hybridizations with parent plasmid (pATl53), the single component on 2D-ge 1 analys is was the ret i cul ocyte lysate background 46

kD protein. The MW of the hybrid-selected translation products were: M1622, 55 kD; FM564, 38 kD; M444, 21 kD; FM1636, 12 kD.

On 2D-gels, the basic 38 kD translation product, encoded by mRNA hybridizing to FM564, co-migrated with a native brain protein which is a

component of brain synaptic plasma membranes, as well as of cytosol (and which could be an enzyme). The translation products encoded by hybrid­

selected M444 mRNA and by M1622 mRNA did not correspond to any brain protein counterpart. Recent results (C. Lowndes) have shown that the translation product of M444 hybrid-selected mRNA is processed in vitro by dog pancreas microsomes and it may be further post-translationally modified in vivo.

On Northern ana lys is, [32 P]l abe 1 ed cDNAs FM564, M444 and M1622 hybridized to single bands of brain mRNA corresponding to 1.6 kb, 1.4 kb and 2.0 kb, respectively. FM564 hybridized to a ubiquitous mRNA found

in total cellular poly(A)+RNA from kidney, spleen, pancreas and liver,

as well as to free and membrane-bound brain polysomal mRNA. In

contrast, M444 and M1622 hybridized only to brain poly(A)+ RNA and

specifically to that of membrane-bound polysomes.

The mRNA corresponding to M444 increased gradually with develop­

ment, its appearance coinciding with synaptogenesis and the onset of

myel ination in the rat brain. This confirms our previous observation of a developmental increase in the 21 kD translation products (8).

298

M1622 mRNA appeared to be present in lower concentrations at two days than at subsequent ages.

In conclusion, we have isolated and characterized, by hybrid-selec­ted translation, two cloned cDNAs: M444 (encoding a 21 kD polypeptide) and M1622 (encoding a 55 kD polypeptide), whose corresponding mRNAs are specific to brain membrane-bound polysomes. The M444 and, to a lesser extent, the M1622 sequences are developmentally regulated, and the relative abundance of the mRNA suggests an important role in brain development. Their identities and post-translational processing are currently being investigated. Two other cloned cDNAs, FM564 (encoding a basic 38 kD protein) and FM1636 (encoding an acidic 12 kD protein) correspond to ubiquitous mRNAs. The brain protein counterpart of FM564 is a constituent both of cytosol and of synaptic plasma membranes.

We thank the Brain Research Trust, the Worshipful Company of Pewterers and the Wellcome Trust for financial support.

REFERENCES 1. Kelly, P.T. and Cotman, C.W. J. Cell Biol. 79: 173-183, 1978. 2. Bennett, V., Davies, J. and Fowler, W.E. Nature 299: 126-131, 1982. 3. Carlin, R.K., Bartelt, D.C. and Siekevitz, P. J. Cell Biol. 96:

443-448, 1983. 4. Strocchi, P., Brown, B.A., Young, J.D., Bonventre, J.A. and Gilbert,

J.M. J. Neurochem. 37: 1295-1307, 1981. 5. Lim, L., Hall, C., Leung, T., Mahadevan, T. and Whatley, S. J.

Neurochem. 41: 1177-1181, 1983. 6. Lim, L., Hall, C., Leung, T. and Whatley, S. Biochem. J. 244:

677-680, 1984. 7. Kelly, P.T. and Montgomery, P.R. Brain Res. 233: 265-286, 1982. 8. Kelly, P.T., McGuiness, T.L. and Greengard, P. Proc. Natl. Acad.

Sci. USA 81: 945-949, 1984. 9. Hall, C., Mahadevan, L., Whatley, S., Biswas, G. and Lim, L.

Biochem. J. 219: 751-761, 1984. 10. Hall, C. and Lim, L. Biochem. J. 196: 327-336, 1981. 11. Gilbert, J.M. and Strocchi, P. J. Neurochem. 40: 153-159, 1983. 12. Grunstein, M. and Hogness, D.S. Proc. Natl. Acad. Sci. USA 72:

3961-3965, 1975.

27 OPIOID PEPTIDE PRECURSORS IN THE AMPHIBIAN XENOPUS LAEVIS G.J.M. MARTENS, O. CIVELLI AND E. HERBERT Department of Zoology, University of Nijmegen, The Netherlands

Opioid peptides exhibit morphinomimetic activity when injected into animals. The simplest opioid peptides are methionine (met) and leucine (leu)enkephalin, while the others are carboxy terminal extensions of either met- or leu-enkephalin. The sequences of the opioid peptides are contained within the opioid peptide precursor proteins pro-opiomelano­cortin (POMC), proenkephalin (proenkephalin A) or prodynorphin pro-enke­phal in B). The amino acid sequences of these precursors have been determi ned in some mammal i an spec i es by the use of recombi nant DNA technology (1-7). The structures of the prohormones are characterized by the occurrence of repeated sequences of biologically active peptides, melanophore stimulating hormone (MSH) sequences in the case of POMC and enkephalin sequences in proenkephalin and prodynorphin. The repeat units might have been generated during evolution by duplication events. In order to gain insight into the structural organization of the precur­sor proteins during phylogeny and to establ ish the sequences of some opioid peptides in lower vertebrates, we determined the primary struc­tures of POMC and proenkephalin in the South African clawed toad, Xenopus laevis. This amphibian species diverged from the principal line of vertebrate evolution some 350 million yr ago. The amino acid sequence of Xenopus proenkephalin was derived from the nucleotide sequence of a DNA fragment isolated from a genomic library. The primary structure of Xenopus POMC was deduced from the nucleotide sequence of a cloned cDNA isolated from a pituitary cDNA library.

