Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies on nucleosomal histone acetylation and nonhistone chromosomal proteins in relation to chromatin… Davie, James Ronald 1979

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1979_A1 D38.pdf [ 15.02MB ]
Metadata
JSON: 831-1.0094862.json
JSON-LD: 831-1.0094862-ld.json
RDF/XML (Pretty): 831-1.0094862-rdf.xml
RDF/JSON: 831-1.0094862-rdf.json
Turtle: 831-1.0094862-turtle.txt
N-Triples: 831-1.0094862-rdf-ntriples.txt
Original Record: 831-1.0094862-source.json
Full Text
831-1.0094862-fulltext.txt
Citation
831-1.0094862.ris

Full Text

STUDIES ON NUCLEOSOMAL HISTONE ACETYLATION AND NONHISTONE CHROMOSOMAL PROTEINS IN RELATION TO CHROMATIN STRUCTURE BY JAMES RONALD DAVIE Sc., The U n i v e r s i t y of B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of B i o c h e m i s t r y F a c u l t y of M e d i c i n e We a c c e p t t h i s t h e s i s as conforming to t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September 197 9 c) James Ronald Davie, 1979 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1WS Date i i ABSTRACT PART A - CHROMATIN SYNTHESIS Experiments a r e d e s c r i b e d t h a t suggest m i c r o c o c c a l n u c l e a s e s e l e c -t i v e l y e x c i s e s from t r o u t t e s t i s c h r o m a t i n a p o p u l a t i o n of mononucleo-somes c o n t a i n i n g i n c r e a s e d l e v e l s of n e w l y - s y n t h e s i z e d h i s t o n e s and, p o s s i b l y , n e w l y - s y n t h e s i z e d DNA. The r e s u l t s suggest t h a t newly-syn-t h e s i z e d c h r o m a t i n has an i n c r e a s e d s e n s i t i v i t y t o m i c r o c o c c a l n u c l e a s e d i g e s t i o n . PART B - STRUCTURE OF TRANSCRIPTIONALLY ACTIVE CHROMATIN D i f f e r e n t n u c l e a s e s were used to probe the s t r u c t u r e of t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n . M i c r o c o c c a l n u c l e a s e and d e o x y r i b o n u c l e a s e I I , enzymes which p r e f e r e n t i a l l y d i g e s t i n t e r -n u c l e o s o m a l l i n k e r DNA, have b o t h been used s u c c e s s f u l l y t o f r a c t i o n -a t e c h r o m a t i n i n t o t r a n s c r i p t i o n a l l y a c t i v e and i n a c t i v e r e g i o n s . D e o x y r i b o n u c l e a s e I , an enzyme which d i g e s t s b o t h i n t r a - and i n t e r -n u c l e o s o m a l DNA, s e l e c t i v e l y d e s t r o y s t r a n s c r i p t i o n a l l y competent genes. A f t e r t r o u t t e s t i s n u c l e i o r c h r o m a t i n was d i g e s t e d w i t h one of t h e above n u c l e a s e s , nucleosomes a s s o c i a t e d w i t h t h e n u c l e a s e s e n s i t i v e r e g i o n s ( t r a n s c r i p t i o n a l l y a c t i v e r e g i o n s ) were s e l e c t i v e l y e l u t e d from t h e d i g e s t e d n u c l e i by t h e a d d i t i o n o f low s a l t (0.1 M o r 0.2 M N a C l ) ; a l t e r n a t i v e l y , nucleosomes were i s o l a t e d from the d i g e s t p r o d u c t s by v i r t u e of t h e i r s o l u b i l i t y i n 0.1 M NaCl o r 2 mM MgCl^. The accumulated r e s u l t s suggest t h a t nucleosomes c o n t a i n i n g h i g h l y a c e t y l a t e d h i s t o n e H4 and normal l e v e l s o f t h e a c e t y l a t e d . s p e c i e s of h i s t o n e s H3, H2A and i i i H2B are associated with t r a n s c r i p t i o n a l l y competent chromatin regions. Furthermore, the internucleosomal l i n k e r DNA associated with these regions contains low l e v e l s of histone Hi, and high l e v e l s of HMG-T1, HMG-T2 and HMG-T3, and lower l e v e l s of other nonhistone chromosomal proteins. The bulk of chromatin, which i s t r a n s c r i p t i o n a l l y i n a c t i v e , i s associated with nucleosomes containing low l e v e l s of acetylated H4 ( i . e . unacetylated and monoacetylated H4 are the major species), and the internucleosomal l i n k e r DNA i s associated mainly with histone HI. The r o l e of H4 a c e t y l a t i o n i n t r a n s c r i p t i o n a l l y competent chromatin may be to reduce in t e r a c t i o n s between core p a r t i c l e s and thus render the extended chromatin region accessible to RNA polymerases. Conversely, unacetylated H4 may be involved i n maintaining higher l e v e l s of compaction of the chromatin. PART C - EFFECT OF SODIUM N-BUTYRATE ON HISTONE ACETYLATION The biochemical mechanisms underlying the increased a c e t y l a t i o n of histones found i n butyrate - treated t i s s u e culture c e l l s were investigated. The r e s u l t s c l e a r l y indicate that the increased a c e t y l a t i o n of histone i n vivo i s most probably due to an i n h i b i t i o n of deacetylase enzyme a c t i v i t y caused by butyrate. This i n h i b i t i o n i s e n t i r e l y r e v e r s i b l e and appears to be a general phenomenon, since butyrate increases l e v e l s of acetylated H3 and H4 i n a l l t i s s u e culture c e l l types (Xenopus l a e v i s embryonic c e l l s (X58), r a t a s c i t e s c e l l s (IRC8), mouse f i b r o b l a s t s (3T3), baby hamster kidney c e l l s (BHK) and Friend i v e r y t h r o l e u k e m i c c e l l s ) s t u d i e d . V TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES x i LIST OF FIGURES x i i i ACKNOWLEDGEMENT x v i i i DEDICATION x i x INTRODUCTION 1 I . The H i s t o n e s 1 I I . H i s t o n e M o d i f i c a t i o n s 9 (a) A c e t y l a t i o n 10 (b) M e t h y l a t i o n 12 (c) P h o s p h o r y l a t i o n 13 (d) N - P h o s p h o r y l a t i o n 17 (e) A d e n o s i n e - D i p h o s p h o - R i b o s y l a t i o n (ADP-r i b o s y l a t i o n ) 17 I I I . The Nucleosome 19 (a) H i s t o n e - H i s t o n e I n t e r a c t i o n s 19 (b) H i s t o n e - DNA I n t e r a c t i o n s 21 IV. Nucleosomal DNA S i z e D i v e r s i t y 22 V. S t r u c t u r e of t h e Nucleosome 26 V I . Nucleosome Models 28 V I I . H i g h e r Order Chromatin S t r u c t u r e : R o l e of HI 29 V I I I . T r a n s c r i p t i o n a l l y A c t i v e Chromatin 30 IX. R e p l i c a t i o n of Chromatin 34 X. Nonh i s t o n e Chromosomal P r o t e i n s 37 X I . H i s t o n e A c e t y l a t i o n : C o r r e l a t i o n w i t h T r a n s c r i p t i o n a l A c t i v i t y of Chromatin 39 v i TABLE OF CONTENTS - Q Page XII. The Present Investigation 40 MATERIALS AND METHODS 42 PARTS A AND B I. Materials and Abbreviations 42 (a) Materials 42 (b) Abbreviations 42 II. Cell Incubations 43 III. Preparation of Nuclei and Chromatin 44 IV. Enzymatic Digestion of Nuclei or Chromatin and Fractionation of the Digest Products 45 (a) Preparation of mononucleosomes from micrococcal nuclease digested nuclei 45 (b) Preparation of nucleosome subfractions from micrococcal nuclease digested nuclei (Sanders' procedure) 46 (i) Digestion of nuclei 46 ( i i ) Stepwise elution of nucleosomes 46 ( i i i ) Fractionation of the salt-eluted products 47 (c) Preparation of nucleosome subfractions from micrococcal nuclease digested nuclei (Levy and Dixon's procedure) 47 (i) Digestion of nuclei 47 ( i i ) Preparation and fractionation of nucleosomes 47 (d) Fractionation of DNase II digested chromatin (Gottesfeld's procedure) 48 (i) Digestion of chromatin 48 ( i i ) Fractionation of the chromatin d_jgest products. 48 ( i i i ) Studies of the efficiency of Mg , histone or RNase to precipitate S2 associated nucleosomal material 49 (e) Preparation of mononucleosomes from DNase I digested nuclei 49 (f) Preparation of nucleosome subfractions from DNase I digested nuclei (Sanders' procedure) 50 (i) Digestion of nuclei 50 ( i i ) Stepwise elution of nucleosomes 50 ( i i i ) Fractionation of the salt-eluted products 50 y i i TABLE OF CONTENTS Page V. DNase I D i g e s t i o n o f L a b e l l e d Mononucleosomes 51 V I . R e a c t i o n o f Nucleosomes or DNase I D i g e s t e d Nucleosomes w i t h N - E t h y l m a l e i m i d e under D e n a t u r i n g C o n d i t i o n s 51 V I I . G e l E x c l u s i o n Chromatography of Unknowns PI and P I I 52 V I I I . TCA P r e c i p i t a b i l i t y T e s t s o f Unknowns PI and P I I 52 IX. High V o l t a g e Paper E l e c t r o p h o r e s i s 53 X. H i s t o n e E x t r a c t i o n 53 XI. Q u a n t i t a t i o n o f the H i s t o n e Sample 54 X I I . G e l E l e c t r o p h o r e s i s o f P r o t e i n s (a) S t a r c h g e l e l e c t r o p h o r e s i s 55 (b) Acid^/urea g e l e l e c t r o p h o r e s i s 55 (c) SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s 56 (d) Two-dimensional g e l e l e c t r o p h o r e s i s 57 X I I I . E x t r a c t i o n o f H i s t o n e s from 15% Po l y a c r y l a m i d e - S D S G e l s 58 XIV. S o l u b i l i z a t i o n o f P o l y a c r y l a m i d e G e l s 58 XV. A n a l y s i s o f DNA Fragments Produced by N u c l e a s e D i g e s t i o n . 58 (a) P r e p a r a t i o n o f DNA fragments 59 (b) Non-denaturing p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of DNA 59 (c) D e n a t u r i n g p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of DNA 60 PART C I. M a t e r i a l s and A b b r e v i a t i o n s 62 (a) M a t e r i a l s . 62 (b) A b b r e v i a t i o n s 62 I I . C e l l L i n e s and C u l t u r e C o n d i t i o n s 62 I I I . P r e p a r a t i o n o f H i s t o n e s 64 IV. P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s 64 v i i i TABLE OF CONTENTS Page V. Assay of In V i t r o H i s t o n e A c e t y l a s e and D e a c e t y l a s e A c t i v i t y 65 PART A - CHROMATIN SYNTHESIS RESULTS AND DISCUSSION 67 I . P a r t i a l C h a r a c t e r i z a t i o n of Newly S y n t h e s i z e d Chromatin .67 (a) C h a r a c t e r i z a t i o n of m i c r o c o c c a l n u c l e a s e d i g e s t p r o d u c t s 67 (b) DNA s y n t h e s i s 69 (c) H i s t o n e s y n t h e s i s and h i s t o n e a c e t y l a t i o n 74 (d) P a r t i a l c h a r a c t e r i z a t i o n of unknowns PI and P I I . 77 PART B - STRUCTURE OF TRANSCRIPTIONALLY ACTIVE CHROMATIN RESULTS AND DISCUSSION 82 I . C h a r a c t e r i z a t i o n of Nucleosome S u b t r a c t i o n s and Chromosomal P r o t e i n s R e l e a s e d by M i c r o c o c c a l N u c l e a s e D i g e s t i o n of N u c l e i 82 (a) D e t e r m i n a t i o n of t h e l e v e l s of a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h mononucleosomes . 82 1. K i n e t i c s of [ 1 4 C ] a c e t a t e i n c o r p o r a t i o n i n t o monomer and multimer f r a c t i o n s 83 2. E f f e c t of e x t e n s i v e n u c l e a s e d i g e s t i o n on t h e r e l a t i v e [ 1 4 C ] a c e t a t e i n c o r p o r a t i o n i n t o monomer and multimer f r a c t i o n s 84 3. Comparison of t h e l e v e l s of a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h mononucleosomes and u n d i g e s t e d n u c l e i 89 (b) Chromatin f r a c t i o n a t i o n (Sanders' procedure) .... 93 1. Q u a n t i t a t i o n of DNA c o n t e n t i n s a l t - e x t r a c t e d c h r o m a t i n f r a c t i o n s 95 2. C h a r a c t e r i z a t i o n of t h e DNA fragments a s s o c i a t e d w i t h s a l t - e x t r a c t e d c h r o m a t i n f r a c t i o n s 97 3. Q u a n t i t a t i o n of t h e a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h s a l t - e x t r a c t e d c h r o m a t i n f r a c t i o n s 101 4. Q u a n t i t a t i o n of t h e a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h column f r a c t i o n a t e d s a l t -e x t r a c t e d nucleosome f r a c t i o n s 109 5. A n a l y s i s of chromosomal p r o t e i n s a s s o c i a t e d w i t h s a l t - e x t r a c t e d c h r o m a t i n f r a c t i o n s .... H 6 (c) Chromatin f r a c t i o n a t i o n (Levy and Dixon's p r o c e d u r e ) 118 1. Q u a n t i t a t i o n of DNA c o n t e n t i n t h e c h r o m a t i n f r a c t i o n s 120 ,ix TABLE OF CONTENTS Page 2. Q u a n t i t a t i o n o f the a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h the chro m a t i n f r a c t i o n s 120 (d) Summary 123 I I . C h a r a c t e r i z a t i o n of Nucleosome S u b t r a c t i o n s and Chromosomal P r o t e i n s R e l e a s e d from DNase I I D i g e s t e d Chromatin 127 (a) Q u a n t i t a t i o n o f DNA c o n t e n t i n the ch r o m a t i n f r a c t i o n s 127 (b) K i n e t i c s o f DNase I I ch r o m a t i n d i g e s t i o n 130 (c) C h a r a c t e r i z a t i o n of t h e DNA fragments a s s o c i a t e d w i t h each c h r o m a t i n f r a c t i o n 130 (d) D e t e r m i n a t i o n o f the l e v e l s o f a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h the c h r o m a t i n f r a c t i o n s 135 (e) Behaviour o f S ^ n u c l e o s o m a l m a t e r i a l i n the pr e s e n c e o f Mg , RNase or h i s t o n e 142 ( f ) Summary 144 I I I . C h a r a c t e r i z a t i o n o f Nucleosome S u b t r a c t i o n s and Chromosomal P r o t e i n s R e l e a s e d by DNase I D i g e s t i o n of N u c l e i 146 (a) C h a r a c t e r i z a t i o n of nucleosomes r e l e a s e d from DNase I d i g e s t e d n u c l e i 147 (1) D i g e s t p r o d u c t s r e l e a s e d from DNase I d i g e s t e d n u c l e i 147 (2) Q u a n t i t a t i o n o f a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h n u c l e o s o m a l f r a c t i o n s 153 (b) C h a r a c t e r i z a t i o n o f nucleosome s u b f r a t i o n s and chromosomal p r o t e i n s r e l e a s e d from DNase I d i g e s t e d n u c l e i (Sanders' p r o c e d u r e ) 157 (1) Q u a n t i t a t i o n o f t h e DNA c o n t e n t i n s a l t -e x t r a c t e d c h r o m a t i n f r a c t i o n s 158 (2) C h a r a c t e r i z a t i o n o f the DNA fragments a s s o c i a t e d w i t h s a l t - e x t r a c t e d c h r o m a t i n f r a c t i o n s 161 (3) C h a r a c t e r i z a t i o n o f the p r o t e i n s a s s o c i a t e d w i t h s a l t - e x t r a c t e d c h r o m a t i n f r a c t i o n s 168 (4) Q u a n t i t a t i o n o f the a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h the s a l t - e x t r a c t e d c h r o m a t i n f r a c t i o n s 172 X TABLE OF CONTENTS Page (5) B i o - G e l A- 0.5 column f r a c t i o n a t i o n of s a l t - e x t r a c t e d nucleosome f r a c t i o n s 174 ( i ) Column f r a c t i o n a t i o n of t h e chromosomal p r o t e i n s a s s o c i a t e d w i t h t h e s a l t - e x t r a c t e d c h r o m a t i n f r a c t i o n s 174 ( i i ) Q u a n t i t a t i o n of t h e a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h n u c l e o s o m a l s u b f r a c t i o n s 183 (6) D e t e r m i n a t i o n of t h e degree of a c e t y l group i n c o r p o r a t i o n into, t h e s a l t - e x t r a c t e d f r a c t i o n s 193 (c) DNase I d i g e s t i o n of mononucleosomes 196 (d) Summary 2 0 2 PART C - EFFECT OF SODIUM N-BUTYRATE ON HISTONE ACETYLATION RESULTS 205 I . E f f e c t s of B u t y r a t e on E r y t h r o l e u k e m i c C e l l H i s t o n e s ... 206 I I . E f f e c t s of B u t y r a t e on Other C e l l L i n e s 209 I I I . H i s t o n e A c e t y l a s e A c t i v i t y i n T r o u t T e s t i s C e l l S u spensions and i n E r y t h r o l e u k e m i c C e l l L y s a t e s 213 IV. H i s t o n e D e a c e t y l a s e A c t i v i t y i n C e l l L y s a t e s 215 • - . DISCUSSION 218 CONCLUDING REMARKS 223 BIBLIOGRAPHY 232 x i . LIST OF TABLES INTRODUCTION T a b l e Page I. Nomenclature and P r o p e r t i e s o f H i s t o n e s 2 I I . P h o s p h o r y l a t i o n Sequences i n H i s t o n e s 15 I I I . DNA Content of Nucleosomes 23 IV. P r o p e r t i e s of Endonucleases Used 27 PART A - CHROMATIN SYNTHESIS V. Q u a n t i t a t i o n of N e w l y - S y n t h e s i z e d DNA A s s o c i a t e d w i t h Nucleosome F r a c t i o n s 71 V I . Q u a n t i t a t i o n o f N e w l y - S y n t h e s i z e d H i s t o n e A s s o c i a t e d w i t h Nucleosome F r a c t i o n s 75 V I I . Q u a n t i t a t i o n o f TCA P r e c i p i t a b l e M a t e r i a l A s s o c i a t e d w i t h Unknowns PI and P I I 81 PART B - STRUCTURE OF TRANSCRIPTIONALLY ACTIVE CHROMATIN V I I I . Q u a n t i t a t i o n of t h e L e v e l s of H i s t o n e A c e t y l a t i o n A s s o c i a t e d w i t h Nucleosomal F r a c t i o n s 86 IX. Q u a n t i t a t i o n of [ 1 4 C ] A c e t a t e - L a b e l l e d H i s t o n e F r a c t i o n s ... 90 X. Q u a n t i t a t i o n of DNA Content i n S a l t - E x t r a c t e d Chromatin F r a c t i o n s R e l e a s e d from M i c r o c o c c a l N u c l e a s e D i g e s t e d N u c l e i (Sanders' Procedure) ^6 X I . Q u a n t i t a t i o n o f A c e t y l a t e d S p e c i e s of H i s t o n e H4 i n S a l t - E x t r a c t e d Nucleosome F r a c t i o n s (Sanders' Procedure) ... X I I . S p e c i f i c A c t i v i t i e s of H i s t o n e H3 and H4 i n S a l t -E x t r a c t e d Nucleosome F r a c t i o n s 108 x i i LIST OF TABLES T a b l e Page X I I I . Q u a n t i t a t i o n of A c e t y l a t e d S p e c i e s o f H i s t o n e H4 i n Column F r a c t i o n a t e d , S a l t - E x t r a c t e d Nucleosome F r a c t i o n s 114 XIV. Q u a n t i t a t i o n o f DNA Content i n Chromatin F r a c t i o n s (Levy and Dixon's Procedure) 121 XV. Q u a n t i t a t i o n o f A c e t y l a t e d S p e c i e s of H i s t o n e H4 i n t h e Chromatin F r a c t i o n s (Levy and Dixon's Procedure) 124 XVI. Q u a n t i t i a t i o n o f DNA Content i n F r a c t i o n s R e l e a s e d from DNase I I D i g e s t e d Chromatin 129 +2 XVII. E f f i c i e n c y o f Mg , H i s t o n e or RNase i n P r e c i p i t a t i o n of S2 A s s o c i a t e d Nucleosomal M a t e r i a l 143 X V I I I . RNase P r e c i p i t a t i o n o f S2 A s s o c i a t e d Nucleosomal M a t e r i a l 145 XIX. Q u a n t i t a t i o n of DNA Content i n S a l t - E x t r a c t e d Chromatin F r a c t i o n s R e l e a s e d from DNase I D i g e s t e d N u c l e i (Sanders' Procedure) 159 XX. Q u a n t i t a t i o n of A c e t y l a t e d S p e c i e s of H i s t o n e H4 i n S a l t - E x t r a c t e d Nucleosome F r a c t i o n s R e l e a s e d from DNase I D i g e s t e d N u c l e i 175 XXI. Q u a n t i t a t i o n of A c e t y l a t e d S p e c i e s o f H i s t o n e H4 i n S a l t - E x t r a c t e d Nucleosome F r a c t i o n s R e l e a s e d from E i t h e r M i c r o c o c c a l N u c l e a s e o r DNase I D i g e s t e d N u c l e i 190 XXII. S p e c i f i c A c t i v i t i e s o f H i s t o n e s H3 and H4 i n S a l t -E x t r a c t e d Nucleosome F r a c t i o n s 195 X X I I I . S p e c i f i c A c t i v i t i e s of H i s t o n e s A s s o c i a t e d w i t h Mononucleosomes B e f o r e and A f t e r DNase I D i g e s t i o n 199 XXIV. N - E t h y l [ 3H] M a l e i m i d e R e a c t i v i t y o f t h e C y s t e i n y l R e s i d u e of H i s t o n e H3 A s s o c i a t e d w i t h E i t h e r U n d i g e s t e d Mononucleosomes o r DNase I D i g e s t e d Mononucleosomes i n V a r i o u s D e n a t u r i n g S o l u t i o n s 201 x i i i LIST OF FIGURES INTRODUCTION F i g u r e Page 1. Amino a c i d sequence o f h i s t o n e H2A 4 2. Amino a c i d sequence o f h i s t o n e H2B 5 3. Amino a c i d sequence o f h i s t o n e H3 6 4. Amino a c i d sequence o f h i s t o n e H4 7 5. Amino a c i d sequence o f h i s t o n e HI 8 PART A - CHROMATIN SYNTHESIS 6. B i o - G e l A-5m column s e p a r a t i o n o f nucleosomes o b t a i n e d from an 8-min m i c r o c o c c a l n u c l e a s e d i g e s t o f t r o u t t e s t i s n u c l e i 68 7. [ 3H] Thymidine i n c o r p o r a t i o n i n t o DNA o f nucleosomes as a f u n c t i o n o f time 70 8. [ 3H] Thymidine i n c o r p o r a t i o n i n t o DNA o f multimers and monomers as a f u n c t i o n o f time 72 9. [ 3H] L y s i n e and [ 1 I +C] a c e t a t e i n c o r p o r a t i o n i n t o h i s t o n e s of nucleosomes as a f u n c t i o n o f time .. 75 10. EDTA s o l u b i l i z e d m a t e r i a l from n u c l e i i s o l a t e d from [ 3H] l y s i n e - and [ l t +C] a c e t a t e - l a b e l l e d t r o u t t e s t i s c e l l s 78 11. Sephadex G25 g e l e x c l u s i o n chromatography of unknowns PI and P I I 79 PART B - STRUCTURE OF TRANSCRIPTIONALLY ACTIVE CHROMATIN 12. [ l t +C] A c e t a t e i n c o r p o r a t i o n i n t o h i s t o n e s of n u c l e o -somes as a f u n c t i o n of time 84 13. [ l l fC] A c e t a t e i n c o r p o r a t i o n i n t o h i s t o n e s of mult-imers and monomers as a f u n c t i o n of' time 85 x i v LIST OF FIGURES Page M i c r o c o c c a l n u c l e a s e d i g e s t i o n o f n u c l e i i s o l a t e d from [ 1 1 +C] a c e t a t e - l a b e l l e d t r o u t t e s t i s c e l l s ... 87 A c e t y l a t e d components of whole t r o u t t e s t i s h i s t o n e s and h i s t o n e s from nucleosomes 92 E x p e r i m e n t a l p r o c e d u r e f o r the s t e p w i s e i s o l a t i o n o f n u c l e o s o m a l s u b f r a c t i o n s r e l e a s e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i by each s u c c e s s i v e l y h i g h e r NaCl c o n c e n t r a t i o n 94 P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f the DNA fragments g e n e r a t e d by m i c r o c o c c a l n u c l e a s e d i g e s t i o n of t r o u t t e s t i s n u c l e i and r e l e a s e d by s t e p w i s e i n c r e a s e s i n NaCl c o n c e n t r a t i o n s 98 P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f the DNA fragments r e l e a s e d by m i c r o c o c c a l n u c l e a s e i n t o the SO and SSI f r a c t i o n s 99 L o c a l i z a t i o n o f a c e t y l a t e d h i s t o n e s p e c i e s s e p a r a t e d on a c i d - u r e a g e l s G e l scans of h i s t o n e s a n a l y z e d on a c i d - u r e a g e l s . B i o - G e l A-5m s e p a r a t i o n o f nucleosomes o b t a i n e d from e x t r a c t i o n of m i c r o c o c c a l n u c l e a s e d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0.1 M NaCl i n B u f f e r D .. B i o - G e l A-5m s e p a r a t i o n of nucleosomes o b t a i n e d from e x t r a c t i o n o f m i c r o c o c c a l n u c l e a s e d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0.4 M NaCl i n B u f f e r D .. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f DNA fragments a s s o c i a t e d w i t h column f r a c t i o n a t e d , s a l t - e x t r a c t e d nucleosome f r a c t i o n s 15% PAGE SDS s e p a r a t i o n o f p r o t e i n s r e l e a s e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d t r o u t t e s t i s n u c l e i by s u c c e s s i v e l y h i g h e r NaCl c o n c e n t r a t i o n s . E x p e r i m e n t a l p r o c e d u r e f o r i s o l a t i o n o f a 0.1 M NaCl s o l u b l e n u c l e o s o m a l s u b f r a c t i o n r e l e a s e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i 119 G e l scans o f h i s t o n e s a n a l y z e d on a c i d - u r e a g e l s . 122 E x p e r i m e n t a l p r o c e d u r e f o r the i s o l a t i o n o f a t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n f r a c t i o n 128 102 104 110 111 113 117 XV LIST OF FIGURES _ Page 28. Time c o u r s e o f c h r o m a t i n f r a c t i o n a t i o n (I) 131 29. Time c o u r s e o f chr o m a t i n f r a c t i o n a t i o n ( I I ) 132 30. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of the DNA fragments s o l u b i l i z e d by DNase I I d i g e s t i o n o f t r o u t t e s t i s c h r o m a t i n 134 31. H i s t o n e p r o f i l e s on an a c i d / u r e a / p o l y a c r y l a m i d e g e l 136 32. G e l scans o f the h i s t o n e H4 r e g i o n s o f an a c i d / u r e a g e l ... 138 33. Two-dimensional PAGE s e p a r a t i o n o f h i s t o n e s from chromatin f r a c t i o n s , s t a i n e d w i t h Coomassie b l u e 140 34. B i o - G e l A-5m s e p a r a t i o n o f nucleosomes i s o l a t e d from DNase I d i g e s t e d n u c l e i 148 35. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f s i n g l e - s t r a n d e d DNA fragments a s s o c i a t e d w i t h column f r a c t i o n a t e d nucleosome f r a c t i o n s 150 36. B i o - G e l A-5m s e p a r a t i o n o f nucleosome l a b e l l e d w i t h [ 1 I + C ] a c e t a t e and [ 3 H ] l y s i n e i s o l a t e d from DNase I d i g e s t e d n u c l e i 152 37. B i o - G e l A-0.5m f r a c t i o n a t i o n o f n u c l e o s o m a l components .... 154 38. H i s t o n e p r o f i l e s on an acidr-urea .polyacrylamide g e l 155 39. B i o - G e l A-5m s e p a r a t i o n o f nucleosomes o b t a i n e d from e x t r a c t i o n o f DNase I d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0.1 M NaCl i n B u f f e r D 162 40. B i o - G e l A-5m s e p a r a t i o n of nucleosomes o b t a i n e d from e x t r a c t i o n o f DNase I d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0.2 M NaCl i n B u f f e r D 163 41. B i o - G e l A-5m s e p a r a t i o n o f nucleosomes o b t a i n e d from e x t r a c t i o n o f DNase I d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0.4 M NaCl i n B u f f e r D 164 42. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f s i n g l e - s t r a n d e d DNA fragments g e n e r a t e d by DNase I d i g e s t i o n o f t r o u t t e s t i s n u c l e i and r e l e a s e d by s t e p w i s e i n c r e a s e s i n NaCl c o n c e n t r a t i o n s 167 43. 15% PAGE SDS s e p a r a t i o n o f p r o t e i n s r e l e a s e d from DNase I d i g e s t e d t r o u t t e s t i s n u c l e i by s u c c e s s i v e l y h i g h e r NaCl c o n c e n t r a t i o n s 169 x v i LIST OF FIGURES „ Page 44. G e l scans o f h i s t o n e s a n a l y z e d on a c i d - u r e a g e l s 173 45. B i o - G e l A-0.5m s e p a r a t i o n o f nucleosomes o b t a i n e d from e x t r a c t i o n of m i c r o c o c c a l n u c l e a s e d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0.1 M NaGl i n B u f f e r D 176 46. B i o - G e l A-0.5m s e p a r a t i o n o f nucleosomes o b t a i n e d from e x t r a c t i o n o f DNase I d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0.1 M NaCl i n B u f f e r D 177 47. 15% PAGE SDS s e p a r a t i o n o f chromosomal p r o t e i n s a s s o c i a t e d w i t h column f r a c t i o n a t e d , 0.1 M NaCl e x t r a c t e d riucieosome f r a c t i o n s 180 48. Two-dimensional PAGE s e p a r a t i o n of h i s t o n e s from s a l t - e x t r a c t e d nucleosome f r a c t i o n s 185 49. B i o - G e l A-0.5m s e p a r a t i o n o f nucleosomes o b t a i n e d from e x t r a c t i o n o f DNase I d i g e s t e d t r o u t t e s t i s n u c l e i 0.2 M NaCl i n B u f f e r D 187 50. G e l scans of h i s t o n e s a n a l y z e d on a c i d - u r e a g e l s 188 51. Two-dimensional PAGE s e p a r a t i o n o f h i s t o n e s from s a l t - e x t r a c t e d nucleosome f r a c t i o n s and from u n d i g e s t e d n u c l e i 192 52. B i o - G e l A-0.5m column f r a c t i o n a t i o n o f DNase I d i g e s t e d mononucleosomes 197 PART C - EFFECTS OF SODIUM N-BUTYRATE ON HISTONE ACETYLATION 53. H i s t o n e p a t t e r n s of F r e i n d e r y t h r o l e u k e m i c c e l l s 207 54. O p t i c a l scans on a c i d - u r e a g e l s 208 55. E f f e c t o f b u t y r a t e on h i s t o n e a c e t y l a t i o n i n v a r i o u s c e l l t ypes 211 56. O p t i c a l scans of h i s t o n e s from v a r i o u s c e l l types a n a l y z e d on a c i d - u r e a a c r y l a m i d e g e l s 212 57. H i s t o n e a c e t y l a s e a c t i v i t y i n s u s p e n s i o n s o f t r o u t t e s t i s c e l l s 214 58. H i s t o n e a c e t y l a s e a c t i v i t y i n c e l l - f r e e e x t r a c t s o f F r i e n d e r y t h r o l e u k e m i c c e l l s 216 x v i i . LIST OF FIGURES Page 59. H i s t o n e d e a c e t y l a s e a c t i v i t y i n c e l l - f r e e e x t r a c t s of F r i e n d e r y t h r o l e u k e m i c c e l l s 217 x v i i i ACKNOWLEDGEMENT I wish to thank my s u p e r v i s o r , Dr. P e t e r Candido, f o r h i s comments, encouragement, e n t h u s i a s t i c i n t e r e s t and f r i e n d s h i p d u r i n g the cou r s e o f t h i s work. I a l s o w i s h to thank the many p e o p l e who gave me a d v i c e and h e l p , e s p e c i a l l y J . Ingman-Baker, N.T.N. Wong, J . H e w i t t , and Drs. B. A l i n e , B. B h u l l a r , R. Reeves and C. R i c h a r d s o n . The r e s e a r c h was s u p p o r t e d by g r a n t s to Dr. P. Candido from the M e d i c a l Research C o u n c i l . x i x DEDICATION to My P a r e n t s My B r o t h e r s , C h r i s , P h i l , Ian and Stephen and My S i s t e r s , E l i z a b e t h and Brenda 1 INTRODUCTION The fundamental r e p e a t i n g s t r u c t u r a l u n i t of c h r o m a t i n from a l l e u c a r y o t i c c e l l s s t u d i e d c o n s i s t s of a p a i r of each of t h e nucleosomal h i s t o n e s , H2A, H2B, H3 and H4, complexed w i t h 145 base p a i r s o f DNA. These n u c l e o s o m a l c o r e p a r t i c l e s a r e j o i n e d by DNA known as l i n k e r DNA to which n o n - h i s t o n e s such as HI and H5 can b i n d ( 9 2 ) . When examined by e l e c t r o n m i c r s c o p y , c h r o m a t i n has a "beads on a s t r i n g " appearance ( 1 ) , t h e "beads" b e i n g the nucleosomes. The nucleosomes c o n t a i n i n g the n u c l e o s o m a l h i s t o n e s , HI and about 200 base p a i r s o f DNA can be e x c i s e d from c h r o m a t i n by m i l d d i g e s t i o n w i t h m i c r o c o c c a l n u c l e a s e . F u r t h e r d i g e s t i o n w i t h m i c r o c o c c a l n u c l e a s e " t r i m s " t h e n u c l e o s o m a l DNA w i t h t h e l o s s of 40 to 60 base p a i r s o f DNA a l o n g w i t h the a s s o c i a t e d HI y i e l d i n g the n u c l e o s o m a l c o r e p a r t i c l e . L. The H i s t o n e s The h i s t o n e s , a group o f b a s i c p r o t e i n s , a r e t h e major s t r u c t u r a l p r o t e i n s of c h r o m a t i n . The nomenclature and some p r o p e r t i e s of t h e h i s t o n e s a r e summarized i n T a b l e I . H i s t o n e H5 i s s i m i l a r to HI and i s found i n t h e n u c l e a t e d e r y t h r o c y t e s o f b i r d s , amphibians and f i s h . E x a m i n a t i o n o f t h e p r i m a r y s t r u c t u r e of t h e nu c l e o s o m a l h i s t o n e s p r e s e n t e d i n F i g u r e s 1 to 4 i l l u s t r a t e s t h e unequal d i s t r i b u t i o n of the 2 TABLE I H i s t o n e A l t e r n a t e Nomenclatures M o l e c u l a r Weight NH^-term i n u s HI I , F l , KAP, l y s i n e - r i c h 21,000 A c e t y l - S e r H2A I l b i , F2a2, LAK ) s l i g h t l y 14,000 A c e t y l - S e r ) l y s i n e H2B I I b 2 , F2b, KAS ) r i c h 13,000 Pro H3 I I I , F3, ARE ) a r g i n i n e 15,300 A l a ) H4 IV, F 2 a l , GRK ) r i c h 11,300 A c e t y l - S e r H5 V, F2C, KSA, e r y t h r o c y t e - 20,000 Thr s p e c i f i c 3 b a s i c amino a c i d s . There i s a predominance of t h e s e amino a c i d s a t the a m i n o - t e r m i n a l p o r t i o n of t h e n u c l e o s o m a l h i s t o n e s , whereas the carboxy-t e r m i n a l p o r t i o n c o n t a i n s a l a r g e number of h y drophobic amino a c i d s c a p a b l e of f o l d i n g i n t o a g l o b u l a r s t r u c t u r e . U n l i k e t h e nucleosomal h i s t o n e s , b o t h t h e amino- and c a r b o x y - t e r m i n a l r e g i o n s of H i a r e h i g h l y p o s i t i v e l y charged ( F i g . 5 ) . One s t r i k i n g f e a t u r e of the h i s t o n e s i s t h e e v o l u t i o n a r y i n v a r i a b i l i t y of t h e i r amino a c i d sequences. H i s t o n e s H3 and H4 a r e t h e most c o n s e r v e d , w i t h H4 showing the l e a s t v a r i a b i l i t y o f a l l p r o t e i n s sequenced ( 2 ) . Though H2A and H2B show v a r i a b i l i t y i n t h e a m i n o - t e r m i n a l p o r t i o n , t h e n e t p o s i t i v e charge has been r e t a i n e d . The c a r b o x y - t e r m i n a l p o r t i o n s of t h e n u c l e o s o m a l h i s t o n e s have been u n u s u a l l y h i g h l y conserved d u r i n g e v o l u t i o n ( 3 - 5 ) . These r e g i o n s a r e i n v o l v e d i n important h i s t o n e - h i s t o n e i n t e r a c t i o n s which form the n u c l e o s o m a l c o r e p a r t i c l e . HI i s the l e a s t c o nserved of th e h i s t o n e s . E x a m i n a t i o n of H i ' s amino a c i d sequence sugg e s t s t h a t i t s b i o l o g i c a l f u n c t i o n d i f f e r s from t h a t of the n u c l e o s o m a l h i s t o n e s . HI i s not i n v o l v e d i n n u c l eosomal f o r m a t i o n but may f u n c t i o n i n t h e maintenance o f the h i g h e r o r d e r s t r u c t u r e of c h r o m a t i n . 4 , , . . : , A C - S E R - G L Y - A R G - G L Y - L Y S - G L N - G L Y - G L Y - L Y S - A L A - A R G - A L A - L Y S - A L A - L Y S - T H R - A R G - S E R - S E R - A R G -1 1 0 2 0 ' - A L A - G L Y - L E U - G L N - - P H E - P R O - V A L - G L Y - A R G - V A L - H I S - A R G - L E U - L E U - A R G - L Y S - G L Y - A S N - T Y R - A L A - G L U -- 3 0 4 0 - A R G - V A L - G L Y - A L A - G L Y - A L A - P R O - V A L - T Y R - L E U - A L A - A L A - V A L - L E U - G L U - T Y R - L E U - T H R - A L A - G L U - I L E -5 0 6 0 - L E U - G L U - L E U - A L A - - G L Y - A S N - A L A - A L A - A R G - A S P - A S N - L Y S - L Y S - T H R - A R G - • I L E - I L E - P R O - A R G - H I S - L E U -: 7 0 8 0 - G L N - L E U - A L A - ! L E - - A R G - A S N - A S P - G L U - G L U - L E U - A S N - L Y S - L E U - L E U - G L Y - L Y S - V A L - T H R - I L E - A L A - G L N -9 0 1 0 0 - G L Y - G L Y - V A I - L E U - - P R O - A S N - I L E - G L N - A L A - V A L - L E U - L E U - P R O - L Y S - L Y S - - T H R - G L U - S E R - H I S - H I S - L Y S -1 1 0 1 2 0 - A L A - L Y S - G L Y - L Y S - C O O I 1 ) 1 2 9 FIG. 1. Amino a c i d sequence of h i s t o n e H2A, i n d i c a t i n g t h e p r e s e n c e of a c e t y l - N - s e r i n e a t t h e a m i n o - t e r m i n a l and of e p s i l o n - N - a c e t y l l y s i n e a t l y s i n e - 5 . S e r i n e r e s i d u e s a t p o s i t i o n s 1 and 19 a r e p o t e n t i a l s i t e s of p h o s p h o r y l a t i o n ( 2 ) . 