The Xenopus genomi c 1 i brary contained partial MboI -di gested blood cell DNA cloned into the BamH1 site of the vector AJ1 and was construc­ted by Drs. Y. Chien and I.B. Dawid (NIH, Bethesda, MD, USA). The 1 i brary was screened under reduced stringency of hybridi zat ion condi­tions (8) using a nick-translated human proenkephalin cDNA probe (918 bp), which contains the full protein-coding region (provided by Dr. M. Comb, University of Oregon, Eugene, OR, USA). The eDNA library was constructed from 2 119 of pituitary poly(A)+RNA isolated from blaek-

300

adapted Xenopus laevis, and contained approximately 6000 transformants. The library was screened with a synthetic oligodeoxyribonucleotide (tetradecamer) corresponding to the His-Phe-Arg-Trp-Gly sequence of a -MSH. Approximately 0.5-1% of the clones were found to be positive. DNA sequence analysis was performed by the dideoxy chain termination method (9) using M13 mp8 and mp9 subclones as templates.

The partial amino acid sequence of Xenopus proenkephalin could be deduced from the nucleotide sequences of a DNA fragment isolated by probing the Xenopus genomic library with a human proenkephalin cDNA probe. Xenopus proenkephalin contains seven enkephalin sequences, namely five copies of met-enkephal in, one copy of met-enkephal in-Arg­Gly-Tyr and one copy of met-enkephalin-Arg-Phe, but no leu-enkephalin. Mamma 1 ian proenkepha 1 ins contai n four copies of met-enkepha 1 in and one copy each of met-enkephalin-Arg-Gly-Leu, met-enkephalin-Arg-Phe and leu­enkephalin. The absence of a leu-enkephalin sequence in Xenopus proen­kephalin suggests that met-enkephalin is the primordial duplication unit duri ng proenkepha 1 in evo 1 ut ion, and that one met-enkepha 1 in sequence relatively recently switched to a leu-enkephalin sequence in mammalian proenkephalins (by the switch of one nucleotide). This finding reinforces the concept that the proenkephalin gene evolved by a series of duplication and rearrangement events. The structural organization of the proenkephal in proteins are remarkably similar between Xenopus and mammals. As in mammals, all enkephalin sequences in Xenopus proenkeph­alin are flanked on both sides by pairs of basic amino acids, which are believed to be recognition sites of proteolytic cleavage enzymes (10).

Similar to the isolation of the Xenopus proenkephalin clone, we attempted to isolate a clone for Xenopus POMC by screening the genomic library with a probe prepared by nick-translation of a mouse POMC cDNA clone. However, even under low-stringency hybridization conditions, no reproducible signal could be detected either in the library screening or in Southern analyses of genomic DNA. Hence, we tried a different approach involving the screening of a 1 ibrary of reduced complexity (cDNA library) with a different probe (synthetic oligodeoxyribonucleo­tide). The cDNA library was constructed from mRNA isolated from Xenopus pituitary glands, a tissue which produces large amounts of POMC. The synthetic probe was a pool of tetradecamers corresponding to the penta-

301

peptide sequence His-Phe-Arg-Trp-Gly in a-MSH. The sequence His-Phe-Arg -Trp is responsible for the melanophore-stimulating activity of the hormone (11) and it is conserved in all melanotropic peptides. More­over, on the basis of chymotryptic peptide mapping of newly-synthesized a-MSH, in combination open with selective amino acid incorporation studies, we could establish that this sequence is present in Xenopus a-MSH.

Several hybridization-positive clones could be isolated from the Xenopus pituitary eDNA library using the tetradecamer probe. The structure of Xenopus POMC was deduced from the nucleotide sequences of these clones. The sequences of a, a, and y -MSH, corti cotropi n-l ike intermediate lobe peptide (CLIP) and a-endorphin, are present in Xenopus POMC, and their locations within the prohormone are very similar to those of the mammalian counterparts. Thus, as in the case of proenkeph­alin, it appears that the structural organization of POMe is remarkably conserved duri ng phylogeny. The spacer regi ons between the bi oact i ve domains, however, share an extremely low degree of homology, although these regions are almost identical in length between Xenopus and mamma 1 s. Again, the bi oact i ve pept ides in Xenopus POMe are fl anked by pairs of basic amino acids. Most of these pairs appear to be cleavage sites of specific processing enzymes, as determined in in vitro biosynthetic studies (12). It was very interesting to note that the N­terminal amino acid of Xenopus a-MSH is not serine, as in mammal ian a-MSH, but alanine. This indicates that diacetyl-a-MSH, present in rat pituitary (13), cannot be found in Xenopus. The N-terminal amino acid of a-MSH is acetylated and this acetylation greatly enhances the biolog­ical activity of the hormone (11). Whether a Ser/Ala substitution affects the acetylation reaction and/or the bioactivity of a-MSH is at present unknown. The fact that Xenopus 13-endorphi n, as other known endorphins, contains a met-enkephalin sequence at its N-terminus, suggests that this peptide could display opiate-like activity in Xenopus.

In conclusion, the structural organization of the opioid peptide precursor proteins proenkephalin and POMe, and the structures of their component bioactive domains are remarkably well conserved during vertebrate evolution. This suggests that opioid peptides might well

302

have a function in lower vertebrates. In addition to the evolution of the genes for opioid peptide precursors, evolution of the genes recep­tors and processing enzymes may have created families of receptors and enzymes having different affinities for the opioid peptides and their precursors. Thus, more specialized peptide-receptor interactions could be generated to meet the demands of progressively more complex organisms.

This work was supported by NIH Grants AM16879, AM30155 and DA02736 to E.H. G.J.M.M. is supported by the Netherlands Organization for the Advancement of Pure Research (ZWO).

REFERENCES 1. Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A.C.Y.,

Cohen, S.N. and Numa, S. Nature 278: 423-427, 1979. 2. Cochet, M., Chang, A.C.Y. and Cohen, S.N. Nature 297: 335-339,

1982. 3. Whitfeld, P.L., Seeburg, P.H. and Shine, J. DNA 1: 133-143, 1982. 4. Notake, M., Tobimatso, T., Watanabe, Y., Takahashi, H., Mishina,

M. and Numa, S. FEBS Lett. 156: 67-71, 1983. 5. Noda, M., Teranishi, Y., Takahashi, H., Toyosato, M., Notake, M.,

Nakanishi, S. and Numa S. Nature 297: 431-434, 1982. 6. Comb, M., Rosen, H., Seeburg, P.H., Adelman, J. and Herbert, E.

DNA Z: 213-229, 1983. 7. Horikawa, S., Takai, T., Toysato, M., Takahashi, H., Noda, M.,

Kakidani, H., Kubo, S., Hirose, T., Inayama, S., Hayashida, H., Miyata, J. and Numa, S. Nature 306: 611-614, 1983.

8. Benton, W.D. and Davis, R.W. Science 196: 180-182, 1977. 9. Sanger, F., Nickleu, S. and Coulson, A.R. Proc. Natl. Acad. Sci.

USA 74: 5463-5467, 1977. 10. Douglass, J., Civelli, O. and Herbert, E. Ann. Rev. Biochem. 53:

665-715, 1984. 11. Schwyzer, R. and Eberle, A. In: Frontiers of Hormone Research (Ed.

Tj.B. Wimersma Greidanus), Karger, Basel, 1977, pp. 18-25. 12. Martens, G.J.M., Jenks, B.G. and van Overbeeke, A.P. Eur. J.

Biochem. 122: 1-10, 1982. 13. Rudman, D., Chawla, R.K. and Hollins, B.M. J. Biol. Chern. 254:

10102-10108, 1979.

28 SENSITIVE HYBRIDIZATION TECHNIQUES AS POWERFUL TOOLS IN MOLECULAR GENETICS TO IDENTIFY BRAIN-SPECIFIC GENE PRODUCTS T.A. RHYNER, A.A. BORBELy 1 AND J. MALLET Laboratoire de Neurobiologie Cellulaire et MolEkulaire, CNRS, Gif-sur­Yvette, France and 1 Pharmako 1 ogi sches Inst itut der Uni vers i t:1t ZUri ch, ZUrich, Switzerland

As a consequence of the compl exity of the nervous system, many characteristic proteins are only expressed at a low abundance and their detection and purification often represents a formidable task. Only a few dozen brain-specific proteins have so far been characterized and many functionally identified molecules have not yet been purified to homogeneity.

The recent advances of molecular genetics provide a new and powerful approach to identify and, thereafter, to purify relevant proteins. Along this line, Milner and Sutcliffe (1) have recently generated a cDNA library of adult male rat poly(A)+ mRNA and have selected, by Northern blot analysis, clones corresponding to mRNAs which are specifically expressed in brain. This analysis also revealed interesting characteristics of nervous tissue-specific mRNAs.

In this paper, we describe an alternative strategy which allows a rapid screening of cDNA clones encoding low abundance transcripts. As a first step toward determining whether specific transcripts can be i dent i fi ed that are induced by sl eep-depri vat i on, cDNA was generated from sleep-deprived forebrain mRNA and selected by hybridization in liquid medium with cerebellum mRNA. This subtracted cDNA population was used to construct a 1 ibrary. In a first series of experiments, this library was screened by colony hybridization with forebrain cDNA probes subtracted with cerebellum mRNA. Transcripts which were specific to this structure could be identified, although they were expressed at a very low level.

Total poly(A)+RNA from nervous tissue was isolated according to standard techniques (2,3). The integrity of the poly(A)+ RNA prepara­tions was verified by their ability to be efficiently transcribed into cDNA (see below) and translated in a reticulocyte lysate (4).

A standard procedure was used for single-strand (ss) cDNA synthesis

304

(5), except that 4 mM sod i urn pyrophosphate was added to the react ion mixture. After RNA hydrolysis, ss-cDNA was subjected to an RNA-driven hybridization (Rot=1500 M.s/l) for 60 hr at 68 0 in a sodium phosphate/ sodium chloride buffer containing 10 ].Jg/ml poly(U). The unhybridized cDNA was purified by chromatography on hydroxylapatite (HAP). Double­strand (ds) cDNA synthesi sand bacteri a 1 transformation was performed according to Wickens et al. (6) and Hanahan (7). cDNA probes were synthes i zed and subtracted as descri bed above. Co 1 ony hybri d i zat ions were carried out as described by Hanahan and Meselson (8), under stringent conditions.

The strategy used to generate subtracted cDNA libraries is based on specific cDNA-mRNA hybridization followed by purification of the unhybridized cDNA by HAP chromatography. For the subtraction to work efficiently, it is necessary that the cDNAs be present initially in single-stranded form. However, reverse transcriptase may also catalyze the synthesis of anticomplementary DNA. Under standard conditions, most of the transcribed cDNA has a second strand DNA counterpart, resulting in subtraction artifacts.

The effect of sod i urn pyrophosphate and act i nomyc in D, wh i ch have been reported to reduce anticomplementary DNA synthesis of viral RNA templates (9,10), were tested. Sodium pyrophosphate, at an optimal concentration of 4 mM, prevented nearly all the second strand synthesis and, consequently, < 5% of the cDNA eluted as ds-DNA from HAP. A short transient hairpin-like structure occurred, however, at the 3' end of the ss-cDNA, which could be used after the subtraction procedure for priming the second strand cDNA synthesis with high efficiency. In contrast, actinomycin D (50-150 ].Jg/ml) was not very potent in reducing the synthesis of ds-cDNA, either alone or in combination with pyrophosphate.