5 Ac P Ac P Ac i i — i — Ac il'i'-pRO-GLU-PRO-ALA-LYS-SER-ALA-PRO-ALA-PRO-LYS-LYS-GLY-SlIR-LYS-LYS-ALA-VAL-THR-LYS-1 ' Io~ ~ 2~n~ '-}:- ™!l™lLY ll1} S P' R"- Y' L- Y ^  LVS -ARG -LYS -ARG -SE R -ARG -LYS -GLU -SER -TYR -SER -VAL -TYR -VAL-3 0 <fn" - T Y R -L Y S - V A L -L E U - L Y S - G L N - V A L - H I S - P R O - A S P - T H R - G L Y - I L E - S E R - S E R - L Y S - A L A - M E T - G L Y - I L E - M E T -. : 5 G 6 0 - A S N - S E R - P H E - V A L - A S N - A S P - I L E - P H E - G L U - A R G - I L E - A L A - G L Y - G L U - A L A - S E R - A R G - L E U - A L A - H I S - T Y R -7 0 8 0 - A S N - L Y S - A R G - $ E R - T H R - 1 L E - T H R - S E R - A R G - G L U - 1 L E - G L N - T H R - A L A - V A L - A R G - L E U - L E U - L E U - P R O - G L Y -9 0 1 0 0 - G L U - L E U - A L A - L Y S - H I S - A L A - V A L - S E R - G L U - G L Y - T H R - L Y S - A L A - V A L - T H R - L Y S - T Y R - T H R - S E R - S E R - L Y S - C Q O H ) '. H O 1 2 0 1 2 5 FIG. 2. Amino acid sequence of histone H2B, indicating the presence of multiple epsilon-N-acetyllysine residues at positions 5, 12, 15 and 20. Sites of phosphorylation at serine residues 6, 14, 32 and 36 are also shown (2). 6 flc_P Ac Ac H N - A L A - A R G - T H R - L Y S - G L N - T H R - A L A - A R G - L Y S - S E R - T H R - G L Y - G L Y - L Y S - A L A - P R O - A R G - L Y S - G L N - L E U -Ac ME P - A L A - T H R - L Y S - A L A - A L A - A R G - L Y S - S E R - A L A - P R O - A L A - T H R - G L Y - G L Y - V A L - L Y S - L Y S - P R O - H I S - A R G - T Y R -3 0 '10 - A R G - P R O - G L Y - T H R - V A L - A L A - L E U - A R G - G L U - I L E - A R G - A R G - T Y R - G L N - L Y S - S E R - T H R - G L U - L E U - - L E U - I L E -• 5 0 6 0 - A R G - L Y S - L E U - P R O - P H E - G L N - A R G - L E U - V A L - A R G - G L U - I L E - A L A - G L N - A S P - P H E - - L Y S - T H R - A S P - - L E U - A R G -7 0 8 0 - P H E - G L N - S E R - S E R - A L A - V A L - M E T - A L A - L E U - G L N - G L U - A L A - C Y S - G L U - A L A - T Y R - L E U - V A L - G L Y - - L E U - P H E -9 0 1 0 0 - G L U - A S P - T H R - A S N - L E U - C Y S - A L A - I L E - H I S - A L A - L Y S - A R G - V A L - T H R - I L E - M E T - • P R O - L Y S - A S P - I l E - G l N -no- ' 120 - L E U - A L A - A R G - A R G - I L E - A R G - G L Y - G L U - A R G - A L A - C O O H | 1 3 0 1 3 5 FIG. 3. Amino a c i d sequence of h i s t o n e H3, i n d i c a t i n g s i t e s of a c e t y l a t i o n a t l y s i n e r e s i d u e s 9 (which i s a l s o a s i t e of m e t h y l a t i o n i n some H3 m o l e c u l e s ) , 14, 18 and 23. P h o s p h o r y l a t i o n may o c c u r a t s e r i n e r e s i d u e s i n p o s i t i o n s 10 and 28. L y s i n e - 2 7 i s s u b j e c t to m e t h y l a t i o n ( 2 ) . Ac Ac Ac Ac i i i i i i A C ' S E R - G L Y - A R G - G L Y - L Y S - G L Y - G L Y - L Y S - G L Y - L E U - G L Y - L Y S - G L Y - 6 L Y - A L A - L Y S - A R G - I I I S - A R G - L Y S -1 1 0 2 0 - V A L - L E U - A R G - A S P - A S N - I L E - G L N - G L Y - I L E - T H R - L Y S - P R O - A L A - I L E - A R G - A R G - L E U - A L A - A R G - A R G - G L Y -3 0 4 0 - G L Y - V A L - L Y S - A R G - I L E - S E R - G L Y - L E U - I L E - T Y R - G L U - G L U - T H R - A R G - G L Y - V A L - L E U - L Y S - V A L - P H E - I - E U -5 0 6 0 - G L U - A S N - V A L - I L E : - A R G - A S P - A L A - V A L - T H R - T Y R - T H R - G L U - H I S - A L A - L Y S - A R G - L Y S - T H R - V A L - T H R - A L A 7 0 8 0 - ^ E T - A S P - V A L - V A L - T Y R - A L A - L E U - L Y S - A R G - G L N - G L Y - A R G - T H R - L E U - T Y R - G L Y - P H E - G L Y - G L Y - C O O H J 1 9 0 1 0 0 1 0 2 FIG. 4. Amino acid sequencer, of histone H4, indicating multiple sites of acetylation at lysine residues 5, 8, 12 and 16. The amino-terminal serine residue is stably acetylated and transiently phosphorylated. Lysine-20 is a site of methylation (2). 8 5 10 15 AcAla-Clu-Val-Ala-Pro-Ala-Pro-Ala-Ala-Ala-Ala-Pro-Ala-Lys-Ala-i 20 25 30 Pro-Lys-Lys-Lys-Ala-Ala-Ala-Lys-Pro-Lys-Lys-Ser-Gly-Pro - A l a -35 40 45 Val-Gly-Glu-Leu-Ala-Gly-Lys-Ala-Val-Ala-Ala7Ser-Lys-Clu-Arg-50 55 ' 60 Ser-Gly-Val-Ser-Leu-Ala-Ala-Leu-Lys-Lys-Ser-Leu-Ala-Ala-Gly-65 70 75 Gly-Tyr-Asp-Val-Glu-Lys-Asn-Asn-Ser-Arg-Val-Lys-Ile-Ala - V a l -80 85 90 Lys-Ser-Leu-Val-Tlir-Lys-Gly-Thr-Leu-Val-Glu-Thr-Lys-Gly-Thr-95 100 105 Gly-Ala-Ser-Gly-Ser-Phe-Lys-Leu-Asn-Lys-Lys-Ala-Val-Clu-Ala-110 115 120 Lys-Lys-Pro-Ala-Lys-Lys-Ala-Ala-Ala-Pro-Lys-Ala-Lys-Lys-Val-125 130 135 Ala-Ala-Lys-Lys-Pro-Ala-Ala-Ala-Lys-Lys-Pro-Lys-Lys-Val - A l a -140 145 150 Ala-Lys-Lys-Ala-Val-Ala-Ala-Lys-Lys-Ser-Pro-Lys-Lys-Ala-Lys-155 160 165 Lys-Pro-Ala-Thr-Pro-Lys-Lys-Ala-Ala-L^ys^-S^e^P^r^-Lys^-Lys-Alu-170 175 1 8 0 Thr-Lys-Ala-Ala-Lys-Pro-Lys-Ala-Ala-Lys-Pro-Lys-Lys-Ala-Ala-185 190 Lys-Ser-Fro-Lys-Lys-Val-Lys-Ly s-Pro-A 1 n-Ala-Ala-l.y s-Ly s FIG. 5. The complete sequence of t r o u t t e s t i s H i showing the p o s i t i o n s of t h e r e p e a t i n g p e n t a p e p t i d e s u b u n i t (, ) and of t h e s i t e s of p h o s p h o r y l a t -i o n ( ) (241). 9 I I . H i s t o n e M o d i f i c a t i o n s The h i s t o n e s undergo a v a r i e t y of p o s t - s y n t h e t i c m o d i f i c a t i o n s such as a c e t y l a t i o n , p h o s p h o r y l a t i o n , m e t h y l a t i o n and A D P - r i b o s y l a t i o n . The m o d i f i c a t i o n s a r e sequence s p e c i f i c and o c c u r i n a s p e c i f i c r e g i o n of t h e h i s t o n e m o l e c u l e . M o d i f i c a t i o n s of t h e n u c l e o s o m a l h i s t o n e s o c c u r i n t h e h i g h l y b a s i c a m i n o - t e r m i n a l p a r t s of t h e m o l e c u l e w h i l e t h e m o d i f i c a t i o n s to HI o c c u r i n b o t h the amino-and c a r b o x y - t e r m i n a l p o r t i o n s . The m o d i f i c a t i o n s a l l o w f o r d r a m a t i c changes i n the c h e m i c a l n a t u r e of t h e p r o t e i n ' s s i d e c h a i n s . For example, a c e t y l a t i o n of a l y s y l s i d e c h a i n w i l l n e u t r a l i z e i t s p o s i t i v e c h a r g e . Thus, the m o d i f i c a t i o n s a l t e r t h e p r o p e r t i e s of t h e s i d e c h a i n i n t h e same manner as a r a d i c a l amino a c i d s u b s t i t u t i o n . 10 (a) A c e t y l a t i o n H i s t o n e s can be e n z y m a t i c a l l y a c e t y l a t e d i n two d i s t i n c t w a y S j ( 6 ) . In the f i r s t t y pe, a c e t y l a t i o n o c c u r s a t th e 2-amino group of the amino-t e r m i n a l s e r i n e o f h i s t o n e s HI, H2A and H4. T h i s t y p e of a c e t y l a t i o n f o l l o w s soon a f t e r s y n t h e s i s and i s e s s e n t i a l l y i r r e v e r s i b l e . The second t y p e o c c u r s a t t h e e-amino groups of the l y s y l r e s i d u e s a t t h e a m i n o - t e r m i n a l p o r t i o n of the n u c l e o s o m a l h i s t o n e s . U n l i k e t h e f i r s t t ype of a c e t y l a t i o n , N - a c e t y l a t i o n i s a r a p i d , r e v e r s i b l e m o d i f i c a t i o n (7, 8) which o c c u r s l a r g e l y i n t h e S phase of the c e l l c y c l e . N - a c e t y l a t i o n o c c u r s a t s p e c i f i c l y s y l r e s i d u e s i n t h e a m i n o - t e r m i n a l p o r t i o n of n u c l e o s o m a l h i s t o n e s ( F i g . 1-4). Comparison of the amino a c i d sequences around the a c e t y l a t e d l y s y l r e s i d u e s s u g g e s t s t h a t the a c e t y l a t i o n s i t e s can be c l a s s i f i e d i n t o two t y p e s . Type A, an e - a c e t y l a t e d l y s i n e i s s urrounded by amino a c i d s w i t h s m a l l n e u t r a l s i d e c h a i n s , and type B, th e l y s i n e i s p a r t of a L y s - A r g , Arg-Lys or L y s - L y s p a i r . The sequence about the a c e t y l a t e d l y s y l r e s i d u e may a c t as r e c o g n i t i o n s i t e s f o r t h e enzymes r e s p o n s i b l e f o r t h e a d d i t i o n and removal of the a c e t y l groups, namely the h i s t o n e a c e t y l t r a n s f e r a s e s and d e a c e t y l a s e s ( 9 ) . These enzymes may a l s o r e c o g n i z e a secondary f e a t u r e of t h e h i s t o n e s , i . e . the n u c l e o s o m a l h i s t o n e c o r e complex. H i s t o n e a c e t y l t r a n s f e r a s e s and d e a c e t y l a s e s have been p u r i f i e d ( 2 ) . S t u d i e s of t h e s e enzymes suggest t h a t many d i f f e r e n t t y p e s of h i s t o n e a c e t y l t r a n s f e r a s e s and d e a c e t y l a s e s may be i n v o l v e d i n t h e p r o c e s s of r a p i d a d d i t i o n and removal of the a c e t y l groups. 11 N e u t r a l i z a t i o n o f the p o s i t i v e l y charged l y s y l groups by a c e t y l a t i o n would reduce the i o n i c i n t e r a c t i o n s between, the a m i n o - t e r m i n a l p o r t i o n of the h i s t o n e and the n e g a t i v e l y charged phosphate backbone of the DNA. Such a l t e r a t i o n s of the DNA-histone i n t e r a c t i o n , and hence a c e t y l a t i o n , may be r e q u i r e d f o r c h r o m a t i n assembly, removal of h i s t o n e s from DNA and t r a n s c r i p t i o n of c h r o m a t i n . The b i o l o g i c a l r o l e o f h i s t o n e a c e t y l a t i o n i n c h r o m a t i n assembly has been s t u d i e d by L o u i e and Dixon (10). K i n e t i c s t u d i e s i n d i c a t e d t h a t the newly s y n t h e s i z e d H4 m o l e c u l e s were s u b j e c t e d to s t e p w i s e a c e t y l a t i o n s f o l l o w e d by d e a c e t y l a t i o n . M u l t i p l e a c e t y l a t i o n s o f H4 would a l l o w the p o s i t i v e charge d e n s i t y of the a m i n o - t e r m i n a l p o r t i o n o f the m o l e c u l e to d e c r e a s e , a l l o w i n g f o r the c o r r e c t a l i g n m e n t of the h i s t o n e w i t h DNA. Removal of the a c e t y l groups would then a l l o w the c o r r e c t i o n i c bonds to be made between the l y s y l group and phosphate backbone of DNA. H i s t o n e a c e t y l a t i o n may be r e q u i r e d f o r removal of the h i s t o n e from DNA. D u r i n g spermatogenesis i n t r o u t t e s t i s , the h i s t o n e s a r e r e p l a c e d by a h i g h l y b a s i c p r o t e i n , protamine. The p o s s i b l e involvement of h i s t o n e a c e t y l a t i o n d u r i n g t h i s replacement p r o c e s s has been suggested by Candido and Dixon (11). The c o r r e l a t i o n between h i s t o n e a c e t y l a t i o n and t r a n s c r i p t i o n a l a c t i v i t y w i l l be d i s c u s s e d i n a l a t e r s e c t i o n . 12 (b) M e t h y l a t i o n Of t h e f i v e major h i s t o n e s o n l y H3 and H4 a r e m e t h y l a t e d . The m o d i f i c a t i o n o c c u r s a t t h e N^-amino group o f l y s i n e s w i t h a r e s u l t i n g change i n b a s i c i t y and h y d r o p h o b i c i t y but no change i n charge a t p h y s i o l o g i c a l pH. The m e t h y l groups a r e t r a n s f e r r e d e n z y m a t i c a l l y to a l y s y l r e s i d u e i n t h e m o l e c u l e ' s a m i n o - t e r m i n a l p o r t i o n , and the m o d i f i c a t i o n i s s t a b l e ( 1 2 ) . H4 i s m e t h y l a t e d a t o n l y one s i t e , the l y s i n e a t p o s i t i o n 20 (13-16), w h i l e H3 can be m e t h y l a t e d a t two major s i t e s , L y s 9 and Lys 27, and some-times a t two minor s i t e s ( 1 7 ) , Lys 4 and Lys 36. The m o d i f i e d l y s y l r e s i d u e may c o n t a i n from one to t h r e e methyl groups. In d i f f e r e n t organs of the r a t (14), E h r l i c h a s c i t e s tumor c e l l s ( 1 8 ) , t r o u t t e s t i s ( 1 3 ) , c a l f thymus (19), and carp t e s t i s (20), H4 i s m o d i f i e d m a i n l y as the N - d i m e t h y l l y s i n e , and H3 i s m o d i f i e d as the N^-monomethyl l y s i n e , N^-dimethyl l y s i n e and N ^ - t r i m e t h y l l y s i n e w i t h the N^-dimethyl l y s i n e p r e d o m i n a t i n g . However, H4 from pea s e e d l i n g i s not m e t h y l a t e d , and H3 from t h e same sou r c e e x i s t s as th e N^-mono-and N^-dimethyl l y s i n e w i t h no N ^ - t r i m e t h y l l y s i n e d e t e c t a b l e (21,22). The m e t h y l group i s t r a n s f e r r e d e n z y m a t i c a l l y w i t h S-adenosylmethionine as the methyl donor. M e t h y l a s e s have been i s o l a t e d from H e l a S-3 c e l l s ( 2 3 ) , c h i c k e n embryo n u c l e i ( 2 4 ) , and r a t b r a i n c h r o m a t i n ( 2 5 ) . The methylases have been found i n t h e n u c l e u s (24, 25) and may be c h r o m a t i n bound ( 2 5 ) . 13 When the h i s t o n e s a r e ch r o m a t i n bound, o n l y H3 and H4 a r e used as s u b s t r a t e s ( 2 5 ) , but the s p e c i f i c i t y i s l o s t when s o l u b l e h i s t o n e s a r e used as s u b s t r a t e s . Thus, c h r o m a t i n bound H3 and H4 may be the t r u e s u b s t r a t e s f o r the m e t h y l a s e s . The temporal sequence o f H3 and H4 m e t h y l a t i o n a f t e r s y n t h e s i s has been examined i n E h r l i c h a s c i t e s tumor c e l l s (18) and t r o u t t e s t i s ( 1 2 ) . M e t h y l a t i o n o c c u r r e d more s l o w l y than h i s t o n e s y n t h e s i s , and the m e t h y l a t i o n p r o b a b l y o c c u r r e d a f t e r t h e h i s t o n e was bound t o DNA. H4 m e t h y l a t i o n p r o b a b l y f o l l o w s t h e s t e p w i s e a c e t y l a t i o n s and d e a c e t y l a t i o n s ( 1 2 ) . M e t h y l a t i o n may be i n v o l v e d i n the f i n a l arrangement o f h i s t o n e s H3 and H4 on newly r e p l i c a t e d DNA (18) and might be i n v o l v e d i n h i s t o n e i n t e r -a c t i o n s w i t h o t h e r m o l e c u l e s such as h i s t o n e phosphokinases ( 1 2 ) . (c) P h o s p h o r y l a t i o n A l l f i v e major h i s t o n e s from t r o u t t e s t i s (6) and mammalian c e l l s (26,27) a r e p h o s p h o r y l a t e d w i t h t h e p o s s i b l e e x c e p t i o n o f H2B (26,27). The e x t e n t o f p h o s p h o r y l a t i o n d u r i n g the c e l l c y c l e v a r i e s f o r each h i s t o n e (26-31). F o r t h e n u c l e o s o m a l h i s t o n e s , the s i t e s o f p h o s p h o r y l a t i o n a l l o c c u r i n the b a s i c a m i n o - t e r m i n a l p o r t i o n of the m o l e c u l e ( F i g . 1-5, T a b l e I I ) , .but f o r HI the p h o s p h o r y l a t i o n s i t e s o c c u r i n b o t h the a m i n o - t e r m i n a l or c a r b o x y - t e r m i n a l p o r t i o n s o f t h e m o l e c u l e . 14 Temporal s t u d i e s of h i s t o n e p h o s p h o r y l a t i o n u s i n g s y n c h r o n i z e d Chinese hamster o v a r y c e l l s (26) and H e l a S-3 c e l l s (27) suggest t h a t . R l and 113 a r e p h o s p h o r y l a t e d a t d i f f e r e n t times d u r i n g t h e c e l l c y c l e w h i l e H2A.and I!4 ' a r e p h o s p h o r y l a t e d a t u n i f o r m r a t e s d u r i n g t h e c e l l c y c l e . K i n e t i c s t u d i e s of t h e p h o s p h o r y l a t i o n o f H2A and H4 i n t r o u t t e s t i s i n d i c a t e t h e s e h i s t o n e s a r e p h o s p h o r y l a t e d s h o r t l y a f t e r s y n t h e s i s (32). Phos-p h o r y l a t i o n of H4 d i d not o c c u r a p p r e c i a b l y u n t i l a f t e r a s e r i e s of a c e t y l a t i o n s and d e a c e t y l a t i o n s w h i l e H2A was p h o s p h o r y l a t e d s h o r t l y a f t e r s y n t h e s i s f o l l o w e d by d e p h o s p h o r y l a t i o n . H3 d i f f e r s from t h e o t h e r n u c l eosomal h i s t o n e s i n t h a t i t i s phos-p h o r y l a t e d to a g r e a t e r e x t e n t d u r i n g m i t o s i s than d u r i n g o t h e r p a r t s of t h e c e l l c y c l e (.26, 31). D u r i n g the c e l l c y c l e of Chinese hamster c e l l s , H3 was p h o s p h o r y l a t e d d u r i n g i n t e r p h a s e but t h e l e v e l s of p h o s p h o r y l a t e d H3 i n c r e a s e d d r a m a t i c a l l y d u r i n g m i t o s i s . The p h o s p h o r y l a t e d H3 was d e p h o s p h o r y l a t e d when t h e c e l l s l e f t anaphase. HI has been the most s t u d i e d h i s t o n e w i t h r e g a r d s t o i t s temporal p h o s p h o r y l a t i o n p a t t e r n . The p h o s p h o r y l a t i o n of HI o c c u r s a t from one to f o u r s i t e s ( T a b l e I I ) . As w i t h H3, HI i s p h o s p h o r y l a t e d a t d i f f e r e n t times d u r i n g the c e l l c y c l e (26, 31) w i t h maximal p h o s p h o r y l a t i o n o c c u r r i n g d u r i n g m i t o s i s . During t h e c e l l c y c l e H i i s p h o s p h o r y l a t e d i n d i f f e r e n t r e g i o n s of t h e m o l e c u l e (31, 33). D u r i n g i n t e r p h a s e , H i i s p h o s p h o r y l a t e d i n t h e c a r b o x y - t e r m i n a l p o r t i o n , but d u r i n g maximal p h o s p h o r y l a t i o n i n m i t o s i s , HI i s p h o s p h o r y l a t e d a t b o t h amino-and c a r b o x y - t e r m i n a l p o s i t i o n s . 15 TABLE I I P h o s p h o r y l a t i o n Sequences i n H i s t o n e s H i s t o n e Sequence Source Group 1: H2A PO, Ac A c - S e r - G l y - A r g - G l y - L y s 1 5 t r o u t l i v e r t e s t i s , i n v i v o r a t H4 •PO. Ac .4 i A c - S e r - G l y - A r g - G l y - L y s 1 5 t r o u t t e s t i s Group 2: H2B Ac PO, . t4 - A l a - L y s - S e r - A l a - P r o 6 t r o u t t e s t i s H3 (1) Me PO. t t4 - A r g - L y s - S e r - T h r - G l y - t r o u t t e s t i s , c a l f (2) 10 Ac PO. i .4 - A r g - L y s - S e r - A l a - P r o 28 thymus n u c l e i i n v i t r o t r o u t t e s t i s , c a l f thymus n u c l e i i n v i t r o Group 3: PO, r a t l i v e r m vxvo |4 . rr—. T T I / i \ T A T A -i o ^ . 1 T . T . T T r a t l i v e r h i s t o n e Hi (1) - L y s - A r g - L y s - A l a - S e r - G l y - P r o - P r o - V a l , . _ . k i n a s e I ( c y c l i c AMP) i n v i t r o ,4 r a t l i v e r h i s t o n e (2) - G l y - A l a - S e r - G l y - S e r - P h e - L y s k i n a s e ( n o n - c y c l i c 106 AMP, i n v i t r o ) (3) - A l a - A l a - L y s - L y s - S e r - P r o - L y s t r o u t t e s t i s 157 Data c o l l e c t e d from Dixon et a l . (6) 16 -S t u d i e s of HI p h o s p h o r y l a t i o n d u r i n g the c e l l c y c l e o f Physarum  poly c e p h a l u m suggest t h a t the degree of p h o s p h o r y l a t i o n d u r i n g t h e c e l l c y c l e d i f f e r s from t h a t o f mammalian c e l l s (28, 2 9 ) . HI was p h o s p h o r y l a t e d d u r i n g G2 and reached a peak at e a r l y prophase. The p h o s p h o r y l a t e d HI was s u b s e q u e n t l y d e p h o s p h o r y l a t e d as the c e l l s p r o g r e s s e d to metaphase. The r e s u l t s suggested t h a t HI must be c o m p l e t e l y p h o s p h o r y l a t e d b e f o r e chromosome c o n d e n s a t i o n , and thus may a c t as a " m i t o t i c t r i g g e r " . J. A l t e r -n a t i v e l y , i n mammalian c e l l s p h o s p h o r y l a t i o n of HI d u r i n g i n t e r p h a s e may be a p r e p a r a t i v e step f o r chromosome c o n d e n s a t i o n not a " m i t o t i c t r i g g e r " ( 3 1 ) , o r i t may be i n v o l v e d i n t h e compaction of h e t e r o c h r o m a t i n (34) w h i l e p h o s p h o r y l a t i o n d u r i n g m i t o s i s may be r e l a t e d t o chromosome c o n d e n s a t i o n ( 2 7 ) . Enzymes r e s p o n s i b l e f o r h i s t o n e p h o s p h o r y l a t i o n have been i s o l a t e d (35-38). The k i n a s e s may be e i t h e r cAMP-dependent (350 o r independent (36-38), and i n some i n s t a n c e s the k i n a s e s demonstrate h i s t o n e s p e c i f i c i t y (37, 38). The b i o l o g i c a l r o l e of h i s t o n e p h o s p h o r y l a t i o n remains l a r g e l y a mystery. P r o b a b l y t h e r o l e o f p h o s p h o r y l a t i o n f o r h i s t o n e s H4, H2A and H2B d i f f e r s from t h a t of H3 and HI. P h o s p h o r y l a t i o n of H4 and H2B may be i n v o l v e d i h t h e c o r r e c t p o s i t i o n i n g and b i n d i n g of t h e newly s y n t h e s i z e d h i s t o n e to DNA (32) w h i l e H3 and HI may be i n v o l v e d i n chromosome c o n d e n s a t i o n d u r i n g m i t o s i s (26, 27, 31, 33, 39). 17 (d) N-Phosphorylation Phosphorylation can occur at the lysyl residue of HI (N -phosphoryl lysine), and the hi s t i d y l of H4 (3-phosphoryl histidine) (40). The modification i s acid-labile and alkali-stable and would be destroyed by acid-extraction procedures commonly used for isolating histones. Histone kinases responsible for the N-phosphorylation have been isolated from regenerating rat liver (40) and Walker-256 carcinosarcoma cells (41). One kinase with pH optimum of 9.5 could phosphorylate the His of H4 while another with a pH optimum of 6.5 could phosphorylate the lysine of HI. Both kinases were unaffected by cAMP (41). In regenerating rat. liver only pre-existing H4 molecules were phosphorylated at the peak of DNA synthesis (42) . The modification has a h a l f - l i f e of two hours and has been suggested to be involved in the replication of DNA (40, 42). (e) Adenosine - Diphospho - Ribosylation ( ADP-ribosylation) Histones are one of the many types of proteins to be ADP-ribosylated. Hi is the major acceptor of ADP-ribose groups (43-46, 102) while the "core" histones are ADP-ribosylated only to a minor extent. In rat (44) and Hela cells (47) H2B, H2A and H3 were found to be modified while ADP-ribosylation of H4 was not detectable. 18 i The l i n k a g e of t h e ADP-ribose g r o u p ( s ) w i t h HI i s a l k a l i - l a b i l e (48, 4 9 ) . For HI from r a t l i v e r t h e ADP-ribose group i s l i n k e d to a s e r i n e r e s i d u e (102, 4 3 ) , but f o r HI from t r o u t t e s t i s t h e l i n k a g e i s to the -C00H o f g l u t a m i c a c i d ( 4 8 ) . The HI m o l e c u l e i s A D P - r i b o s y l a t e d i n bo t h t h e amino- and c a r b o x y - t e r m i n a l p o r t i o n s ( 4 8 ) . The number of ADP r i b o s e u n i t s a t t a c h e d to HI v a r i e s w i t h the source-and may be from 1 t o 15 (49, 50, 46, 4 8 ) . The number of u n i t s a t t a c h e d may depend t o some degree on t h e a c t i v i t y of p o l y ADP-ribose g l y c o h y d r o l a s e (50), an enzyme r e s p o n s i b l e f o r t h e d e g r a d a t i o n of t h e p o l y (ADP-ribose) c h a i n , w i t h i n a p a r t i c u l a r t i s s u e (46). HI has been r e p o r t e d t o be c r o s s - l i n k e d by a s i n g l e c h a i n of p o l y (ADP-ribose) 15 u n i t s l o n g i n H e l a c e l l s and HBL-100 c e l l s (a r a t mammary c e l l l i n e ) (46). The a c t i v i t y o f t h e g l y c o h y d r o l a s e i n t h e HBL-100 c e l l s was v e r y low. However, t h e a c t i v i t y of g l y c o h y d r o l a s e from r a t MTW-9 c e l l s (a r a t mammary tumor c e l l l i n e ) was r e l a t i v e l y h i g h , and t h e average number of u n i t s a t t a c h e d t o HI was reduced to 1.5. The d e g r a d a t i o n was not due to the p r e s e n c e of p h o s p h o d i e s t e r a s e , another enzyme which w i l l degrade the ADP-ribose polymer ( 4 9 ) . The enzyme r e s p o n s i b l e f o r t h e s y n t h e s i s o f p o l y ( A D P - r i b o s e ) , p o l y (ADP-ribose) polymerase, has been i s o l a t e d from s e v e r a l s o u r c e s i n c l u d i n g r a t l i v e r ( 50), t r o u t t e s t i s ( 50), and H e l a c e l l s (51, 47, 52). The enzyme, which has an almost a b s o l u t e requirement f o r DNA (50), i s t i g h t l y a s s o c i a t e d w i t h c h r o m a t i n (49) and c a t a l y z e s t h e s u c c e s s i v e a d d i t i o n of ADP r i b o s e u n i t s from NAD to form a polymer. The enzyme i s a s s o c i a t e d w i t h 19 t h e l i n k e r DNA r e g i o n between nucleosome c o r e p a r t i c l e s (47, 51, 52) and has been suggested to be a s s o c i a t e d w i t h template " a c t i v e " c h r o m a t i n (51). The b i o l o g i c a l f u n c t i o n o f t h i s m o d i f i c a t i o n i s p o o r l y u n d e r s t o o d , but i t has been suggested to be i n v o l v e d i n chromosome c o n d e n s a t i o n (45, 53). . ; I I I . The Nucleosome S i n c e t h e d i s c o v e r y i n 1974 t h a t c h r o m a t i n hag a b a s i c r e p e a t i n g s t r u c t u r a l u n i t ( c a l l e d the nucleosome)(1, 54, 55), many r e s e a r c h groups have p r e s e n t e d e v i d e n c e t o e l u c i d a t e the f i n e s t r u c t u r e o f t h i s r e p e a t i n g u n i t . A l t h o u g h t h e f i n e s t r u c t u r e o f the nucleosome w i t h r e g a r d s to c o n f o r m a t i o n o f t h e h i s t o n e s w i t h i n the nucleosome, h i s t o n e - h i s t o n e i n t e r a c t i o n s , and histone-DNA i n t e r a c t i o n s i s not f u l l y u n d e r s t o o d , many known a s p e c t s o f nucleosome s t r u c t u r e have enabl e d r e s e a r c h e r s t o p r e s e n t models o f t h i s r e p e a t i n g c h r o m a t i n u n i t . (a) H i s t o n e - H i s t o n e I n t e r a c t i o n s The h i s t o n e - h i s t o n e i n t e r a c t i o n s a r e im p o r t a n t i n maintenance o f t h e h i s t o n e c o r e complex. The h i s t o n e complement o f the complex c o n t a i n s two m o l e c u l e s each o f t h e h i s t o n e s H2A, H2B, H3 and H4 (1). The co r e h i s t o n e c o m p o s i t i o n has been examined and c o n f i r m e d by c r o s s - l i n k i n g s t u d i e s o f c h r o m a t i n (46) and o f i s o l a t e d n u c l e o s o m a l c o r e p a r t i c l e s (57). The co r e h i s t o n e complex has been i s o l a t e d f r e e o f DNA at h i g h i o n i c s t r e n g t h (58-61) and has been r e p o r t e d t o e x i s t i n s o l u t i o n as a t e t r a m e r , (H2A) 20 (H2B)(H3)(H4), (59, 61) o r an octamer, (H2A)^(H2B)^(H3) (H4)^ (56, 60, 62). E i c k b u s h and M o u d r i a n a k i s (62) f a v o r t h e octamer form of t h e h i s t o n e complex and suggest t h a t t h e d i s c r e p a n c y may be due t o i s o l a t i n g t h e complex i n an u n p u r i f i e d form. S t u d i e s on t h e i n t e r a c t i o n s o f the h i s t o n e s , and c r o s s - l i n k i n g s t u d i e s suggest the p r e f e r e n t i a l a s s o c i a t i o n o f H3 and H4 to form a (H.3) ^ t^) 2 t e t r a m e r (63-66), and t h e p r e f e r e n t i a l a s s o c i a t i o n of H2A and H2B to form a (H2A)(H2B) dimer (67, 68, 69, 62, 70", 63). NMR s t u d i e s have demonstrated t h e involvement of the c a r b o x y - t e r m i n a l p o r t i o n s of t h e h i s t o n e s i n t h e h i s t o n e - h i s t o n e i n t e r a c t i o n s . T h i s r e g i o n o f t h e m o l e c u l e adopts a ' g l o b u l a r s t r u c t u r e (71) w h i l e t h e a m i n o - t e r m i n a l p o r t i o n o f t h e m o l e c u l e e x i s t s as a random c o i l .(71). The a m i n o - t e r m i n a l p o r t i o n of t h e m o l e c u l e i s v u l n e r a b l e t o d i g e s t i o n by t r y p s i n when e i t h e r a s s o c i a t e d w i t h t h e h i s t o n e c o r e complex o r bound to DNA (59, 72). NMR s t u d i e s suggest t h e involvement o f r e s i d u e s 31-95 f o r H2A and 37-114 f o r H2B i n t h e f o r m a t i o n of the (H2A) (H2B) dimer (62, 71). R e s i d u e s 42-120 f o r H3 and 38-102 f o r H4 a r e i n v o l v e d i n the t e r t i a r y s t r u c t u r e of t h e (H.3) ^ Olb) ^ t e t r a m e r (71, 73). The i n t e r a c t i o n s o f H2A o r H2B w i t h H3 o r H4 have been s t u d i e d by u s i n g c h e m i c a l c r o s s l i n k i n g (67, 68, 70). Through t h e use of z e r o l e n g t h c r o s s l i n k e r s ( 7 0 ) , H2B has been found to c o n t a i n s e p a r a t e b i n d i n g s i t e s f o r H2A and H4. The c a r b o x y - t e r m i n a l h a l f of H2B i n t e r a c t s w i t h the c a r b o x y -t e r m i n a l p o r t i o n o f H4 w h i l e t h e a m i n o - t e r m i n a l p o r t i o n of H2B i n t e r a c t s w i t h 21 H2A. The i n t e r a c t i o n s o f the h i s t o n e components of the (H2A) (H2B) dimer o r (113) ^ (M) ^ t e t r a m e r a r e l a r g e l y h y d r o p h o b i c i n n a t u r e (62) w h i l e the i n t e r a c t i o n s between the two (H2A) (H2B) dimers and the (H.3) ^ (H4)2 t e t r a m e r to form t h e h i s t o n e octamer a r e p o s s i b l y m a i n t a i n e d by H-bonds. (b) H i s t o n e - DNA I n t e r a c t i o n s The n u c l e o s o m a l DNA i s compacted about seven f o l d (74). The DNA i s wrapped around the h i s t o n e c o r e about 1 3 / 4 t u r n s (75), w i t h each h i s t o n e c o r e complexing w i t h 140 base p a i r s of DNA i n a s i n g l e l e f t - h a n d e d , non-interwound DNA s u p e r c o i l (76). The n u c l e o s o m a l h i s t o n e s d e v o i d o f t h e i r a m i n o - t e r m i n a l amino a c i d s a r e c a p a b l e of compacting t h e DNA i n t o a n u c l e o p r o t e i n complex ( 7 7 ) . D i g e s t i o n o f c h r o m a t i n w i t h t r y p s i n r e s u l t s i n t h e r e l e a s e o f 20 to 30 amino a c i d s from t h e a m i n o - t e r m i n a l p o r t i o n o f the nucleosomal h i s t o n e s (72). I f t h e 11 S, n u c l e o s o m a l c o r e p a r t i c l e , i s d i g e s t e d w i t h t r y p s i n , the s e d i -m e n t a t i o n c o e f f i c i e n t changes to 9.7 S w i t h an accompanying i n c r e a s e d s e n s i t i v i t y to m i c r o c o c c a l n u c l e a s e (77, 78). Thus the a m i n o - t e r m i n a l p o r t i o n s o f t h e n u c l e o s o m a l h i s t o n e s may not be n e c e s s a r y f o r the f o l d i n g o f DNA i n t o a n u c l e o p r o t e i n complex but may be i n v o l v e d i n the maintenance of the n a t i v e n u c l e o s o m a l c o r e p a r t i c l e s t r u c t u r e . H3 and H4 a r e v e r y important i n t h e o r g a n i z a t i o n o f the DNA around the h i s t o n e c o r e complex (79, 80). H3 and H4,.when a s s o c i a t e d w i t h 140 22 base p a i r s of DNA, can.produce n u c l e o p r o t e i n complexes which a r e m o r p h o l o g i c a l l y s i m i l a r to nucleosomes ( 7 9 ) . H4 p l a y s a key r o l e i n the c o r r e c t a s s o c i a t i o n of DNA w i t h the h i s t o n e c o r e ( 8 0 ) . IV. Nucleosomal DNA S i z e D i v e r s i t y When c h r o m a t i n i s m i l d l y d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e , the DNA l i n k e r r e g i o n i s most s u s c e p t i b l e to a t t a c k . The d i g e s t p r o d u c t s may be s e p a r a t e d by g e l e l e c t r o p h o r e s i s ^ t o y i i l d . . . a r e p e a t of m u l t i p l e s of t h e lowest u n i t l e n g t h , about 200 base p a i r s ( 5 4 ) . A f t e r f u r t h e r d i g e s t i o n , &• the n u c l e o s o m a l DNA i s trimmed to about 140 base p a i r s , t h e l e n g t h of DNA a s s o c i a t e d w i t h t h e n u c l e o s o m a l c o r e p a r t i c l e (81, 8 2 ) . Extreme d i g e s t c o n d i t i o n s r e s u l t i n d i g e s t i o n of t h e i n t r a n u c l e o s o m a l DNA l e n g t h y i e l d i n g fragments o f about 127, 118, 108, 99, 80, 60 and 52 base p a i r s i n l e n g t h ( 8 0 ) . U s i n g m i l d d i g e s t c o n d i t i o n s , t h e nu c l e o s o m a l DNA l e n g t h has been found t o v a r y w i t h d i f f e r e n t s o u r c e s (83-88) ( T a b l e I I I ) . I t has been suggested t h a t t h e DNA r e p e a t l e n g t h d e c r e a s e d w i t h i n c r e a s i n g t r a n s c r i p t i o n a l a c t i v i t y of t h e c e l l s t u d i e d (.66). However, s t u d i e s by G o t t e s f e l d and Melton (85) demonstrate t h e l e n g t h of t h e nu c l e o s o m a l DNA remains the same f o r b o t h t r a n s c r i b e d and n o n t r a n s c r i b e d r e g i o n s of c h r o m a t i n . I n some ca s e s t h e DNA r e p e a t l e n g t h of a c t i v e l y d i v i d i n g c e l l s was s h o r t e r than t h a t of t h e mature c e l l ( 8 3 ) , but t h i s i s not a g e n e r a l c h a r a c t e r i s t i c of ch r o m a t i n from a c t i v e l y d i v i d i n g c e l l s ( 8 6 ) . 23 TABLE I I I DNA Content of Nucleosomes: C e l l type DNA r e p e a t l e n g t h (base p a i r s ) A s p e r g i l l u s Yeast R a b b i t c o r t i c a l neuron Neurospora Physarum Tetrahymena m i c r o n u c l e u s C e l l s grown i n c u l t u r e CHO HeLa hepatoma teratoma P815 myoblast C V l , e x p o n e n t i a l l y growing o r c o n f l u e n t BHK r a t k i d n e y p r i m a r y c u l t u r e myotube C6, e x p o n e n t i a l l y growing or c o n f l u e n t Rat bone marrow Rat f e t a l l i v e r '.. Rat l i v e r Rat k i d n e y S y r i a n hamster l i v e r S y r i a n hamster k i d n e y C h i c k o v i d u c t 154 165, 163 162 170 171, 173 175 177 183, 188 188 188 188 189 189 190 191 193 198 192 193 198, 196 196 196 196 196 24 (TABLE I I I Continued) C e l l t y p e DNA r e p e a t l e n g t h (base p a i r s ) Tetrahymena macronucleus 202 R a b b i t c e r e b e l l a r neuron 200 R a b b i t n o n a s t r o c y t i c g l i a l c e l l s 200 S t y l o n y c h i a m i c r o n u c l e u s 202 C h i c k e n e r y t h r o c y t e 207, 212 Sea u r c h i n g a s t r u l a 218 S t y l o n y c h i a macronucleus 220 Sea u r c h i n sperm 241 F o r r e f e r e n c e s , see Chambon (92) 25 What causes the n u c l e o s o m a l DNA s i z e d i v e r s i t y ? A l t h o u g h the r o l e o f nu c l e o s o m a l DNA l e n g t h v a r i a b i l i t y i s unknown, s u g g e s t i o n s f o r the cause o f the v a r i a b i l i t y have been made. V a r i a b i l i t y i n t h e nucleosomal DNA might r e s u l t from t h e v a r i a b i l i t y i n the p r i m a r y sequence o f HI (.81-84, 89) which b i n d s t o t h e nu c l e o s o m a l DNA b e f o r e trimming o f the nucleosomal DNA takes p l a c e (90) . Weintraub (83) has r e p o r t e d t h a t d u r i n g e r y t h r o p o i e s i s i n t h e c h i c k , the nucleosomal DNA l e n g t h i n c r e a s e s from 190 base p a i r s to 212 base p a i r s . Accompanying the i n c r e a s e i n n u c l e o s o m a l DNA l e n g t h i s an i n c r e a s e i n the l e v e l s o f t h e r e d b l o o d c e l l s p e c i f i c h i s t o n e , H5 (a h i s t o n e s i m i l a r to H i but more b a s i c ) . Thus, t h e pre s e n c e o f H5 may cause the change i n nu c l e o s o m a l DNA l e n g t h . A l t e r n a t i v e l y , Wilhelm et a l . (.91) doubt t h a t t h e r e i s a d i r e c t c o r r e l a t i o n between t h e p r i m a r y sequence o f HI o r H5 and the n u c l e o s o m a l DNA r e p e a t l e n g t h . The sequence v a r i a b i l i t y o f the nuc l e o s o m a l h i s t o n e s , H2A and H2B, might i n f l u e n c e the s i z e o f the nuc l e o s o m a l DNA (92, 93), There i s doubt as to whether o r not H2A and H2B a r e r e s p o n s i b l e . S p a d a f o r a et a l . have r e c o n s t i t u t e d chromatin by complexing the nu c l e o s o m a l h i s t o n e s from e i t h e r c a l f thymus, Ch i n e s e hamster o v a r y c e l l s or sea u r c h i n g a s t r u l a c e l l s w i t h SV40 DNA form I by s t e p w i s e d i a l y s i s from 2 M NaCl ( 9 4 ) . In a l l c a s e s , a l t h o u g h the H2A and H2B sequence v a r i e d , t h e DNA r e p e a t l e n g t h remained the same. C a u t i o n s h o u l d be taken w i t h t h e s e types o f r e c o n s t i t u t i o n experiments because i n a c c u r a c i e s i n the DNA-histone i n t e r a c t i o n s i n d u c e d by t h e method of r e c o n s t i t u t i o n used may l e a d t o a r t i f a c t u a l r e s u l t s . 26" V_. S t r u c t u r e o f t h e Nucleosome The b a s i c s t r u c t u r e o f t h e nucleosome c o r e p a r t i c l e i s a h i s t o n e c o r e surrounded by DNA. N o l l suggested the DNA was on t h e e x t e r i o r o f the p a r t i c l e from d i g e s t i o n s t u d i e s u s i n g DNase I ( 9 5 ) . T h i s enzyme has the a b i l i t y t o d i g e s t b o t h i n t e r - and i n t r a n u c l e o s o m a l DNA a t s i m i l a r r a t e s . The DNA d i g e s t p r o d u c t s , when de n a t u r e d , e x h i b i t e d a 10 base r e p e a t l a d d e r when s e p a r a t e d by g e l e l e c t r o p h o r e s i s . T h i s r e p e a t l e n g t h c o r r e s p o n d s to the p i t c h o f the DNA h e l i x . I d e a l l y , one would want to know the exact p o s i t i o n o f c u t t i n g s i t e s o f t h e n u c l e o s o m a l c o r e DNA w i t h t h i s enzyme and, t h e r e f o r e , be a b l e t o suggest where histone-DNA i n t e r a c t i o n s i n t e r f e r e d w i t h DNase I a t t a c k . By l a b e l l i n g t h e 5' ends of t h e nucleosomal c o r e p a r t i c l e s 32 w i t h P, v a r i o u s workers have shown t h a t s i t e s 30, 60, 70, 80 and 110 e x h i b i t a lower a c c e s s i b i l i t y t o d i g e s t i o n (96-100). U s i n g h i g h r e s o l u t i o n g e l e l e c t r o p h o r e s i s , L u t t e r r e p o r t e d t h a t t h e DNase I c u t t i n g s i t e s were m u l t i p l e s o f about 10.4 bases i n s t e a d o f 10 (101). Endonucleases o t h e r t h a n DNase I ( T a b l e IV) were examined f o r t h e i r i n t r a n u c l e o s o m a l c o r e DNA c u t t i n g p a t t e r n s . A l l o f t h e endonucleases cut a t a 10-nucleogide i n t e r v a l (96-99) but the s u s c e p t i b i l i t y o f the s i t e s d i f f e r e d . A l t h o u g h the c o n f o r m a t i o n o f t h e DNA w i t h i n t h e nucleosome i n f l u e n c e s i t s n u c l e a s e s u s c e p t i b i l i t y , t h e a c t u a l r a t e o f c l e a v a g e a t a g i v e n s i t e may be a f u n c t i o n o f the enzyme's c a t a l y t i c and p h y s i c o c h e m i c a l p r o p e r t i e s . However, a l l t h e endonucleases d i g e s t e d s i t e s 30, 60, 80 and 110 base p a i r s from t h e 5' end of the nu c l e o s o m a l c o r e DNA w i t h decreased 27. TABLE IV P r o p e r t i e s o f Endonucleases Used Source Beef pancreas Hog s p l e e n S t a p h y l o c o c c u s (DNase I) (DNase I I ) aureus ( M i c r o c o c c a l n u c l e a s e ) Mr I s o e l e c t r i c p o i n t pH optimum A c t i v a t o r s P r o d u c t 31,000 4.7 - 5.0 7.0 M g + 2 5' - Phosphate t e r m i n a l 38,000 10.2 4.8 EDTA 3' -Phosphate t e r m i n a l 16,800 9.6 9.2 C a + 2 3 L Phosphate t e r m i n a l 28 frequencies relative to other sites. Presumably, this variation could be attributed to the orientation of the sugar phosphate bonds to the histone core. From these observations the pitch of the DNA has been estimated to be 10.33 - 10.40 base pairs (10.0)"'.. Also, the results support suggestions that the DNA is wound around the histone core smoothly, without interruption of base-stacking interactions (75). Small angle neutron scattering measurements (103, 104) and X-ray scattering (105) data of the nucleosome further consolidate the endonuclease results that DNA is on the outside of the histone core. The results , • o •-indicate the histone core is 32 A in radius surrounded by a DNA rich shell' o o 20 A thick, giving the nucleosome core particle an average radius of 52 A. Crystals of the nucleosome core particle have been studied by electronmi-croscopy (106) and X-ray diffraction (75) . The nucleosome core particle o has dimensions of 110 x 110 x.'57 A, and is somewhat wedge-shaped. The DNA is wound into about 1 3/4 turns of a f l a t superhelix with a pitch of . '.~ o o about 28 A and an average diameter of about 90 A. The pitch of the super-helix is small enough that interactions between two DNA turns might take place i f aided by cations and/or histone salt bridges (i.e. amino-terminals of the nucleosomal histones). VI. Nucleosomal Models Romberg's model (1) for the nucleosome', consisting of an histone octamer, (H2A)2 (H2B.)2 (H3)„2 (HA)^ surrounded by DNA, has been basically unaltered. The model has been further detailed byWeintraub (76) to -explain transcription and replication processes in the presence of • 29 nucleosomes ( t o be d i s c u s s e d i n a l a t e r s e c t i o n ) . Based p a r t l y on t h e e x i s t e n c e i n s o l u t i o n of an h e t e r o t y p i c t e t r a m e r h i s t o n e complex, (H2A)(H2B) (H3)(H4), (61, 59) Weintraub has p r e s e n t e d a model f o r t h e n u c l e o s o m a l c o r e p a r t i c l e t h a t c o n s i s t s of two i s o l o g o u s l y p a i r e d h e t e r o t y p i c p r o t e i n t e t r a m e r s surrounded by a s i n g l e non-interwound, l e f t - h a n d e d DNA s u p e r c o i l 140 base p a i r s l o n g and about 95 base p a i r s i n c i r c u m f e r e n c e . The nucleosome i s o r g a n i z e d about a dyad a x i s of symmetry, a s p e c i f i c a t i o n t h a t i s s u p p o r t e d by X-ray d i f f r a c t i o n d a t a (75) and endonuclease d i g e s t i o n s t u d i e s (100). A l t h o u g h the e x i s t e n c e of an h e t e r o t y p i c t e t r a m e r h i s t o n e complex i n s o l u t i o n has been q u e s t i o n e d ( 6 2 ) , no e v i d e n c e i s a v a i l a b l e to d i s a p p r o v e t h a t t h e h i s t o n e octamer when bound to DNA cannot s p l i t i n t o s ymmetrical h a l v e s , each c o n t a i n i n g t h e h e t e r o t y p i c h i s t o n e t e t r a m e r . T h i s model has the a b i l i t y t o e x p l a i n how t r a n s c r i p t i o n o r r e p l i c a t i o n of the DNA might occur w i t h o u t h i s t o n e d i s p l a c e m e n t . V I I . Higher Order Chromatin S t r u c t u r e : Role of HI The "beads on a s t r i n g " form of c h r o m a t i n c o n s i s t s of the n u c l e o s o m a l c o r e p a r t i c l e s j o i n e d by t h e l i n k e r DNA and r e p r e s e n t s the mostf.extended form of c h r o m a t i n . Compaction of the l i n k e r a l l o w s t h e f o r m a t i o n of f i b e r s , o 100 A i n d i ameter (79, 74, 107, 108). F u r t h e r compaction r e s u l t s i n t h e o appearance of a 300 A d i ameter f i b e r (79, 109-111) or " s o l e n o i d s " (110). S t a b i l i z a t i o n o f the s o l e n o i d a l s t r u c t u r e r e q u i r e s HI (75, 110, 109, 112) and d i v a l e n t c a t i o n s . Of t h e f i v e main h i s t o n e s , HI i s the o n l y h i s t o n e not i n v o l v e d i n the f o r m a t i o n of t h e n u c l e o s o m a l c o r e p a r t i c l e . HI i s a s s o c i a t e d w i t h about 35-40 base p a i r s l e n g t h of DNA i n t h e l i n k e r r e g i o n ( 9 0 ) . The main f u n c t i o n 30 a s s i g n e d t o t h i s h i s t o n e i s a c r o s s l i n k i n g r o l e i n the maintenance o f a h i g h e r o r d e r c h r o m a t i n s t r u c t u r e (113, 109, 112, 75, 114). DNase I I appears to have t h e a b i l i t y t o r e c o g n i z e h i g h e r o r d e r c h r o m a t i n s t r u c t u r e s (79, 115, 116). N u c l e i o r chrom a t i n d i g e s t e d w i t h DNase I I a t a p p r o p i a t e i o n i c s t r e n g t h s ( 1 mM C a C l ^ o r 150 mM NaCl) w i l l produce d o u b l e - s t r a n d e d DNA p r o d u c t s w i t h a 100 base p a i r p e r i o d i c i t y , i n d i c a t i n g n u c l e a s e d i g e s t i o n between and w i t h i n t h e nucleosome c o r e p a r t i c l e s has o c c u r r e d (115). I o n i c c o n d i t i o n s of t h i s s o r t a r e r e q u i r e d f o r t h e maintenance o f h i g h e r o r d e r c h r o m a t i n s t r u c t u r e s (110, 112, 75, 113, 117, 109). DNase I I d i g e s t i o n o f c h r o m a t i n a t lower i o n i c s t r e n g t h r e s u l t s i n d i g e s t i o n o f o n l y t h e l i n k e r DNA r e g i o n . V I I I . T r a n s c r i p t i o n a l l y A c t i v e Chromatin A l t h o u g h t h e b u l k o f ch r o m a t i n appears t o c o n s i s t of a r e p e a t o f nucleosomes, does a c t i v e l y t r a n s c r i b e d c h r o m a t i n have the same n u c l e o s o m a l s t r u c t u r e ? D i g e s t i o n s t u d i e s o f n u c l e i o r ch r o m a t i n by m i c r o c o c c a l n u c l e a s e suggest t h a t a c t i v e l y t r a n s c r i b e d c h r o m a t i n i s a s s o c i a t e d w i t h nucleosomes. H y b r i d i z a t i o n s t u d i e s u s i n g e i t h e r t o t a l DNA (118), cDNA t o c y t o p l a s m i c p o l y a d e n y l a t e d mRNA (119) o r cDNA t o a s p e c i f i c mRNA i n d i c a t e t h a t t h e nucleosomes c o n t a i n most i f n ot a l l o f t h e genomic sequences (118) , and, i n t h e ca s e o f an a c t i v e l y t r a n s c r i b i n g gene such as the ovalbumin gene i n hen o v i d u c t , the nucleosomes p r o t e c t sequences from d i g e s t i o n (120, 121, 122). Nucleosomes a l s o p r o t e c t sequences c o d i n g f o r a c t i v e l y t r a n s c r i b i n g r i b o s o m a l RNA genes (123-126). U n l i k e o t h e r a c t i v e l y t r a n s c r i b i n g genes 31 i s t u d i e d , t h e r i b o s o m a l RNA gene's p r o t e c t i o n by nucleosomes d e c r e a s e s with, i n c r e a s i n g t r a n s c r i p t i o n a l a c t i v i t y (.123, 126). F u r t h e r e l a b o r a t i o n o f t h e s e s t u d i e s demonstrated t h a t m i c r o c o c c a l n u c l e a s e p r e f e r e n t i a l l y e x c i s e s a c t i v e l y t r a n s c r i b i n g c h r o m a t i n . U s i n g a cDNA (ovalbumin) probe, sequences c o d i n g f o r the ovalbumin gene were found to be p r e f e r e n t i a l l y a s s o c i a t e d w i t h mononucleosomes ge n e r a t e d by m i c r o c o c c a l n u c l e a s e d i g e s t i o n o f hen o v i d u c t n u c l e i (122, 121). Dinucleosomes, t r i n u c l e o s o m e s , and h i g h e r r e p e a t s a l l had d i m i n i s h i n g l e v e l s o f the ovalbumin DNA sequences. However, genes t h a t were not b e i n g t r a n s c r i b e d ( i . e . t h e g l o b i n gene i n hen o v i d u c t ) d i d not e x h i b i t t h e p r e f e r e n t i a l e x c i s i o n by m i c r o c o c c a l n u c l e a s e . The s t r u c t u r e of a c t i v e l y t r a n s c r i b i n g genes has been s t u d i e d by e l e c t -r o n m i c r o s c o p y . As t h e t r a n s c r i p t i o n a l a c t i v i t y o f t h e r i b o s o m a l RNA gene i n c r e a s e s ( e s t i m a t e d from t h e number o f n a s c e n t RNA c h a i n s a t t a c h e d ) , the f r e q u e n c y of nucleosomes d e c r e a s e s . F o r v e r y a c t i v e rRNA genes, the f r e q u e n c y of nucleosomes i s e i t h e r v e r y low o r n e g l i g i b l e (.127-129) . A l t h o u g h t h i s would appear to c o n f l i c t w i t h t h e b i o c h e m i c a l e v i d e n c e (124), s e v e r a l a u t h o r s (92, 127) s p e c u l a t e t h a t t h e compact nucleosomal s t r u c t u r e t r a n s f o r m s i n t o a more "open" c o n f o r m a t i o n w i t h t h e h i s t o n e s s t i l l p r e s e n t on the DNA o f f e r i n g p r o t e c t i o n a g a i n s t m i c r o c o c c a l n u c l e a s e o r DNase I I a t t a c k (see Nucleosome m o d e l s ) . McKnight et a l . ( 2 1 2 ) have p r e s e n t e d e v i d e n c e t h a t t h e n u c l e o s o m a l h i s t o n e s a r e found on t r a n s c r i b e d n o n r i b o s o m a l c h r o m a t i n . The a u t h o r s demonstrated through the use o f immunoelectron m i c r o s c o p y u s i n g a n t i -%2 h i s t o n e immunoglobulins t h a t the h i s t o n e s , H2B and H3, remain a s s o c i a t e d w i t h t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n . The c h r o m a t i n r e g i o n c o u l d t h e r e f o r e be t r a n s c r i b e d w i t h o u t l o s s o f t h e h i s t o n e s as s u g g e s t e d by i n v i t r o t r a n s c r i p t i o n s t u d i e s o f BK v i r u s c h r o m a t i n (.130) and SV40 ch r o m a t i n (131)„ and by e x a m i n a t i o n o f t h e SV40 t r a n s c r i p t i o n a l complex (132). U n l i k e m i c r o c o c c a l n u c l e a s e and DNase I I which p r e f e r e n t i a l l y d i g e s t t h e l i n k e r DNA r e g i o n , DNase I d i g e s t s b o t h i n t r a - and i n t e r n u c l e o s o m a l DNA w i t h e q u a l f r e q u e n c y . A l s o u n l i k e the o t h e r two n u c l e a s e s , DNase I w i l l p r e f e r e n t i a l l y d i g e s t t r a n s c r i p t i o n a l l y competent genes (120, 133-136). D i g e s t i o n o f n u c l e i from c h i c k e r y t h r o c y t e s , o r hen o v i d u c t w i t h DNase I r e s u l t s i n the p r e f e r e n t i a l d i g e s t i o n o f the g l o b i n (133). o r ovalbumin gene (120, 134) ;, r e s p e c t i v e l y . A l t e r n a t i v e l y . , the g l o b i n gene from hen o v i d u c t o r t h e ovalbumin gene from c h i c k e n e r y t h r o c y t e s were not p r e f e r e n t i a l l y d i g e s t e d by DNase I. A l t h o u g h t h e b u l k o f c h r o m a t i n i s d i g e s t e d by DNase I , p r o d u c i n g a 10 base s i n g l e - s t r a n d DNA r e p e a t when examined by g e l e l e c t r o p h o r e s i s , t h i s m a t e r i a l from t r a n s c r i p t i o n a l l y incompetent genes has the a b i l i t y t o h y b r i d i z e e i t h e r t o t o t a l u n d i g e s t e d fragmented DNA o r to a s p e c i f i c cDNA probe. Thus, the DNA o f a t r a n s c r i p t i o n a l l y competent gene must be d i g e s t e d t o a c i d -s o l u b l e o l i g o n u c l e o t i d e s o r to s m a l l fragments e i t h e r u n able t o form s t a b l e duplexes,,', or a b l e to form s t a b l e d u p l e x e s .but a t a much reduced r a t e (120). S e l e c t i v e d i g e s t i o n by DNase I of t r a n s c r i p t i o n a l l y a c t i v e i n t e g r a t e d a d e n o v i r u s genes i n t r a n s f o r m e d hamster c e l l s (135)<,-.•, and o f DNA sequences t r a n s c r i b e d i n t o p o l y A mRNA from t r o u t t e s t i s (136) has a l s o been r e p o r t e d . 33" The s e l e c t i v e d i g e s t i o n by DNase I does not change w i t h t h e t r a n s c r i p t i o n a l a c t i v i t y o f t h e gene (134). Thus, the nucleosomes a s s o c i a t e d w i t h a t r a n s c r i p t i o n a l l y competent gene e x i s t i n an a l t e r e d c o n f o r m a t i o n s e n s i t i v e t o DNase I a t t a c k (133). To f u r t h e r demonstrate the e x i s t e n c e of nucleosomes w i t h an a l t e r e d c o n f o r m a t i o n , mononucleosomes c o n t a i n i n g sequences of a t r a n s c r i p t i o n a l l y competent genes were examined f o r DNase I s e n s i t i v i t y . Mononucleosomes o b t a i n e d from c h i c k e n e r y t h r o c y t e n u c l e i had t h e a b i l i t y t o h y b r i d i z e to a cDNA ( g l o b i n ) probe but when d i g e s t e d by DNase I , mononucleosomes c o n t a i n i n g t h e g l o b i n gene were s e l e c t i v e l y a t t a c k e d . , However, i d e n t i c a l e xperiments u s i n g mononucleosomes c o n t a i n i n g ovalbumin gene sequences from hen o v i d u c t n u c l e i were not s e l e c t i v e l y d i g e s t e d by DNase I . T h i s d i s c r e p a n c y might be r e s o l v e d i f mononucleosomes from hen o v i d u c t d i d not c o n t a i n HMG p r o t e i n s ( h i g h m o b i l i t y group p r o t e i n s : a group of n o n h i s t o n e chromosomal p r o t e i n s ) w h i l e t h e mononucleosomes from c h i c k e r y t h r o c y t e s d i d as HMG p r o t e i n s have been suggested to be i n v o l v e d i n m a i n t a i n i n g t h e DNase I s e n s i t i v e s t a t e (137). Chromatin has been f r a c t i o n a t e d by many d i f f e r e n t p r o c e d u r e s w i t h t h e g o a l of s e p a r a t i n g t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n from the b u l k of chrom-a t i n ( f o r a r e v i e w see Ref. 138). Of t h e methods r e p o r t e d , t e c h n i q u e s u t i l -i z i n g s e l e c t i v e e x c i s i o n of t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n by DNase I I (139, 140) o r m i c r o c o c c a l n u c l e a s e (142-144, 122) have been the most s u c c e s s - ... f u l . G o t t e s f e l d has r e p o r t e d a method f o r f r a c t i o n a t i n g c h r o m a t i n by d i g e s t -i o n of c h r o m a t i n w i t h DNase I I f o l l o w e d by t h e s e l e c t i v e p r e c i p i t a t i o n o f t h e 34 d i g e s t p r o d u c t s w i t h 2 mM MgCl^. The M g s o l u b l e f r a c t i o n was e n r i c h e d i n DNA sequences complementary t o p o l y A - c o n t a i n i n g c y t o p l a s m i c RNA (141). +2 Mononucleosomes a s s o c i a t e d w i t h t h e Mg - s o l u b l e f r a c t i o n had a h i g h e r s e d i m e n t a t i o n c o e f f i c i e n t than t h a t of nucleosomes a s s o c i a t e d w i t h t h e b u l k of c h r o m a t i n (14 v s 11 S) (130). The 14 S mononucleosomes had a h i g h e r s e n s i t i v i t y t o DNase I d i g e s t i o n than d i d 11 S mononucleosomes. Levy W. e t a l . have used m i c r o c o c c a l n u c l e a s e t o d i g e s t t r o u t t e s t i s n u c l e i . The d i g e s t p r o d u c t s were r e l e a s e d by l y s i s o f t h e n u c l e i w i t h EDTA f o l l o w e d by t h e s e l e c t i v e p r e c i p i t a t i o n o f t h e p r o d u c t s w i t h 0.1 M NaCl (142-144). The 0.1 M NaCl s o l u b l e f r a c t i o n was e n r i c h e d i n t r a n s c r i b e d sequences, and t h i s f r a c t i o n c o n s i s t e d o n l y of mononucleosomes t h a t c o n t a i n e d t h e n u c l e o s o m a l h i s t o n e s , H6 (a n o n h i s t o n e chromosomal p r o t e i n ) , no HI and 140 base p a i r s of DNA (143). IX. R e p l i c a t i o n o f Chromatin S e v e r a l problems i n u n d e r s t a n d i n g the mechanism of c h r o m a t i n r e p l i c a t i o n a r e s i m i l a r to t h o s e encountered f o r the t r a n s c r i p t i o n p r o c e s s . F o r example, a r e nucleosomes a s s o c i a t e d w i t h DNA when i t i s b e i n g r e p l i c a t e d ? The mode of s e g r e g a t i o n of t h e nucleosomes a t the r e p l i c a t i o n f o r k and whether o r not newly s y n t h e s i z e d h i s t o n e s a r e assembled w i t h o u t " o l d " h i s t o n e s t o form t h e h i s t o n e octamer a r e a l s o important q u e s t i o n s . 35 T r a n s m i s s i o n e l e c t r o n m i c r o s c o p i c t e c h n i q u e s have been used t o s t u d y c h r o m a t i n r e p l i c a t i o n i n t h e S phase genome of c e l l u l a r b l a s t o d e r m D r o s o p h i l i a m e l a n o g a s t e r embryos (145). In t h i s study, nucleosomes were not removed o r d i s s o c i a t e d p r i o r to r e p l i c a t i o n . P u l s e l a b e l l i n g s t u d i e s u s i n g [ 3 H ] - t h y m i d i n e c o n f i r m e d t h e p r e s e n c e of nucleosomes on t h e newly s y n t h e s i z e d DNA (146-148). The nucleosomes a s s o c i a t e d w i t h newly s y n t h e s i z e d were e x c i s e d by m i c r o c o c c a l n u c l e a s e a t t w i c e t h e r a t e o f nucleosomes from t h e b u l k o f c h r o m a t i n (146, 148). However, t h i s enhanced s e n s i t i v i t y d e c r e a s e d as t h e t h e nucleosomes presumably a t t a i n e d t h e h i g h e r o r d e r . c h r o m a t i n s t r u c t u r e (146). The nucleosomes a s s o c i a t e d w i t h newly s y n t h e s i z e d d i f f e r e d i n s t r u c t u r a l f e a t u r e s from nucleosomes a s s o c i a t e d w i t h t h e b u l k of c h r o m a t i n , i n t h a t they had a s m a l l e r DNA r e p e a t l e n g t h and d i f f e r e d b o t h i n m i c r o c o c c a l n u c l e a s e and DNase I d i g e s t p a t t e r n s of t h e i r c o r e p a r t i c l e s (148). Thus, l i k e t h e nucleosomes a s s o c i a t e d w i t h a c t i v e l y t r a n s c r i b i n g c h r o m a t i n , t h e nucleosomes a s s o c i a t e d w i t h newly s y n t h e s i z e d c h r o m a t i n have an a l t e r e d c o n f o r m a t i o n . The s e g r e g a t i o n o r d i s t r i b u t i o n o f t h e nucleosomes a t t h e r e p l i c a t i o n f o r k i s c o n s e r v a t i v e . I n t h e p r e s e n c e o f t h e p r o t e i n s y n t h e s i s i n h i b i t o r , c y c l o h e x i m i d e , t h e DNA s y n t h e s i z e d i s d i g e s t e d a t t w i c e t h e r a t e o f p a r e n t a l c h r o m a t i n (146, 149). I f t h e nucleosomes o r i e n t a t e d themselves i n a d i s p e r s i v e manner a t the r e p l i c a t i o n f o r k , t h e f r e q u e n c y of m i c r o c o c c a l n u c l e a s e a t t a c k would i n c r e a s e about f i v e f o l d due to t h e i n c r e a s e d s p a c i n g between t h e nucleosomes.- (146) . 3.6 The nucleosomes a s s o c i a t e d w i t h newly s y n t h e s i z e d DNA c o n s i s t o f o n l y "newly s y n t h e s i z e d h i s t o n e s which a r e not mixed w i t h " o l d " p r e v i o u s l y s y n t h e s i z e d h i s t o n e s . The r e p l i c a t i n g DNA o f SV40 was r e p o r t e d t o a s s o c i a t e p r e f e r e n t i a l l y w i t h newly s y n t h e s i z e d h i s t o n e s (150, 151). A l s o t h e a s s o c i a t i o n o f newly s y n t h e s i z e d h i s t o n e s w i t h newly s y n t h e s i z e d DNA, and the a s s o c i a t i o n o f " o l d " h i s t o n e s w i t h t h e p a r e n t a l DNA from E h r l i c h a s c i t e s tumor c e l l s has been r e p o r t e d ( 1 5 2 ) . The p r e f e r e n t i a l a s s o c i a t i o n o f o n l y newly s y n t h e s i z e d h i s t o n e s t o form a nucleosome has been demonstrated by L e f f a k e t a l . (153). In t h i s s t u d y c h i c k myoblast organ c u l t u r e s were p u l s e d f o r one hour w i t h dense amino a c i d s and [ 3H ] - l y s i n e . The nucleosomes were i s o l a t e d and the h i s t o n e s c r o s s - l i n k e d t o form a c r o s s - l i n k e d octamer c o r e h i s t o n e complex. A n a l y s i s o f t h e c r o s s - l i n k e d octamer by d e n s i t y g r a d i e n t , e q u i l i b r i u m c e n t r i f u g a t i o n demonstrated the c o n s e r v a t i v e assembly o f o n l y newly s y n t h e s i z e d h i s t o n e s t o form t h e nucleosome. Through t h e p r o c e s s e s o f c o n s e r v a t i v e s e g r e g a t i o n o f the nucleosomes a t t h e r e p l i c a t i o n f o r k and c o n s e r v a t i v e assembly of the nucleosomes, i n f o r m a t i o n about s t r u c t u r a l c h r o m a t i n r e g i o n s , i . e . a t r a n s c r i b i n g r e g i o n , might be p e r p e t u a t e d d u r i n g subsequent c e l l g e n e r a t i o n s . The assembly p r o c e s s o f h i s t o n e s . w i t h DNA to form a nucleosome seems to r e q u i r e an assembly f a c t o r i n v i v o . The f a c t o r i s an a c i d i c , t h e r m o s t a b l e . p r o t e i n t h a t has been i s o l a t e d from a s u p e r n a t a n t e x t r a c t o f Xenopus l a e v i s 37 eggs (154, 155), mouse f i b r o b l a s t n u c l e i and wheat embryos (155). The f a c t o r b i n d s the f o u r n u c l e o s o m a l h i s t o n e s i n equimolar amounts (155). R e c e n t l y a n i c k i n g - c l o s i n g enzyme has been found t o be a b l e t o assemble nucleosomes under p h y s i o l o g i c a l c o n d i t i o n s ' i n t h e absence of..the assembly f a c t o r (156). The n i c k i n g - c l o s i n g enzyme w i l l assemble nuclesomes w i t h a g r e a t e r e f f i c i e n c y than t h e assembly f a c t o r . I t i s not c l e a r a t t h i s t ime whether or not t h e assembly f a c t o r i s a s p e c i a l i z e d p r o t e i n c a p a b l e of a i d i n g t h e n i c k i n g - c l o s i n g enzyme i n nucleosome assembly i n o n l y c e r t a i n systems such as Xenopus o o c y t e s where r a p i d assembly o f nucleosomes from the s t o r e d h i s t o n e s i s r e q u i r e d . X. N o n h i s t o n e Chromosomal P r o t e i n s A l a r g e v a r i e t y o f n o n h i s t o n e chromosomal p r o t e i n s a r e a l s o bound t o ch r o m a t i n . These p r o t e i n s have been c l a s s i f i e d i n t o two groups based on t h e i r e l e c t r o p h o r e t i c m o b i l i t y on 20% p o l y a c r y l a m i d e g e l s a t pH 2.4 (224). They a r e termed low m o b i l i t y group (LMG) o r h i g h m o b i l i t y group (HMG) p r o t e i n s . The f o u r main HMG p r o t e i n s , HMG-1, HMG-2, HMG-14 and HMG-17, from c a l f thymus have been, e i t h e r ' p a r t i a l l y or . t o t a l l y ; s e q u e n c e d . The :HMG p r o t e i n s comprise a s m a l l f r a c t i o n o f t h e t o t a l chromosomal p r o t e i n s , b e i n g about 3% by weight of DNA (158). HMG-T, a p r o t e i n i s o l a t e d from t r o u t t e s t i s , has a s i m i l a r a m i n o - t e r m i n a l sequence t o HMG-1 and HMG-2 (159). A l s o , H6, f o r m e r l y thought to be a h i s t o n e i n t r o u t t e s t i s , has a sequence 38 s i m i l a r t o HMG-14 and HMG-17 (160, 168). Res e a r c h groups have i n v e s t i g a t e d whether the low l e v e l s o f t h e HMG p r o t e i n s were a s s o c i a t e d w i t h s p e c i a l i z e d r e g i o n s o f c h r o m a t i n ( i . e . a c t i v e l y t r a n s c r i b i n g c h r o m a t i n ) . HMG-T from t r o u t t e s t i s n u c l e i and HMG-1 and HMG-2 from c a l f thymus o r mouse b r a i n n u c l e i were r e a d i l y r e l e a s e d i n t o t h e s u p e r n a t a n t a f t e r m i c r o c o c c a l n u c l e a s e d i g e s t i o n (161). The r e s u l t s suggest t h a t the p r o t e i n s a r e a s s o c i a t e d w i t h t h e l i n k e r DNA r e g i o n (161, 162). However, when r a b b i t thymus n u c l e i were d i g e s t e d w i t h m i c r o -c o c c a l n u c l e a s e , o n l y s u b f r a c t i o n s of HMG-1 and HMG-2 (perhaps' m o d i f i e d forms) were r a p i d l y , r e l e a s e d (163) w h i l e HMG-14, 17 and a s u b p o p u l a t i o n o f HMG-1 and HMG-2 were a p p a r e n t l y t i g h t l y bound to t h e nucleosome (158, 163). H6 remained bound t o t h e nucleosome a f t e r t r o u t t e s t i s n u c l e i were d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e (162) , but H6 was r e l e a s e d when t r o u t t e s t i s n u c l e i were d i g e s t e d w i t h DNase I (160, 161). DNase I d i g e s t i o n o f c a l f thymus n u c l e i (161), mouse b r a i n n u c l e i (161) o r duck e r y t h r o c y t e c h r o m a t i n (164) r e l e a s e d HMG-1 and HMG-2, but t h e r e l e a s e o f t h e s e p r o t e i n s was not observed a f t e r DNase I d i g e s t i o n o f r a b b i t thymus or r a t l i v e r n u c l e i (165). The d i s c r e p a n c y may be due t o t h e n u c l e a r i s o l a t i o n and d i g e s t i o n c o n d i t i o n s used. The p o s s i b i l i t y e x i s t s t h a t t h e s e p r o t e i n s might be a s s o c i a t e d w i t h t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n by v i r t u e o f t h e f a c t t h a t HMG p r o t e i n s t h a t a r e bound to l i n k e r DNA a r e r a p i d l y r e l e a s e d a f t e r m i c r o c o c c a l n u c l e a s e d i g e s t i o n . Moreover, n u c l e o s o m a l bound HMG p r o t e i n s a r e r e l e a s e d a f t e r DNase I d i g e s t i o n u s i n g c o n d i t i o n s known t o s e l e c t i v e l y d i g e s t a c t i v e genes. I n t e r e s t i n g l y , HMG-14 and HMG-17 have been suggested t o be r e q u i r e d f o r maintenance of a DNase I s e n s i t i v e , t r a n s c r i p t i o n a l l y a c t i v e gene (137). 39, HMG-1 and HMG-2 have been i s o l a t e d and c h a r a c t e r i z e d from s e v e r a l s o u r c e s i n c l u d i n g c a l f thymus, c a l f k i d n e y , c a l f l i v e r , c h i c k e n thymus, c h i c k r e d b l o o d c e l l s and duck red b l o o d c e l l s (157). The p r o t e i n s were found t o be v e r y s i m i l a r d e s p i t e the s o u r c e . I f t h e p r o t e i n s do p l a y a r o l e i n m a i n t a i n i n g c h r o m a t i n i n a t r a n s c r i p t i o n a l l y a c t i v e form, HMG-1 and HMG-2 would presumably be i n v o l v e d as s t r u c t u r a l or n o n s p e c i f i c gene r e g u l a t o r s . A24 i s a unique chromosomal p r o t e i n c o n t a i n i n g u b i q u i t i n a t t a c h e d t o h i s t o n e H2A by an i s o p e p t i d e l i n k a g e (166). A24 has been found t o be a s s o c i a t e d w i t h nucleosomes (166). U b i q u i t i n has been i d e n t i f i e d i n t r o u t t e s t i s but the p r e s e n c e of A24 has n o t y e t been r e p o r t e d (167). X I . H i s t o n e A c e t y l a t i o n : C o r r e l a t i o n w i t h T r a n s c r i p t i o n a l  A c t i v i t y o f Chromatin The h i s t o n e s a r e m o d i f i e d p o s t - s y n t h e t i c a l l y by a c e t y l a t i o n , p h o s p h o r y l a t i o n and m e t h y l a t i o n . A c e t y l a t i o n a t t h e £-lysyl r e s i d u e s of th e n u c l e o s o m a l h i s t o n e s has been suggested t o p l a y a r o l e i n gene r e g u l a t i o n . S t u d i e s i n v o l v i n g hormonal s t i m u l a t i o n such as t h e a l d o s t e r o n e - s t i m u l a t e d k i d n e y (169), t h e u t e r u s a f t e r estradiol-17|-inje£;tion (170) and c o r t i s o l -s t i m u l a t e d l i v e r (2) have shown i n c r e a s e d a c e t y l a t i o n of h i s t o n e H4 and, i n some c a s e s , H3. In a l l c a s e s the i n c r e a s e d h i s t o n e a c e t y l a t i o n preceded i n c r e a s e d RNA s y n t h e s i s . Other agents t h a t s t i m u l a t e d RNA 40 s y n t h e s i s such as polyaraines. and m i t o g e n i c agents a l s o i n c r e a s e d t h e l e v e l of h i s t o n e a c e t y l a t i o n ( 2 ) . H i g h l y a c e t y l a t e d forms: of the h i s t o n e s have been r e p o r t e d t o be a s s o c i a t e d w i t h t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n . H i s t o n e s H3 and H4 a s s o c i a t e d w i t h t h e SV40 minichromosome a r e h i g h l y a c e t y l a t e d w h i l e t h o s e a s s o c i a t e d w i t h t h e minichromosome o f a n o n - t r a n s f o r m i n g mutant have d e c r e a s e d l e v e l s of t h e a c e t y l a t e d h i s t o n e s p e c i e s (171). A l s o , t h e t r a n s c r i p t i o n a l l y a c t i v e macronucleus from Tetrahymena p y r i f o r m i s c o n t a i n s h i g h l y a c e t y l a t e d h i s t o n e s w h i l e t h e t r a n s c r i p t i o n a l l y i n e r t m i c r o n u c l e u s has low l e v e l s of t h e a c e t y l a t e d h i s t o n e s (172). X I I . The P r e s e n t I n v e s t i g a t i o n At t h e b e g i n n i n g o f t h i s p r o j e c t , s t u d i e s of h i s t o n e m o d i f i c a t i o n s ( i . e . a c e t y l a t i o n ) i n r e l a t i o n t o nucleosome s t r u c t u r e had not y e t been r e p o r t e d . The t r o u t t e s t i s system was i d e a l f o r t h i s i n v e s t i g a t i o n and t h e s i t e s , k i n e t i c s and c e l l u l a r l o c a l i z a t i o n s of h i s t o n e a c e t y l a t i o n have .been w e l l c h a r a c t e r i z e d . The l a r g e q u a n t i t i e s of t i s s u e a v a i l a b l e , easy p r e p a r a t i o n of n u c l e i and c h r o m a t i n , and a b i l i t y of c e l l s u s p e n s i o n s t o i n c o r p o r a t e r a d i o a c t i v e p r e c u r s o r s p r o v i d e more r e a s o n s f o r s e l e c t i n g t h i s system. P a r t s A and B of t h i s t h e s i s r e p o r t on t h e l e v e l s of t h e a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h nucleosomes, and t h e l e v e l s o f a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h nucleosome s u b t r a c t i o n s . The r e s u l t s 41 i n d i c a t e t h a t t h e mononucleosomes. a s s o c i a t e d w i t h t h e bulk, o f c h r o m a t i n c o n t a i n t h e f u l l spectrum o f a c e t y l a t e d h i s t o n e s p e c i e s found i n whole c h r o m a t i n . However, n u c l e o s o m e s ; a s s o c i a t e d w i t h n u c l e a s e - s e n s i t i v e ( m i c r o c o c c a l n u c l e a s e , DNase II: o r DNase I) regions; of c h r o m a t i n ( p r o b a b l y r e g i o n s of c h r o m a t i n i n v o l v e d i n r e p l i c a t i o n (148) and/or t r a n s c r i p t i o n (140, 141, 142, 133, 121, 122)) c o n t a i n e d h i g h l e v e l s of t h e a c e t y l a t e d s p e c i e s of h i s t o n e H4-. The o t h e r n u c l e o s o m a l h i s t o n e s H2A, H3 and H2B c o n t a i n e d normal l e v e l s o f t h e i r r e s p e c t i v e a c e t y l a t e d forms. The l i n k e r DNA a s s o c i a t e d w i t h t h e n u c l e a s e - s e n s i t i v e c h r o m a t i n r e g i o n s c o n t a i n e d non-h i s t o n e chromosomal p r o t e i n s w i t h HMG-T p r e d o m i n a t i n g w h i l e t h e l i n k e r DNA a s s o c i a t e d w i t h the b u l k of c h r o m a t i n c o n t a i n e d p r e d o m i n a n t l y h i s t o n e H i . P a r t C o f t h i s t h e s i s r e p o r t s on t h e b i o c h e m i c a l mechanisms u n d e r l y i n g t h e i n c r e a s e d a c e t y l a t i o n o f h i s t o n e s found i n b u t y r a t e - t r e a t e d t i s s u e c u l t u r e c e l l s . The r e s u l t s c l e a r l y i n d i c a t e t h a t t h e i n c r e a s e d a c e t y l a t i o n of h i s t o n e i n v i v o i s most p r o b a b l y due t o an i n h i b i t i o n of d e a c e t y l a s e enzyme a c t i v i t y caused by b u t y r a t e . T h i s i n h i b i t i o n i s e n t i r e l y r e v e r s i b l e and a p p e a r s t o be a g e n e r a l phenomenon, s i n c e b u t y r a t e i n c r e a s e s l e v e l s o f a c e t y l a t e d H3 and H4 i n a l l t i s s u e c u l t u r e c e l l t y p e s t e s t e d . 