We then constructed a subtracted cDNA 1 ibrary with poly(A)+ RNA originating from sleep-deprived rats (11). Subtractions were performed with cerebellum mRNA because this brain area is mostly involved in functions such as planning, initiation and control of movement (12), and there is no evidence of its involvement in sleep. For the subtraction of a compl ex ss-cDNA popul at ion deri ved from forebrai n, hybridi zat ion has to be performed at high Rot values. An Rot of 1500 M.s/l is satis­factory for the hybridization of brain cDNA transcribed from cytoplasmic

305

(13) and nuclear (14) poly(A)+ RNA. Under these conditions, we could remove about 90% of a total forebrain cDNA population using an excess of cerebellum poly(A)+RNA. This selected cDNA population could then be cloned with high efficiency in E. co7i (200 clones/ng ds-cDNA) using plasmid pBR322 as vector. As a result of the drastic hybridization conditions, inserts ranging from only 250 to 500 bp were obtained. Nevertheless, these lengths were sufficient for specific and accurate screening by colony hybridization.

To test the specificity of the subtraction procedure, we hybridized clones from this library with a cerebellum ss-cDNA probe. About 6% of the clones gave a positive signal. To ensure that this small percentage did not result from the short size of the inserts of the subtracted 1 i brary, we al so screened a non-subtracted control forebrain 1 i brary. This control library was constructed with a pseudo-subtracted forebrain cDNA popul at ion (i. e. incubated for 60 hr at 68 ° in the absence of cerebellum mRNA). The corresponding inserts ranged also from 250 to 500 bp in length. About 50% of the clones hybridized with the cerebellum cDNA probe. This result shows that most forebrain transcripts, also expressed in cerebe 11 urn, were eli mi nated by our subtract i on procedure prior to cloning.

Figure 1-1 shows the hybridization pattern of one hundred randomly chosen clones from the subtracted library hybridized with a forebrain cDNA probe. Interestingly, the six clones which were vi sual i zed with this probe were also detected using a cerebellum cDNA probe (results not shown). To improve the sensitivity of the screening procedure, these clones were screened with a cDNA probe subtracted with cerebellum poly(A)+RNA. The six clones that were labeled with the total forebrain probe still exhibited a faint signal but, in addition, five clones were revealed that encode transcripts not expressed at detectable levels in cerebellum (Fig. 1-3). Figure 1-3 also shows that the use of subtracted probes greatly decreases hybridization backgrounds. Since the sub­tracted and unsubtracted probes had the same specific activity, the question arose as to whether the decreased background resulted from the shortening of the probe that occurred during the subtraction procedure (see above). This was tested by incubating the un subtracted probe for 60 hr at 68°. Under these conditions, the background signals were not

306

,

Fig. 1. Detection of forebrain transcripts by colony hybridization. The same 100 clones from the subtracted forebrain cDNA 1 ibrary were placed on the three filters. Filters were hybridized with forebrain cDNA probe (3 x 10 5 cpm/ml), using total tissue poly(A)+RNA as template (left panel); the same probe (6 x 105 cpm/ml) incubated 50 hr at 68° in the subtraction buffer (pseudo-subtraction) (central panel); the same probe subtracted with cerebellum total poly(A) RNA (5 x 105 cpm/ml) (right panel). The underl ined areas correspond to clones transformed with the unmodified pBR322 plasmid (background). Exposure time, 24 hr (left panel); 18 hr (central panel) and 6 days (right panel).

significantly reduced (Fig. 1-3), although the size of the cDNA probe had decreased.

Clearly, as previously reported (15-18), this result shows that the use of adsorbed probes greatly enhances the sensitivity of the screening procedures. A quant i tat i ve assessment was then performed by Northern blot analysis, using a previously characterized tyrosine hydroxylase cDNA clone (19): transcripts present in a tissue at an abundance as low as 0.0003% can still be detected by colony hybridization with subtracted probes (results not shown).

Applications of this methodology are numerous and should allow the ident i fi cat i on of clones correspondi ng to functionally important proteins, such as neurotransmitter synthesizing enzymes, receptors, and ionic channels. Rat choline acetyl transferase cDNA clones have recently been isolated through this approach in our laboratory (Berrard et al., in preparation). This method should also allow the identification of transcripts which are stage- or tissue-specific, or whose expression in mature brain is physiologically or pharmacologically modulated. More

307

specifically, the search for possible transcripts induced by sleep­deprivation can now be approached using this strategy. Isolation of specific sequences permits access to the protein itself, and allows for the study of its structure, function and regulation. By such means, it may be hoped that many important proteins will be uncovered that would not have been accessible by conventional biochemical techniques.

We gratefully acknowledge the helpful discussions and technical assistance of all our laboratory colleagues. This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the Fondation pour la Recherche Medicale Francaise, the Association pour la Recherche sur 1 e Cancer and Rhone- Poul enc Sante. TAR recei ved a fell owsh i p from the Fonds National Suisse de la Recherche Scientifique.

REFERENCES 1. Milner, R.J. and Sutcliffe, J.G. Nucleic Acids Res. 11: 5497-5520,

1983. 2. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J.

Biochem. 18: 5294-5299, 1979. 3. Aviv, H. and Leder, P. Proc. Natl. Acad. Sci. USA 69: 1408-1412,

1972. 4. Merrick, W.C. Meth. Enzymol. !Ql: 606-615, 1983. 5. Buell, G.N., Wickens, M.P., Payvar, F. and Schimke, R.T. J. Biol.

Chem. 253: 2471-2482, 1978. 6. Wickens, M.P., Buell, G.N. and Schimke, R.T. J.Biol. Chem. 253:

2483-2495, 1978. 7. Hanahan, D. J. Mol. Biol. 1§2: 557-580, 1983. 8. Hanahan, D. and Meselson, M. Gene 10: 63-67, 1980. 9. Myers, J.C. and Spiegelman, S. Proc. Natl. Acad. Sci. USA 75:

5329-5333, 1978. 10. Ruprecht, R.M., Goodman, N.C. and Spiegelman, S. Biochim. Biophys.

Acta 294: 192-203, 1973. 11. Borbely, A.A. and Neuhaus, H.U. J. Compo Physiol. 133: 71-87, 1979. 12. Allen, G.I. and Tsukahara, N. Physiol. Rev. 54: 957-1006, 1974. 13. Hastie, N.D. and Bishop, J.O. Cell~: 761-774, 1976. 14. Hahn, W.E., Van Ness, J. and Maxwell, I.H. Proc. Natl. Acad. Sci.

USA 75: 5544-5547, 1978. 15. Timberlake, W.E. Dev. Biol. 78: 497-510, 1980. 16. Scott, M.R.D., Westphal, K.-H. and Ribgy, P.W.J. Cell 34: 557-567,

1983. 17. Kavathas, P., Sukhatme, V.P., Herzenberg, L.A. and Parnes, J.P.

Proc. Natl. Acad. Sci. USA 81: 7688-7692, 1984. 18. Hedrick, S.M., Cohen, 0.1., Nielsen, E.A. and Davis, M.M. Nature

308: 149-153, 1984. 19. Lamouroux, A., Faucon Biguet, N., Samolyk, D., Privat, A., Salomon,

J.C., Pujol, J.F. and Mallet, J. Proc. Natl. Acad. Sci. USA 79: 3881-3885, 1982.

29 MOLECULAR CLONING AND NUCLEOTIDE SEQUENCES OF cDNA AND GENOMIC DNA FOR THE RAT BRAIN S-100 PROTEIN Y. TAKAHASHI, R. KUWANO, H. USUI, T. MAEDA AND T. IWANAGA 1

Department of Neuropharmacology, Brain Research Institute and 1 Department of Anatomy, School of Medicine, Niigata University, Japan

S-100 protein is a brain-specific Ca ++ -binding protein and is localized mainly in astrocytes in the CNS. We previously observed the in vitro translation of mRNA for the S subunit of this protein in a reticulocyte cell-free system (1). Recently, we succeeded in cloning DNA complementary to rat brain mRNA for the S-100 S subunit and deter­mined the nucleotide sequence of this cDNA (2). In addition to cDNA, we describe here the successful cloning and nucleotide sequence determina­tion of genomic DNA for the S subunit of the S-100 protein.

The procedures for cloning and nucleotide sequence determination of S-100 cDNA were previously reported (2). A rat genomic 1 ibrary was kindly given by Dr. J. Bonner. The phage containing genomic DNA was inoculated into E. co7i strain DP50SupF and screened by in situ plaque hybridization (3) with nick-translated S-100 cDNA as a probe. DNA fragments of the S-100 gene were subcloned into the EcoRI and Hi ndII I sites of the pBR322 for subsequent structural analyses. Restri ct ion enzyme digests were analyzed by el ectrophoresi s on polyacryl amide and agarose gels and their sequences were determined by the method of Maxam and Gilbert (4).

The sequence of our S-100 cDNA clone contained 1488 bp which included the 276 bp of the complete coding region, 120 bp of the 5'noncoding region and 1092 bp of the 3'noncoding region containing two polyadenylation signals (2). In addition, the poly(A) tail was also found. The amino acid sequence, deduced from the nucleotide sequence, was homologous to the amino acid sequence of the S subunit of bovine S-100 except for four residues showing species differences.

We used the full-l ength cDNA clone to probe a rat Hae I II genomi c library by plaque hybridization and isolated several genomic clones (Fig. 1). DNA fragments of the positive clones were sublconed into the EcoRI and Hindlll sites of pBR322 and used for further structural analysis. The restriction enzyme map of the S-100 gene, deduced by

309

.. 1/1 I ...

Fig. 1. Southern blot analysis of the rat genomic DNA library. The rat HaeIII gene 1 ibrary was screened by plaque hybridization using S-100 cDNA as probe. DNA from four genomic clones was digested with EcoRI and HindII!. Left: DNA digests were electrophoresed on a 1% agarose gel and stained with ethidium bromide. Right: Southern blot analysis of the same gel carried out using [32P]cDNA probes. HindIII digests of DNA were used as MW markers.

analysis of these cloned DNA fragments, is shown in Fig. 2A. Southern blot ana lys is of the EcoRI fragments of rat total genomi c DNA, us i ng cDNAs as probes, gave bands of essentially the same si ze as those calculated for the cloned genomic DNA (Fig. 28). These results provide evidence for a single chromosomal locus for the S-100 gene.

We detected the approximate location of exons in the S-100 gene by Southern blot analysis of various restriction fragments of the cloned DNA, using nick-translated [32p]cDNA as probe, and then compared their sequence to that of the cDNA. Thus, we coul d locate at 1 east two introns and three exons showing typical GT and AG junctions as shown in Fig. 2A. Interestingly, within intron 1 of the S-100 gene, we could identify the 10 sequence initially described by Sutcliffe (5). It must be mentioned that the subunit of the S-100 protein is not neuron-spe­cific. We have not yet been able to determine the exact transcription initiation site, but analyses are in progress using S nuclease protec­tion mapping and primer extension. Additionally, we have examined rat cerebellar sections by in situ hybridization using nick-translated

310

A

I

-- 15II: I: 151 I: :c: 0: 15! EE 'tiE E'tI E 0 'tI mm .5m m.5m u c

IDID :tID 1D:t1D W :f II. I I III I I n 2

II

-lkb

• B

origin

• 23 kb

- - 9.4

6.5 • If .. 4.3 • 2.3 • 2.0

Fig. 2. Organization of the rat S-100 «(3 subunit) gene. (A) Restric­tion nuclease map and localization of exons and introns. The cleavage sites of BamHI, HindIII and EcoRI restriction nucleases are shown. The localization of exons (I to III, filled boxes) and introns (1 and 2) were determined as described in the text. (B) Southern blot analysis of rat total genomic DNA. Total genomic DNA was digested with EcoRI, HindIII and BamHI restriction endonucleases and hybridized to cDNA probes covering (a) the coding region and (b) the 3'-noncoding region. MW markers were as in Fig. 1.