42 MATERIALS, AND METHODS PARTS A AND B. I . M a t e r i a l s and A b b r e v i a t i o n s (a) M a t e r i a l s A l l c h e m i c a l s o b t a i n e d c o m m e r c i a l l y were of t h e h i g h e s t p u r i t y o r reagent grade. S p e c i a l reagents- were o b t a i n e d as. f o l l o w s : m i c r o c o c c a l n u c l e a s e (E-C. 3.1.4.7.) from Sigma; d e o x y r i b o n u c l e a s e I (E.C. 3.1.4.5.) from Sigma; d e o x y r i b o n u c -l e a s e I I (E-C. 3.1.4.6.) from Sigma; a c r y l a m i d e from B i o Rad o r Matheson, Coleman and B e l l ; N, N' -methylene b i s a c r y l a m i d e from Eastman Kodak; N, N' - d i a l l y l t a r t a r d i a m i d e . . f roiri Bio. Rad;. :.> N, N, N', N 1 - t e t r a m e t h y l e t h y l e n e d i a m i n e from Canal I n d u s t r i a l Corp.; u r e a from Schwarz/Mann; a g a r o s e from Calbiochem; s t a r c h from Connaught L a b o r a t o r i e s and E l e c t r o s t a r c h Company; M i n i c o n B15 c o n c e n t r a t o r from Amicon*, aqueous c o u n t i n g s c i n t i l l a n t from Amersham/Searle; NCS t i s s u e s o l u b i l i z e r from Amersham/Searle; g l a s s f i b r e f i l t e r s from. Reeve A n g e l and p r o t e i n a s e K from Beckman I n s t r u m e n t s , I n c . R a d i o a c t i v e compounds: - L - [ 1 1 +C] l y s i n e (342 mCi/mmole) , D L - [ 4, 5 - 3H] l y s i n e (25 or 40 Ci/mmole), [methyl - 3H] t h y m i d i n e (47 Ci/mmole), and sodium [ 1 - 1 !*C] a c e t a t e (60.1 mCi/ mmole) - were o b t a i n e d from Amersham/Searle Corp. N - [ e t h y l -2 - 3H] e t h y l maleimide (150-300 mCi/mmole) was: o b t a i n e d from New England N u c l e a r . 43 (b) A b b r e v i a t i o n s T r i s : B u f f e r A: B u f f e r B: B u f f e r C: B u f f e r D: B u f f e r E: TCA: SDS: EDTA: EGTA: T r i s (hydroxymethyl) aminomethane 50 mM T r i s - H C l , pH 7.4, 25 mM K C l , 0.25 M s u c r o s e and 1 mM MgCl,, 50 mM T r i s - H C l , pH 7.4, 25 mM K C l , 0.25 M s u c r o s e , 1 mM M g C l 2 and 15 mM B-mercaptoethanol p h o s p h a t e - b u f f e r e d s a l i n e , pH 7.2, c o n t a i n i n g 0.14 M NaCl, 2.7 mM K C l , 9 mM Na^HPO,, 1.5 mM KH HPO^, 0.9 mM C a C l 2 and 0.5 mM MgC_ 2 50 mM T r i s - H C l , pH 7.4, 25 mM K C l , 0.25 M s u c r o s e and 2 mM M g C l 2 10 mM T r i s - H C l , pH 7.4, 3 mM MgCl and 10 mM NaCl T r i c h l o r o a c e t i c a c i d Sodium d o d e c y l s u l f a t e ( E t h y l e n e d i n i t r i l o ) t e t r a a c e t i c a c i d ( d i s o d i u m s a l t ) . . • ..... E t h y l e n e g l y c o l - b i s - ( B - a m i n o e t h y l e t h e r ) N, N' -t e t r a a c e t i c a c i d PMSF: - P h e n y l m e t h y l s u l f o n y l f l u o r i d e NEM: - N - E t h y l m a leimide TEMED: - N, N, N', N ' - T e t r a m e t h y l e t h y l e n e d i a m i n e DNA: - D e o x y r i b o n u c l e i c a c i d RNA: - R i b o n u c l e i c a c i d RNase: - P a n c r e a t i c r i b o n u c l e a s e DNase I : - D e o x y r i b o n u c l e a s e I DNase I I : - D e o x y r i b o n u c l e a s e I I ACS: - Aqueous c o u n t i n g s o l u t i o n PAGE: - P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s I I . C e l l I n c u b a t i o n s N a t u r a l l y m a t u r i n g t r o u t t e s t i s (Sun V a l l e y T r o u t Farm, M i s s i o n B.C.) o r t e s t i s o b t a i n e d from rainbow t r o u t (Salmo g a i r d n e r i i ) i n which 44 . spermatogenesis had been in d u c e d by twice-weekly i n j e c t i o n s o f salmon p i t u i t a r y e x t r a c t (186) were used. The t i s s u e was s c i s s o r - m i n c e d i n 3 to 4 volumes o f e i t h e r B u f f e r A, 0.1% g l u c o s e or B u f f e r C. A c e l l s u s -p e n s i o n was p r e p a r e d by g e n t l e hand h o m o g e n i z a t i o n ( t h r e e complete s t r o k e s ) i n a P o t t e r - E l v e h j e m homogenizer w i t h a T e f l o n p e s t l e as d e s c r i b e d by L o u i e and Dixon (10). The c e l l s were c e n t r i f u g e d a t 1,000 x g f o r 10 min and resuspended i n 2 volumes o f Waymouth's medium (187) w i t h 10 mM T r i s - H C l b u f f e r (pH 7.2) i n s t e a d o f phosphate b u f f e r . The l a t t e r b u f f e r was u s u a l l y used f o r i n c u b a t i o n times e x c e e d i n g 4 h. A l s o added to the s u s p e n s i o n s were 100 u n i t s / m l of p e n i c i l l i n and s t r e p t o m y c i n , and a drop o f p h e n o l r e d . The s u s p e n s i o n was p r e i n c u b a t e d f o r 10 min a t 15 to 16° C b e f o r e the a d d i t i o n o f one or more o f t h e f o l l o w i n g r a d i o a c t i v e l a b e l s : sodium [ 1- 1 I +C] a c e t a t e to e i t h e r 5, 15 or 50 y C i / m l f i n a l c o n c e n t r a t i o n , L - [ 1 L f C ] l y s i n e to 7.5 y C i / m l f i n a l c o n c e n t r a t i o n , L - [ 4, 5 (n)- 3 H ] l y s i n e m o n o h y d r o c h l o r i d e t o 33.3 o r 100 y C i / m l f i n a l c o n c e n t r a t i o n and [ m e t h y l - 31I] thymidine to 50 y C i / m l . f i n a l c o n c e n t r a t i o n . A f t e r v a r i o u s i n c u b a t i o n t i m e s , the c e l l s were c o l l e c t e d by no • centrfftfgation?._tll9000:'':xp;g:":fdrlf0r-ten. CGells-:were.-either-:frozen^at- - 80 C or used immediately. I l l P r e p a r a t i o n o f N u c l e i . a n d Chromatin T e s t i s n u c l e i and c h r o m a t i n were p r e p a r e d e s s e n t i a l l y as d e s c r i b e d by by Honda et a l . (188) •. except t h a t i n some i n s t a n c e s , t h e n u c l e i were t r e a t e d 45 w i t h t h e p r o t e a s e i n h i b i t o r , p h e n y l m e t h y l s u l f o n y l f l u o r i d e (0.1 mM) p r i o r t o c h r o m a t i n p r e p a r a t i o n . The c o n c e n t r a t i o n of t h e n u c l e i was determined by c o u n t i n g t h e n u c l e i on a hemocytometer. A l t e r n a t i v e l y , a 10.0 y l a l i q u o t of t h e n u c l e a r s u s p e n s i o n was added to a s o l u t i o n c o n t a i n i n g 4 M u r e a and 2 M N a Cl and the absorbance of t h e s o l u t i o n a t 260 nm was measured. The c o n c e n t r a t i o n of t h e c h r o m a t i n sample was d e t e r m i n e d by a d d i n g 10 ul!.. of the c h r o m a t i n p r e p a r a t i o n to 0.99 ml bf 1 N NaOH, and t h e absorbance of t h e s o l u t i o n a t .260 nm was measured. IV Enzymatic D i g e s t i o n of N u c l e i o r Chromatin and F r a c t i o n a t i o n of t h e D i g e s t P r o d u c t s (a) P r e p a r a t i o n of mononucleosomes from m i c r o c o c c a l n u c l e a s e d i g e s t e d  n u c l e i 8 Washed n u c l e i (5 x 10 / ml) were i n c u b a t e d w i t h 300 A^^Q u n i t s / m l of m i c r o c o c c a l n u c l e a s e (Sigma) a t 37°C i n e i t h e r B u f f e r B, 1 mM C a C l ^ o r B u f f e r A (has 3-mercaptoethanol o m i t t e d ) , 1 mM C a C l 2 f o r 8, 15 o r 30 min. The d i g e s t i o n was t e r m i n a t e d by making the s o l u t i o n 10 mM i n EDTA and p l a c i n g i t on i c e . A f t e r c e n t r i f u g a t i o n a t 12,000 x g f o r 15 min, t h e d i g e s t e d n u c l e i were resuspended v i g o r o u s l y i n 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and c e n t r i f u g e d f o r 30 min a t 12,000 x g. The s u p e r -n a t a n t was made e i t h e r 7% i n s u c r o s e or was a p p l i e d d i r e c t l y t o a B i o - G e l A-5m column (90 x 1.5 cm) e q u i l i b r a t e d w i t h 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA a t 4°C as d e s c r i b e d by Shaw et a l . (189). 46 P r e p a r a t i o n o f nucleosome s u b f r a c t i o n s and m i c r o c o c c a l n u c l e a s e  d i g e s t e d n u c l e i (. Sanders' p r o c e d u r e ) ( i ) D i g e s t i o n o f n u c l e i Washed n u c l e i were resuspended to 40 A.,./ml i n B u f f e r D c o n t a i n i n g 1 mM C a C l ^ . The n u c l e a r s u s p e n s i o n was i n c u b a t e d w i t h 100 A 2gQ u n i t s / m l o f m i c r o c o c c a l n u c l e a s e (Sigma) at 25°C f o r 4 min. The d i g e s t i o n was t e r m i n a t e d by making the s o l u t i o n 1 mM i n EGTA, pH 7.0 and p l a c i n g i t on i c e . The n u c l e i were c o l l e c t e d by c e n t r i f u g a t i o n a t 3,000 x g f o r 10 min and the s u p e r n a t a n t , SO, p l a c e d on i c e . ( i i ) S t epwise e l u t i p n o f nucleosomes The n u c l e i were resuspended i n B u f f e r D c o n t a i n i n g 0.1 M N a C l and i n c u b a t e d f o r 20 min.t a t 0°C as d e s c r i b e d by Sanders (190). The n u c l e a r s u s p e n s i o n was c e n t r i f u g e d a t 3,000 x g f o r 10 min-., and the s u p e r n a t a n t , SSI, removed. The above s t e p s ( r e s u s p e n s i o n o f the n u c l e i , i n c u b a t i o n , and c e n t r i f u g a t i o n ) were s e q u e n t i a l l y r e p e a t e d u s i n g B u f f e r D c o n t a i n i n g 0.2 M, 0.4 M and f i n a l l y , 0.6 M N a C l . The s u p e r n a t a n t s were s a v e d a f t e r each c e n t r i f u g a t i o n y i e l d i n g SS2, SS4 and SS6 r e s p e c t i v e l y . A l t e r n a t i v e l y , d i g e s t e d n u c l e i were resuspended d i r e c t l y i n B u f f e r D c o n t a i n i n g 0.4 M N aCl, i n c u b a t e d 20 min ; w ; a t 0°C, c e n t r i f u g e d and the s u p e r n a t a n t , SS4T, removed. ( i i i ) F r a c t i o n a t i o n o f t h e s a l t - e l u t e d p r o d u c t s S u p e r n a t a n t s SSI and SS.4T were each a p p l i e d t o a B i o - G e l A-5m column (90 x 1.5 cm) e q u i l i b r a t e d w i t h 10 mM T r i s -H C l , pH 7.5, 0.7 mM EDTA and 0.4 M NaCl a t 4°C as d e s c r i b e d by Shaw et a l . (190). A l t e r n a t i v e l y , s u p e r n a t a n t SSI was a p p l i e d t o a s i m i l a r column e q u i l i b r a t e d w i t h 10 mM T r i s - H S l , pH 7.5, 0.7 mM EDTA and 0.1 M NaCl. ) P r e p a r a t i o n of nucleosome s u b f r a c t i o n s from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i (Levy and Dixon's pr o c e d u r e ) ( i ) D i g e s t i o n of n u c l e i The c o n d i t i o n s used f o r d i g e s t i n g t h e n u c l e i were the same as d e s c r i b e d i n S e c t i o n b ( i ) . The s u p e r n a t a n t i s termed S I . ( i i ) P r e p a r a t i o n and f r a c t i o n a t i o n o f nucleosomes The n u c l e i were resuspended i n 0.2 mM EDTA and i n c u b a t e d f o r 20 min a t 0°C. The n u c l e a r s u s p e n s i o n was c e n t r i f u g e d a t 12,000 x g f o r 20 min, and t h e s u p e r n a t a n t , S2, was removed. The r e s u l t i n g p e l l e t was termed P2. F i v e M NaCl was added s l o w l y to the s u p e r n a t a n t S2 t o a f i n a l c o n c e n t r a t i o n of ,0.1 M. The ;supernatant was ': ... i n c u b a t e d f o r 20 min a t J O ° C and then c e n t r i f u g e d a t 17,000 x g f o r 10 min. The s u p e r n a t a n t , 4,8 S3, and p e l l e t , P3, were b o t h r e s c u e d . The s u p e r n a t a n t s were d i a l y z e d o v e r n i g h t a g a i n s t 10 mM NH.NCO a t 4°C and l y o p h i l i z e d . F r a c t i o n a t i o n of DNase I I d i g e s t e d c h r o m a t i n ( G o t t e s f e l d ' s  p r o c e d u r e ) ( i ) D i g e s t i o n o f c h r o m a t i n I n i t i a l l y , c h r o m a t i n (10 A „ , n / m l ) suspended i n 25 mM sodium a c e t a t e (pH 6.6) was d i g e s t e d w i t h DNase I I a t 100 u n i t s / m l f o r 5 min a t 24°C as d e s c r i b e d by G o t t e s f e l d e t a l . (141). I n l a t e r experiments, c h r o m a t i n was d i g e s t e d w i t h DNase I I a t 4 enzyme u n i t s p e r u n ^ t ^ o r ^ m^- n ( o r from 4 min to 30 min f o r d i g e s t i o n c o u r s e s t u d i e s ) a t 24°C as d e s c r i b e d by G o t t e s f e l d and B u t l e r (139). The d i g e s t i o n was t e r m i n a t e d by r a i s i n g t h e pH t o 7.5 by add-i n g 50 mM T r i s (pH 10) to t h e s o l u t i o n and p l a c i n g i t on i c e . The s o l u t i o n was c e n t r i f u g e d a t 12,000 x g f o r 20 min and the s u p e r n a t a n t , S I , and p e l l e t , P I , p l a c e d on i c e . ( i i ) F r a c t i o n a t i o n o f t h e c h r o m a t i n d i g e s t p r o d u c t s To the s u p e r n a t a n t , S I , 1 M MgCl^ was added t o 2 mM. The s o l u t i o n was i n c u b a t e d 30 min on i c e p r i o r to c e n t r i f u g a t i o n a t 12,000 x g f o r 20 min. The s u p e r n a t a n t , S2, and p e l l e t , P2, were p l a c e d on i c e . I n some i n s t a n c e s S2 was f u r t h e r f r a c t i o n a t e d by the a d d i t i o n o f 1 M MgCl^ to 22 mM, and c e n t r i f u g e d a t 17,000 x g f o r 10 min y i e l d i n g the s u p e r n a t a n t , S3, and p e l l e t , P3. A l t e r n a t i v e l y +2 the Mg - s o l u b l e (S2) f r a c t i o n was d i v i d e d i n t o two p o r t - , i o n s , one of which was t r e a t e d w i t h 10 yg o f p a n c r e a t i c 49 -RNase per ml f o r 10 min a t 37°C, the o t h e r o f which was t r e a t e d f o r 20 min a t 24°C. A f t e r c e n t r i f u g a t i o n (12,000 x g " f o r 30 min), the p e l l e t (P3) was saved, and the s u p e r n a t a n t (S3) was r e d i g e s t e d w i t h a f u r t h e r 10 pg o f RNase per ml f o r 20 min (37°C) or 40 min (24°C). The p e l l e t (P4) and s u p e r n a t a n t (S4) were r e c o v e r e d a f t e r c e n t r i f u g a t i o n a t 17,000 x g f o r 30 min. +2 ( i i i ) S t u d i e s on the e f f i c i e n c y o f Mg , h i s t o n e o r RNase to p r e c i p i t a t e S2 a s s o c i a t e d n u c l e o s o m a l m a t e r i a l To the s u p e r n a t a n t , S2, e i t h e r RNase (10 ug/ml), whole h i s t o n e (10 ug/ml), RNase (10 ug/ml) p r e t r e a t e d w i t h d i e t h y l p y r o c a r b o n a t e (0.1%) or M g C ^ (22 mM) was added. The s o l u t i o n s were i n c u b a t e d f o r 2 to 20 min a t 37°C. A c o n t r o l s o l u t i o n of S2 was i n c u b a t e d f o r 60 min a t 37°C. A f t e r c e n t r i f u g a t i o n a t 12,000 x g f o r 20 min, t h e s u p e r -natant,, S3, and p e l l e t , P3, were saved. P r e p a r a t i o n o f mononucleosomes from DNase . I . d i g e s t e d n u c l e i . Washed n u c l e i (20 A /ml) were i n c u b a t e d w i t h 2.5 ug/ml ZbU of DNase I a t 25°C i n B u f f e r E from 2 to 15 min. The d i g e s t i o n was t e r m i n a t e d by p l a c i n g the n u c l e a r s u s p e n s i o n on i c e . A f t e r c e n t r i f u g a t i o n a t 3,000 x g f o r 10 min, t h e d i g e s t e d n u c l e i were resuspended v i g o r o u s l y i n 10 mM T r i s - H C l , pH7.5, 0.7 mM EDTA, and c e n t r i f u g e d a t 12,000 x g f o r 30 min. The su p e r -n a t a n t was a p p l i e d t o a B i o - G e l A-5m column (90 x 1.5 cm) 50 e q u i l i b r a t e d with. 10 mM T r i s - H C l , 0.7 mM EDTA, pH 7.5, a t 4°C as d e s c r i b e d by Shaw et a l . (189). P r e p a r a t i o n o f nucleosome s u b t r a c t i o n s from DNase I d i g e s t e d n u c l e i (Sanders' p r o c e d u r e ) ( i ) D i g e s t i o n o f n u c l e i Washed n u c l e i (20 A„-_/ml) were i n c u b a t e d w i t h l y g / m l z oU o f DNase I (Sigma) at 15°C i n B u f f e r D f o r 10 t o 60 min,-:. ( u s u a l l y 10 m i n " ) . The d i g e s t i o n was t e r m i n a t e d by p l a c i n g t h e s o l u t i o n on i c e . The n u c l e i were c o l l e c t e d by c e n t r i f u g a t i o n a t 3,000 X g f o r 10 min, and t h e s u p e r n a t a n t , SO, p l a c e d on i c e . ( i i ) Stepwise e l u t i o n o f nucleosomes The s t e p w i s e e l u t i o n o f nucleosomes w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f NaCl was t h e same as d e s c r i b e d i n S e c t i o n ( b ) ( i i ) . In a d d i t i o n , d i g e s t e d n u c l e i were resuspended d i r e c t l y i n B u f f e r D c o n t a i n i n g 0.2 M NaCl, i n c u b a t e d 20 min - a t 0°C, c e n t r i f u g e d , and t h e s u p e r n a t a n t , SS2T, removed. ( i i i ) F r a c t i o n a t i o n o f t h e s a l t - e l u t e d p r o d u c t s S u p e r n a t a n t s SSI or SS4T were each a p p l i e d t o a B i o - G e l A-5m column (90 x 1.5 cm) e q u i l i b r a t e d w i t h 10 mM T r i s -H C l , pH 7.5, 0.7 mM EDTA and 0.4 M NaCl at 4°C as d e s c r i b e d by Shaw et a l . (189). A l t e r n a t i v e l y , s u p e r n a t a n t s SSI, SS4 or SS2T were each 51 a p p l i e d t o a B i o - G e l A-0.5m column (40 x 1.5 cm) e q u i l i b r a t e d w i t h e i t h e r 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and 0.4 M NaCl o r 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and 0.1 M N a C l , a t 4°C. V DNase I D i g e s t i o n o f L a b e l l e d Mononucleosomes T h i r t y u n i - t s °f mononuclesomes i n a 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA b u f f e r , l a b e l l e d w i t h f 1 4 C ] - a c e t a t e and [ 3 H ] - l y s i n e , were c o n c e n t -r a t e d t o a f i n a l volume o f 1.5 ml (20 A „ , n u n i t s / m l ) u s i n g a M i n i c o h B15 . z oU c o n c e n t r a t o r . To 1.0 ml (20 A 0,_) of mononucleosomes, 156 y l of a s o l u t i o n c o n t a i n i n g 40 mM N a C l , 20 mM M g C l 2 (NaCl:5.3 mM, MgCl 2:2 mM f i n a l c o n c e n t -r a t i o n ) was s l o w l y added w i t h r a p i d s t i r r i n g . The mononucleosomes s u s -p e n s i o n was d i g e s t e d w i t h 20 ug/ml of DNase I f o r 3 min a t 37°C. The d i g e s t i o n was t e r m i n a t e d by the a d d i t i o n o f EDTA t o 15 mM and t h e a d d i t i o n of u r e a t o 6 M. To 0.5 ml (10 A o c „ ) of mononucleosomes, 78 y l of 40 mM Z D U N a C l , 20 mM M g C l 2 (5.3 mM and 2 mM f i n a l c o n c e n t r a t i o n s f o r NaCl and M g C l 2 > r e s p e c t i v e l y ) was added w i t h r a p i d s t i r r i n g . T h i s sample d i d not have DNase I added t o i t , but was i n c u b a t e d f o r 3 min a t 37°C. The s u s p e n s i o n was made 15 mM i n EDTA and 6 M i n u r e a . The samples were a p p l i e d t o a B i o - G e l A-0.5m column (90 x 1.0 cm) e q u i l i b r a t e d w i t h 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and 6 M u r e a , a t 4°C. VI R e a c t i o n o f Nucleosomes or DNase I D i g e s t e d Nucleosomes w i t h N-E t h y l m a l e i m i d e under D e n a t u r i n g C o n d i t i o n s Nucleosomes were d i g e s t e d w i t h DNase I as d e s c r i b e d above. An a l i q u o t o f e i t h e r t h e d i g e s t m i x t u r e o r u n d i g e s t e d nucleosomes was removed and added to the a p p r o p i a t e amount of NaCl (0.5 M) and/or u r e a (6 M). In 52 a d d i t i o n , an a l i q u o t from t h e d i g e s t e d o r u n d i g e s t e d nucleosome s u s p e n s i o n s was added t o 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA. A l l s o l u t i o n s were made to t h e same f i n a l volume. An a l i q u o t was removed and r e a c t e d w i t h N - e t h y l [ 3H Imaleimide (New England N u c l e a r , d i l u t e d to 80 mCi/ mmol) as d e s c r i b e d by Wong and Candido (191). V I I G e l E x c l u s i o n Chromatography o f Unknowns P I and P I I L y o p h i l i z e d samples o f e i t h e r P I o r P I I were r e d i s s o l v e d i n 100 mM NH^HCO^. and a p p l i e d to a Sephadex G'^ 25 column (30 X 1.5 cm) e q u i l i b r a t e d w i t h 100 mM NH^HC0 3 a t 4°C. P o o l e d f r a c t i o n s from t h e Sephadex G-25 column were d i a l y z e d a g a i n s t 50 mM NH^HCO^ a t 4°C o v e r n i g h t y and l y o p h i l i z e d . The l y o p h i l i z e d samples were r e d i s s o l v e d i n 100 mM NH^HCO^ and a p p l i e d to a Sephadex G-10 column (26 X 1 cm) e q u i l i b r a t e d w i t h 100 mM NH^HCO^^ a t 4°C. V I I I TCA P r e c i p i t a b i l i t y T e s t s of Unknowns PI and P I I To a 0.5 ml o f a l a b e l l e d PI o r P I I sample 25^1 of a 2 mg/ml s o l u t i o n o f whole h i s t o n e sample was added as c a r r i e r . The s o l u t i o n was made 10% i n TCA by a d d i n g an e q u a l volume o f 20% TCA. The r e s u l t i n g p r e c i p i t a t e was c o l l e c t e d on a g l a s s f i b r e f i l t e r and s e q u e n t i a l l y washed w i t h 3 volumes o f 20% TCA, 3 volumes o f c o l d e t h a n o l , and 1 volume o f e t h e r . The f i l t e r was d r i e d , and counted f o r r a d i o a c t i v i t y a f t e r a d d i t i o n o f 8.0 ml o f ACS (Amersham/Searle). A l s o , 0.1 ml sample o f l a b e l l e d PI o r P I I was added to a g l a s s f i b r e f i l t e r . The f i l t e r was d r i e d , and r a d i o a c t i v i t y was 53 <• det e r m i n e d a f t e r a d d i n g 8 ml o f ACS to the s c i n t i l l a t i o n v i a l . IX H i g h V o l t a g e Paper E l e c t r o p h o r e s i s H i g h v o l t a g e paper e l e c t r o p h o r e s i s o f P I , P I I o r l y s i n e was c a r r i e d out on Whatman 3MM paper a t pH 6.5 f o r 2 h a t 1,000 v o l t s . The pH 6.5 b u f f e r c o n s i s t e d o f p y r i d i n e / a c e t i c a c i d / w a t e r , 100:4:900. X H i s t o n e E x t r a c t i o n H i s t o n e s were a c i d e x t r a c t e d i n t h e f o l l o w i n g ways: (1) N u c l e i were e x t r a c t e d t w i c e w i t h a t l e a s t 4 volumes of 0.2 N HC1 (0°C, 30 min) and t h e e x t r a c t was p r e c i p i t a t e d w i t h 9 volumes o f c o l d a c e t o n e . The p r e c i p i t a t e was d r i e d under N 2. H i s t o n e s from nucleosomes were p r e p a r e d t h e same way a f t e r p r e c i p i t a t i o n of t h e nucleosomes w i t h M g C l 2 a t 10 mM f i n a l c o n c e n t r a t i o n . (2) S o l u t i o n s o f nucleosomes were c o n c e n t r a t e d w i t h an Amicon u l t r a f i l t r a t i o n u n i t w i t h e i t h e r a UM 10 o r PM 30 membrane, f o l l o w e d by f u r t h e r c o n c e n t r a t i o n i n a M i n i c o n B15 c e l l (Amicon). A l t e r n a t i v e l y , nucleosomes were p r e c i p i t a t e d by t h e a d d i t i o n o f M g C l 2 to 10 mM. N u c l e i , c h r o m a t i n , n u c l e o s o m a l p r e c i p i t a t e s and c o n c e n t r a t e d n u c l e o s o m a l s o l u t i o n s were e x t r a c t e d w i t h 0.2 M H 0SO. (30 min on i c e ) , and i n s o l u b l e m a t e r i a l was removed by .54 c e n t r i f u g a t i o n (3,000 x g f o r 10 m i n ) . H i s t o n e s were p r e c i p i t a t e d from t h e e x t r a c t s w i t h 4 volumes of 95% e t h a n o l a t -20°C; t h e p r e c i p i t a t e s were c o l l e c t e d by c e n t r i f u g a t i o n and r e d i s s o l v e d i n d i s t i l l e d water. (3) S u p e r n a t a n t s and p o o l e d column f r a c t i o n s were d i a l y z e d o v e r -n i g h t a t 4°C a g a i n s t 10 mM NH^HCO^, and l y o p h i l i z e d . L y o p h i l i z e d samples, n u c l e i and n u c l e o s o m a l p r e c i p i t a t e s were e x t r a c t e d w i t h 0.4 N H^SO^ (30 min on i c e ) , and i n s o l u b l e m a t e r i a l removed by c e n t r i f u g a t i o n (3,000 x g f o r 10 m i n ) . The a c i d e x t r a c t s were d i a l y z e d o v e r n i g h t a t 4°C a g a i n s t 0.1 N a c e t i c a c i d , l y o p h i l i z e d and r e d i s s o l v e d i n d i s t i l l e d water. Of t h e t h r e e methods, t h e t h i r d method was t h e most e f f i c i e n t i n e x t r a c t i o n . The o t h e r methods q u i t e o f t e n had l o s s e s of t h e a r g i n i n e -r i c h h i s t o n e s , H3 and H4, which, a p p a r e n t l y , r e s u l t s from i n e f f i c i e n t p r e c i p i t a t i o n o f H3 and H4 when ac e t o n e o r , e s p e c i a l l y , e t h a n o l i s added t o the a c i d e x t r a c t . When i n e f f i c i e n t p r e c i p i t a t i o n o c c u r r e d , t h e s e l e c t i v e l o s s of t h e m o d i f i e d h i s t o n e s p e c i e s was never o b s e r v e d . XI. Q u a n t i t a t i o n of the H i s t o n e Sample The c o n c e n t r a t i o n o f a h i s t o n e s o l u t i o n was determined by a t u r b i d i t y a ssay (192). The ass a y tube c o n t a i n e d 10 u l of t h e h i s t o n e s o l u t i o n , 0.79 ml o f d i s t i l l e d water and 0.4 ml of 50% TCA. The c o n t e n t s o f t h e tube were mixed immediately. A f t e r i n c u b a t i o n f o r 13 t o 15 min, t h e t u r b i d i t y of t h e s o l u t i o n was m o n i t o r e d a t 400 nm< (.1 A-^QQ = 107.5yg/ml f i n a l c o n c e n t r a t i o n ) . X I I . G e l E l e c t r o p h o r e s i s o f P r o t e i n s Four g e l systems have been used t o s e p a r a t e , i d e n t i f y and/or q u a n t i t a t e chromosomal p r o t e i n s . The f i r s t two systems a r e used f o r b a s i c chromosomal p r o t e i n s . (a) S t a r c h g e l e l e c t r o p h o r e s i s H i s t o n e samples ( d i s s o l v e d i n 0.2. N HC1) were a p p l i e d t o the s l o t o f a u r e a / l a c t a t e s t a r c h g e l (193) and e l e c t r o p h o r e s i s was c a r r i e d out as d e s c r i b e d by L o u i e and Dixon (10) . G e l s were h o r i z o n t a l l y t r i s e c t e d , and m i d d l e s l i c e s were s t a i n e d 40 min-.-w i t h 0.1% Amido B l a c k c o n t a i n i n g CoC^- and d e s t a i n e d i n N H 2 S 0 4 (193). (b) A c i d / u r e a g e l e l e c t r o p h o r e s i s H i s t o n e s were s e p a r a t e d on 20cm s l a b p o l y a c r y l a m i d e g e l s by the system o f Panyim and C h a l k l e y (194). The f o l l o w i n g volumes of s t o c k s o l u t i o n s : 6.25 ml TEMED s o l u t i o n (43.2% a c e t i c a c i d , 4% TEMED) and 25 ml a c r y l a m i d e s o l u t i o n (30% a c r y l a m i d e , 0.4% b i s a c r y l a m i d e ) - were added t o 19.8 g o f u r e a (6.25 M f i n a l ) . The s o l u t i o n was s t i r r e d over low h e a t , made to 49.5 ml f i n a l w i t h d i s t i l l e d water, and f i l t e r e d . The s o l u t i o n was degassed b e f o r e t h e a d d i t i o n of 0.625 ml ammonium p e r s u l f a t e s o l u t i o n (10% 56 ammonium p e r s u l f a t e ) and p o l y m e r i z e d i n a s l a b g e l . H i s t o n e samples were d i s s o l v e d i n 20 to 30 y l of sample b u f f e r (0.9 N a c e t i c a c i d , 15% s u c r o s e ) . The samples were e l e c t r o p h o r e s e d u s i n g an a c e t i c a c i d r u n n i n g b u f f e r (0.9 N a c e t i c a c i d ) f o r 30 to 40 h a t 170 V a t 4°C. The g e l s were s t a i n e d w i t h 0.25% Coomassie b l u e i n m e t h a n o l / a c e t i c a c i d / w a t e r (5:1:5 v o l / v o l ) and d e s t a i n e d by d i f f u s i o n i n m e t h a n o l / a c e t i c a c i d / w a t e r ( 2 : 1 : 5 v o l / v o l ) . (c) SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s F i f t e e n p e r c e n t p o l y a c r y l a m i d e - S D S s l a b g e l s were made u s i n g a m o d i f i e d Laemmli p r o c e d u r e ( 5 9 ) . The f o l l o w i n g volumes of s t o c k s o l u t i o n s were used t o p r e p a r e t h e lower 15% p o l y a c r y l a m i d e -SDS s e p a r a t i n g g e l : - 15 ml of a c r y l a m i d e s o l u t i o n (30% a c r y l a m i d e , 0.4% b i s a c r y l a m i d e ) , 7.5 ml of T r i s b u f f e r . .(1.5 M T r i s - H C l , pH 8.8), 0.3 ml of SDS s o l u t i o n (10% SDS), 0.15 ml of ammonium p e r s u l f a t e s o l u t i o n (10% ammonium p e r s u l f a t e ) and 7.05 ml of d i s t i l l e d water - were combined, degassed and p o l y m e r i z e d under t -b u t a n o l i n a s l a b g e l a f t e r t h e a d d i t i o n of 10 y l TEMED. A l t e r n a t i v e l y , t h e s e p a r a t i n g lower g e l was p r e p a r e d - u s i n g 0.6% N, N' - d i a l l y t a r t a r d i a m i d e i n s t e a d of 0.4% b i s a c r y l a m i d e i n t h e a c r y l a m i d e s o l u t i o n . T h i s a l l o w e d ease of s o l u b i l i z a t i o n of t h e g e l a f t e r e l e c t r o p h o r e s i s . The f o l l o w i n g volumes o f s t o c k s o l u t i o n s were used to p r e p a r e t h e upper 3% po l y a c r y l a m i d e - S D S s t a c k i n g g e l : - 1.5 ml of a c r y l a m i d e s o l u t i o n (30% a c r y l a m i d e , 57 0.8% b i s a c r y l a m i d e ) , 3.75 ml of T r i s b u f f e r (0.5 M T r i s - H C l , pH 6.8), 0.15 ml of SDS s o l u t i o n (10%." SDS) , 0.15 ml of ammonium p e r s u l f a t e s o l u t i o n (10% ammonium p e r s u l f a t e ) , and 9.45 ml of d i s t i l l e d water - were combined, degassed and p o l y m e r i z e d a f t e r the a d d i t i o n o f 10 y l of TEMED. The p r o t e i n sample was added t o a sample b u f f e r c o n t a i n i n g 0.125 M T r i s - H C l , pH 6.8, 4% SDS, 10% 3-mercaptoethanol and 20% g l y c e r o l . A f t e r t h e a d d i t i o n o f t h e r u n n i n g b u f f e r (0.0495 M T r i s , 0.384 M g l y c i n e , 0.1% SDS), e l e c t r o p h o r e s i s was c a r r i e d out a t .130 V f o r 6 t o 7 h. The g e l s were s t a i n e d w i t h 0.25% Coomassie b l u e i n m e t h a n o l / a c e t i c a c i d / water (2:1:5 v o l / v o l ) . Two-dimensional g e l e l e c t r o p h o r e s i s H i s t o n e s were s e p a r a t e d on 20 cm s l a b a c i d / u r e a p o l y a c r y l a m i d e g e l s by t h e system o f Panyim and C h a l k l e y (194, see above ( b ) ) . G e l s were run a t 170 V f o r 30 to 40 h a t 4°C. F u r t h e r r e s o l u t i o n was a c h i e v e d by r u n n i n g samples i n a second d i m e n s i o n u s i n g t h e sodium d o d e c y l s u l f a t e (SDS) system o f Laemmli (226) as m o d i f i e d by Weintraub e t a l . ( 5 9 , see above ( c ) ) . G e l s from t h e f i r s t d i m e n s i o n were e q u i l i b r a t e d f o r 20 to 30 min i n B u f f e r 0 o f 0 ' F a r r e l l ( 1 9 5 ) (10% g l y c e r o l / 5 % g-mercaptoethanol/2.3% SDS/62.5 mM T r i s - H C l , pH 6.8) and then a p p l i e d h o r i z o n t a l l y t o the top of the SDS s l a b g e l . The s l i c e was s e a l e d w i t h m e l t e d 1% ag a r o s e i n B u f f e r 0 and e l e c t r o p h o r e s i s was c a r r i e d out a t 130 V f o r 7 h. 58 ... X I I I E x t r a c t i o n of H i s t o n e from .15% P o l y a c r y l a m i d e - SDS G e l s S t a i n e d h i s t o n e bands were d i s s e c t e d and e l u t e d w i t h 10 volumes of 50 mM NH^HC0 3, 0.05% SDS, pH 7.8 t o 8.0 as d e s c r i b e d by Weiner et a l . (196). The sample was l y o p h i l i z e d and r e d i s s o l v e d i n 50 mM NH^HCO ,. 0.05% SDS. The h i s t o n e s were p r e c i p i t a t e d by t h e a d d i t i o n of 9 volumes of a c e t o n e , and t h e p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n a t 12,000 X g f o r 10 min. The p e l l e t was t h e n r e d i s s o l v e d i n d i s t i l l e d water and r e p r e c i p i t a t e d w i t h 9 volumes of a c e t o n e . The p r e c i p i t a t e was c e n t r i f u g e d , d r i e d under N 2, and d i s s o l v e d i n 0.2; N HC1. The a c e t o n e p r e c i p i t a t i o n removed t h e SDS and Coomassie b l u e from t h e e x t r a c t e d h i s t o n e s . T h i s was found t o be n e c e s s a r y f o r t h e subsequent r e s o l u t i o n of t h e m o d i f i e d s p e c i e s on s t a r c h g e l s . XIV S o l u b i l i z a t i o n of P o l y a c r y l a m i d e G e l s S t a i n e d , l a b e l l e d p r o t e i n bands e l e c t r o p h o r e s e d on 15% p o l y a c r y l a m i d e -SDS g e l s c o n t a i n i n g b i s a c r y l a m i d e as t h e c r o s s ^ l i n k e r were d i s s e c t e d , s l i c e d and p l a c e d i n a s c i n t i l l a t i o n v i a l . One ml of NCS t i s s u e s o l u b i l i z e r (Amersham/Searle) was added t o t h e v i a l , and i n c u b a t e d 2 days a t room temperature. A l t e r n a t i v e l y , l a b e l l e d p r o t e i n s e l e c t r o p h o r e s e d on 15% p o l y a c r y l a m i d e - S D S c o n t a i n i n g , N, N ' - d i a l l y l t a r t a r d i a m i d e as c r o s s - l i n k e r were s o l u b i l i z e d u s i n g 2 ml of 2% p e r i o d i c a c i d . The s l i c e s were i n c u b a t e d a t room t e m p e r a t u r e f o r 2 days. The s o l u b i l i z e d g e l s l i c e s were counted i n 10 ml of aqueous c o u n t i n g s c i n t i l l a n t (Amersham). XV A n a l y s i s o f DNA Fragments Produced by N u c l e a s e D i g e s t i o n (a) P r e p a r a t i o n bf DNA fragments 59 DNA fragments were p r e p a r e d f o r e l e c t r o p h o r e s i s i n t h r e e ways: ( i ) t h e DNA was i s o l a t e d b e f o r e a d d i t i o n o f sample b u f f e r , i i ) t h e DNA a s s o c i a t e d p r o t e i n was d i g e s t e d b e f o r e a d d i t i o n of sample b u f f e r o r i i i ) t h e sample b u f f e r was added d i r e c t l y . ( i ) Samples of nucleosomes were p r e c i p i t a t e d w i t h 10 mM MgCl^, o r l y o p h i l i z e d a f t e r d i a l y s i s a g a i n s t 10 mM NH^HCO^- The samples were r e d i s s o l v e d i n 1 ml of a s o l u t i o n c o n t a i n i n g 1 M N a C l , 1% SDS and 20 mM EDTA. An e q u a l volume of c h l o r o f o r m / i s o a m y l a l c o h o l (24:1) was added, s t i r r e d , i n c u b a t e d 10 min a t room temperature and c e n t r i f u g e d a t 3,000 x g f o r 10 min. The aqueous phase was c a r e f u l l y removed and r e - e x t r a c t e d as d e s c r i b e d above. The DNA was p r e c i p i t a t e d by a d d i n g 2 v o l of c o l d e t h a n o l to t h e aqueous phase a t -20°C. ( i i ) A l y o p h i l i z e d sample was r e d i s s o l v e d i n a s m a l l volume (10 y l ) of a s o l u t i o n c o n t a i n i n g 25 mM EDTA and 1% SDS;,, f o l l o w e d by the a d d i t i o n of p r o t e i n a s e K (Merck) t o 8.3 mg/ml. Samples were i n c u b a t e d a t 37°C f o r 3 to 4 h. ( i i i ) Sample b u f f e r s used w i l l be d e s c r i b e d i n t h e f o l l o w i n g t e x t , (b) Non-denaturing p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of DNA Three o r f i v e p e r c e n t p o l y a c r y l a m i d e - SDS s l a b g e l s were made u s i n g a m o d i f i e d L o e n i n g (197) p r o c e d u r e . To p r e p a r e the 60 , 3% p o l y a c r y l a m i d e - S D S g e l s , the f o l l o w i n g volumes o f s t o c k s o l u t i o n s were used: - 7.5 ml of a c r y l a m i d e s o l u t i o n (20% a c r y l a m i d e , 2.2% b i s a c r y l a m i d e ) , 5.0 ml of 10 x c o n c e n t r a t e d TBE b u f f e r (10 x TBE = 0.9 M T r i s , 0.9 M b o r a t e , 30 mM EDTA, pH 8.3), 0:5 ml- of° SDS sol u t i o n ' . ( 1 0 % - SDS) ,-.0.-4 ml of ammonium p e r s u l f a t e s o l u t i o n (10% ammonium p e r s u l f a t e ) , and 36.6 ml o f d i s t i l l e d water. These were combined, and the g e l p o l y m e r i z e d a f t e r the a d d i t i o n o f 40 y l TEMED. To make 5% p o l y a c r y l a m i d e - SDS. s l a b g e l s , t h e volume o f the a c r y l a m i d e s o l u t i o n was i n c r e a s e d to 12.5 ml, and t h e volume o f d i s t i l l e d water reduced t o 31.6 ml. In some i n s t a n c e s , t h e c o n c e n t r a t i o n of TBE b u f f e r i n t h e g e l was reduced t o 50 mM T r i s , 50 mM b o r i c a c i d (pH 8.3) and 0.17 mM EDTA f i n a l . The DNA sample was r e d i s s o l v e d i n sample b u f f e r c o n t a i n i n g 1 ml o f 10 x c o n c e n t r a t e d TBE b u f f e r , 1% SDS, 20 mM EDTA and 10% s u c r o s e . A f t e r the a d d i t i o n of r u n n i n g b u f f e r (10.0 f o l d o r 17.9 f o l d d i l u t e d 10 x c o n c e n t r a t e d TBE b u f f e r , 0.1% SDS), the 3% p o l y a c r y l a m i d e g e l s were e l e c t r o p h o r e s e d a t 100 V f o r 3 to 4 h, and the 5% g e l s were e l e c t r o p h o r e s e d a t 150 V f o r 4- h. The g e l s were s t a i n e d w i t h e t h i d i u m bromide (10 yg/ml) f o r 15 min. D e n a t u r i n g p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f DNA Ten p e r c e n t p o l y a c r y l a m i d e - SDS s l a b g e l s were p r e p a r e d by the c o m b i n a t i o n of the f o l l o w i n g s t o c k s o l u t i o n s : - 5 ml of 10 x c o n c e n t r a r e d TBE b u f f e r (0.9 M T r i s , 0.9 M b o r a t e (pH 8.3), 30 mM EDTA), 0.5 ml of ammonium p e r s u l f a t e s o l u t i o n (6.4% ammonium p e r s u l f a t e ) , 0.5 ml of SDS s o l u t i o n ( 1 0 % ) , and 61 26 ml of a c r y l a m i d e s o l u t i o n (19% a c r y l a m i d e , 1% b i s a c r y l a m i d e ) The s o l u t i o n was added s l o w l y t o 21.0 g u r e a (7 M f i n a l c oncent r a t i o n ) , and t h e u r e a was d i s s o l v e d over low hea t . The r e s u l t i n g s o l u t i o n was made to 50 ml w i t h d i s t i l l e d water, f i l t e r e d and degassed. A f t e r t h e a d d i t i o n o f 20 p i TEMED, t h e s o l u t i o n was poured and p o l y m e r i z e d . The sample b u f f e r was pr e p a r e d by d i s s o l v i n g 1 g o f SDS (10% f i n a l c onc-e n t r a t i o n ) and 2 g (20% f i n a l c o n c e n t r a t i o n ) i n t o 1 ml o f 10 x c o n c e n t r a t e d TBE b u f f e r . The b u f f e r s o l u t i o n was made to 10 ml by t h e a d d i t i o n o f 99% d e i o n i z e d formamide. The DNA sample was d i s s o l v e d i n 20 p i o f sample b u f f e r , b o i l e d and immediately p l a c e d on i c e . A f t e r t h e a d d i t i o n o f e l e c t r o -p h o r e s i s r u n n i n g b u f f e r (10 f o l d d i l u t e d 10 x c o n c e n t r a t e d TBE b u f f e r , 0.1% SDS), t h e samples were e l e c t r o p h o r e s e d a t 200 V f o r 4 to 5 h. The g e l was s t a i n e d w i t h e t h i d i u m bromide (10 pg/ml f o r 15 m i n ) . 6.2 PART C I. MATERIALS AND ABBREVIATIONS (a) M a t e r i a l s A l l c h e m i c a l s o b t a i n e d c o m m e r c i a l l y were o f the h i g h e s t p u r i t y o r re a g e n t grade. In a d d i t i o n to t h e s p e c i a l r e a g e n t s mentioned i n M a t e r i a l s and Methods ( P a r t A and P a r t B 1 ( a ) ) , o t h e r s p e c i a l r e a g e n t s were o b t a i n e d as f o l l o w s : sodium p e n i c i l l i n -G and d i h y d r o s t r e p t o m y c i n from GIBCO, c u l t u r e medium from GIBCO. R a d i o a c t i v e compounds: - s o d i u m t l - 1 k C l a c e t a t e (60.1 mCi/ mmole) was o b t a i n e d from Amersham/Searle Corp., and sodium [ 3H1 a c e t a t e ( 500 mCi/mmole) and [ a c e t y l - 3H] - a c e t y l coenzyme A ( 500 mCi/mmole) were o b t a i n e d from New England N u c l e a r . (b) A b b r e v i a t i o n s The f o l l o w i n g a b b r e v i a t i o n s were used throughout t h e t e x t : TMK: - 50 mM T r i s - H C l , pH 7.4, 25 mM K C l and 1 mM M g C l 2 PBS: - phosphate - b u f f e r e d s a l i n e , pH 7.2 c o n t a i n i n g 0.14 M NaCl, 2.7 mM K C l , 9 mM Na 2HP0 4, 1.5 mM KH 2P0 4, 0.9 mM C a C l 2 and 0.5 mM M g C l 2 I I . C e l l L i n e s and C u l t u r e C o n d i t i o n s The o r i g i n s and growth c h a r a c t e r i s t i c s o f t h e v a r i o u s c e l l l i n e s used i n t h i s study a r e g i v e n i n t h e f o l l o w i n g r e f e r e n c e s : F r i e n d e r y t h r o l e u k e m i c 63 c e l l s , c l o n e 745A (198); mouse 3T3 c e l l s (199); hamster BHK-21 c e l l s (200); r a t IRC8 a s c i t e s c e l l s (201); .Xenopus X58 amphibian c e l l s (202). A l l c e l l l i n e s ( except f o r X58) were m a i n t a i n e d and passaged u s i n g s t a n d a r d t i s s u e c u l t u r e p r o c e d u r e s ( 2 0 3 ) i n a medium c o n t a i n i n g 90% D u l b e c c o ' s m o d i f i e d E a g l e ' s medium, 10% f e t a l c a l f serum and 100 ,lg/ml each of sodium p e n i c i l l i n - G and d i h y d r o s t r e p t o m y c i n (DME/FCS). The amphibian X58 c e l l l i n e was m a i n t a i n e d i n t h e above medium d i l u t e d by 33% w i t h s t e r i l e g l a s s -d i s t i l l e d water. A l l c u l t u r e s were p e r i o d i c a l l y m o n i t o r e d and found to be f r e e o f mycoplasm c o n t a m i n a t i o n . When r e q u i r e d , b u t y r i c a c i d ( n e u t r a l i z e d w i t h c o n c e n t r a t e d NaOH) was added to c u l t u r e s to a f i n a l c o n c e n t r a t i o n of 5 mM. When c e l l c u l t u r e s were l a b e l l e d w i t h i s o t o p i c p r e c u r s o r s , c e l l s were i n c u b a t e d i n c u l t u r e medium (DME/FCS) l a c k i n g t h e p r e c u r s o r to maximize _de novo i n c o r p o r a t i o n . C e l l c ounts were made u s i n g a hemacytometer, and c e l l v i a b i l i t y was determined by t r y p a n b l u e dye e x c l u s i o n (203). I t s h o u l d a l s o be mentioned t h a t a f t e r treatment of F r i e n d c e l l s w i t h 5 mM b u t y r a t e f o r 24 h, t h e r a t e of r e c o v e r y (and v i a b i l i t y ) i s s l i g h t l y l e s s than i n comparable c e l l s never exposed to b u t y r a t e . A d d i t i o n a l experiments, however, a l s o i n d i c a t e t h a t t h e g r e a t m a j o r i t y of b u t y r a t e - t r e a t e d c e l l s do r e c o v e r n o r m a l l y a f t e r such t r e a t m e n t . Thus, the b u t y r a t e e f f e c t appears to be r e v e r s i b l e when s h o r t - t e r m t r e a t m e n t s a r e used. 64 I I I P r e p a r a t i o n o f Histones. H i s t o n e s were i s o l a t e d from a l l c e l l t ypes by t h e f o l l o w i n g m o d i f i c a t i o n of t h e method of Marushige and Bonner (20A): f r o z e n (-80°C) c e l l p e l l e t s 8 9 (10 -10 c e l l s ) were homogenized i n 2 ml of a s t a n d a r d s a l i n e c i t r a t e s o l u t i o n (0.075 M NaCl, 0.024 M Na c i t r a t e (pH 8.0)) u s i n g a g l a s s - T e f l o n hand homogenizer. The sample was c e n t r i f u g e d a t 3,000.x g f o r 10 min,., and t h e p e l l e t was rehomogenized i n TMK as above. A f t e r c e n t r i f u g a t i o n as b e f o r e , t h e n u c l e a r p e l l e t was homogenized i n 2 ml o f 10 mM T r i s - H C l (pH 7.4) and l a y e r e d onto 3 ml of 1 M s u c r o s e i n 10 mM T r i s - H C l (pH 7.4). . A f t e r c e n t r i f u g a t i o n f o r 20 min :. at 17,000 x g, t h e g e l a t i n o u s p e l l e t of c h r o m a t i n was e x t r a c t e d by t h e a d d i t i o n of 0.1 t o 0.3 ml of 0.4 N ^ S O ^ f o r 5 - 1 5 min-on i c e . The e x t r a c t was c e n t r i f u g e d a t 12,000 x g f o r 10 min.-.,, and t h e s u p e r n a t a n t was mixed w i t h 4 v o l - o f 95% e t h a n o l . H i s t o n e s were a l l o w e d t o p r e c i p i t a t e o v e r n i g h t a t - 20°C. The p r e c i p i t a t e d h i s t o n e s were c o l l e c t e d by c e n t r i f u g a t i o n a t 3,000 x g, r e d i s s o l v e d i n 0.1 - 0.3 ml of 0.1 M a c e t i c a c i d , d i a l y z e d a g a i n s t t h e same s o l u t i o n f o r 1 hi ; and l y o p h i l i z e d . IV P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s A c i d - u r e a g e l s were r u n as: d e s c r i b e d by Panyim and C h a l k l e y (.194) u s i n g s l a b g e l s i n s t e a d of tubes: (see M a t e r i a l s arid Methods P a r t A and B X I I ( b ) ) . The 1.5 mm s l a b s were run a t 170 V f o r 20 t o 30 h a t 4°C. 65 V Assay of In V i t r o H i s t o n e A c e t y l a s e and D e a c e t y l a s e A c t i v i t y N a t u r a l l y m a t u r i n g t r o u t t e s t i s (Sun V a l l e y T r o u t Farm, M i s s i o n , B.C.) were s c i s s o r - m i n c e d i n 3 t o 4 volumes of PBS. A c e l l s u s p e n s i o n was prepared: by g e n t l e hand homogenization ( t h r e e complete s t r o k e s ) i n a P o t t e r - E l v e h j e m homogenizer w i t h T e f l o n p e s t l e as d e s c r i b e d by L o u i e and Dixon ( 1 0 ) . The c e l l s were c e n t r i f u g e d a t 1,000 x g f o r 10 min, and r e -suspended i n 2 volumes of PBS c o n t a i n i n g 1 0.4 "volumesof: :Waymouth's'medium (187) w i t h 10 mM T r i s - H C l (pH 7.2) i n s t e a d of phosphate b u f f e r , 100 u n i t s / ml o f p e n i c i l l i n and s t r e p t o m y c i n and a drop of p h e n o l r e d . The s u s p e n s i o n was s u b d i v i d e d , and to one s u s p e n s i o n b u t y r i c a c i d ( n e u t r a l i z e d w i t h c o n c e n t r a t e d NaOH) was added to a f i n a l c o n c e n t r a t i o n of 1 mM. The s u s p e n s i o n s were p r e i n c u b a t e d f o r 10 min a t 16°C b e f o r e t h e a d d i t i o n o f sodium [ l - 1 1 + C ] a c e t a t e t o 50 uCi/ml f i n a l c o n c e n t r a t i o n . A l i q u o t s were removed a f t e r v a r i o u s i n c u b a t i o n t i m e s , p l a c e d i n an excess of PBS and c o l l e c t e d by c e n t r i f u g a t i o n (3,000 x g f o r 10 m i n ) . The h i s t o n e s were a c i d e x t r a c t e d as d e s c r i b e d above. An a l i q u o t o f t h e a c i d e x t r a c t was added t o 1 ml of ACS (Amersham/Searle) and counted f o r r a d i o a c t i v i t y . For a s s a y s of h i s t o n e a c e t y l a s e a c t i v i t y i n c r u d e c e l l l y s a t e s ( e r y -t h r o l e u k e m i c c e l l s ) , e q u a l numbers of c o n t r o l or butyrate-grown ( f o r 24 h i n 5 mM b u t y r a t e ) c e l l s were homogenized i n 3 v o l of TMK ( s e e a b o v e ) . To t h e s e homogenates (100 u l per r e a c t i o n ) , 10 y l of [ 3 H ] - a c e t y l - C o A (100 yCi/ml) were added, and th e r e a c t i o n was i n c u b a t e d a t room temperature (22°C) f o r up to 60 min. A t v a r i o u s t i m e s , r e a c t i o n s were stopped by t h e a d d i t i o n o f 5 y l of c o n c e n t r a t e d HC1 to t h e 110 y l i n c u b a t i o n m i x t u r e s . P r o t e i n was p r e c i p i t a t e d from t h e s e samples w i t h c o l d 20% TCA and c o l l e c t e d 66 on g l a s s f i b r e f i l t e r s . The f i l t e r s were washed s u c c e s s i v e l y w i t h 20% TCA, 95% e t h a n o l , e t h e r and d r i e d . R a d i o a c t i v i t y was determined on an Isocap s c i n t i l l a t i o n c o u n t e r ( N u c l e a r C h i c a g o ) . S i m i l a r experiments t o t h e above were a l s o performed u s i n g p u r i f i e d n u c l e i from e r y t h r o l e u k e m i c c e l l s f o r the d e t e r m i n a t i o n o f a c e t y l a s e a c t i v i t y . In t h i s c a s e , a f t e r t h e c e l l s were homogenized, t h e n u c l e i were p e l l e t e d by c e n t r i f u g a t i o n , resuspended i n TMK, p e l l e t e d a second time and assayed f o r a c e t y l a s e a c t i v i t y i n TMK, as d e s c r i b e d above. For a s s a y of h i s t o n e d e a c e t y l a s e a c t i v i t y , e q u a l numbers of e i t h e r c o n t r o l or butyrate-grown e r y t h r o l e u k e m i c c e l l s (5 mM b u t y r a t e f o r 24 h) were homogenized i n TMK as d e s c r i b e d above. Each l y s a t e was then d i v i d e d i n t o t h r e e a l i q u o t s . One of t h e a l i q u o t s was b o i l e d f o r 1 min to a c t as a background c o n t r o l . To a second a l i q u o t , sodium b u t y r a t e was added t o a f i n a l c o n c e n t r a t i o n o f 5 mM. The t h i r d a l i q u o t was assayed w i t h o u t f u r t h e r treatment and s e r v e d f o r the d e t e r m i n a t i o n o f t o t a l d e a c e t y l a s e a c t i v i t y . Each a l o q u o t was i n c u b a t e d w i t h an e q u a l amount of [ 3 H ] -a c e t a t e - l a b e l l e d h i s t o n e ( u s u a l l y 50,000 cpm) p r e p a r e d by l a b e l l i n g Q 9 F r i e n d c e l l s i n v i v o w i t h f ^ H ] - a c e t a t e (2 x 10 c e l l s were i n c u b a t e d f o r 2 h w i t h 150 y C i / m l of [ 3 H ] - a c e t a t e ) . These h i s t o n e s were p u r i f i e d as d e s c r i b e d above. The m i x t u r e s (140 y l ) were i n c u b a t e d a t room temperature (22°C) f o r 4 h and t e r m i n a t e d by t h e a d d i t i o n o f 10 y l of c o n c e n t r a t e d H C l . T h i s a l s o s e r v e d t o c o n v e r t any a c e t a t e r e l e a s e d t o t h e p r o t o n a t e d form. The [ 3 H ] - a c e t i c a c i d r e l e a s e d was then e x t r a c t e d from t h e m i x t u r e s w i t h 0.5 ml o f e t h y l a c e t a t e , and t h e e t h y l a c e t a t e phases were counted i n Bray's s c i n t i l l a t i o n f l u i d (205). 66a PART A - CHROMATIN SYNTHESIS 67 RESULTS AND DISCUSSION I P a r t i a l C h a r a c t e r i z a t i o n o f N e w l y - S y n t h e s i z e d Chromatin The c o n d i t i o n s used f o r l a b e l l i n g DNA and h i s t o n e s from t r o u t t e s t i s c e l l s u s p e n s i o n s have been p r e v i o u s l y d e s c r i b e d by L o u i e and Dixon (206) and Candido (207). I n t h e p r e s e n t study, t h e same c o n d i t i o n s were used to examine nucleosome s y n t h e s i s from l a b e l l e d h i s t o n e and DNA. However, the., major o b j e c t i v e was to study one of t h e p o s t - s y n t h e t i c m o d i f i c a t i o n s of n u c l e o s o m a l h i s t o n e s , namely a c e t y l a t i o n . (a) C h a r a c t e r i z a t i o n of m i c r o c o c c a l n u c l e a s e d i g e s t p r o d u c t s F o l l o w i n g the d i g e s t i o n o f t r o u t t e s t i s n u c l e i w i t h m i c r o c o c c a l n u c l e a s e , t h e p r o d u c t s were f r a c t i o n a t e d on a B i o -G e l A-5m column . as d e s c r i b e d by Shaw et a l . (189). A t y p i c a l column p r o f i l e i s shown i n F i g . 6A. Two main peaks a r e o b t a i n e d ; t h e l a r g e r peak, e l u t i n g a t o r near the v o i d volume, i s termed t h e m u l t i m e r f r a c t i o n and i s due to fragments of c h r o m a t i n which have been o n l y p a r t i a l l y d i g e s t e d . As shown i n F i g . 6B, t h i s f r a c t i o n c o n t a i n s t h e f u l l complement of h i s t o n e s . The second peak, termed the monomer f r a c t i o n , c o r r e s p o n d s t o th e 11 S p a r t i c l e s which have been p r e v i o u s l y c h a r a c t e r i z e d by Honda e t a l . (208) by s u c r o s e g r a d i e n t c e n t r i f u g a t i o n and by s e d i m e n t a t i o n v e l o c i t y a n a l y s i s . The monomer f r a c t i o n c o n t a i n s o n l y t r a c e amounts of h i s t o n e s Hl» :. ( F i g . 6B) .. ::'Fig. 6C shows an;SDS g e l p a t t e r n of h i s t o n e s from monomer p a r t i c l e s f o l l o w i n g more e x t e n s i v e d i g e s t i o n (30 min 68 80 160 FRACTION NO. FIG. 6. A, Bio-Gel A-5m (90 X 1.5 cm) separation of nucleosomes obtained from an 8-min micrococcal nuclease digest (300 &26Q u n i t s / m l ) °f trout t e s t i s n u c l e i . B, PAGE SDS separation (15% g e l , Coomassie blue stained) of proteins from multimer and monomer peak f r a c t i o n s of the separation i l l u s t r a t e d i n A. C, PAGE SDS separation of proteins from monomer nucleo-somes obtained from a 30-min micrococcal nuclease digest (300 A260 units/ml) following f r a c t i o n a t i o n on Bio-Gel A-5m as i n A above. 69 v e r s u s 8 min, w i t h 300 A „ , n u n i t s / m l ) w i t h n u c l e a s e . Here Z o U HI i s t o t a l l y a b s e n t . The l o s s o f HI seems to be a s s o c i a t e d w i t h d i g e s t i o n o f t h e DNA l i n k e r r e g i o n between n u c l e o s o m a l c o r e p a r t i c l e s (90, 189, 209, 210). Shaw et a l . (189) have e x t e n s i v e l y c h a r a c t e r i z e d t h e n u c l e a s e d i g e s t p r o d u c t s of c h i c k e n e r y t h r o c y t e c h r o m a t i n u s i n g t h e s e t e c h n i q u e s , and the above r e s u l t s a r e i n agreement w i t h t h e i r f i n d i n g s . A s m a l l peak o f m a t e r i a l a b s o r b i n g a t 260 nm i s seen emerging l a t e r from t h e column and p r o b a b l y c o n s i s t s o f s m a l l o l i g o n u c l e o t i d e s . (b) DNA s y n t h e s i s T r o u t t e s t i s c e l l s were i n c u b a t e d w i t h [ 3 H ] - t h y m i d i n e f o r v a r i o u s t i m e s . The i s o l a t e d n u c l e i were d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e u s i n g m i l d d i g e s t i o n c o n d i t i o n s (300 A„^„ . 260 u n i t s / m l f o r 8 m i n ) . The d i g e s t p r o d u c t s from each i n c u b a t i o n time were f r a c t i o n a t e d on a B i o - G e l A-5m column ( F i g . 7 ) , and the s p e c i f i c a c t i v i t i e s o f t h e peak f r a c t i o n s ( i n counts p e r min per A_,„) were c a l c u l a t e d f o r b o t h mutimer. arid monomer 260 f r a c t i o n s ( T a b l e V ) . The i n c o r p o r a t i o n o f [ 3 H ] - t h y m i d i n e i n t o monomer and mu l t i m e r f r a c t i o n s f o l l o w s a s i m i l a r time c o u r s e ( F i g . 8 ) , but t h e s p e c i f i c a c t i v i t y o f t h e monomer peak i s c o n s i s t a n t l y h i g h e r than t h a t o f t h e multime r peak. 70 BO 2.0 6 . 0 4.0 2.0 6J0 4J0 2.0 V 2 0 10 E Q. 3 0 * 20 10 4 0 8 0 120 FRACTION NO. 3 0 2 0 10 4 0 8 0 120 FRACTION NO. FIG. 7. [ 3H] Thymidine i n c o r p o r a t i o n i n t o DNA of nucleosomes as a f u n c t i o n of time. T r o u t t e s t i s c e l l s were i n c u b a t e d w i t h [ m e t h y l - 3Hl thymidine (50 y C i / m l ) ; a t v a r y i n g time i n t e r v a l s , a l i q u o t s were removed and n u c l e i were i s o l a t e d . The n u c l e i were d i g e s t e d f o r 8 min w i t h m i c r o c o c c a l n u c l e a s e (300 A ^ u n i t s / m l ) , and the d i g e s t p r o d u c t s were s e p a r a t e d on a B i o - G e l A-5m column (90 X 1.5 cm). R a d i o a c t i v i t y was determined by m i x i n g 1.0 ml column f r a c t i o n s w i t h 8 ml of ACS (Amersham/Searle) and c o u n t i n g them on a U n i l u x l i q u i d s c i n t i l l a t i o n c o u n t e r . L a b e l l i n g was f o r A, 5 min.-; B, 30 min ; C, 60 min. 71 TABLE V Q u a n t i t a t i o n o f Newly - S y n t h e s i z e d DNA A s s o c i a t e d w i t h Nucleosome  F r a c t i o n s . (Data from F i g . 7) I n c u b a t i o n time Peak f r a c t i o n M u l t i m e r Monomer R a t i o : monomer/ multimer cpm/ A 260 5 mint 30 min 60 min. 3391 5120 5397 5326 8295 7994 1.6 1.6 1.5 72 . CO I o o CO I O 8 2 30 60 T I M E (minutes) FIG. 8. [ H] Thymidine i n c o r p o r a t i o n i n t o DNA o f multimers and monomers as a f u n c t i o n of time. [ 3H] Thymidine t o A „ r a t i o s o f l a b e l l e d peak f r a c t i o n s o f monomers and mu l t i m e r s i s o l a t e d on B i o - G e l A-5m columns i n F i g . 7 were p l o t t e d v e r s u s i n c u b a t i o n time,• m o n o m e r s m u l t i m e r s . 73. The i n c o r p o r a t i o n o f l a b e l reaches, a p l a t e a u a f t e r 30 minutes i n c u b a t i o n t i m e f o r both, monomer and multimer f r a c t i o n s . T h i s would suggest t h e c e s s a t i o n o f DNA s y n t h e s i s w i t h , p o s s i b l y , an accompanying lo s s , o f c e l l v i a b i l i t y . The r e s u l t s h o u l d be examined w i t h c a u t i o n a s w i t h i n c r e a s i n g i n c u b a t i o n t i m e s , c e l l clumping was e v i d e n t . C e l l clumping would i n t e r f e r e w i t h u n i f o r m i n c o r p o r a t i o n o f t h e l a b e l . One p o s s i b l e e x p l a n a t i o n f o r t h e h i g h e r s p e c i f i c a c t i v i t y of t h e monomer peak f r a c t i o n would be t h e p r e f e r e n c e o f m i c r o c o c c a l n u c l e a s e f o r A/T r i c h r e g i o n s (124). I f t h e l i n k e r DNA c o n t a i n i n g A/T r i c h regions, were s e l e c t i v e l y d i g e s t e d , t h e mononucleosomes would c o n t a i n g r e a t e r amounts o f t h y m i d i n e r e s i d u e s per DNA fragment l e n g t h t h a n t h a t o f polynucleosomes. The g r e a t e r s p e c i f i c a c t i v i t y f o r t h e monomer f r a c t i o n may a l s o r e f l e c t a n u c l e a s e s e n s i t i v e s t a t e f o r t h e newly r e p l i c a t e d c h r o m a t i n as suggested by S e a l e (148). I t i s d o u b t f u l t h a t t h e n u c l e a s e ' s a b i l i t y t o s e l e c t i v e l y e x c i s e nucleosomes a s s o c i a t e d w i t h newly s y n t h e s i z e d c h r o m a t i n i s a major c o n t r i b u t o r t o t h e ob s e r v e d r a t i o s i n s p e c i f i c a c t i v i t i e s . As t h e time o f i n c u b a t i o n i n c r e a s e s , t h e n u c l e a s e - s e n s i t i v e nucleosomes c o n t a i n i n g t h e newly s y n t h -e s i z e d , l a b e l l e d DNA would be t r a n s f o r m e d i n t o nucleosomes w i t h c h a r a c t e r i s t i c s s i m i l a r t o t h o s e a s s o c i a t e d with, t h e b u l k of c h r o m a t i n . The l a b e l l e d DNA a s s o c i a t e d w i t h n u c l e a s e -s e n s i t i v e nucleosomes s h o u l d d e c r e a s e w i t h t i m e . T h i s r e s u l t 74 was not ob s e r v e d . (c) H i s t o n e s y n t h e s i s and h i s t o n e a c e t y l a t i o n N u c l e i i s o l a t e d from t r o u t t e s t i s c e l l s t h a t were l a b e l l e d w i t h [ 3 H ] - l y s i n e and [ l l f C ] - a c e t a t e f o r v a r i o u s i n c u b a t i o n times were d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e (300 A„, r i/ml f o r 8 m i n ) . The ZOU d i g e s t p r o d u c t s were f r a c t i o n a t e d on a B i o - G e l A-5m column ( F i g . 9 ) . The p r o f i l e s were s i m i l a r f o r a l l i n c u b a t i o n time p o i n t s . The s p e c i f i c a c t i v i t i e s o f t h e n e w l y - s y n t h e s i z e d h i s t o n e s ( 3H co u n t s per min p e r A 0,_) were c a l c u l a t e d f o r t h e peak f r a c t i o n of t h e monomer and o l i g o m e r f r a c t i o n s ( T a b l e V I ) . A l t h o u g h the monomer f r a c t i o n c o n t a i n s reduced amounts of t h e l y s i n e - r i c h h i s t o n e HI, the s p e c i f i c a c t i v i t y o f t h i s f r a c t i o n i s g r e a t e r than t h a t of t h e multimer f r a c t i o n a t e a r l y t i m e s o f i n c u b a t i o n (5 min and 30 m i n ) . T h i s experiment was performed o n l y once so t h e v a l i d i t y o f t h e measurements i s u n c e r t a i n . However, t h e t r e n d i s i n agreement w i t h the i d e a t h a t n e w l y - s y n t h e s i z e d h i s t o n e s a r e i n i t i a l l y a s s o c i a t e d w i t h nucleosomes. a s s o c i a t e d w i t h n u c l e a s e s e n s i t i v e r e g i o n s o f . ... c h r o m a t i n y and l a t e r they become a s s o c i a t e d w i t h nucleosomes c h a r a c t -e r i s t i c o f t h o s e a s s o c i a t e d w i t h t h e b u l k o f c h r o m a t i n . The monomer and multime r f r a c t i o n s a r e b o t h l a b e l l e d w i t h t h e [ ^ C ] - a c e t a t e l a b e l ( F i g . 9). i n d i c a t i n g t h a t t h e h i s t o n e s c o m p r i s i n g t h e n u c l e o s o m a l f r a c t i o n s a r e m o d i f i e d by a c e t y l a t i o n . I t s h o u l d be noted t h a t b o t h n e w l y - s y n t h e s i z e d h i s t o n e s and p r e -e x i s t i n g h i s t o n e s a r e s u s c e p t i b l e t o \ t h i s m o d i f i c a t i o n . T h i s 75 A j'i j j 1 A - — - *"-0 40 80 120 160 FRACTION NO. O 4.0 <D CM i 8.0 ll A ; A 40 80 120 FRACTION NO. 40 80 120 FRACTION NO. c -K\ IA ! I \ y \ ': ! \ i / 20 9 FIG. 9-w 1 3Hj Lys.lne and I l l jCj. acetate incorporation into histones of nucleosomes: as a function of time. Trout testis, cells, were incubated with: L[ 4,5 (n) - 3H] ly sine monohydrochloride (100 pCi/mlX and sodium I l — lkC] acetate (5 yCi/ml); at varying time intervals, aliquots were removed and nuclei were isolated. The nuclei were digested for 8min with: micrococcal. nuclease (.300 A ^ ^ Q units/ml), and. the digest products wer separatedv on a Bio-Gel A-5m column (90 x 1.5 cm). Radioactivity was determined in the same manner as described for Fig. 7. Labelling was for A , 5 m i n B , 30 min ; C, 60 min. 76 TABLE VI Q u a n t i t a t i o n o f N e w l y - S y n t h e s i z e d H i s t o n e A s s o c i a t e d w i t h  Nucleosome F r a c t i o n s (Data from F i g . 9) I n c u b a t i o n time Peak f r a c t i o n M u l t i m e r Monomer R a t i o : monomer / multimer 5 min; 30 min.? 60 min ^H' cpm/A 260 328 913 2179 397 930 1993 1.2 1.0 0.9 77 m o d i f i c a t i o n w i l l be d i s c u s s e d f u r t h e r i n l a t e r s e c t i o n s . (d) P a r t i a l c h a r a c t e r i z a t i o n of unknowns PI and P I I Two peaks o f r a d i o a c t i v i t y a r e seen e l u t i n g from the columns ( F i g . 9) a f t e r t h e monomer f r a c t i o n . The f i r s t peak, P I , e l u t i n g i n f r a c t i o n s 130 to 160 c o n t a i n s b o t h t h e 3H and 1 1 +C l a b e l s . The second peak, P I I , e l u t i n g i n f r a c t i o n s 160 to 190 c o n t a i n s p r e d o m i n a n t l y t h e % l a b e l . PI and P I I d i d n o t c o - e l u t e w i t h the A 2£Q a b s o r b i n g m a t e r i a l , which i s ( p r e s u m a b l y : o l i g o n u c l e o t i d e s . [ 3 H ] - l y s i n e was found t o e l u t e between PI and P I I (not shown). The unknowns, PI and P I I , . c o u l d be r e l e a s e d i n t o s o l u t i o n a f t e r u n d i g e s t e d n u c l e i were resuspended i n EDTA ( F i g . 1 0 ) , but the n u c l e i had to be i s o l a t e d from f r e s h l y l a b e l l e d c e l l s . L a b e l l e d c e l l s f r o z e n a t -80°C p r i o r t o m i c r o c o c c a l n u c l e a s e d i g e s t i o n d i d not y i e l d PI and P I I . A d d i t i o n o f t h e p r o t e a s e -i n h i b i t o r , p h e n y l m e t h y l s u l f o n y l f l u o r i d e , t o t h e b u f f e r s had_ho e f f e c t on;the appearance .of PI.and P I I . PI.was p o o l e d , d i a l y z e d a g a i n s t 50 mM NH^HCO^, l y o p h i l i z e d and a p p l i e d t o a Sephadex G-25 column. The sample e l u t e d a t t h e i n c l u d e d volume ( F i g . 11A) w i t h b o t h and 3 H l a b e l s c o - e l u t i n g . P I I , t r e a t e d i d e n t i c a l l y t o P I , a l s o e l u t e d a t t h e i n c l u d e d volume ( F i g . 11B). 7:8 o c f <0 0 4 CM 0.2 i \ ' i at M \ 10 40 80 120 FRACTION NO. 160 x Q. o O 1 5 CO I o X. E Q. o af FIG. 10. EDTA s o l u b i l i z e d m a t e r i a l from n u c l e i i s o l a t e d from [ 3H] l y s i n e -and [ l l*C] a c e t a t e - l a b e l l e d t r o u t t e s t i s c e l l s . T r o u t t e s t i s c e l l s were i n c u b a t e d w i t h L [ 4 , 5 ( n ) ~ H ] l y s i n e m o n o h y d r o c h l o r i d e (100 y C i / m l ) and The sodium [ 1 - C] a c e t a t e (50 y C i / m l ) f o r 4 hi",., and n u c l e i i s o l a t e d , n u c l e i were i n c u b a t e d f o r 15 min- a t 37 C, c e n t r i f u g e d , and resuspended w i t h 10 mM T r i s , pH 7.5, 0.7 mM EDTA. The s o l u b i l i z e d m a t e r i a l was s e p a r a t e d on a B i o - G e l A-5m column (90 X 1.5 cm). R a d i o a c t i v i t y was determined i n the same manner as d e s c r i b e d f o r F i g . 7. 79 CM I O E Q. o s— CO Q. U 0 40 FRACTION NO. FIG. 11. Sephadex G25 gel exclusion chromatography of unknowns PI and PII. Trout testis cells were incubated with L [4,5(n) - 3H] lysine monohydrochloride (100 yCi/ml) and sodium [1-1I+C] acetate (15 yCi/ml) for 5 min, and nuclei isolated. The nuclei were digested with micrococcal nuclease, and the digest products separated on a Bio-Gel A-5m column as described in the legend to Fig. 10. The unknowns PI (fractions 130 to 160) and PII (fractions 160 to 190) were pooled, dialyzed against 50 mM NH^ NCO , and lyophilized. The samples were separately redissolved in 1 ml 100 NH.HCO-, and applied to a Sephadex G25 column (30 x 1.5 cm) equilibrated with 100 mM NH^ HCO^ . Radioactivity was determined in the same manner as described for Fig. 7 except 0.1 ml column fractions were mixed with 5 ml of ACS (Amersham/Searle). A, PI; B, PII. 8CU The p o o l e d PI and P I I f r a c t i o n s from t h e Sephadex G-25 colums were a p p l i e d s e p a r a t e l y t o a Sephadex G-10 column a f t e r d i a l y s i s , l y o p h i l i z a t i o n , and r e s o l u b i l i z a t i o n . Both PI and P I I e l u t e d a t t h e v o i d volume. From t h e known m o l e c u l a r weight e x c l u s i o n l i m i t s of b o t h columns, PI and PIT would be a p p r o x i m a t e l y 400 d a l t o n s . When PI and P I I were a n a l y z e d by h i g h v o l t a g e paper e l e c t r o p h o r e s i s , t h e y d i d not m i g r a t e as f r e e l y s i n e . T h i s i s i n agreement w i t h the. f a c t that, n e i t h e r PI nor P I I c o -e l u t e d w i t h [ 3H] - l y s i n e when f r a c t i o n a t e d on a B i o - G e l A-5m column. A l s o , t h e [ 1 1 + C ] l a b e l a s s o c i a t e d w i t h PI was not f r e e [ 1 !*C] - a c e t a t e , as i t was not v o l a t i l e a f t e r a c i d i f i c a t i o n . Both PI and P I I were t e s t e d f o r TCA p r e c i p i t a b i l i t y ( T a b l e V I I ) . The m a j o r i t y o f m a t e r i a l from PI o r P I I was not r e t a i n e d on t h e g l a s s f i b r e f i l t e r s when t r e a t e d w i t h 20% TCA. Thus, the l a b e l i s n o t f r e e h i s t o n e . The i d e n t i t i e s of PI and P I I remain unknown. The r e s u l t s suggest t h a t the. m a t e r i a l s have a m o l e c u l a r weight o f a p p r o x i m a t e l y 400 and t h a t l y s i n e , a c e t a t e o r h i s t o n e i s n o t a s s o c i a t e d w i t h e i t h e r of t h e unknowns. 81 TABLE V I I Q u a n t i t a t i o n of TCA P r e c i p i t a b l e M a t e r i a l A s s o c i a t e d w i t h Unknowns PI and P I I Unknowns PI and P I I were o b t a i n e d from n u c l e i i s o l a t e d from t r o u t t e s t i s c e l l s p r e v i o u s l y i n c u b a t e d w i t h L - [ 1 1 +C] l y s i n e (7.5 uCi/ml) f o r 60 min. PI and P I I were o b t a i n e d from a B i o - G e l A-5m column i n t h e same manner as d e s c r i b e d i n F i g . 11. Each 0.5 ml sample w i t h added u n l a b e l l e d h i s t o n e c a r r i e r (50 ug) was made 10% i n TCA. The p r e c i p i t a t e " w a s c o l l e c t e d on a g l a s s f i b r e f i l t e r . ; ; and s u b s e q u e n t l y washed w i t h 20% TCA, e t h a n o l and e t h e r . A 0.1 ml a l i q u o t o f PI o r P I I was a p p l i e d d i r e c t l y t o a g l a s s f i b r e f i l t e r . The f i l t e r d i s c s were d r i e d and counted i n 8 ml o f ACS (Amersham/ S e a r l e ) . C o n t r o l TCA P r e c i p i t a t e cpm/0.5 ml PI . 765 26 P I I 395 47 81a PART B - STRUCTURE OF TRANSCRIPTIONALLY ACTIVE CHROMATIN 82 RESULTS AND DISCUSSION B o t h b i o c h e m i c a l s t u d i e s and e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s (128, 212, 213) suggest t h a t nucleosomes a r e a s s o c i a t e d w i t h t r a n s c r i p t i o n a l l y a c t i v e as w e l l as i n a c t i v e r e g i o n s o f c h r o m a t i n . Because DNase I s e l e c t i v e l y d i g e s t s t r a n s c r i p t i o n a l l y competent r e g i o n s of c h r o m a t i n , the nucleosomes a s s o c i a t e d w i t h t h e s e r e g i o n s a r e b e l i e v e d t o e x i s t i n an a l t e r e d c o n f o r m a t i o n (120, 133, 135). P o s s i b l e m o l e c u l a r mechanisms f o r i n d u c i n g such c o n f o r m a t i o n a l changes i n c l u d e h i s t o n e a c e t y l a t i o n and the a s s o c i a t i o n of s p e c i f i c n o n h i s t o n e p r o t e i n s w i t h t r a n s c r i p t i o n a l l y competent c h r o m a t i n r e g i o n s . I n t h e s e s t u d i e s v a r i o u s methods were used t o f r a c t i o n a t e c h r o m a t i n i n t o t e m p l a t e a c t i v e and i n a c t i v e f r a c t i o n s w i t h the major purpose of examining t h e l e v e l s of t h e a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h each nucleosome f r a c t i o n . By u s i n g n u c l e a s e probes (DNase I , DNase I I and m i c r o c o c c a l n u c l e a s e ) , the s t r u c t u r e of c h r o m a t i n from the two s t a t e s was compared. I . C h a r a c t e r i z a t i o n o f Nucleosome S u b t r a c t i o n s and Chromosomal P r o t e i n s R e l e a s e d by M i c r o c o c c a l N u c l e a s e D i g e s t i o n of N u c l e i (a) D e t e r m i n a t i o n of t h e l e v e l s of a c e t y l a t e d h i s t o n e s p e c i e s  a s s o c i a t e d w i t h mononucleosomes I n t h i s s t u d y t h e l e v e l s o f t h e a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h mononucleosomes t h a t were e x c i s e d from c h r o m a t i n a f t e r d i f f e r e n t e x t e n t s o f m i c r o c o c c a l n u c l e a s e d i g e s t i o n were examined. The c o n t e n t of a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d 83 w i t h mononucleosomes was a l s o compared t o t h o s e a s s o c i a t e d w i t h whole c h r o m a t i n . 