[3H]cDNA (6), and found positive signals in astrocytes, which also showed positive immunohistochemical staining using peroxidase-antiper­oxidase antibodies following treatment with the antiserum against the (3 subunit of S-100 (Fig.3).

In the future, we plan to examine cell-specific gene expression in the eNS using S-100 cDNA and genomic DNA as probes.

311

Fig. 3. In situ hybridization of S-100 mRNA. [3H]cDNA was hybridized to fixed rat cerebellar tissue sections following the procedure of McAllister et al. (6). Magnification, x 715.

REFERENCES 1. Masuda, T., Sakimura, K., Yoshida, Y., Kuwano, R., Isobe, T.,

Okuyama, T. and Takahashi, Y. Biochim. Biophys. Acta 740: 249-254, 1983.

2. Kuwano, R., Usui, H., Maeda, T., Fukui, T., Yamanari, N., Ohtsuka, E., Ikehara, M. and Takahashi, Y. Nucleic Acids Res. lZ: 7455-7465, 1984.

3. Benton, W.O. and Davis, R.W. Science 196: 180-182, 1977. 4. Maxam, A.M. and Gilbert, W. Meth. Enzymol. 65: 499-560, 1980. 5. Sutcliffe, J.G., Milner, R.J., Bloom, F.E. and Lerner, R.A.

Proc. Natl. Acad. Sci. USA 79: 4942-4946, 1982. 6. McAllister, L.B., Scheller, R.H., Kandel, E.R. and Axel, R. Science

222: 800-808, 1983.

AUTHOR INDEX

Ambrosini, M.V. Arveiler, B.

Ball, S.P. Bernocchi, G. Berod, A. Bl anot, F. Blum, M. Boni, C. Borbely, A.A. Boue, J. Brown, I.R. Buda, N. Buono, C.

Camerino, G. Cerbone, A. Chaudhari, N. Ci ve 11 i, O. Comb, M. Coutelle, Ch.

Davies, K.E. DeGennaro, L.J. Dorkins, H.R.

Faucon Biquet, N Forrest, S.

Gennarini, G. Gi nzburg, I. Gioio, A.E. Girma, B. Giuditta, A. Giuffrida, A.M. Goridis, C. Griffin, W.S.T.

Haas, C.A. Hahn, W. E. Hall, C. Herbert, E.

268 131

123 197

57 57

112 57

303 131 174

57 268

131 268 10

299 90

123

123 71

123

57 123

294 81

1 57

1,42,268 182 294 142

71 10

160,296 90,299

Hirn, M. Hirsch, M.R. Horellou, Ph. Hunt, T. Hyden, H.

Ittel, M.E. Iwanaga, T.

Julien, J.-F.

Kaplan, B.B. Kenwrick, S.J. Kil imann, M.W. Kuenzle, C.C. Kuwano, R.

Lamberti, C. Lamouroux, A. Lavenir, I. Leung, T. Lim, L. Liston, D. Littauer, U.Z.

Maeda, T. Mallet, J. Mandel, J. L. Mandel, P. Mares, V. Martens, G.J.M. Masmoudi, A. Mattei, J.F. Mattei, M.G. McGlade, s. McKinnon, R.D. Menna, T. Milner, R.J. Morelli, F. Morrison, M.R.

Niedergang, c.

294 294

57 42

280

233 308

57

1,42 123

71 211 308

268 57

123 160,296 160,296

90 81

308 57,308

131 233 247 299 233 131 131 123

23 268 32

268 142

233

314

Oberle, 1. 131 Smith, T. 123 Owens, G. 10 Speer, A. 123

Subba Rao, K. 224 Sutcliffe, J.G. 23

Perrone, C. 1,42,268 Powell, J. 57

Takahashi, Y. 308 Thomas, G. 90

Reinis, S. 256 Thomassin, H. 233 Rhyner, T.A. 303 Thorne, B. 90 Roberts, J. 112 Tsou, A.-P. 23 Rosen, H. 90

Usui, H. 308 Sadile, A. 268 Santella, L. 42 Scherini, E. 197 Whatley, S. 160 Seasholtz, A. 90 White, C. L. 142 Serra, 1. 182 Wilcox, J.N. 112 Sikela, J. 10 Wilson, L. 123

SUBJECT INDEX

A

Acetylation, of histones, 187,189,191

ACTH, 91 Actin, 162 ADP-ribosylation, function in

cell proliferation, 240-241 differentiation, 242-243 DNA repair, 215, 239 enzymology of, 234-238 histone modification, 188

Adrenal medulla in situ hybridization, 66-67 tyrosine hydroxylase mRNA

1 eve 1 s, 65 Aging, effect on

brain DNase activity, 227 DNA synthesis, 250-252 neuronal RNA content, 283

Alzheimer's disease, effects on brain RNA levels, 150-155 mRNA translation activity,

151,153 RNase activity, 154-155 neuropathology of, 147-149

06- alkylguanine-DNA alkyltransferase, 221

Amino acids, as ADP-ribose acceptors, 239

Anguilla anguilla, 202-204 AtT-20 cells, transfection of, 94 Axon, squid giant, 42-54 Axoplasmic RNA

complexity of, 49-51 poly(A) content, 51 translation activity, 44-45 translation products, 45-47,

48-49 Astrocytes,

cerebellar S100 protein, 310 DNase activity, 228

B

Becker muscular dystrophy, 123-128 1B236 protein

developmental expression, 38

messenger RNA, 23-25 neuroanatomical distribution,

37-38 structure of, 33 multiple molecular forms, 34-37

c Catecholamines

effects on POMC gene expression, 93

molecular genetics of, 57-68 cDNA, specific for

IB236, 33 N-CAM, 294 M44 and M1622 proteins, 297 POMC, 112, 301 proteolipid protein, 39-40 S100 protein, 308-310 synapsin, 76 tubulin, 83-84 tyrosine hydroxylase, 58-61