1. K i n e t i c s of [ - ^ C ] - a c e t a t e i n c o r p o r a t i o n i n t o monomer  and mutimer f r a c t i o n s F i g . 12 shows t h e column p r o f i l e s of n u c l e a s e d i g e s t p r o d u c t s o b t a i n e d from t h e . n u c l e i o f t e s t i s c e l l s w hich were l a b e l l e d f o r v a r i o u s times w i t h [ 1 I + C ] - a c e t a t e . Both t h e monomer and mu l t i m e r peaks a r e seen t o be l a b e l l e d , and t h e p r o f i l e s do n o t change q u a l i t a t i v e l y d u r i n g the c o u r s e of t h e i n c u b a t i o i i from 5 min t o 5 h. The s p e c i f i c a c t i v i t i e s of the peaks ( i n counts per min per A.,^) f o r each time p o i n t a r e p l o t t e d i n F i g . 13. ZoU The i n c o r p o r a t i o n o f [ ^ C ] - a c e t a t e i n t o monomer and mutimer f o l l o w s a s i m i l a r time c o u r s e . The s p e c i f i c a c t i v i t y of t h e monomer peak, however, i s c o n s i s t e n t l y h i g h e r than t h a t o f t h e multime r peak ( T a b l e V I I I ) , t h e r a t i o s f o r monomer t o mutimer b e i n g 1.1 t o 1.2. 2. E f f e c t o f e x t e n s i v e n u c l e a s e d i g e s t i o n on t h e r e l a t i v e  [ l C ] - a c e t a t e i n c o r p o r a t i o n i n t o monomer and multimer  f r a c t i o n s When n u c l e i l a b e l l e d w i t h [ 1 I + C ] - a c e t a t e a r e d i g e s t e d more e x t e n s i v e l y w i t h m i c r o c o c c a l n u c l e a s e , t h e r e l a t i v e s p e c i f i c a c t i v i t i e s of monomer and mul t i m e r peaks remain unchanged ( F i g . 1 4 ) . Here, n u c l e i have been d i g e s t e d f o r . e i t h e r .8 or. .30 min w i t h 84 1.2 0.8 0.4 3 CM 2.4 1.6 0.8 40 80 120 FRACTION NO. 4 0 2 0 o E 160 4 0 1.2 0.8 0.4 S CN 1 0.4 Q2 J V v f 4 0 80 120 FRACTION NO. 4 0 20 x E & 40 20 FIG. 12. [ 1 I fC] A c e t a t e i n c o r p o r a t i o n i n t o h i s t o n e s o f nucleosomes as a f u n c t i o n o f time. T r o u t t e s t i s c e l l s were i n c u b a t e d w i t h sodium [1- 1'*C] a c e t a t e ( 5 y C i / m l ) ; a t v a r y i n g time i n t e r v a l s , a l i q u o t s were removed and n u c l e i were i s o l a t e d . The n u c l e i were d i g e s t e d f o r 8 min w i t h m i c r o c o c c a l n u c l e a s e (300 ^fro u n i t s / m l ) > a n d the d i g e s t p r o d u c t s were s e p a r a t e d on a B i o - G e l A-5m column (90 x 1.5 cm). R a d i o a c t i v i t y was determined i n the same manner as d e s c r i b e d f o r F i g . 7. L a b e l l i n g was f o r : A, 5 min.; B, 30 min.; C, 2 h:. .; D, 4 h . 85 2 4 TIME (hours) FIG. 13. [ C] A c e t a t e i n c o r p o r a t i o n i n t o h i s t o n e s of multimers and monomers as a f u n c t i o n o f time. [1'*C] A c e t a t e t o r a t i o s o f l a b e l l e d peak f r a c t i o n s o f monomers and mu l t i m e r s i s o l a t e d on B i o - G e l A-5m columns i n F i g . 12 were p l o t t e d v e r s u s i n c u b a t i o n time. • monomers; • m u l t i m e r s . 86-TABLE V I I I Q u a n t i t a t i o n o f t h e L e v e l s of H i s t o n e A c e t y l a t i o n  A s s o c i a t e d w i t h Nucleosomal F r a c t i o n s ( d a t a from F i g . 13) Peak f r a c t i o n R a t i o : monomer/ I n c u b a t i o n t i m e M u l t i m e r Monomer multime r c p m / A 2 6 Q •5 min 309 327 1.1 30 min 343 386 1.1 2 h 764 846 1.1 5 h 982 1149 1.2 87 \ t o X E 8-w FRACTION NO. FRACTION NO. FIG. 14. M i c r o c o c c a l n u c l e a s e d i g e s t i o n o f n u c l e i i s o l a t e d from I l l f C ] a c e t a t e - l a b e l l e d t r o u t t e s t i s c e l l s . T r o u t t e s t i s c e l l s were i n c u b a t e d w i t h sodium [ l - ^ C ] a c e t a t e (50 yCi/ml) f o r 90 min.,, and n u c l e i were i s o l a t e d . The n u c l e i were d i g e s t e d f o r (A) 8 min w i t h m i c r o c o c c a l n u c l e a s e (300 A^^Q u n i t s / m l ) or (B) 30 min , w i t h m i c r o c o c c a l n u c l e a s e (300 A ^ u n i t s / m l ) , and t h e nucleosomes were s e p a r a t e d on a B i o - G e l A-5m column (90 x 1.5cm). R a d i o a c t i v i t y was determined i n t h e same manner as d e s c r i b e d f o r F i g . 7. 88 300 A„,„ u n i t s / m l of n u c l e a s e . I n t h e 30 min sample, 260 the monomer f r a c t i o n r e p r e s e n t s 62% o f t h e m a t e r i a l ( e x c l u d i n g , t h e low m o l e c u l a r weight peak c e n t e r e d a t F r a c t i o n 140). The r a t i o o f [ l l f C ] - a c e t a t e ( i n c o u n t s per min per A„,„) i n monomer v e r s u s multimer i s s t i l l 260 1.2 as i t i s i n t h e 8-min d i g e s t . S i n c e H i does not c o n t a i n N - a c e t y l groups and s i n c e i t s N - a c e t y l group i s not l a b e l l e d under t h e c o n d i t i o n s used (214), v a r i a b i l i t y i n t h e HI c o n t e n t of t h e mononucleosomes w i l l not a f f e c t t h e i r s p e c i f i c a c t i v i t i e s , i . e . [ 1 1 +C] a c e t a t e counts per min per A 0,_. Hence t h e h i g h e r s p e c i f i c a c t i v i t y o f monomers must be due t o t h e l o s s o f n u c l e o t i d e s , presumably from t h e l i n k e r DNA between n u c l e o s o m a l c o r e p a r t i c l e s . T h i s would c o r r e s p o n d t o a 20% d e c r e a s e i n t h e n u c l e o t i d e l e n g t h r e l a t i v e t o t h e nu c l e o s o m a l DNA, i . e . r o u g h l y 40 base p a i r s . T h i s l o s s of t h e i n t e r n u c l e o s o m a l DNA seems t o be a s s o c i a t e d w i t h t h e p a r t i a l l o s s of HI-, as seen i n t h e 8-min d i g e s t o f F i g . 6B. The HI r e m a i n i n g may be bound t o t h e ends of th e n u c l e o s o m a l DNA. F u r t h e r d i g e s t i o n o f t h e s e mono-nucleosomes would l e a d t o "trim m i n g " o f t h e nucleosome DNA. and complete l o s s of HI ( F i g . 6C). A l t h o u g h t h e complete l o s s o f HI was ob s e r v e d , t h e l o s s o f t h e a d d i t i o n a l n u c l e o t i d e s d u r i n g trimming would presumably n o t be e x t e n s i v e enough t o r e s u l t i n a measurable change 89-. i n absorbance. A l t h o u g h no attempt was. made i n t h e s e studies, t o examine t h e trimming o f nucleosome DNA i n d e t a i l , i t i s i n t e r e s t i n g t o n o t e t h a t W h i t l o c k and Simpson (210) e s t i m a t e d t h e l e n g t h o f DNA protected by HI t o be 40 t o 50 base p a i r s . 3. Comparison o f t h e l e v e l s o f a c e t y l a t e d h i s t o n e s p e c i e s  a s s o c i a t e d w i t h mononucleosomes and u n d i g e s t e d n u c l e i In o r d e r t o compare t h e e x t e n t of l a b e l l i n g of s p e c i f i c h i s t o n e s from monomers and from whole n u c l e i , h i s t o n e s were e x t r a c t e d , s e p a r a t e d by SDS g e l e l e c t r o p h o r e s i s , and a n a l y s e d f o r r a d i o a c t i v i t y . As shown i n T a b l e IX, t h e s p e c i f i c a c t i v i t i e s of H3, H2B + H2A, and H4 a r e v e r y s i m i l a r i n h i s t o n e s e x t r a c t e d from e i t h e r whole n u c l e i o r from c h r o m a t i n monomers a f t e r 8 or 30 min .» o f n u c l e a s e d i g e s t i o n at 300 &2(,o u n i t s / m l . I n d i v i d u a l h i s t o n e s from monomers were a l s o examined f o r t h e i r c o n t e n t o f m o d i f i e d s p e c i e s by s t a r c h g e l e l e c t r o p h o r e s i s on u r e a / a l u m i n u m / l a c t a t e g e l s . T h i s t e c h n i q u e r e s o l v e s , most o f t h e a c e t y l a t e d and p h o s p h o r y l a t e d components o f p u r i f i e d t r o u t t e s t i s h i s t o n e f r a c t i o n s and t h e i r c h a r a c t e r i z a t i o n has: been d e s c r i b e d p r e v i o u s l y (214-216). A photograph o f such a g e l i n which H2A, H2B, H3 and H4 from monomers a r e 90-TABLE IX Q u a n t i t a t i o n of I ^ C ] A c e t a t e — L a b e l l e d H i s t o n e F r a c t i o n s H i s t o n e s l a b e l l e d with. Illf'C] a c e t a t e were e x t r a c t e d from mononucleo-somes o r from u n d i g e s t e d n u c l e i , and s e p a r a t e d on 15% p o l y a c r y l a m i d e SDS g e l s . The g e l s l i c e s were scanned a t 550 nm a f t e r s t a i n i n g w i t h Coomassie b l u e and co u n t e d a f t e r s o l u b i l i z a t i o n . H i s t o n e f r a c t i o n T o t a l n u c l e a r Nucleosome monomers h i s t o n e 8-min: ;.< d i g e s t 30-min d i g e s t P e r c e n t cpm/A,.^ i n Each H i s t o n e F r a c t i o n H3 44 52 47 H2A + H2B a 10 9 13 H4 46 39 40 a H i s t o n e s , H2A and H2B, when e x t r a c t e d from n u c l e i w i t h 0.2N HC1 and p r e c i p i t a t e d w i t h a c e t o n e , c o - e l e c t r o p h o r e s e d on 15% p o l y a c r y l a m i d e SDS g e l s . 91 compared t o t h e c o r r e s p o n d i n g f r a c t i o n s from whole c h r o m a t i n , i s shown i n F i g . 15. I n H2A, t h e un m o d i f i e d , m o n o a c e t y l , and p h o s p h o r y l a t e d components a r e v i s i b l e . H2B i s seen t o c o n t a i n t h e u n m o d i f i e d s p e c i e s and an a d d i t i o n a l component which c o u l d be e i t h e r m o n o a c e t y l a t e d H2B or some c o n t a m i n a t i n g , u n m o d i f i e d H2A. H2B from monomers does c o n t a i n a c e t y l a t e d s p e c i e s , however, s i n c e l a b e l l e d a c e t y l groups a r e p r e s e n t i n t h i s f r a c t i o n ( d a t a not shown). The H3 c o n t a i n s u n m o d i f i e d and m o n o a c e t y l a t e d s p e c i e s , and t h e H4 c o n t a i n s u n m o d i f i e d , mono-, d i - , t r i - , and t e t r a a c e t y l a t e d s p e c i e s . The l a t t e r bands a r e f a i n t and somewhat smeared. The s e p a r a t i o n o f t h e m o d i f i e d s p e c i e s on t h e s e samples i s n o t as d i s t i n c t as t h a t o b t a i n e d w i t h h i s t o n e s i s o l a t e d by l a r g e r s c a l e column p r o c e d u r e s (214). T h i s i s e v i d e n t l y due t o t h e presence of r e s i d u a l SDS i n t h e s e samples, which were o b t a i n e d by p r e p a r a t i v e SDS g e l e l e c t r o p h o r e s i s . Furthermore, chromatography on B i o - G e l P10 columns, t h e method o f c h o i c e f o r l a r g e h i s t o n e samples, l e a d s t o some f r a c t i o n -a t i o n of a c e t y l a t e d s p e c i e s from u n m o d i f i e d ones (214), and t h e r e s u l t i n g enrichment o f m o d i f i e d s p e c i e s makes t h e i r v i s u a l i z a t i o n on g e l s much e a s i e r . I t i s e v i d e n t from F i g . 15, however, t h a t t h e s t a r c h 92 FIG. 15. A c e t y l a t e d components of whole t r o u t t e s t i s h i s t o n e s and h i s t o n e s from nucleosomes. H i s t o n e s i s o l a t e d by a c i d - e x t r a c t i o n from nucleosomes or n u c l e i were s e p a r a t e d by p o l y a c r y l a m i d e - SDS g e l e l e c t r o p h o r e s i s and s t a i n e d w i t h Coomassie b l u e . The p r o t e i n bands were d i s s e c t e d , and SDS and Coomassie b l u e were removed from the h i s t o n e s by a c e t o n e e x t r a c t i o n b e f o r e a p p l i c a t i o n to a urea/aluminum l a c t a t e s t a r c h g e l . E l e c t r o p h o r e s i s was f o r 9 h. a t 35 mA. The m i d d l e s l i c e o f t h e g e l was s t a i n e d w i t h 0.125% Amido b l a c k c o n t a i n i n g C o C l 2 f o r 30 min and d e s t a i n e d w i t h 0.5 M H SO^. a, c, e, and g a r e h i s t o n e s e x t r a c t e d from n u c l e i and b, d, f , and h a r e h i s t o n e s e x t r a c t e d from nucleosomes. " T o t a l " r e p r e s e n t s whole h i s t o n e s e x t r a c t e d from nucleosomes. Cathode i s a t t h e bottom. 93 g e l p a t t e r n s o f h i s t o n e s from whole c h r o m a t i n and from monomers a r e v e r y s i m i l a r . The above s t u d i e s , t h e r e f o r e , i n d i c a t e t h a t c h r o m a t i n s u b u n i t s i s o l a t e d from m i c r o c o c c a l n u c l e a s e d i g e s t i o n c o n t a i n t h e major a c e t y l a t e d h i s t o n e s p e c i e s n o r m a l l y found i n whole c h r o m a t i n . In a d d i t i o n , t h e p h o s p h o r y l a t e d form o f H2A i s a l s o p r e s e n t . The l e v e l s of t h e s e m o d i f i e d s p e c i e s i n chromatin monomers a r e a l s o v e r y c l o s e t o t h o s e found i n whole c h r o m a t i n . However, t h e s e r e s u l t s do n o t r u l e out t h e p o s s i b i l i t y t h a t major d i f f e r e n c e s may e x i s t i n t h e c o n t e n t o f t h e h i g h l y m o d i f i e d s p e c i e s ( i . e . s p e c i e s t h a t c o n t a i n t h r e e or f o u r a c e t y l groups; or which a r e b o t h a c e t y l a t e d and phosphorylated)., s i n c e t h e s e a r e p r e s e n t a t v e r y low l e v e l s . , and have not been q u a n t i t a t e d i n t h e s e s t u d i e s . Chromatin f r a c t i o n a t i o n (Sanders' p r o c e d u r e ) R e c e n t l y M. Sanders (190) has r e p o r t e d a p r o c e d u r e f o r t h e f r a c t i o n a t i o n o f c h r o m a t i n . The p r o c e d u r e i n v o l v e s s t e p w i s e t r e a t m e n t s o f m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i w i t h i n c r e a s i n g s a l t c o n c e n t r a t i o n s ( j i g . 16). U s i n g t h i s p r o c e d u r e , t h e c o n t e n t o f t h e h i s t o n e a c e t y l a t e d s p e c i e s a s s o c i a t e d w i t h each nucleosome s u b t r a c t i o n / and t h e n o n - h i s t o n e chromosomal p r o t e i n s 94 MICROCOCCAL NUCLEASE DIRKSTEH < SUPERNATANT (SO) SUPERNATANT (SS2) SUPERNATANT (SS3) SUPERNATANT (SS4) SUPERNATANT (SS6) PELLET (PO) 1. Resuspend in Buffer D plus 0.2 M NaCl 2. Incubate 20 min., 0°C 3. Centrifuge PELLET (P2) Repeat as above with Buffer D plus 0.3 M NaCl, then successively with 0.4 M and 0.6 M NaCl PELLET (P3) P e l l e t (P4) PELLET (P6) FIG. 16. E x p e r i m e n t a l p r o c e d u r e f o r t h e i s o l a t i o n of n u c l e o s o m a l sub-f r a c t i o n s from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i by s u c c e s s i v e l y h i g h e r NaCl c o n c e n t r a t i o n s . 95 r e l e a s e d were examined. 1. Q u a n t i t a t i o n o f DNA c o n t e n t i n s a l t - e x t r a c t e d c h r o m a t i n  f r a c t i o n s F o l l o w i n g t h e d i g e s t i o n of t r o u t t e s t i s n u c l e i w i t h m i c r o c o c c a l n u c l e a s e , t h e c h r o m a t i n was f r a c t i o n a t e d by s e q u e n t i a l n u c l e a r e x t r a c t i o n w i t h i n c r e a s i n g concen-t r a t i o n s o f sodium c h l o r i d e ( F i g . 16) as d e s c r i b e d by Sanders (190), except t h a t t h e i n i t i a l NaCl concen-t r a t i o n was 0.1 M i n s t e a d of 0.2 M, arid t h e 0 . 3 M NaCl e x t r a c t i o n s t e p was o m i t t e d . The p e r c e n t a g e of t o t a l a b s o r b i n g m a t e r i a l r e l e a s e d i n each f r a c t i o n ( T a b l e X) was i n c l o s e agreement w i t h t h e y i e l d s o b t a i n e d by Sanders. F u r t h e r -more, Sanders (190) r e p o r t e d t h a t t h e p e r c e n t a g e o f m a t e r i a l r e l e a s e d i n t o each f r a c t i o n was independent of t h e e x t e n t o f d i g e s t i o n . T h e r e f o r e , each f r a c t i o n r e p r e s e n t s a d i s t i n c t n u c l e o s o m a l p o p u l a t i o n . The DNA r e l e a s e d i n SO was almost t o t a l l y a c i d s o l u b l e . Most o f t h e r e m a i n i n g a c i d s o l u b l e m a t e r i a l was r e -l e a s e d i n SSI and SS2. 96 TABLE X Q u a n t i t a t i o n of DNA Content i n S a l t - E x t r a c t e d Chromatin F r a c t i o n s  R e l e a s e d from M i c r o c o c c a l N u c l e a s e D i g e s t e d N u c l e i  (Sanders' Procedure) The A^gQ a b s o r b i n g m a t e r i a l r e l e a s e d i n t h e d i f f e r e n t s a l t - e x t r a c t e d nucleosome f r a c t i o n s a f t e r m i c r o c o c c a l n u c l e a s e d i g e s t i o n of t r o u t t e s t i s n u c l e i was q u a n t i t a t e d by a d d i n g 100 y l of t h e f r a c t i o n to 0.9 ml of 0.6 M NaCl i n B u f f e r D and measuring the absorbance a t 260 nm. The p e r c e n t a g e of a c i d s o l u b l e m a t e r i a l i n each f r a c t i o n was measured by a d d i n g 100 y l of t h e f r a c t i o n to 0.9 ml of I N p e r c h l o r i c acid... and measuring the absorbance a t 260 nm of t h e s u p e r n a t a n t a f t e r c e n t r i f u g a t i o n . F r a c t i o n T o t a l A „ , n i n P e r c e n t A 0 , n i n 260 260 Each F r a c t i o n A c i d S o l u b l e M a t e r i a l % SO 13.4 83.8 551 7.2 58.5 552 7.8 ' 30.0 SS4 43.4 2.9 SS6 7.8 0.6 97 2. C h a r a c t e r i z a t i o n o f t h e DNA fragments a s s o c i a t e d w i t h  s a l t - e x t r a c t e d c h r o m a t i n f r a c t i o n s The DNA s i z e c l a s s e s from each s a l t - e x t r a c t e d f r a c t i o n were examined on 3% n o n d e n a t u r i n g p o l y a c r y l a m i d e g e l s ( F i g . 17). In agreement w i t h Sanders' r e s u l t s , t h e average DNA fragment s i z e i n c r e a s e d w i t h each s u c c e s s i v e l y h i g h e r NaCl c o n c e n t r a t i o n . The DNA i n SSI and SS2 c o n s i s t e d almost e n t i r e l y o f monomer s i z e fragments. Fragments o f l e s s than monomeric s i z e were a l s o o b s e r v e d i n t h e SSI and to a l e s s e r e x t e n t i n SS2. The SS4 f r a c t i o n c o n t a i n e d v e r y l i t t l e monomer; i t c o n s i s t e d m o s t l y o f dimer and l e s s e r amounts o f t r i m e r , t e t r a m e r , pentamer and hexamer fragments. Fragments g r e a t e r than octamer i n l e n g t h predominated i n t h e SS6 f r a c t i o n . The DNA s i z e s f o r SSI and SS2 were f u r t h e r c h a r a c t e r i z e d on 5% n o n d e n a t u r i n g p o l y a c r y l a m i d e g e l s , and t h e i r s i z e s were c a l i b r a t e d u s i n g Fnu DI ge n e r a t e d fragments of 0X174 DNA as r e f e r e n c e markers. The DNA from the SSI shows a t l e a s t s i x d i f f e r e n t fragments ( F i g . 18) w i t h a 146 base p a i r fragment p r e d o m i n a t i n g . A l a r g e r fragment o f 170 base p a i r s i s a l s o e v i d e n t i n 9 8 a b e d FIG. 17. Polyacrylamide gel electrophoresis of the DNA fragments generated by micrococcal nuclease digestion of trout testis nuclei and released by stepwise increases in NaCl concentrations. DNA (0.2 A , ) obtained from the stepwise eluted fractions (see "Materials and Methods ) was analyzed on a 3% polyacrylamide SDS gel. The gel was stained with ethidium bromide and photographed under ultraviolet light. a, b, c and d are SSI, SS2, SS4 and SS6, respectively. l a and lb are the 146 base pair monomer and 170 base pair monomer, respectively; 2, 3 and 4 are dimer, trimer and tetramer, respectively. S i s a 124 base pair fragment. 99 a b c d e f g h i . FIG. 18. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of t h e DNA fragments r e l e a s e d by m i c r o c o c c a l n u c l e a s e i n t o t h e SO and SSI f r a c t i o n s (see " M a t e r i a l s and Methods"). ( a ) , d i g e s t e d n u c l e i were e x t r a c t e d with. B u f f e r D p l u s 0.1 M N a C l . The SSI f r a c t i o n was f r a c t i o n a t e d on a B i o - G e l A-5m column ( F i g . 21) and t h e monomer peak p o o l e d . The DNA ( 0 . 0 5 ^^Q) was t h e n a p p l i e d t o t h e 5% p o l y a c r y l a m i d e SDS g e l . (b) t o ( f ) , DNA r e l e a s e d i n t o SO from two d i f f e r e n t p r e p a r a t i o n s of m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i . (b) and (c) , DNA (0.2 and 1.2 A^^Q, r e s p e c t i v e l y ) r e l e a s e d i n t o SO from one p r e -p a r a t i o n , (d) , (_e) and ( f ) , DNA ( 0 . 2 , 0.6 and 1.2 A r e s p e c t i v e l y ) r e l e a s e d i n t o SO from another p r e p a r a t i o n . (g) and T h ) , DNA (0.2 and 0.6 A^^Q, r e s p e c t i v e l y ) r e l e a s e d i n SSI. S i z e e s t i m a t e s a r e based on a c a l i b r a t i o n u s i n g p r e c i s e l y d e f i n e d Fnu DI g enerated fragments of 0X174 DNA. 100 t h e SSI DNA. The 146 base p a i r and 170 base p a i r DNA fragments may c o r r e s p o n d t o t h e mononucleosomes MN^ and MN^ r e s p e c t i v e l y , r e p o r t e d by Bakayev et- a l . (30). • The DNA fragments of l e n g t h l e s s t h a n 146 base p a i r s c o rresponded t o s i z e s o f 124, 102, 92 and 75 base p a i r s . S i m i l a r fragments have been o b s e r v e d by N o l l and Romberg (217), A x e l et a l . (218), Bakayev et a l . (90) and W h i t l o c k (97) and a r e p r o b a b l y t h e r e s u l t of i n t e r n a l nucleosome c l e a v a g e s . The DNA from SS2 c o n s i s t e d p r e d o m i n a n t l y of a 170 base p a i r fragment. L e s s e r amounts of fragments c o r r e s p o n d i n g t o t h o s e r e l e a s e d i n SSI were a l s o o b s erved ( d a t a not shown). The a c i d s o l u b i l i t y measurements f o r SO ( T a b l e X) suggested t h a t some a c i d p r e c i p i t a b l e m a t e r i a l e x i s t e d i n t h i s f r a c t i o n . T h e r e f o r e , t h e DNA o f t h e SO f r a c t i o n was examined on 5% n o n - d e n a t u r i n g p o l y a c r y l a m i d e g e l s ( F i g . 18). Low amounts of DNA fragments i d e n t i c a l t o t h o s e r e l e a s e d i n SSI were o b s e r v e d . T h i s may r e -p r e s e n t l e a k a g e from damaged n u c l e i o f m a t e r i a l which i s u s u a l l y e x t r a c t e d i n t h e 0.1 M NaCl f r a c t i o n . 101; In agreement w i t h Sanders' o b s e r v a t i o n s (.190), the; r e s u l t s a r e c o n s i s t e n t w i t h t h e i n t e r p r e t a t i o n t h a t t h e f r a c t i o n o f c h r o m a t i n which i s most s e n s i t i v e t o m i c r o c o c c a l n u c l e a s e i s e l u t e d a t t h e lowest NaCl c o n c e n t r a t i o n s . Q u a n t i t a t i o n of t h e a c e t y l a t e d h i s t o n e s p e c i e s  a s s o c i a t e d w i t h s a l t - e x t r a c t e d c h r o m a t i n f r a c t i o n s To q u a n t i t a t e t h e c o n t e n t of t h e m o d i f i e d h i s t o n e s p e c i e s , P a n y i m - C h a l k l e y g e l s (194) were used. T h i s system r e s o l v e s each h i s t o n e and i t s m o d i f i e d forms more e f f e c t i v e l y t h a n s t a r c h - u r e a g e l s (193) and does not r e q u i r e t h e p r i o r s e p a r a t i o n of each h i s t o n e by SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . F i g . 19A shows a photograph of a t y p i c a l Coomassie b l u e s t a i n e d P a n y i m - C h a l k l e y g e l where [lhC] - a c e t a t e l a b e l l e d h i s t o n e s have been s e p a r a t e d . Accompanying t h e s t a i n e d g e l i s an a u t o r a d i o g r a m of t h e g e l ( F i g . 19B). The a u t o r a d i o g r a m i n d i c a t e s t h e l o c a t i o n of t h e a c e t y l a t e d h i s t o n e s p e c i e s . Only h i s t o n e s m o d i f i e d v i a N -a c e t y l a t i o n a r e l a b e l l e d ; HI, which i s m o d i f i e d by a N - a c e t y l a t i o n , i s not l a b e l l e d . A c i d e x t r a c t e d h i s t o n e s from t h e d i f f e r e n t f r a c t i o n s " were a n a l y z e d on P a n y i m - C h a l k l e y g e l s . The h i s t o n e p a t t e r n s were examined f o r d i f f e r e n c e s i n t h e 102 FIG. 19. Localization of acetylated histone species separated on acid-urea gels. A, Trout testis cells were incubated with sodium [l- l l*C] acetate (50 uCi/ml) for 9 h. , and nuclei were isolated. Labelled histones were acid-extracted and applied to an acid-urea gel. Electrophoresis was for 20 h at 170 V (4 C ). The gel was stained with Coomassie blue and destained. B, After the gel was dried, an autoradiogram of the gel was prepared after a three month exposure time. A , unacetylated H4; A 3 4 5 m o n°-> d±-> t r l~> and tetra-acetylated H4, respectively. 1 A2' 103 e x t e n t of m o d i f i c a t i o n ( F i g . 2 0 ) . The most s t r i k -i n g d i f f e r e n c e s were found i n t h e c o n t e n t of HI,, and i n t h e l e v e l s o f H4 a c e t y l a t i o n . The HI c o n t e n t r e l a t i v e t o t h e o t h e r h i s t o n e s i n c r e a s e d from v e r y low i n SSI t o maximal i n SS4. As t h e 146 base p a i r monomer fragment predominates i n th e SSI f r a c t i o n , t h e HI c o n t e n t would be e x p e c t e d to be low s i n c e t h i s nucleosome s p e c i e s i s d e v o i d of HI (189, 217, 219). The HI p r e s e n t would presumably be a s s o c i a t e d w i t h t h e 170 base p a i r fragment as r e p o r t e d by Bakayev et a l . (90) f o r t h e mononucleosome'MN^. , The c o n t e n t of HI i n c r e a s e d i n SS2 w i t h a c o r r e s p o n d i n g i n c r e a s e i n t h e 170 base p a i r fragment. The SS4 f r a c t i o n c o n t a i n e d t h e g r e a t e s t amount of HI r e l a t i v e t o t h e o t h e r f o u r n u c l e o s o m a l h i s t o n e s . T h i s r e s u l t i s t o be expected as HI i s d i s s o c i a t e d from c h r o m a t i n o r n u c l e i between 0.35 and 0.6 M N aCl .(220, 221). The l e v e l o f H4 a c e t y l a t i o n was c o n s i d e r a b l y h i g h e r i n SSI t h a n i n t h e o t h e r fractions,,, and was found t o d e c r e a s e w i t h s e q u e n t i a l e x t r a c t i o n s . As a check a g a i n s t p o s s i b l e s e l e c t i v e l o s s e s of h i s t o n e s d u r i n g e x t r a c t i o n , t h e a c i d e x t r a c t e d h i s t o n e s from SSI, SS2, SS4 and SS6 were examined on 15% p o l y a c r y l a m i d e SDS 104 H2 B K H2A FIG. 20. Gel scans of histones analyzed on acid-urea gels. Histones were acid-extracted from nucleosomes that had been released from micrococcal nuclease digested nuclei by stepwise, increasing NaCl concentrations. Gels were stained with Coomassie blue and scanned i n a Gilford spectrophotometer at 550 nm. MN, micrococcal nuclease. Ao, unacetylated H4; ^»^2 a n C^ ^3' m o n o~> ^ i - and tri-acetylated H4, respectively. 105 g e l s . The measured l e v e l s o f HA r e l a t i v e t o t h e o t h e r t h r e e n u c l e o s o m a l h i s t o n e s remained c o n s t a n t i n a l l f r a c t i o n s ; ( d a t a n o t shown). T h e r e f o r e , t h e o b s e r v e d l e v e l o f HA a c e t y l a t i o n i n t h e SSI f r a c t i o n was not due t o a s e l e c t i v e l o s s o f t h e u n a c e t y l a t e d ( A Q ) and t h e m o n o a c e t y l a t e d (A^) HA s p e c i e s but t o an i n c r e a s e i n t h e l e v e l s of t h e d i - (A^) and t r i -a c e t y l a t e d (A^) HA s p e c i e s . The e x t e n t of HA a c e t y l a t i o n f o r t h e d i f f e r e n t f r a c t i o n s was c a l c u l a t e d as a r a t i o o f t h e c o n t e n t of A 0 and A s p e c i e s d i v i d e d by t h e sum o f the A. and A Z O i o s p e c i e s . The i n v e r s e r e l a t i o n s h i p between the l e v e l s o f HA a c e t y l a t i o n and t h e i n c r e a s i n g c o n c e n t r a t i o n s of NaCl used i n the s e q u e n t i a l e x t r a c t i o n s i s e v i d e n t ( T a b l e X I ) . The degree of a c e t y l group i n c o r p o r a t i o n a s s o c i a t e d w i t h each f r a c t i o n i n a two hour p e r i o d i n v i t r o was determined by r a d i o a c t i v e l a b e l l i n g o f c e l l s u s p e n s i o n s . N u c l e i i s o l a t e d from t r o u t t e s t i s c e l l s l a b e l l e d with: [ J 1*C] - a c e t a t e were d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e and s u b s e q u e n t l y t r e a t e d w i t h i n c r e a s i n g s a l t c o n c e n t r a t i o n s . The h i s t o n e s from f r a c t i o n s : SSI, SS2 and SSA were s e p a r a t e d u s i n g SDS 106 TABLE XI Q u a n t i t a t i o n ' o f A c e t y l a t e d S p e c i e s of H i s t o n e H4 i n S a l t - E x t r a c t e d Nucleosome F r a c t i o n s (Sanders' P r o c e d u r e ) H i s t o n e s p r e p a r e d by a c i d e x t r a c t i o n o f the s a l t - e x t r a c t e d nucleosome f r a c t i o n s were s e p a r a t e d on a c i d - u r e a g e l s . The g e l s were s t a i n e d w i t h Coomassie blue-, and scanned a t 550 nm u s i n g a G i l f o r d s p e c t r o p h o t o m e t e r . A + A * F r a P t l o n - R a t i o o f H4 -? 3 A + A, o 1 SSI SS2 SS4 SS6 0.84 0.59 0.32 0.34 Determined from the A ^ peak h e i g h t s o f the scanned H4 s p e c i e s 107 p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s , and t h e i r s p e c i f i c a c t i v i t i e s i n terms o f Ill*C] cpm/A^^^ were determined ( T a b l e X I I ) . For H4 t h e degree o f a c e t a t e i n c o r p o r a t i o n I n c r e a s e s w i t h t h e i n c r e a s i n g NaCl c o n c e n t r a t i o n s used i n t h e s e q u e n t i a l e x t r a c t i o n s . Thus, t h e l a b e l l i n g o f H4 a c e t y l groups does not c o r r e s p o n d t o t h e l e v e l s o f H4 a c e t y l a t i o n determined by u s i n g scans o f s t a i n e d a c i d - u r e a g e l s ( F i g . 20). Levy et a l . (174) have r e p o r t e d a s i m i l a r r e s u l t . M i c r o c o c c a l n u c l e a s e d i g e s t e d t r o u t t e s t i s c h r o m a t i n was f r a c t i o n a t e d t o y i e l d a f r a c t i o n c o n t a i n i n g h i g h l e v e l s o f t h e a c e t y l a t e d H4 s p e c i e s . A l t h o u g h t h e H4 a s s o c i a t e d w i t h t h i s f r a c t i o n was e n r i c h e d i n a c e t y -l a t e d s p e c i e s , i t was l a b e l l e d t o a l e s s e r e x t e n t w i t h I 1 ^ C ] - a c e t a t e than t h e H4 a s s o c i a t e d w i t h o t h e r f r a c t i o n s c o n t a i n i n g low l e v e l s of t h e a c e t y l a t e d H4 s p e c i e s . Moore et a l . (222) have i n v e s t i g a t e d t h e t u r n o v e r k i n e t i c s o f a c e t y l groups i n e u k a r y o t i c c e l l s . Two ty p e s o f a c e t y l a t i o n were r e p o r t e d : one i n which 50% of t h e a c e t a t e was removed w i t h a h a l f - l i f e of 3 min and t h e r e s t w i t h a much l o n g e r h a l f - l i f e of 30 t o 40 min. Although, t h e much l o n g e r h a l f - l i f e o f a c e t y l groups i n t r o u t t e s t i s (23 hours (11)) makes i t 108 TABLE X I I S p e c i f i c A c t i v i t i e s o f H i s t o n e s H3 and H4 i n S a l t - E x t r a c t e d Nucleosome Fraction'. H i s t o n e s l a b e l l e d w i t h [lkC] a c e t a t e were e x t r a c t e d from s a l t - e l u t e d nucleosome f r a c t i o n s and s e p a r a t e d on 15% p o l y a c r y l a m i d e SDS g e l s c o n t a i n i n g 0.6% N, N' - d i a l l y l t a r t a r d i a m i d e as c r o s s l i n k e r . The g e l s l i c e s were scanned a t 550 nm a f t e r s t a i n i n g w i t h Coomassie b l u e , and counted a f t e r s o l u b i l i z a t i o n . S a l t - E x t r a c t e d S p e c i f i c . A c t i v i t y o f H i s t o n e F r a c t i o n F r a c t x o n • H3 H4 lkC cpm/A 5 5 Q X 10 3 S S 1 1-90 1.93 s s 2 2.07 1.93 SS4 2.44 2.32 109 d i f f i c u l t t o r e l a t e the k i n e t i c s i n t h i s system t o t h a t of c u l t u r e d c e l l s , the lower s p e c i f i c a c t i v i t y o f t h e a c e t y l a t e d H4 a s s o c i a t e d w i t h t h e SSI f r a c t i o n s u g g e s t s t h a t t h e H4 i n t h i s f r a c t i o n may c o n s t i t u t e a s u b p o p u l a t i o n of m o l e c u l e s which undergoes a c e t y l group t u r n o v e r a t a r e l a t i v e l y slow r a t e . 4. Q u a n t i t a t i o n of t h e a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h column f r a c t i o n a t e d s a l t - e x t r a c t e d  nucleosome f r a c t i o n s To determine i f the h i g h l y a c e t y l a t e d H4 s p e c i e s are.! a s s o c i a t e d w i t h the n u c l e o s o m a l (146 and 170 base p a i r ) DNA, t h e SSI f r a c t i o n was f u r t h e r f r a c t i o n a t e d on a B i o - G e l A-5m. column ( F i g . 21). A l s o , t h e t o t a l monomer p o p u l a t i o n was examined f o r the e x t e n t of H4 a c e t y l a t i o n by e x t r a c t i n g m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i w i t h B u f f e r D c o n t a i n i n g 0.4 M NaCl.. and f r a c t i o n a t i n g t h e e x t r a c t on t h e column ( F i g . 22). Both columns were e q u i l i b r a t e d w i t h 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and 0.4 M NaCl t o p r e v e n t a g g r e g -a t i o n of t h e SS4T f r a c t i o n (223) . The f i r s t peak, e l u t i n g a t or near the v o i d volume of both: columns i s termed t h e multimer f r a c t i o n and c o n s i s t s o f fragments g r e a t e r t h a n t r i m e r i n l e n g t h n o 20 40 60 80 FRACTION NO. I FIG. 21.= B i o - G e l A-5m (90 X 1.5 cm) s e p a r a t i o n of nucleosomes o b t a i n e d from e x t r a c t i o n of m i c r o c o c c a l n u c l e a s e d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0.1 M NaCl i n B u f f e r D. I l l 20 40 FRACTION NO. 60 80 FIG. 22. B i o - G e l A-5m (90 X 1.5 cm) s e p a r a t i o n of nucleosomes o b t a i n e d from e x t r a c t i o n of m i c r o c o c c a l n u c l e a s e d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0.4 M NaCl i n B u f f e r D. 112 ( F i g . 23, l a n e a ) . F o r t h e column p r o f i l e shown i n F i g . 21, the second peak c o n t a i n e d o n l y monomer s i z e DNA w i t h t h e 146 base p a i r DNA fragment predominat-i n g ( F i g . 18, l a n e a and F i g . 23, l a n e e ) . The two slower e l u t i n g peaks p r o b a b l y c o n s i s t e d o f p a r t i c l e s w i t h DNA fragments o f up to 124 base p a i r s i n s i z e and o f s m a l l a c i d s o l u b l e o l i g o n u c l e o t i d e s , r e s p e c t i v e l y . However, t h i s i s not known f o r c e r t a i n as t h e DNA s i z e c l a s s e s from t h e s e peaks were n o t e s t i m a t e d by p o l y -a c r y l a m i d e g e l e l e c t r o p h o r e s i s . The same p r o f i l e f o r the SSI f r a c t i o n a t i o n was observed whether t h e column was e q u i l i b r a t e d w i t h 0.4 o r 0.1 M NaCl. The second peak o f t h e column p r o f i l e i n F i g . 