Cell-free translation, of axoplasmic mRNA, 45-47 human brain mRNAs, in

Alzheimer's Disease, 152-153 cerebe 11 urn, 142 Down's Syndrome, 163-165

rat brain mRNAs, 161-163 Cerebellum

DNase activity, 225-226 G-Substrate, localization of,

78-79 neuronal DNA content, 200-209 RNA recovery and activity,

143-147 S100 protein, localization of,

310-311 subtraction hybridization, 304

Cerebral cortex chromatin changes in

development, 175 chromosomal protein post­

translational modifications, 188-195

developmental changes in non­histone nuclear proteins, 176

DNase activity, 225-226

identifier sequences in chromatin, 177-178

Chloramphenicol acetyl transferase, 28, 101-102

Chromatin condensation of, 197-209 chromatin-bound proteins, post­

translation modifications of, 186-188

developmental changes in, 175-176

identifier sequences in, 177-178 organization of, 174 poly(ADPR) polymerase-trans-

ferase in, 235 Chloride ions, 288-289 Chromosomes, studies of

human 11 and 12, 62 human 21, 160-161, 163-165 human X, 123-128, 131-140 mouse 7, 25 mouse X, 26

Colony hybridization, 58, 75, 296 Comparative studies

brain-specific RNA sequence homology, 27-28

diversity of gene expression, 1-8

Purkinje neuron DNA content and condensation, 204-208

Cot, 2-5, 19, 49-51, 272, 304 Corticotropin releasing factor

(CRF), 91, 100 Crass ius auratus

genomic DNA, 3-5 RNA changes in learning, 290-291 RNA sequence complexity, 5-7

Creatine kinase, in vitro trans­lation product of, 162

o

Deiter's neurons, 288-290 Deoxyribonucleic acid, see DNA Depression, 62 Developmental studies

1B236 protein, 38-39 chromosomal protein modifica­

tions, 188-194 DNA synthesis and turnover,

250-254 DNase activity, 226-229 identifier sequences, 178-180

316

neuronal DNA content and con­densation, cerebellum, 200-204

nonhistone nuclear proteins, 176-177

nucleosomal DNA repeat length, 175-176

Dexamethasone effects on proenkephalin mRNA,

100 DNA

changes during, learning, 258-263, 270-274 sleep, 274-276

comparative studies, 3-5, 204-208

cytochemical measurement of, 197-200

neuronal content, 200-204 nucleosomal repeat length,

175-176 renaturation of, 3 repair of, 216-221, 239-240 replication,

mechanisms of, 211-213 replicative enzymes, 213-216

synthesis, appearance and distribution

of DNA synthesizing cells in brain, 201, 248-250

inhibitors, 201, 257-258 synthesis and turnover in

adult brain, 268-270 X-chromosome-specific

sequences, 124-125 DNA ligase, 215 DNA methyl transferase, 215 DNA polymerases, 212, 214, 215,

220, 228-229 DNA polymerase accessory

proteins, 214 DNases

in brain development and aging, 224-232

DNase I, 175 Dopamine S-hydroxylase, 62 Down's syndrome, 163-165 Duchenne muscular dystrophy,

124-128, 167-169 Dynorphin, 90, 299

E Ependymin, 290

Endoglycosidase F, 24 Endorphin, 90, 112, 119-120, 301 Enkephal in

regulation of gene expression, 90-109

phylogenetic aspects of, 299-301

Erjnaceus europaeus, 205-208 Estrogen, effects on POMe gene

expression, 112-120

F

Feulgen reaction, 198 Fragile X syndrome

genetic analysis of, 131-140

G

GABA, 288-289 G-Substrate, 78-79 Gene expression

diversity of, 1-8, 10-11 Gene transfer of

rat tyrosine hydroxylase, 67-68 human proenkephalin, 93-109

Glial cells chromatin proteins,

modifications of, 188-194 Granule cells

DNA content of, 200-204 Growth fraction, 250

H

Heat-shock proteins effect of chromosome 21,

167-169 homology with 68K MAP, 167-170

Heterogeneous nuclear RNA, 178-180 Hibernation, effect on Purkinje

cell DNA content and chromatin compaction, 206-208

High mobility group proteins, 185-186

Hippocampus DNA synthesis in, 248,250 involvement in learning,

284-286, 288 pyramidal cells in aging,

282-283

317

Histones characterization of, 174 developmental aspects of,

182-183 HI histone, 176-238 post-translational

modifications of, 186-195 Hoechst 33342

action of, 199 measurement of nuclear DNA

content, 201-206 Hybridization

cDNA kinetic analysis, 49-51 colony screening, 58,75,83,296,

299,306,308 dot-blot, 16,38,115 Northern-blot, 27-28, 63-64,

77-78, 95-96, 178, 294, 297-301, 303-306

RNA-DNA saturation, 5-7 Southern analysis, 26,125,177,

294,309 Hybridization histochemistry for

specific mRNAs G-substrate, 78-79 POMe, 118 postmortem human brain, 147 S100, 310-311 tyrosine hydroxylase, 66-67 tubulin, 86-87

Hybridization-selection for synapsin I, 76 membrane-bound polysomal mRNA,

297 Hydroxylapatite chromatography,

4-5, 304 Hypothalamus

POMe gene expression, 112-120

Identifier sequence(s) in brain-specific transcriptional

regulation, 28-30, 177 1B236 gene introns, 25 neuronal chromatin, 177-178 hnRNA, 178-180 S100 gene introns, 309 nucleotide sequence of, 180 tissue specificity of, 20

Immunohistochemistry in situ hybridization, see

hybridization histochemistry

J

Joseph disease, 147

L

Labeling index, 250 Learning

autoradiographic studies, 262-263

DNA synthesis and, 258-262, 271-274

DNA synthesis inhibitors effects on, 257-258

ependyma changes in, 290-291 RNA base ratio changes in,

284-288, 291 RNA synthesis, 284-285 neuronal membrane, changes in,

288-290 S100 protein changes in,

288-291 types of:

active avoidance, 271 habituation, 272 passive avoidance, 258 reversal of handedness, 272,