22 was s u b d i v i d e d i n t o i n t e r m e d i a t e and monomeric f r a c t i o n s . F r a c t i o n s 30 to 41 ( t h e i n t e r m e d i a t e f r a c t i o n ) c o n t a i n e d dimer, some t r i m e r and v e r y l i t t l e monomer ( F i g . 23, l a n e b ) . The monomer f r a c t i o n (42 to 51) c o n t a i n e d m o s t l y monomer and some dimer ( F i g . 23, l a n e c ) . H i s t o n e s were a c i d / e x t r a c t e d from SSI monomeric f r a c t i o n and from t h e SS4T m u l t i m e r i c , i n t e r m e d i a t e and monomeric f r a c t i o n s . The h i s t o n e s were s e p a r a t e d on P a n y i m - C h a l k l e y g e l s , and t h e r e l a t i v e degree of H4 a c e t y l a t i o n was e s t i m a t e d ( T a b l e X I I I ) . c 113 FIG. 23. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f DNA fragments a s s o c i a t e d w i t h column f r a c t i o n a t e d , s a l t - e x t r a c t e d nucleosome f r a c t i o n s . S a l t - e x t r a c t e d f r a c t i o n s SSI and SS4T were p r e p a r e d as d e s c r i b e d i n " M a t e r i a l s and Methods" and f r a c t i o n a t e d on B i o - G e l A-5m columns as p r e s e n t e d i n F i g . 21 and F i g . 22. The column f r a c t i o n s were p o o l e d as f o l l o w s : multimer (21-25), i n t e r m e d i a t e (30-41), monomer (42-51), and o l i -g o n u c l e o t i d e (57-63). DNA (0.2 A ^ Q ) o b t a i n e d from the f r a c t i o n s (see " M a t e r i a l s and Methods") was a n a l y z e d on a 3% p o l y a c r y l a m i d e - SDS g e l . The g e l was s t a i n e d w i t h e t h i d i u m bromide and photographed under u l t r a v i o l e t l i g h t . a, b, c and d a r e m u l t i m e r , i n t e r m e d i a t e , monomer and o l i g o n u c l e o t i d e f r a c t i o n s from SS4T f r a c t i o n a t e d nucleosomes, r e s p e c t i v e l y . e and f a r e monomer and o l i g o n u c l e o t i d e f r a c t i o n s from SSI f r a c t i o n a t e d nucleosomes, r e s p e c t i v e l y . 1, 2 and 3 a r e monomer, dimer and t r i m e r , r e s p e c t i v e l y . ,114: TABLE X I I I Q u a n t i t a t i o n of A c e t y l a t e d S p e c i e s of H i s t o n e H4 i n  Column F r a c t i o n a t e d , S a l t - E x t r a c t e d Nucleosome  F r a c t i o n s M i c r o c o c c a l n u c l e a s e d i g e s t e d t r o u t t e s t i s n u c l e i were e x t r a c t e d w i t h e i t h e r 0.1 M NaCl i n B u f f e r D (SSI) o r 0.4 M NaCl i n B u f f e r D (SS4T). The s a l t - e x t r a c t e d nucleosome f r a c t i o n s were f u r t h e r f r a c t i o n a t e d u s i n g a B i o -G e l A-5m column e q u i l i b r a t e d w i t h 0.4 M N a C l , 10 mM T r i s , pH 7.5, and 0.7 mM EDTA. Monomer f r a c t i o n s f o r SS4T were p o o l e d and a c i d e x t r a c t e d . The h i s t o n e s were s e p a r a t e d on a c i d - u r e a g e l s , s t a i n e d w i t h Coomassie b l u e , and scanned at 550 nm u s i n g a G i l f o r d s p e c t r o p h o t o m e t e r . F r a c t i o n R a t i o o f H4 A + A I o SSI monomer 0.97 SS4T multim e r 0.45 SS4T i n t e r m e d i a t e 0.51 SS4T monomer 0.58 * Determined from the A 550 peak h e i g h t s o f the scanned H4 s p e c i e s . 115 The p r o f i l e s , f o r t h e SSI t o t a l and SSI monomer f r a c t i o n s , were s i m i l a r f o r h i s t o n e s H3, H2A, H2B and H4 (d a t a not shown). The e x t e n t o f H4 a c e t y l a t i o n f o r t h e SSI monomer ( T a b l e X I I I ) was s l i g h t l y h i g h e r t h a n t h a t o f th e SSI t o t a l ( T a b l e X I ) . F o r t h e SS4T f r a c t i o n , t h e l e v e l s o f H4 a c e t y l a t i o n i n c r e a s e d as t h e chr o m a t i n fragment s i z e d e c r e a s e d . The SS4T monomer f r a c t i o n d i d not c o n t a i n l e v e l s o f H4 a c e t y l a t i o n as h i g h as t h o s e o b served f o r t h e SSI monomer. T h i s was t o be expected; s i n c e t h e monomers c o n t a i n i n g i n c r e a s e d l e v e l s o f a c e t y l a t e d H4 would be d i l u t e d w i t h i n t h e t o t a l monomer p o p u l a t i o n o f t h i s f r a c t i o n . The r e s u l t s i n d i c a t e t h a t low s a l t e l u t i o n (0.1 M NaCl) o f m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i r e l e a s e s a p o p u l a t i o n o f monomers c o n t a i n i n g i n c r e a s e d l e v e l s o f h i g h l y a c e t y l a t e d H4 from n u c l e a s e s e n s i t i v e r e g i o n s of c h r o m a t i n . These r e s u l t s a r e c o n s i s t e n t w i t h t h e o b s e r v a t i o n by Simpson (.178) t h a t n u c l e o -somal c o r e p a r t i c l e s produced e a r l y i n t h e c o u r s e of m i c r o c o c c a l n u c l e a s e d i g e s t i o n o f b u t y r a t e - t r e a t e d n u c l e i ( B u t y r a t e t r e a t m e n t l e a d s t o t h e enrichment o f t h e a c e t y l a t e d h i s t o n e s p e c i e s o f H3 and H4) had more 116 h i g h l y a c e t y l a t e d H4 than t h o s e produced l a t e r i n t h e d i g e s t i o n . 5. A n a l y s i s , of chromosomal p r o t e i n s a s s o c i a t e d w i t h  s a l t - e x t r a c t e d c h r o m a t i n f r a c t i o n s The t o t a l p r o t e i n c o n t e n t o f each o f t h e s u p e r -n a t a n t f r a c t i o n s was a n a l y z e d on 15% p o l y a c r y l a m i d e SDS g e l s ( F i g . 2 4 ) . HMG-T, a p r o t e i n homologous t o HMG-1 and HMG-2 i n mammals (161, 124), i s prominent i n th e SO f r a c t i o n . T h i s o b s e r v a t i o n a g r e e s w i t h r e s u l t s o f Levy W. et a l . (152) t h a t the a c t i o n o f m i c r o c o c c a l n u c l e a s e l e a d s t o t h e p r e f e r e n t i a l s o l u b i l i z a t i o n of HMG-T. The SO f r a c t i o n a l s o c o n t a i n s minor amounts o f a p r o t e i n w i t h an apparent m o l e c u l a r weight o f 22,000, low l e v e l s o f nuc l e o s o m a l h i s t o n e s , and a few non-h i s t o n e p r o t e i n s i n the 40-90,000 m o l e c u l a r weight range (not shown i n F i g . 24). The low l e v e l o f nucl e o s o m a l h i s t o n e s i s c o n s i s t e n t w i t h t h e low amount of n u c l e o s o m a l DNA p r e s e n t ( F i g . 1 8 ) . SSI c o n t a i n s c o r e h i s t o n e s and low l e v e l s o f H i and HMG-T. The SSI monomer ( p r e d o m i n a n t l y 146 base p a i r fragments), o b t a i n e d from f r a c t i o n a t i o n o f SSI on a B i o - G e l A^5m column e q u i l i b r a t e d w i t h 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and 0.1 M NaCl c o n t a i n e d no HMG-T or HI when examined on p o l y a c r y l a m i d e SDS g e l s (data 117 S O S S - \ S S 2 S S 4 S S 6 H I 2 9 K • + 2 2 K M N i H 3 , H 2 B < H 2 A H 4 FIG. 24. 15% PAGE SDS s e p a r a t i o n o f p r o t e i n s r e l e a s e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d t r o u t t e s t i s n u c l e i by s u c c e s s i v e l y h i g h e r NaCl concentra-t i o n s . The s a l t - e x t r a c t e d f r a c t i o n s were d i a l y z e d a g a i n s t 10 mM NH.HCO o v e r n i g h t and samples c o r r e s p o n d i n g to 0.6 A^ftO w e r e l y o p h i l i z e d . The samples were r e d i s s o l v e d i n sample b u f f e r c o n t a i n i n g SDS and a p p l i e d to t h e g e l . The g e l was s t a i n e d w i t h Coomassie b l u e . SO, f r a c t i o n r e l e a s e d by t h e i n i t i a l d i g e s t i o n ; SSI - SS6, f r a c t i o n s s u c c e s s i v e l y r e l e a s e d by B u f f e r D c o n t a i n i n g 0.1 M - 0.6 M NaCl, r e s p e c t i v e l y ; MN, m i c r o c o c c a l n u c l e a s e . 118 not shown). Ag a i n , t h e s e r e s u l t s a g r e e w i t h t h e s u g g e s t i o n by Levy W. et a l . (.162). t h a t HMG-T i s a s s o c i a t e d w i t h t h e l i n k e r DNA. SS2 is; s i m i l a r t o SSI except t h a t t h e co n t e n t o f H i is; h i g h e r and t h e c o n t e n t o f HMG-T i s lower. T r a c e s o f n o n h i s t o n e p r o t e i n s i n t h e 40-90,000 m o l e c u l a r weight range were a l s o v i s i b l e on t h e o r i g i n a l g e l f o r b o t h t h e SSI and SS2 f r a c t i o n s . SS4 and SS6 c o n t a i n e d t h e n u c l e o s o m a l h i s t o n e s , l a r g e amounts o f HI (HI b e i n g h i g h e s t i n SS4), no HMG-T and an u n i d e n t i f i e d p r o t e i n o f 29,000 apparent m o l e c u l a r • w e i g h t . These r e s u l t s suggest t h a t t h e i n t e r n u c l e o s o m a l l i n k e r DNA o f n u c l e a s e s e n s i t i v e r e g i o n s o f ch r o m a t i n may c o n t a i n HMG-T,, and p o s s i b l y l e s s e r amounts o f o t h e r n o n h i s t o n e p r o t e i n s . Furthermore, t h e n u c l e o -somes i n t h e s e r e g i o n s c o n t a i n h i g h l y a c e t y l a t e d H4. Chromatin f r a c t i o n a t i o n . ( L e v y and Dixon's p r o c e d u r e ) Levy and Dixon (142). have d e s i g n e d a c h r o m a t i n f r a c t i o n a t i o n p r o c e d u r e which p a r t i a l l y p u r i f i e s a t r a n s c r i p t i o n a l l y a c t i v e n u c l e o s o m a l s u b t r a c t i o n ( F i g . 2 5 ) . The p r o c e d u r e i n v o l v e s m i l d m i c r o c o c c a l n u c l e a s e d i g e s t i o n o f t r o u t t e s t i s n u c l e i ^ , and 119 MICROCOCCAL NUCLEASE DIGESTED NUCLEI CENTBIFUGE r S I S2 1 . Slowly add S M NaCl to 0 . 1 M f i n a l 2 . Incubate 20 min. O 'C 3 . C e n t r i f u g e S3 1 P I 1. Reauapend i n .2 mM EDTA 2. Incubate 20 a i n . , 0°C 3. Centrifuge P3 FIG. 25. E x p e r i m e n t a l p r o c e d u r e f o r i s o l a t i o n of a 0.1 M NaCl s o l u b l e n u c l e o s o m a l s u b t r a c t i o n r e l e a s e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i . 120 subsequent l y s i s of the p e l l e t e d , d i g e s t e d n u c l e i w i t h EDTA. The d i g e s t p r o d u c t s a r e r e l e a s e d i n t o t h e s u p e r n a t a n t which i s f u r t h e r f r a c t i o n a t e d by t h e a d d i t i o n of 0.1 M NaCl a f t e r removal of the i n s o l u b l e m a t e r i a l by c e n t r i f u g a t i o n . The s a l t - s o l u b l e m a t e r i a l (S3) was m o s t l y monomeric i n s i z e and was e n r i c h e d over 7 f o l d i n DNA sequences complementary t o p o l y a d e n y l a t e d RNA. 1. Q u a n t i t a t i o n of DNA c o n t e n t i n t h e c h r o m a t i n f r a c t i o n s The p e r c e n t a g e of t o t a l a b s o r b i n g m a t e r i a l r e l e a s e d i n each f r a c t i o n was q u a n t i t a t e d ( T a b l e XIV). The y i e l d s were s i m i l a r t o t h o s e r e p o r t e d by Levy and Dixon (142) except t h a t t h e p e r c e n t a g e of m a t e r i a l they o b t a i n e d i n S2 was much lower. The d i s c r e p a n c y may be due to t h e i r use of t r o u t t e s t i s i n a - l a t e s t a g e o f m a t u r a t i o n . T e s t i s a t t h i s s t a g e w i l l have some DNA packaged as n u c l e o p r o t a m i n e which i s r e s i s t a n t t o m i c r o c o c c a l n u c l e a s e d i g e s t i o n (188). 2. Q u a n t i t a t i o n of a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d  w i t h t h e c h r o m a t i n f r a c t i o n s The a c i d e x t r a c t e d h i s t o n e s from f r a c t i o n s P2, P3 and S3 were a n a l y z e d on a c i d - u r e a g e l s , and t h e scans of the s t a i n e d g e l of t h e l a t t e r two a r e shown i n F i g . 26. The c o n t e n t of HI was low r e l a t i v e t o t h e c o r e h i s t o n e s f o r t h e S3 f r a c t i o n but was much h i g h e r f o r the P3 and P2 f r a c t i o n s . T h i s i s c o n s i s t e n t w i t h t h e f a c t 121 •A TABLE XIV Q u a n t i t a t i o n of DNA Content i n Chromatin F r a c t i o n s (Levy and Dixon's Procedure) ~~ The A 2 6 Q a b s o r b i n g m a t e r i a l r e l e a s e d i n t h e d i f f e r e n t c h r o m a t i n f r a c t i o n s a f t e r m i c r o c o c c a l n u c l e a s e d i g e s t i o n of t r o u t t e s t i s n u c l e i was q u a n t i t a t e d by a d d i n g 100 y l of t h e f r a c t i o n t o 0.9 ml of 0.6 M NaCl i n B u f f e r D and measuring the absorbance a t 260 nm. F r a c t i o n T o t a l A n £ n i n 260 Each F r a c t i o n SI S2 S3 9.8 38.5 7.1 122 FIG. 26. G e l scans of h i s t o n e s a n a l y z e d on a c i d - u r e a g e l s . H i s t o n e s were a c i d - e x t r a c t e d from the 0.1 M NaCl s o l u b l e - (S3) and 0.1 M NaCl i n s o l u b l e nucleosomes (P3) t h a t had been r e l e a s e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i . G e l s were s t a i n e d w i t h Coomassie b l u e and scanned i n a G i l f o r d s p e c t rophotometer a t 550 nm. A , u n a c e t y l a t e d H4; A^, A^, A^ and A^, mono-, d i - , t r i - and t e t r a - a c e t y ? a t e d H4, r e s p e c t i v e l y ; 123 • t h a t S3 f r a c t i o n s c o n t a i n p r e d o m i n a n t l y mono-nucleosomes (142) . The c o n t e n t of t h e a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d w i t h each f r a c t i o n was examined. F o r a l l the f r a c t i o n s the c o n t e n t of t h e a c e t y l a t e d s p e c i e s of H2A, H2B and H3 d i d not d i f f e r . However, t h e l e v e l o f the H4 a c e t y l a t e d s p e c i e s was h i g h e r i n S3 than i n t h e o t h e r f r a c t i o n s ( T a b l e XV). F o r b o t h c h r o m a t i n f r a c t i o n a t i o n p r o c e d u r e s (Sanders' and L e v y ' s ) , t h e 0.1 M NaCl s o l u b l e nucleosome f r a c t i o n i s e n r i c h e d i n t h e a c e t y l a t e d h i s t o n e H4 s p e c i e s . The r e s u l t s suggest t h a t b o t h p r o c e d u r e s p r e f e r e n t i a l l y s o l u b i l i z e t h e n u c l e o s o m a l s u b f r a c t i o n t h a t may be a s s o c i a t e d w i t h t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n . Summary The i n i t i a l s t u d i e s ( S e c t i o n 1 ( a ) ) demonstrated t h a t mononucleosomes c o n t a i n e d normal l e v e l s o f the major a c e t y l a t e d h i s t o n e s p e c i e s compared t o those o f t h e t o t a l n u c l e a r h i s t o n e p o p u l a t i o n . L a b e l l i n g s t u d i e s u s i n g [ 1 I + C ] - a c e t a t e a l s o suggested t h a t t h e degree of a c e t y l a t i o n was s i m i l a r f o r t h e h i s t o n e s i s o l a t e d e i t h e r from mononucleosomes or n u c l e i . At t h a t s t a g e i t was not r e a l i z e d t h a t t h e t o t a l p o p u l a t i o n o f a c e t y l a t e d h i s t o n e s s p e c i e s was n o t e q u a l l y r e p r e s e n t e d by t h e l a b e l , [ l t + C ] - a c e t a t e : - t h a t i s , h i s t o n e s ( e s p e c i a l l y H4) 124. TABLE XV Q u a n t i t a t i o n of A c e t y l a t e d S p e c i e s of H i s t o n e H4 i n t h e  Chromatin F r a c t i o n s (Levy and Dixon's  Pr o c e d u r e ) H i s t o n e s p r e p a r e d by a c i d e x t r a c t i o n of nucleosome f r a c t i o n s were s e p a r a t e d on a c i d - u r e a g e l s . The g e l s were s t a i n e d w i t h Coomassie b l u e , and scanned a t 550 nm u s i n g a G i l f o r d s p e c t r o p h o t o m e t e r . A. + A * F r a c t i o n R a t i o o f H4 2 3 A + A, o 1 P2 P3 S3 0 .32 0 .40 0 .68 * Determined from t h e A peak h e i g h t s of t h e scanned H4 s p e c i e s . .125 a s s o c i a t e d w i t h a n u c l e o s o m a l s u b t r a c t i o n a s s o c i a t e d w i t h t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n were l a b e l l e d t o a l e s s e r e x t e n t than n u c l e o s o m a l s u b t r a c t i o n s a s s o c i a t e d w i t h t h e b u l k o f c h r o m a t i n . F o r l a t e r experiments t h e P a n y i m - C h a l k l e y g e l system was used. T h i s method enabled t h e q u a n t i t a t i o n o f t h e l e v e l s o f the a c e t y l a t e d h i s t o n e s p e c i e s w i t h o u t t h e use of l a b e l l i n g t e c h n i q u e s . Treatment o f m i c r o c o c c a l n u c l e a s e : d i g e s t e d n u c l e i w i t h 0:4 M NaCl w i l l s o l u b i l i z e most of t h e d i g e s t p r o d u c t s , as does l y s i s of t h e n u c l e i w i t h EDTA. Comparison of t h e a c e t y l a t e d h i s t o n e . s p e c i e s a s s o c i a t e d w i t h mononucleosomes w i t h t h o s e a s s o c i a t e d w i t h m u l t i m e r s demonstrated t h a t t h e monomers were;, enriched-.::••'c;^ i n t h e a c e t y l a t e d H4 s p e c i e s (A^ and A^, T a b l e X I I I ) . As t h e t o t a l h i s t o n e p o p u l a t i o n would be expected t o c o n t a i n predominantly, high, level's of:'the A^ and A^. s p e c i e s , o f H4<-. s i m i l a r t o SS4 and SS6 ( T a b l e X I ) , t h e mononucleosomes would c o n t a i n a g r e a t e r l e v e l of t h e A^ and A^ s p e c i e s of H4 than t h a t of t h e H4 e x t r a c t e d from u n d i g e s t e d n u c l e i . The a c t u a l degree of enrichment i n t h e a c e t y l a t e d H4 s p e c i e s a s s o c i a t e d w i t h mononucleosomes would be dependent on t h e e x t e n t of m i c r o c o c c a l n u c l e a s e d i g e s t i o n . F o r example, t h e mononucleo-12 6 somes o b t a i n e d from an e x t e n s i v e m i c r o c o c c a l n u c l e a s e d i g e s t i o n o f n u c l e i would c o n t a i n p r e d o m i n a n t l y t h e A and A s p e c i e s of H4 ° 1 w h i l e a r e l a t i v e l y m i l d d i g e s t i o n would produce mononucleosomes e n r i c h e d i n t h e A^ and A^ s p e c i e s o f H4. T h e r e f o r e , i t would be d o u b t f u l t h a t a d i f f e r e n c e i n t h e c o n t e n t o f t h e minor a c e t y l a t e d h i s t o n e s s p e c i e s ( i . e . H4 A^ and A^) a s s o c i a t e d w i t h mononucleosomes or n u c l e i would have been d e t e c t a b l e i n the e a r l i e r experiments ( s e c t i o n 1 ( a ) ) where q u a n t i t a t i o n o f the minor a c e t y l a t e d h i s t o n e s p e c i e s was n o t p o s s i b l e . Both f r a c t i o n a t i o n p r o c e d u r e s used g e n e r a t e d a nucleosome f r a c t i o n s o l u b l e i n 0.1 M N a Cl t h a t c o n t a i n e d e n r i c h e d l e v e l s o f t h e a c e t y l a t e d H4 s p e c i e s . The 0.1 M NaCl s o l u b l e nucleosomes g e n e r a t e d by the Sanders' or Levy's p r o c e d u r e s were a l s o s i m i l a r i n t h a t t h e nucleosome f r a c t i o n s c o n t a i n e d low l e v e l s o f Hl^^and the n u c l e o s o m a l DNA fragment was of monomeric s i z e . As t h e 0.1 M N a C l s o l u b l e mononucleosomes o b t a i n e d by t h e Levy's p r o c e d u r e r e p r e s e n t a t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n f r a c t i o n and as r e c e n t r e p o r t s (.121, 122) have demonstrated t h a t a c t i v e genes a r e c l e a v e d i n t o mononucleosomes more r a p i d l y than i s b u l k c h r o m a t i n by m i c r o c o c c a l n u c l e a s e , t h e 0.1 M NaCl s o l u b l e mononucleosome f r a c t i o n t h a t was o b t a i n e d by t h e Sanders' p r o c e d u r e p r o b a b l y c o r r e s p o n d s t o nucleosomes:. a s s o c i a t e d w i t h a t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n f r a c t i o n . 127 Thus, a c e t y l a t i o n o f H4 may p l a y a key r o l e i n m a i n t a i n i n g t h e s t r u c t u r e o f t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n . Non-h i s t o n e p r o t e i n s ( i . e . HMG—T) may a l s o be i n v o l v e d i n t h e maintenance o f t h i s s t r u c t u r e , a s t r u c t u r e t h a t seems l i k e l y t o be an extended s t a t e o f c h r o m a t i n . C h a r a c t e r i z a t i o n of Nucleosome S u b f r a c t i o n s and Chromosomal P r o t e i n s R e l e a s e d from DNase I I D i g e s t e d Chromatin R e c e n t l y , G o t t e s f e l d and h i s co-workers have shown t h a t t h e DNase I I f r a c t i o n a t i o n p r o c e d u r e o f Marushige and Bonner (225), c o u p l e d w i t h + 2 Mg p r e c i p i t a t i o n , ; , ( F i g . 27) r e s u l t s i n t h e i s o l a t i o n o f a c h r o m a t i n f r a c t i o n (S2) e n r i c h e d i n a c t i v e l y t r a n s c r i b i n g sequences (140, 141). Lev y - W i l s o n et a l . , a p p l y i n g t h i s p r o c e d u r e t o D r o s o p h i l a c e l l s , o b s e r v e d a . 40% i n c r e a s e i n t h e i n c o r p o r a t i o n o f l a b e l l e d a c e t a t e i n t o h i s t o n e s o f t h e t e m p l a t e - a c t i v e f r a c t i o n (173). The p a t t e r n s o f h i s t o n e a c e t y l a t i o n i n t h e d i f f e r e n t f r a c t i o n s were examined. (a) Q u a n t i t a t i o n o f DNA c o n t e n t i n the c h r o m a t i n f r a c t i o n s The p e r c e n t a g e o f a b s o r b i n g m a t e r i a l a s s o c i a t e d w i t h each f r a c t i o n i s p r e s e n t e d i n T a b l e XVI. A l t h o u g h t h e d i g e s t c o n d i t i o n s (.5 min,, w i t h 100 u n i t s / m l DNase I I ) used were i d e n t i c a l t o t h o s e of G o t t e s f e l d et a l . (.141) , t h e p e r c e n t a g e of A „ , n a b s o r b i n g m a t e r i a l i n S2 was lower f o r t r o u t t e s t i s , t h a n f o r r a t l i v e r . The r e s u l t s suggest t h a t r a t l i v e r c h r o m a t i n c o n t a i n s a g r e a t e r p e r c e n t a g e o f t r a n s c r i p t i o n a l l y - a c t i v e c h r o m a t i n t h a n does t r o u t t e s t i s c h r o m a t i n . 128, PI DNase II DIGESTED CHROMATIN CENTRIFUGE P2 S I 1 . Slowly add 1 M MgCU to 2 mM final 2 . Incubate 2 0 , min. at 0°C 3. Centrifuge S 2 ("ACTIVE" CHROMATIN FRACTION) FIG. 27. E x p e r i m e n t a l p r o c e d u r e f o r t h e i s o l a t i o n of a t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n f r a c t i o n r e l e a s e d from DNase I I d i g e s t e d c h r o m a t i n . 129 TABLE XVI Q u a n t i t a t i o n of DNA Content In F r a c t i o n s R e l e a s e d from D K W T T D i g e s t e d Chromatin '. ' ~ The A 2 g Q a b s o r b i n g m a t e r i a l r e l e a s e d i n the d i f f e r e n t f r a c t i o n s a f t e r DNase I I d i g e s t i o n o f t r o u t t e s t i s c h r o m a t i n was q u a n t i t a t e d by adding 100 y l of t h e f r a c t i o n t o 0.9 ml of 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and measuring the absorbance a t 260 nm. The d i s t r i b u t i o n o f DNA among t h e v a r i o u s f r a c t i o n s o b t a i n e d from DNase I I d i g e s t i o n o f r a t l i v e r c h r o m a t i n i s a l s o p r e s e n t e d (141) . Chromatin F r a c t i o n PI S2 P2 T o t a l A 2 6 Q i n e a c h . F r a c t i o n Rat L i v e r T r o u t T e s t i s Chromatin- Chromatin 84.6" + 4.8 11.3 + 3.9 4.1 + 2.5 86.4 6.4 + 1 . 7 6.6 130 -K i n e t i c s of DNase I I d i g e s t i o n F o r t h e f o l l o w i n g experiments, t h e c o n c e n t r a t i o n of DNase I I was lowered to 40 u n i t s / m l , and t h e d i g e s t i o n time was increas-.-. . ed t o 8 min. A t i m e c o u r s e of d i g e s t i o n of t r o u t t e s t i s c h r o m a t i n i s p r e s e n t e d i n F i g . 28. S i m i l a r to r e s u l t s p r e s e n t e d by +2 G o t t e s f e l d e t a l . (139), the 2 mM Mg - s o l u b l e f r a c t i o n , S2, r e a c h e s a s t e a d y v a l u e e a r l y i n t h e d i g e s t w h i l e the m a t e r i a l i n +2 SI i n c r e a s e s . Thus, the 2 mM Mg - s o l u b l e f r a c t i o n forms p a r t of t h e most n u c l e a s e s e n s i t i v e f r a c t i o n of c h r o m a t i n DNA. T r o u t t e s t i s c e l l s c o n t a i n an enzyme s i m i l a r to DNase I I . To a s s a y f o r th e endogeneous DNase I I a c t i v i t y , a sample of c h r o m a t i n was i n c u b a t e d f o r 30 min a t 24°C i n the absence o f exogenous DNase I I . A f t e r t h i s i n c u b a t i o n time, 3.8% of t h e c h r o m a t i n was s o l u b i l i z e d , and 64% of t h i s m a t e r i a l remained +2 s o l u b l e a f t e r t h e a d d i t i o n of 2 mM Mg C h a r a c t e r i z a t i o n o f t h e DNA fragments a s s o c i a t e d w i t h each  c h r o m a t i n f r a c t i o n The m a t e r i a l t h a t remained s o l u b l e a f t e r t h e a d d i t i o n o f +2 2 mM Mg (S2) was f u r t h e r f r a c t i o n a t e d by t h e a d d i t i o n o f 22 mM +2 +2 +2 Mg y i e l d i n g a 22 mM Mg - s o l u b l e f r a c t i o n , S3, and a 22 mM Mg • +2 i n s o l u b l e - f r a c t i o n , P3. The a d d i t i o n o f 22 mM Mg w i l l p r e c i p i t a t e most of the n u c l e o s o m a l m a t e r i a l but not t h e o l i g o n u c l e o t i d e s p r e s e n t i n S2. F i g . 29 p r e s e n t s the p e r -c e n t a g e o f A 9 f t n a b s o r b i n g m a t e r i a l a s s o c i a t e d w i t h each f r a c t i o n 131 FIG. 28. 4 0 t O CD CM 2 o ^ 2 0 UJ O 0 2 0 TIME (minutes) Time c o u r s e of c h r o m a t i n f r a c t i o n a t i o n ( I ) . A f t e r v a r i o u s times of i n c u b a t i o n w i t h DNase I I (4 u n i t s / A , u n i t of DNA), samples were removed, and an a p p r o p r i a t e volume of 0.1 M T r i s - H C l (pH 11) was added to g i v e a f i n a l pH o f 7.5. The c h r o m a t i n was separated, i n t o t h e f i r s t s u p e r n a t a n t (SI) and subsequent f r a c t i o n a t i o n i n t o 2 mM Mg s o l u b l e (S2) and i n s o l u b l e m a t e r i a l as d e s c r i b e d i n " M a t e r i a l s and Methods" ( F i g . 2 7 ) . The A a b s o r b i n g m a t e r i a l was q u a n t i t a t e d by a d d i n g 100 i l l o f the f r a c t i o n to 0:9 ml of 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and m easuring t h e absorbance a t 260 nm. • , S I ; • , S2. 132 o CO CN 2 o O ^ 2 O QC 0 20 TIME (minutes) FIG. 29. Time c o u r s e o f c h r o m a t i n f r a c t i o n a t i o n ( I I ) . S o l u b i l i z e d c h r o m a t i n f r a c t i o n s were o b t a i n e d from DNase I I d i g e s t e d c h r o m a t i n as d e s c r i b e d i n the l e g e n d to F i g . 28. The 2 mM Mg - s o l u b l e f r a c t i o n (S_2J was f u r t h e r f r a c t i o n a t e d by t h e a d d ^ i o n of 22 mM Mg y i e l d i n g a 22 mM Mg - s o l u b l e f r a c t i o n (S3) and a 22 mM Mg - i n s o l u b l e f r a c t i o n ( P3). The A„,_ a b s o r b i n g m a t e r i a l was q u a n t i t a t e d i n t h e same manner as F i g . 28. • , S3; • , P3.' 133 as a f u n c t i o n o f i n c u b a t i o n time. The amount o f A n,. n a b s o r b i n g +2 m a t e r i a l p r e c i p i t a t e d by 22 mM Mg (P3) drops t o a c o n s t a n t v a l u e a f t e r 15 min.. i n c u b a t i o n t i m e w h i l e t h e amount r e m a i n i n g s o l u b l e (S3) i n c r e a s e s s t e a d i l y w i t h time. T h i s r e s u l t i s p r o b a b l y a consequence o f DNase I I d i g e s t i n g t h e l i n k e r DNA wh i c h w i l l i n c r e a s e t h e l e v e l s o f o l i g o n u c l e o t i d e s t h a t a r e s o l u b l e i n + 2 Mg . The endogenous DNase I I g e n e r a t e d m a t e r i a l t h a t i s s o l u b l e +2 i n S2 can be almost c o m p l e t e l y p r e c i p i t a t e d by 22 mM Mg •'/.:.'> . s u g g e s t i n g t h a t t h e l e v e l of o l i g o n u c l e o t i d e s i s low i n S2. The DNA fragment s i z e o f t h e m a t e r i a l a s s o c i a t e d w i t h P2 and P3 was examined on 3% n o n d e n a t u r i n g p o l y a c r y l a m i d e g e l s ( F i g . 30). The n u c l e o s o m a l m a t e r i a l e x c i s e d from c h r o m a t i n by t h e endogenous DNase I I a c t i v i t y c o n t a i n e d DNA fragments g r e a t e r t h a n pentamer i n l e n g t h f o r bo t h t h e P2 and P3 f r a c t i o n s ( F i g . 30, l a n e s a and f ) . As t h e i n c u b a t i o n time i n t h e p r e s e n c e o f exogenous DNase I I i n c r e a s e s , t h e P2 f r a c t i o n s m a i n t a i n a f u l l spectrum o f t h e d i f f e r e n t n u c l e o s o m a l DNA s i z e c l a s s e s with, a s l i g h t a c c u m u l a t i o n of monomeric s i z e DNA. The m a t e r i a l p r e c i p i t a t e d by 22 mM Mg (P3) shows an a c c u m u l a t i o n of t h e monomeric DNA s i z e fragment w i t h i n c r e a s i n g i n c u b a t i o n t i m e . A f t e r a 30 min I n c u b a t i o n , t h e monomeric DNA fragment i s t h e predominant s p e c i e s . The appearance o f t h e monomer DNA fragment i s p r o b a b l y a r e s u l t o f j i h g fed cba F I G . 30. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f t h e DNA fragments s o l u b i l i z e d by DNase I I d i g e s t i o n o f t r o u t t e s t i s c h r o m a t i n . A f t e r v a r i o u s times o f i n c u b a t i o n w i t h exogeneous DNase I I (4 u n i t s / A^^Q u n i t o f DNA) o r endogeneous DNase I I , samples were withdrawn and f r a c t i o n a t e d as d e s c r i b e d + i n " M a t e r i a l s and Methods". +@NA (.0.5 ^ ^ Q ) o b t a i n e d from t h e 2 mM Mg - i n s o l u b l e (P2) and 22 mM Mg - i n s o l u b l e (P3) f r a c t i o n s was a n a l y z e d i n a 3% p o l y a c r y l a m i d e SDS g e l . The g e l was s t a i n e d w i t h e t h i d i u m bromide and photographed under u l t r a v i o l e t l i g h t . b, c, d and e a r e P2 f r a c t i o n s o b t a i n e d from c h r o m a t i n i n c u b a t e d w i t h exogeneous DNase I I f o r 4 min , 8 min , 16 min and 30 min , r e s p e c t i v e l y . g, h, i and j a r e P3 f r a c t i o n s o b t a i n e d from c h r o m a t i n i n c u b a t e d w i t h exogeneous DNase I I f o r 4 min , 8 min , 16 min and 30 min , r e s p e c t i v e l y . a and f a r e P2 and P3 f r a c t i o n s , r e s p e c t i v e l y , o b t a i n e d from c h r o m a t i n i n c u b a t e d 30 min w i t h endogeneous DNase I I . 1 , 2 and 3 a r e monomer, dimer and t r i m e r , r e s p e c t i v e l y . 135 s e q u e n t i a l d i g e s t i o n o f l a r g e r n u c l e o s o m a l fragments. The amount of A^^Q a b s o r b i n g m a t e r i a l p r e c i p i t a t e d by 22 mM +2 Mg (P3) was found to d i m i n i s h to a c o n s t a n t v a l u e a f t e r 15 min d i g e s t i o n ( F i g . 2 9 ) . The l a r g e r v a l u e s a t e a r l y i n c u b a t i o n times may be due to the i n i t i a l r e l e a s e of l a r g e n u c l e o s o m a l r e p e a t s ( i . e . t r i n u c l e o s o m e s ) ( F i g . 30, l a n e g ) . As t h e d i g e s t i o n c o n t i n u e s , t h e l i n k e r DNA a s s o c i a t e d w i t h t h e polynucleosomes would be e x c i s e d which would e v e n t u a l l y r e s u l t i n t h e a c c u m u l a t i o n of mononucleosomes. W i t h the l o s s o f t h e l i n k e r DNA t h e amount +2 of A „ , n a b s o r b i n g m a t e r i a l p r e c i p i t a t e d by 22 mM Mg would d e c r e a s e to a c o n s t a n t v a l u e when the mononucleosomes s h o u l d be t h e major n u c l e o s o m a l s p e c i e s . D e t e r m i n a t i o n of the l e v e l s o f a c e t y l a t e d ' h i s t o n e s p e c i e s  a s s o c i a t e d w i t h the c h r o m a t i n f r a c t i o n s F i g . 31 shows the h i s t o n e p a t t e r n s r e s o l v e d i n a c i d - u r e a +2 g e l s o b t a i n e d from t h e v a r i o u s s t a g e s of t h e DNase Il/Mg f r a c t i o n a t i o n p r o c e d u r e . Most of the h i s t o n e p a t t e r n s demon-s t r a t e low l e v e l s o f t h e h i s t o n e H4 r e l a t i v e t o the o t h e r n u c l e o -somal h i s t o n e s . T h i s was due to i n c o m p l e t e p r e c i p i t a t i o n of t h e H4 m o l e c u l e s when e t h a n o l was added to t h e a c i d - e x t r a c t e d h i s t o n e sample. The h i s t o n e p a t t e r n s from c o n t r o l c h r o m a t i n ( C ) , from t h e u n d i g e s t e d p e l l e t (PI) a f t e r DNase I I t r e a t m e n t , and from 136 Pi FIG. 31. H i s t o n e p r o f i l e s on an a c i d / u r e a / p o l y a c r y l a m i d e g e l . H i s t o n e s were p r e p a r e d from c h r o m a t i n f r a c t i o n s and s e p a r a t e d on a 1.5 mm s l a b g e l by t h e system o f Panyim and C h a l k l e y (194). The g e l was s t a i n e d w i t h Coomassie b l u e . C, h i s t o n e s from u n f r a c t i o n a t e d c h r o m a t i n c o n t r o l ; P I , h i s t o n e s from c h r o m a t i n p e l l e t r e m a i n i n g a f t e r DNase II d i g e s t i o n ; P2, h i s t o n e s from 2 mM Mg -insoluble f r a c t i o n of DNase II - r e l e a s e d m a t e r i a l ; S2, h i s t o n e s from 2 mM Mg - s o l u b l e f r a c t i o n ^ o f DNase II - r e l e a s e d m a t e r i a l ; P3, h i s t o n e s from f r a c t i o n r e n d e r e d 2 mM Mg - i n s o l u b l e by t h e ^ f i r s t RNase d i g e s t i o n of S2; P4, h i s t o n e s from f r a c t i o n r e n d e r e d 2 mM Mg - i n s o l u b l e by t h e second RNase d i g e s t i o n o f S2. The p o s i t i o n s o f t h e h i s t o n e s a r e i n d i c a t e d i n sample C. H i s t o n e H3 i s r e s o l v a b l e i n t o t h r e e components ( u n m o d i f i e d , mono- and d i - a c e t y l a t e d ) . H4 i s r e s o l v a b l e i n t o f o u r components A^, u n m o d i f i e d ; A^, A^, A^ and A^ a r e mono-, d i - , t r i - and t e t r a - a c e t y l a t e d , r e s p e c t i v e l y . RNase, p a n c r e a t i c r i b o n u c l e a s e . 137 t h e 2 mM M g ' ^ - i n s o l u b l e f r a c t i o n . (P2) a r e i n d i s t i n g u i s h a b l e +2 from each o t h e r ( F i g . 31). The 2 mM Mg - s o l u b l e f r a c t i o n (S2) however, i s g r e a t l y e n r i c h e d i n t h e d i a c e t y l a t e d (A^), t r i -a c e t y l a t e d (A^) and t e t r a a c e t y l a t e d (A^) s p e c i e s of H4. The +2 s o l u b i l i t y of t h i s c h r o m a t i n f r a c t i o n i n 2 mM Mg i s b e l i e v e d to be due to i t s h i g h c o n t e n t of RNA (139). Treatment of t h i s f r a c t i o n w i t h p a n c r e a t i c r i b o n u c l e a s e r e s u l t e d i n p r e c i p i t a t i o n of a p p r o x i m a t e l y 35% of the m a t e r i a l ; a sample of h i s t o n e s from t h i s p r e c i p i t a t e (P3) i s a l s o shown i n F i g . 