284-286 swimming adaptation, 290-291 vestibular learning, 286-288 V-maze, 258

Locus coeruleus tyrosine hydroxylase mRNA,

effects of reserpine, 63-66 Linkage analysis

Becker muscular dystrophy, 123-128

Duchenne muscular dystrophy, 123-128

Fra X-mental retardation syndrome, 131-137

X-Linked mental retardation, 138 S-Lipotropin, 91-92, 112 Loligo paelli, 42

M

Membrane-associated mRNA, purification and translation of, 296-298

Mental retardation, genetic

318

studies of, 131-140 Messenger RNA

Alzheimer's disease, effects of, 151-154

axoplasmic, 42-54 bimorphoric species, 16 brain-specific, membrane-bound,

296-298 complexity of, 5-7 Down's syndrome, effects of,

163-165 evolutionary conservation of,

27-28 nonadenylated

characteristics of, 11-13 isolation and cloning of,

14-18 polymorphic species, 26-27 rat brain, translation of, 161 regulation of, 18-20 stability in postmortem tissue,

143-147 Methylation of histones, 187-190

nonhistone chromosomal proteins, 192-194

Microdensitometry, 199-200 Microtubule proteins

microtubule-associated proteins (MAPs), 82-83

TAU factors, 82-83 tubulin isotypes, 82-83

Microtube-associated proteins (MAPs)

Down's syndrome and, 163-165 homology with heat-shock

proteins, 167 localization in synaptosomes,

166-167 Mitochondria, in brain, 47-48,

237-238 Melanocyte stimulating hormone

(MSH), 91-92, 112, 299

N

N-CAM, developmental regulation of mRNA, 294-295

Neomycin, resistance to, 28,95 Neuron-specific enolase, in

vitro translation product of, 162

Nicotinamide, 233,241 Nonadenylated RNA, see mRNA

Nonhistone chromosomal proteins developmental changes in,

176-177 general characteristics and

modifications of, 183-185 high mobility group proteins in

postnatal development, 185-186

post-translational modifica­tions of, 188-194

Nuclear transcription assay, 116,118

Nucleosome basic structure, 174-175 developmental changes in DNA

repeat length, 175-176 identifier sequences in,

177-178

o Olfactory bulbs, appearance and

distribution of DNA synthesizing cells, 248-249, 253

Oligodeoxyribonucleotides, probes for:

G-substrate, 78-79 u-MSH, 300-301 tubulin, 84

Opioid peptides, genes for, 90-92 299

p

319

Peptide(s) synthetic, 34-38 Phenylalanine hydroxylase,

homology with tyrosine hydroxylase, 61-62

Pheochromocytoma (PC 12) cells, 58 Phosphorylation, of brain

chromosomal proteins, 187, 188-194

Polyadenylic acid, content in axoplasm, 51-52

Poly(ADPR) polymerase, 215, 234-243

Poly(A)+RNA, see messenger RNA Polysomes

immunoadsorption of, 74-75 membrane-bound, 161-163,

296-298

Post-translational modification of brain chromosomal proteins, 182-195, 238

Proenkephalin, 91-109, 299-302 Pro-opiomelanocortin (POMC), gene

expression, regulation by estrogen, 112-120

Probes, see cDNA and oligodeoxy­ribonucleotides

Propidium iodide, 199,202 Protein kinase

cAMP-dependent phosphorylation, 61

cGMP-dependent phosphorylation, 71

Proteolipid protein (PLP), 26,39 Purine insertase, 217 Purkinje cells

G-substrate localization, 78-79 nuclear DNA content of,

developmental studies, 200-204

comparative studies, 204-208 Pyramidal neurons, hippocampal

RNA content and base composi­tion, 282

RNA changes in learning, 284-286

R

Rana escu7enta, 205-208 Reticulocyte cell-free system, in

vitro translation in, 44-49, 72, 74-76, 161-169

Reserpine, effects on tyrosine hydroxylase mRNA, 63-66

Restriction fragment length poly­morphism (RFLP), see linkage analysis

Restriction mapping, organization of the S100 (8 subunit) gene, 310

RNAs axoplasmic, 42-54 BC1 and BC2, sequence of, 180 class III, 27-28 heterogeneous nuclear, 178-180 ribosomal, stability of, 144,

150-151, 153 see also mRNA and cDNAs

RNA-DNA hybridization, see hybridization

RNA polymerase(s), 28-30, 177 RNase, levels in Alzheimer's

disease, 154-155 RNase H, 212,214

s

s. aureus V8 protease, 165-166 S100 protein

gene identification and structure of, 308-310

in situ hybridization, 310 learning and, 288-290

Schizophrenia, 62 Sleep

deprivation of, 303-304 effect on brain DNA turnover,

274-276 Stem cells, in brain, 251 Substantia nigra

reserpine effects on tyrosine hydroxylase mRNA, 63-66

Synapsin I, 71-78 Synaptosomes, presence of 68K

MAP, 166-167

T

TAU factors, 82 Topoisomerase, 212,214 Trisomy-21

effect on synthesis heat-shock proteins, 167-169

effect on 68K MAP protein, 163-165

Transfection, see gene transfer

320

Tubulin, gene expression in brain, 81-87

Tyrosine hydroxylase molecular genetics of, 57-68

u Uracil-DNA glycosylase, 217,

219-220

v

Vaccinia virus, use as transformation vehicle, 102-103

Vasopressin, 91

w

Western blot analysis, 35 Wheat germ, cell-free translation

system, 144-145, 151-152, 155

x X-chromosome

chromosome-specific DNA sequences, 124

linkage analysis of Duchenne muscular dystrophy, 125-127

mental retardation, 131-140 restriction fragment length

polymorphisms, 125 Xenopus 7aevis, 299-301