31. A l t h o u g h t h e H4 r e g i o n o f t h e P3 sample i s not v i s i b l e on t h i s photograph, the sample i s l i k e w i s e g r e a t l y e n r i c h e d i n a c e t y l a t e d H4 s p e c i e s . I n t h i s experiment, the y i e l d o f H4 i n the P3 f r a c t i o n i s e s p e c i a l l y low due t o t h e i n e f f i c i e n t e t h a n o l p r e c i p i t a t i o n of t h e a c i d e x t r a c t e d sample; i n o t h e r e x p e r i m e n t s t h e H4 p a t t e r n c o n s i s t e n t l y resembled t h a t of t h e S2 f r a c t i o n w i t h the a d d i t i o n of t h e RNase band. A second t r e a t m e n t of the s u p e r n a t a n t to P3 w i t h RNase l e d to p r e c i p i t a t i o n of a f u r t h e r 10 - 15% o f the o r i g i n a l d i g e s t p r o d u c t s , and t h i s f r a c t i o n , P4, resembled the S2 and P3 f r a c t i o n s i n i t s c o n t e n t of a c e t y l a t e d H4 ( F i g . 31). The r e l a t i v e p r o p o r t i o n s of t h e a c e t y l a t e d H4 components a t each s t a g e o f the f r a c t i o n a t i o n a r e more r e a d i l y seen i n g e l scans o f the H4 r e g i o n ( F i g . 32). In whole c h r o m a t i n , t h e m o n o a c e t y l a t e d s p e c i e s a r e most, abundant, .but t h e d i - and t r i -a c e t y l a t e d s p e c i e s a r e r e a d i l y d e t e c t e d ; t h e t e t r a - a c e t y l a t e d s p e c i e s a r e not r e s o l v e d from the a d j a c e n t H2B band i n t h e s e 138 (—) B FIG. 32.: Gel scans, of the histone H4 regions of an acid/urea gel. The gel is from a different experiment from that illustrated in Fig. 31. A A^, A and A_ are subspecies, of histone H4^  as. described, in the legend to' : Fig. 31. (A) H4 from unfractionated control chromatin; (B) H4 from PI, the chromatin pellet remaining after DNase II digestion; (£)_ H4 from P2, the 2 mM Mg -insoluble fraction of DNase II released material^2 (D and E)_ H4 from P3 and P4, respectively, £§e fractions rendered 2 mM Mg -insoluble by RNase digestion of the 2 mM Mg -soluble fraction. Gels were stained with Coomassie Blue and scanned in a Gilford spectrophotometer at 550. nm. 139 scans ( F i g . 32A). The c h r o m a t i n r e m a i n i n g i n s o l u b l e a f t e r DNase I I +2 d i g e s t i o n (PI) and t h e 2 mM Mg - i n s o l u b l e component of the d i g e s t (P2) a l s o c o n t a i n H4 w i t h t h e above d i s t r i b u t i o n o f a c e t y l bands ( F i g . 32 B and C ) . I n t h e p e l l e t r e s u l t i n g from RNase trea t m e n t of the S2 f r a c t i o n (P3 and P4, F i g . 32, D and E) , t h e A 2 and A., s p e c i e s e q u a l o r exceed t h e A^ i n amount. A^ seems to be t h e most prominent component i n t h e s e f r a c t i o n s , however, as w i l l be seen below, an unknown p o l y p e p t i d e m i g r a t e s v e r y c l o s e t o A^ i n t h e f i r s t d i m e n s i o n , and t h e a c t u a l amount o f A^ p r e s e n t i s approx-i m a t e l y e q u a l to t h e amount of k^- The A^ component, v i s i b l e i n t h e photograph of t h e o r i g i n a l g e l (e.g. F i g . 31, P 4 ) , was n o t r e s o l v e d i n t h e scan. In o r d e r to examine the p o s s i b i l i t y t h a t the H4 r e g i o n of t h e g e l might be contaminated w i t h unknown p r o t e i n s p e c i e s , samples of h i s t o n e s from whole c h r o m a t i n and from f r a c t i o n s S2 and P4 were a n a l y z e d on t w o - d i m e n s i o n a l g e l s i n which components were s e p a r a t e d on an a c i d / u r e a g e l i n t h e f i r s t d i m e n s i o n f o l l o w e d by e l e c t r o p h o r e s i s i n a SDS p o l y a c r y l a m i d e g e l i n the second d i m e n s i o n . As seen i n F i g . 33A a l l h i s t o n e f r a c t i o n s a r e s e p a r a t e d from each o t h e r i n t h i s system. E x a m i n a t i o n of t h e S2 f r a c t i o n c o n f i r m s t h a t the b u l k o f t h e H4 p r e s e n t i s i n t h e a c e t y l a t e d forms ( F i g . 33B). Furthermore, A^ i s now c l e a r l y v i s i b l e and i s p r e s e n t i n amounts a p p r o x i m a t e l y e q u a l to t h o s e of A^. An u n i d e n t i f i e d component of s l i g h t l y lower m o b i l i t y than H4, d e s i g n a t e d 12K, m i g r a t e s between A ? and A^ and i s b a r e l y 140 •<-> mm " ,12K FIG. 33. Two-dimensional PAGE s e p a r a t i o n o f h i s t o n e s from c h r o m a t i n f r a c t i o n s , s t a i n e d w i t h Coomassie b l u e . The f i r s t ( h o r i z o n t a l ) d i m e n s i o n c o n s i s t e d o f an a c i d / u r e a g e l (194), and t h e second ( v e r t i c a l ) d imension c o n s i s t e d o f the p o l y a c r y l a m i d e SDS g e l system o f Laemmli (226) as m o d i f i e d by Weintraub et a l . ( 5 9 ) . F o r t h e second dimension, a g e l s t r i p was ex-c i s e d from a l a n e o f t h e a c i d / u r e a g e l c o n t a i n i n g t h e h i s t o n e s ; t h e s t r i p was e q u i l i b r a t e d i n B u f f e r 0 o f O ' F a r r e l l (195) f o r 20 to 30 min, a p p l i e d to the s u r f a c e o f t h e SDS s l a b g e l , and s e a l e d w i t h melted 1% agarose i n B u f f e r 0. The r e m a i n i n g r e g i o n o f t h e f i r s t - d i m e n s i o n s t r i p was s t a i n e d w i t h Coomassie b l u e and i s shown a t t h e top of t h e c o r r e s p o n d i n g two-d i m e n s i o n a l s e p a r a t i o n . (A) H i s t o n e s from c o n t r o l u n f r a c t i o n a t e d c h r o m a t i n ; (B) h i s t o n e s from t h e 2 mM M g + ^ - s o l u b l e DNase I I f r a c t i o n , S2; (C) h i s t o n e s from t h e m a t e r i a l r e n d e r e d 2 mM M g + ^ - i n s o l u b l e by RNase d i g e s t i o n o f S2 (P4). 12 K and 29K a r e u n i d e n t i f i e d p r o t e i n s . The p o s i t i o n s o f t h e h i s t o n e f r a c -t i o n s a r e shown i n A. Components A0-A4 r e f e r t o s u b s p e c i e s of H4 as de s -c r i b e d i n the legn e d t o F i g . 31. 141 v i s i b l e i n t h e photograph of t h i s g e l ( F i g . 33B). T h i s component has not been d e t e c t e d i n u n f r a c t i o n a t e d c h r o m a t i n . An a d d i t i o n a l u n i d e n t i f i e d component, d e s i g n a t e d 29K, c o i n c i d e s e x a c t l y w i t h d i a c e t y l a t e d H3 i n the f i r s t dimension, b u t ^ m i g r a t e s j u s t ahead of HI i n t h e SDS dimension ( F i g . 33 B and C ) . I t a l s o seems to be a s s o c i a t e d w i t h t h e t e m p l a t e - a c t i v e c h r o m a t i n f r a c t i o n , and on a c i d / u r e a g e l s g i v e s t h e e rroneous i m p r e s s i o n t h a t t h e A^ s p e c i e s of H3 i s e n r i c h e d i n t h i s f r a c t i o n ( s e e, f o r i n s t a n c e , P4 of F i g . 31, a r r o w ) . When the experiment was done, the p o s s i b i l i t y t h a t 12K and 29K components a r o s e from p r o t e o l y t i c d e g r a d a t i o n , i . e . by a contaminant of the DNase I I , c o u l d not be r u l e d out. However, i f t h i s i s so, they a r e e v i d e n t l y a s s o c i a t e d +2 w i t h the 2 mM Mg - s o l u b l e f r a c t i o n . The absence of o t h e r low m o l e c u l a r weight s p e c i e s on the g e l s , however, argues a g a i n s t t h i s p o s s i b i l i t y . A t w o - d i m e n s i o n a l g e l o f t h e P4 c h r o m a t i n f r a c t i o n i s shown i n F i g . 33C. The lower amount of p r o t e i n a p p l i e d to t h e g e l a c c e n t u a t e s the f a c t t h a t A^ and A^ a r e predominant H4 s p e c i e s ; t h e AQ and A^ components were c l e a r l y v i s i b l e on t h e o r i g i n a l gels ;. : but do n o t appear d i s t i n c t i n t h e photograph. The AQ component was not d i s c e r n i b l e even on the o r i g i n a l g e l . The 29K p r o t e i n i s p r e s e n t , but t h e 12K spot i s not v i s i b l e , p o s s i b l y due to t h e lower l o a d i n g . 14,2 The above r e s u l t s c l e a r l y show t h a t th e DNase I I , M g ^ -s o l u b l e f r a c t i o n of c h r o m a t i n i s g r e a t l y e n r i c h e d i n the m u l t i -a c e t y l a t e d s p e c i e s of H4. +2. B e h a v i o u r of S2 n u c l e o s o m a l m a t e r i a l i n t h e p r e s e n c e of Mg ,' RNase or h i s t o n e G o t t e s f e l d e t a l . (139) suggested t h a t t h e s o l u b i l i t y o f t h e +2 S2 a s s o c i a t e d n u c l e osomal m a t e r i a l i n 2 mM Mg was due to t h e p r e s e n c e of n a s c e n t RNA a t t a c h e d to t h e nucleosome. Treatment of the S2 f r a c t i o n w i t h RNase r e s u l t e d i n t h e p r e c i p i t a t i o n of +2 t h e 2 mM Mg - s o l u b l e nucleosomes p o s s i b l y due to t h e d i g e s t i o n of the RNA. The p o s s i b i l t y t h a t RNase, w h i c h i s p o s i t i v e l y charged a t p h y s i o l o g i c a l pH, p r e c i p i t a t e d t h e nucleosomes by v i r t u e o f i t s p o s i t i v e charge as.opposed to i t s enzymatic a c t i v i t y was examined. +2 RNase, h i s t o n e and Mg were s t u d i e d f o r t h e i r a b i l i t y t o p r e c i p i t a t e t h e S2 .nucleosomal m a t e r i a l ( T a b l e X V I I ) . A c o n t r o l was r e q u i r e d because some p r e c i p i t a t i o n o c c u r r e d n o r m a l l y d u r i n g the i n c u b a t i o n p e r i o d . I n t h i s experiment t h e p e r c e n t a g e of S2 m a t e r i a l p r e c i p i t a t e d by RNase was much lower than +2 p r e v i o u s l y o b s e r v e d . B o t h 22 mM Mg and whole h i s t o n e were +2 c a p a b l e of p r e c i p i t a t i n g the n u c l e o s o m a l m a t e r i a l w i t h Mg b e i n g the most e f f e c t i v e . The h i s t o n e s a s s o c i a t e d w i t h the p r e c i p i t a t e d m a t e r i a l were 143 TABLE XVII E f f i c i e n c y o f Mg , -Histone or RNase i n P r e c i p i t a t i o n o f S2 A s s o c i a t e d Nucleosomal M a t e r i a l A 2 mM M g ' ^ - s o l u b l e c h r o m a t i n f r a c t i o n (S2) was o b t a i n e d from DNase I I d i g e s t e d c h r o m a t i n as d e s c r i b e d i n " M a t e r i a l s and Methods". To t h e S2 +2 f r a c t i o n e i t h e r RNase (10 Ug/ml), whole h i s t o n e (10 Ug/ml) o r Mg ;(22 mM) was added. The s o l u t i o n was i n c u b a t e d f o r 20 min a t 3 7 ° C V and c e n t r i f u g e d . A c o n t r o l sample of S2 was i n c u b a t e d f o r 60 min a t 37°C and c e n t r i f u g e d . The a b s o r b i n g m a t e r i a l remained s o l u b l e was q u a n t i t a t e d by a d d i n g 100 u l of t h e s u p e r n a t a n t to 0.9 ml of 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and measuring t h e absorbance a t 260 nm. Agent P e r c e n t A 0 , n i n P r e c i p i t a t e d M a t e r i a l c o n t r o l 10.3 RNase (10 Ug/ml) 18.9 H i s t o n e (10 ug/ml) 47.8 M g + 2 (22 mM) 68.2 144 examined on a c i d - u r e a g e l s (not shown). The p r e c i p i t a t e s t h a t were o b t a i n e d by a d d i n g e i t h e r RNase , whole h i s t o n e , or 22'mM + 2 Mg t o t h e S2 f r a c t i o n c o n t a i n e d s i m i l a r h i g h l e v e l s of t h e a c e t y l a t e d h i s t o n e H4 s p e c i e s , s u g g e s t i n g t h e same n u c l e o s o m a l f r a c t i o n was b e i n g p r e c i p i t a t e d . The S2 f r a c t i o n was i n c u b a t e d w i t h RNase .' f o r v a r i o u s t i m e s , and t h e p e r c e n t a g e o f i n s o l u b l e m a t e r i a l ^^as quant'itat%dr.(Table X V I I I ) . The p e r c e n t a g e o f m a t e r i a l p r e c i p i t a t e d d i d not v a r y a p p r e c i a b l y o v e r the t i m e p e r i o d s t u d i e d . I f t h e p r e c i p i t a t i o n of t h e S2 m a t e r i a l was caused by enzymatic d e g r a d a t i o n of t h e n a s c e n t RNA, an i n c r e a s e of i n s o l u b l e m a t e r i a l with, i n c r e a s i n g i n c u b a t i o n time would have been expected. The r e s u l t s suggest t h a t RNase * p r e c i p i t a t e s t h e n u c l e o s o m a l S2 m a t e r i a l by v i r t u e of i t s p o s i t i v e charge r a t h e r t h a n by d i g e s t i o n of t h e n ascent RNA. Summary +2 The Mg - s o l u b l e DNase I I f r a c t i o n , S2, from r a t l i v e r i s o r g a n i z e d i n t o nucleosomes and i s e n r i c h e d i n n ascent RNA and n o n - h i s t o n e p r o t e i n s . (139). I t has a l s o been shown t h a t g l o b i n genes, a r e e n r i c h e d i n t h e Mg - s o l u b l e f r a c t i o n of F r i e n d c e l l s which, have been i n d u c e d t o s y n t h e s i z e hemoglobin (.228),-,. a l t h o u g h t h e r e i s a disagreement as t o whether the same e n r i c h -145 TABLE X V I I I RNase • P r e c i p i t a t i o n > o f S2 A s s o c i a t e d Nucleosomal M a t e r i a l +2 A 2 mM Mg - s o l u b l e c h r o m a t i n f r a c t i o n (S2) was o b t a i n e d from DNase I I d i g e s t e d c h r o m a t i n as d e s c r i b e d i n " M a t e r i a l s and Methods". RNase ,;' (10 jjg/ml) was added t o the S2 f r a c t i o n . A f t e r i n c u b a t i o n f o r v a r i o u s t i m e s a t 37°C, a l i q u o t s were removed and c e n t r i f u g e d . The A. a b s o r b i n g m a t e r i a l r e m a i n i n g s o l u b l e was q u a n t i t a t e d by adding 100 y l of t h e s u p e r n a t a n t to 0.9 ml of 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and measuring the absorbance a t 260 nm. I n c u b a t i o n Time P e r c e n t A i n ( m i n ) P r e c i p i t a t e d M a t e r i a l s 2 5 10 17.3 19.7 19.6 146 ment o c c u r s i n uninduced c e l l s (228, 229). However, i n c e l l s t h a t have l o s t the c a p a c i t y f o r hemoglobin i n d u c t i o n , no enrichment of g l o b i n sequences i s seen i n t h i s f r a c t i o n (229). +2 The e v i d e n c e so f a r thus s u g g e s t s t h a t t h e Mg - s o l u b l e DNase I I f r a c t i o n (S2) i s e n r i c h e d i n a c t i v e gene sequences. The r e s u l t s suggest t h a t t h e S2 a s s o c i a t e d n u c l e o s o m a l m a t e r i a l i s a s s o c i a t e d w i t h a c h r o m a t i n r e g i o n t h a t i s s e n s i t i v e to DNase I I a t t a c k . The nucleosomes i n t h i s r e g i o n a r e i n i t i a l l y r e l e a s e d as polynucleosomes, but w i t h c o n t i n u e d d i g e s t i o n , the major nucleosome s p e c i e s becomes the mononucleosomes. The t r a n s c r i p t i o n a l l y - a c t i v e n u c l e o s o m a l f r a c t i o n c o n t a i n s t h e f o u r c o r e h i s t o n e s , H2A, H2B, H3 and H4, but o n l y H4 was found to be g r e a t l y e n r i c h e d i n m u t i - a c e t y l a t e d s p e c i e s . The r e s u l t s suggest t h a t DNase I I r e c o g n i z e s and s e l e c t i v e l y e x c i s e s nucleosomes e n r i c h e d i n t h e a c e t y l a t e d H4 s p e c i e s ; t h e s e nucleosomes a r e b e l i e v e d to be d e r i v e d from t r a n s c i p t i o n a l l y competent c h r o m a t i n r e g i o n s . I l l : C h a r a c t e r i z a t i o n o f Nucleosome S u b f r a c t i o n s and Chromosomal P r o t e i n s R e l e a s e d by DNase I D i g e s t i o n of N u c l e i Weintraub and Groudine (133) have demonstrated t h a t t r a n s c r i p t - . i o n a l l y competent genes e x h i b i t an i n c r e a s e d s u s c e p t i b i l i t y t o 14 7 d i g e s t i o n by DNas.e I . The s e n s i t i v i t y of t h e genes, t o DNase I is. a p p a r e n t l y n o t dependent on t h e r a t e a t which i t is; t r a n s c r i b e d (134)... Levy and Dixon (.136) have a l s o examined t h e a b i l i t y o f DNase I t o s e l e c t i v e l y d i g e s t t r a n s c r i p t i o n a l l y - c o m p e t e n t genes.. DNase I d i g e s t i o n o f t r o u t t e s t i s n u c l e i , under c o n d i t i o n s , i n which 10% o f t h e t o t a l DNA was d i g e s t e d , r e s u l t e d i n t h e p r e f e r e n t i a l d e p l e t i o n of t h e DNA sequences b e i n g t r a n s c r i b e d i n t o p o l y a d e n y l a t e d mRNA. The DNase I s e n s i t i v i t y o f t r a n s c r i p t i o n a l l y - c o m p e t e n t genes has l e d t o t h e . p r o p o s a l t h a t t r a n s c r i p t i o n a l l y - c o m p e t e n t c h r o m a t i n c o n t a i n s nucleosomes w i t h an a l t e r e d c o n f o r m a t i o n . The p o s s i b i l i t y t h a t h i s t o n e a c e t y l a t i o n as w e l l as t h e p r e s e n c e o f o t h e r s p e c i f i c p r o t e i n s , might p r o v i d e a mechanism, f o r the proposed s t r u c t u r a l changes was i n v e s t i g a t e d . (a) C h a r a c t e r i z a t i o n of nucleosomes r e l e a s e d from DNase I d i g e s t e d  n u c l e i 1. D i g e s t p r o d u c t s r e l e a s e d from DNase I d i g e s t e d n u c l e i The p r o d u c t s from a DNase I d i g e s t i o n o f n u c l e i were f r a c t i o n a t e d on a B i o - G e l A-5m column e q u i l i b r a t e d with. 10 mM T r i s - H C l , pH 7.5, and 0.7 mM EDTA ( F i g . 34). The column p r o f i l e was s i m i l a r t o the f r a c t i o n a t i o n of d i g e s t products; from n u c l e i d i g e s t e d m i l d l y w i t h m i c r o c o c c a l n u c l e a s e ( j i g . 6). The f r a c t i o n a t e d p r o d u c t s were p o o l e d i n t o f o u r n u c l e o s o m a l s i z e c l a s s e s : m u l t i m e r ( f r a c t i o n s 18 t o 23), i n t e r m e d i a t e (24 to 27), 148 40 FRACTION NO. FIG. 34. B i o - G e l A-5m (90 X 1.5 cm) s e p a r a t i o n of nucleosomes i s o l a t e d from DNase I d i g e s t e d n u c l e i . T r o u t t e s t i s n u c l e i (20.A , n/ml) were d i g e s t e d w i t h DNase I (2.5 ug/ml) f o r 5 min a t 25°C. The d i g e s t e d n u c l e i were c o l l e c t e d by c e n t r i f u g a t i o n , and the d i g e s t p r o d u c t s r e l e a s e d by r e s u s p e n d i n g the n u c l e i i n 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA. 149. monomer (28.to 3 3 ) , and submonomer (34 t o 4 1 i . I t was, assumed t h a t t h e multimer, i n t e r m e d i a t e , and monomer f r a c t i o n s c o r r e s p o n d e d t o : p o l y n u c l e o s o m e s , d i n -ucleosomes,. and mononucleosomes, r e s p e c t i v e l y , as t h e DNA fragments r e s o l v e as smears on n o n d e n a t u r i n g p o l y a c r y l a m i d e g e l s . The s i n g l e - s t r a n d e d DNA fragments a s s o c i a t e d with, each f r a c t i o n were a n a l y z e d on d e n a t u r i n g p o l y a c r y l -amide gels. ( F i g . 35). Each f r a c t i o n shows a p a t t e r n o f bands t h a t a r e m u l t i p l e s o f t e n bases and a prominent band a t 80 n u c l e o t i d e s . The p a t t e r n i s due to t h e n i c k i n g o f t h e n u c l e o s o m a l DNA s t r a n d s by DNase I a t t e n bases a p a r t (230). B e s i d e s c o n t a i n i n g t h e c h a r a c t e r i s t i c p a t t e r n o f bands, t h e l a r g e r n u c l e osomal s i z e c l a s s e s , c o n t a i n DNA fragments l a r g e r t h a n 140 bases. N o l l (230) has demonstrated t h a t although. DNase I a t t a c k s b o t h t h e n u c l e o s o m a l and l i n k e r DNA, the nucleosome s t i l l remains i n t a c t . N o l l has o b t a i n e d mononucleosomes from DNase I d i g e s t e d r a t l i v e r n u c l e i t h a t sediment a t the same r a t e as: i n t a c t 11 S c h r o m a t i n s u b u n i t s . The mononucleosomes c o n t a i n e d s i n g l e - , s t r a n d e d DNA fragments t h a t were m u l t i p l e s o f 10 Bases. However, the d i s t r i B u t i o n o f s i n g l e - s t r a n d e d fragments 150 abed FIG. 35. Polyacrylamide gel electrophoresis of single-stranded DNA fragments associated with column fractionated nucleosome fractions. Nucleosomes obtained from DNase I digested nuclei were fractionated as described in the legend to Fig. 34. The column fractions were pooled as follows: multimer (18-23), intermediate (24-27), monomer (28-33) and sub-monomer (34-41). DNA (0.5 A ) obtained from the fractions (see "Materials and Methods" Section XV a (ij)was analyzed on a 10% polyacrylamide SDS gel containing 7. M urea. After electrophoresis at 200 V for 4 h , the gel was stained with ethidium bromide and photographed under ultraviolet light, a, b, c and d are multimer, intermediate, monomer and submonomer fractions, respectively. 151' p r e s e n t e d r e l a t i v e l y few fragments o f 10 and 20 bases. As fragments no s m a l l e r than 30 o r 40 bases were r e s o l v a b l e on t h e g e l s , N o l l ' s o b s e r v a t i o n was not v e r i f i e d . The p o s s i b i l i t y o f t h e r e l e a s e of f r e e h i s t o n e s from DNase I d i g e s t e d n u c l e i was examined. N u c l e i o b t a i n e d from [ 1 1 +C] a c e t a t e and [ 3H] l y s i n e l a b e l l e d t r o u t t e s t i s c e l l s were d i g e s t e d w i t h DNase I , and t h e d i g e s t p r o d u c t s were f r a c t i o n a t e d on a B i o - G e l A-5m column ( F i g . 36). A l l of t h e l a b e l was a s s o c i a t e d w i t h t h e mul t i m e r , i n t e r m e d i a t e and monomer f r a c t i o n s , s u g g e s t i n g t h a t t h e h i s t o n e s a r e bound t o DNA. S i n c e t h e n u c l e o s o m a l DNA i s n i c k e d by DNase I a t st a g g e r e d i n t e r v a l s ( 9 8 ) , i t s h o u l d be p o s s i b l e f o r t h e n i c k e d DNA s t r a n d s to remain as an i n t a c t d o u b l e -s t r a n d e d DNA fragment i n t h e absence of h i s t o n e s . To remove t h e h i s t o n e s from DNA, sodium l a u r y l s a r c o s i n a t e ( S a r k o s y l ) was used (127). The e f f i c i e n c y o f S a r k o s y l f o r d i s s o c i a t i n g t h e h i s t o n e s was examined by u s i n g mononucleosomes c o n t a i n i n g h i s t o n e s l a b e l l e d w i t h [ 1 1 +C] a c e t a t e i s o l a t e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i . The l a b e l l e d mononucleosomes were t r e a t e d w i t h 1% S a r k o s y l and f r a c t i o n a t e d on a B i o - G e l A-0.5m column 152 o CD CM o 2.0 1.0 i\ Zo* 40 CO co b - b T— X X 20 | • o co 10 1 o < * • i 4 • 20 20 40 FRACTION NO. FIG. 36. B i o - G e l A-5m (90 X 1.5 cm) s e p a r a t i o n o f nucleosomes l a b e l l e d w i t h [^C] a c e t a t e and [ 3H] l y s i n e i s o l a t e d from DNase I d i g e s t e d n u c l e i . T r o u t t e s t i s c e l l s were i n c u b a t e d w i t h L [4, 5 (n) - 3H] l y s i n e mono-h y d r o c h l o r i d e (100 yCi/ml) and sodium [ l - l l f C ] a c e t a t e (50 y C i / m l ) f o r 7 h, and n u c l e i were i s o l a t e d . T h e ^ n u c l e i (20 A^gg/ml) were d i g e s t e d 15 min w i t h DNase I (2.5 pg/ml) a t 25 C, and the d i g e s t p r o d u c t s were s e p a r a t e d on a B i o - G e l A-5m column. R a d i o a c t i v i t y was determined i n the same manner as d e s c r i b e d f o r F i g . 7. 153 e q u i l i b r a t e d with: 10 mM T r i s - H C l , (pll 7.5) , Q.7 mM EDTA, and 1% S a r k o s y l ( F i g . 37A) . ; The lab.el e l u t e s . a f t e r t h e A 2gQ, a b s o r b i n g m a t e r i a l i i n d i c a t i n g t h e i n t a c t n u c l e o s o m a l DNA i s f r e e o f bound h i s t o n e s . U s i n g t h e same p r o c e d u r e , u n l a b e l l e d nucleosomes i s o l a t e d from a DNase I d i g e s t e d n u c l e i were f r a c t i o n -a t e d on t h e B i o - G e l A-0.5m c o l u m n ( F i g . 37B). Only one peak o f A^gQ - a b s o r b i n g m a t e r i a l i s . seen e l u t i n g which s u g g e s t s t h e n i c k e d n u c l e o s o m a l DNA remains i n t a c t i n the absence o f h i s t o n e s . Q u a n t i t i a t i o n o f a c e t y l a t e d h i s t o n e s p e c i e s a s s o c i a t e d  w i t h n u c l e o s o m a l f r a c t i o n s The h i s t o n e s a s s o c i a t e d w i t h t h e DNase I g e n e r a t e d n u c l e o s o m a l f r a c t i o n s were a n a l y z e d f o r t h e i r l e v e l s of a c e t y l a t e d s p e c i e s . T r o u t t e s t i s n u c l e i were d i g e s t e d w i t h DNase I u n t i l 6.6, 8.2 : o r 16.9% of t h e i n i t i a l ^260 - a b s o r D : L n § m a t e r i a l was s o l u b i l i z e d and t h e d i g e s t p r o d u c t s were f r a c t i o n a t e d on a B i o - G e l A-5m column. The h i s t o n e s from t h e multimer f r a c t i o n and t h e monomer f r a c t i o n were a c i d e x t r a c t e d . The h i s t o n e s r e m a i n i n g w i t h t h e n u c l e a r p e l l e t ; a f t e r t h e d i g e s t p r o d u c t s were r e l e a s e d w e r e a l s o a c i d e x t r a c t e d . The h i s t o n e s from t h e 6.6%' and 8.2% d i g e s t f r a c t i o n s ; were s e p a r a t e d on a c i d - u r e a g e l s - ( j i g . - 3 8). Each, f r a c t i o n c o n t a i n e d e q u i v a l e n t p r o p o r t i o n s o f t h e n u c l e o s o m a l h i s t o n e s , H2A, 154 "I t , , ^_ 0 2 0 4 0 60 FRACTION NO. FIG. 37. B i o - G e l A-0.5m (90 x 1.0 em) f r a c t i o n a t i o n o f nu c l e o s o m a l components. (A) Mononucleosomes (^ 10 A ^ Q ) l a b e l l e d w i t h [ l t +C] a c e t a t e were i s o l a t e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i as d e s c r i b e d i n " M a t e r i a l s and Methods". The mononucleosomes were c o n c e n t r a t e d by p r e c i p i t a t i o n w i t h 10 mM MgCl„ and c o l l e c t e d by c e n t r i f u g a t i o n . The mononucleosomes were resuspended i n 0.5 ml o f 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and 1% S a r k o s y l and a p p l i e d t o t h e column. R a d i o a c t i v i t y was deter m i n e d by m i x i n g 0.5 ml column f r a c t i o n s w i t h 8 ml ACS (Amersham/ S e a r l e ) . (B) Nucleosomes (^ 20 A ^ ) were i s o l a t e d from DNase I d i g e s t e d n u c l e i as d e s c r i b e d i n the le g e n d t o F i g . 34. The nucleosomes were c o n c e n t r a t e d , c o l l e c t e d and resuspended as d e s c r i b e d above b e f o r e a p p l i c a t i o n t o t h e column. 155 a b c d e f FIG. 38. H i s t o n e p r o f i l e s on an a c i d - u r e a p o l y a c r y l a m i d e g e l . H i s t o n e s were a c i d - e x t r a c t e d from n u c l e i , polynucleosomes or mononucleosomes t h a t were i s o l a t e d from DNase I d i g e s t e d n u c l e i . The h i s t o n e s were s e p a r a t e d on an a c i d - u r e a p o l y a c r y l a m i d e g e l by t h e system of Panyim and C h a l k l e y (194). The g e l was s t a i n e d w i t h Coomassie b l u e . (a) and ( d ) , h i s t o n e s from n u c l e a r p e l l e t r e m a i n i n g a f t e r DNase I d i g e s t i o n u n t i l 6.6% or 8.2%, r e s p e c t i v e l y , o f t h e DNA was s o l u b i l i z e d , and d i g e s t p r o d u c t s removed; (b) and ( e ) , h i s t o n e s from multimer f r a c t i o n of f r a c t i o n a t e d d i g e s t p r o d u c t s from n u c l e i d i g e s t e d w i t h DNase I u n t i l 6.6% or 8.2% DNA s o l u b i l i z e d , r e s p e c t i v e l y ; (c) and ( f ) , h i s t o n e s from monomer f r a c t i o n of f r a c t i o n a t e d d i g e s t p r o d u c t s from n u c l e i d i g e s t e d w i t h DNase I u n t i l 6.6% o r 8.2% DNA s o l u b i l i z e d , r e s p e c t i v e l y . 156, H2B, H3 and H4. E x a m i n a t i o n o f t h e photograph, and scans o f t h e g e l s l i c e s (not shown) < sug g e s t e d t h e l e v e l s . o f t h e a c e t y l a t e d s p e c i e s f o r h i s t o n e s . H2A, H2B and H4 were s i m i l a r f o r a l l t h e f r a c t i o n s and d i d n o t v a r y w i t h t h e e x t e n t o f d i g e s t i o n i n c l u d i n g t h e 16.9% d i g e s t i o n . A l l f r a c t i o n s c o n t a i n e d p r e d o m i n a n t l y t h e u n a c e t y l a t e d (A^) and m o n o a c e t y l a t e d (A^) species, o f H4. A l s o t h e d i - ( A „ ) , t r i - ( A ~ ) , and t e t r a a c e t y l a t e d (A ) H4 s p e c i e s 4 were v i s i b l e but to a much l e s s e r e x t e n t t h a n t h e A o and A^ s p e c i e s . The l e v e l s o f t h e a c e t y l a t e d s p e c i e s o f h i s t o n e H3 appeared t o d i m i n i s h i n t h e monomer and multime r f r a c t i o n s r e l a t i v e t o t h e l e v e l s o f t h e H3 a c e t y l a t e d s p e c i e s a s s o c i a t e d w i t h t h e p e l l e t . The monomer f r a c t i o n c o n t a i n e d the lowest l e v e l s o f a c e t y l a t e d H3 s p e c i e s . The monomer f r a c t i o n always had de c r e a s e d l e v e l s o f t h e H3 a c e t y l a t e d s p e c i e s r e g a r d l e s s o f t h e ex t e n t o f d i g e s t i o n . The r e l a t i v e l y h i g h l e v e l s o f t h e a c e t y l a t e d H3 s p e c i e s a s s o c i a t e d w i t h t h e p e l l e t may be d e c e i v i n g as n o n h i s t o n e chromosomal p r o t e i n s , a r e known to run i n t h i s p o s i t i o n ( F i g . 33). The l e v e l ! o f HI i s seen t o d e c r e a s e as; t h e n u c l e o -somal DNA s i z e d e c r e a s e s ( F i g . 38). As d i g e s t i o n o f 157 t h e l i n k e r DNA r e s u l t s i n t h e l o s s o f HI, the monomer f r a c t i o n would be expected to c o n t a i n t h e l e a s t amount of HI. T h i s e x p e c t a t i o n i s c o n f i r m e d as the monomer f r a c t i o n c o n t a i n s t h e lowest amount of HI. These s t u d i e s i n d i c a t e t h a t mononucleosomes i s o l a t e d from DNase I d i g e s t e d n u c l e i c o n t a i n t h e f o u r n u c l e o s o m a l h i s t o n e s , H2A, H2B, H3 and H4. The mono-nucleosomes c o n t a i n t h e same l e v e l s o f a c e t y l a t e d s p e c i e s o f h i s t o n e s H2A, H2B and H4 as t h o s e o f p o lynucleosomes but appear t o be d e f i c i e n t i n t h e a c e t y l a t e d s p e c i e s of H3. C h a r a c t e r i z a t i o n o f nucleosome s u b f r a c t i o n s and chromosomal  p r o t e i n s r e l e a s e d from DNase I d i g e s t e d n u c l e i (Sanders' p r o c e d u r e ) The Sanders' f r a c t i o n a t i o n p r o c e d u r e i n v o l v e s d i g e s t i o n of t h e n u c l e i w i t h m i c r o c o c c a l n u c l e a s e f o l l o w e d by successive.' e x t r a c t i o n s of t h e n u c l e i w i t h b u f f e r s o l u t i o n s c o n t a i n i n g i n c r e a s -i n g c o n c e n t r a t i o n s of NaCl ( F i g . 16). P r e v i o u s experiments have been d e s c r i b e d i n which m i c r o c o c c a l n u c l e a s e was used to d i g e s t n u c l e i f o l l o w e d by subsequent f r a c t i o n a t i o n (see S e c t i o n 1 ( b ) ) . The e x p e r i m e n t s were r e p e a t e d w i t h DNase I as t h e d i g e s t i n g n u c l e a s e . The l e v e l s o f a c e t y l a t e d h i s t o n e s p e c i e s and chromo-somal p r o t e i n s a s s o c i a t e d w i t h t h e s a l t r e l e a s e d n u c l e o s o m a l s u b f r a c t i o n s were examined i n the same manner as i n t h e m i c r o c o c c a l n u c l e a s e experiments. 158 Q u a n t i t a t i o n o f t h e DNA c o n t e n t i n s a l t ^ e x t r a c t e d  c h r o m a t i n f r a c t i o n s : F o l l o w i n g t h e d i g e s t i o n o f t h e t r o u t t e s t i s , n u c l e i with: DNase I , the d i g e s t e d n u c l e i , were sequ e n t ^ t a l l y t r e a t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f NaCl as d e s c r i b e d by Sanders ( 1 9 0 ) , except t h a t the i n i t i a l NaCl c o n c e n t r a t i o n was 0.1 M i n s t e a d of 0.2 M and t h e 0.3 M N aCl e x t r a c t i o n s t e p was o m i t t e d . The p e r c e n t a g e of t o t a l ^go a ^ , s o r ^ > : L n S m a t e r i a l r e l e a s e d i n each f r a c t i o n i s p r e s e n t e d i n T a b l e XIX. The v a l u e s o b t a i n e d f o r each s a l t - e x t r a c t e d f r a c t i o n d i d not v a r y a p p r e c i a b l y w i t h t h e e x t e n t of d i g e s t i o n i n t h e range between 1.5% and 30% s o l u b i l i z a t i o n o f t h e DNA. The i n v a r i a b i l i t y of - the amount of DNA a s s o c i a t e d w i t h each s a l t - e x t r a c t e d f r a c t i o n when the n u c l e i were d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e to d i f f e r e n t e x t e n t s has been r e p o r t e d by Sanders. (190) . Sanders suggested t h e s t a b i l i t y o f t h e i n t e r n u c l e o s o m a l i n t e r a c t i o n s was not a l t e r e d s i g n i f i c a n t l y a f t e r t h e c h r o m a t i n had been d i g e s t e d . T h i s i n t e r p r e t a t i o n can be extended t o i n c l u d e DNase I . The p e r c e n t a g e o f A^^Q a b s o r b i n g m a t e r i a l a s s o c i a t e d w i t h each f r a c t i o n from m i c r o c o c c a l n u c l e a s e d i g e s t e d 159 TABLE XIX Q u a n t i t a t i o n . Q £ DNA Content i n S a l t - E x t r a c t e d Chromatin F r a c t i o n s  R e leased from DNase I D i g e s t e d N u c l e i ( S anders' ~ ~ Procedure) T h e A 2 6 0 a b s o r b i n S m a t e r i a l r e l e a s e d , i n t h e d i f f e r e n t s a l t - e x t r a c t e d nucleosome f r a c t i o n s a f t e r DNase I d i g e s t i o n of t r o u t t e s t i s n u c l e i was q u a n t i t a t e d by adding 100 y l of the f r a c t i o n t o 0.9 ml o f 0.6 M NaCl i n B u f f e r D and measuring the absorbance a t 260 nm. F r a c t i o r i T o t a l A 2 6 Q i n Each F r a c t i o n % SO 5.0 SSI 3.2 SS2 2 . 7 s s 4 26.8 SS6 22.7 160 n u c l e i o r DNase I d i g e s t e d n u c l e i were q u i t e d i f f e r e n t ( T a b l e X and X I X ) . These d i f f e r e n c e s may be due t o s e v e r a l f a c t o r s . F i r s t l y , m i c r o c o c c a l n u c l e a s e w i l l i n t r o d u c e DNA c u t s t h a t a r e a d j a c e n t on b o t h s t r a n d s w h i l e DNase I w i l l i n t r o d u c e s t a g g e r e d c u t s (101). S e c o n d l y , m i c r o c o c c a l n u c l e a s e p r e f e r e n t i a l l y d i g e s t s t h e l i n k e r r e g i o n w h i l e DNase I d i g e s t s b o t h t h e l i n k e r and n u c l e o s o m a l DNA a t comparable r a t e s (230). T h i r d l y , t h e p r e f e r e n t i a l d i g e s t i o n of t h e l i n k e r DNA by m i c r o -c o c c a l n u c l e a s e w i l l r e l e a s e chromosomal p r o t e i n s such as HMG-T which a r e n o t r e l e a s e d by DNase I d i g e s t i o n (162). One o r more of t h e above f a c t o r s might r e s u l t i n t h e g r e a t e r ease of low s a l t s o l u b i l i z a t i o n of nucleosomes from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i . The combined p e r c e n t a g e of t o t a l a b s o r b i n g ' ; m a t e r i a l a s s o c i a t e d w i t h SSI (DNase I) and SS2 (DNase I) i s 5.9. T h i s v a l u e i s s l i g h t l y lower than t h e p e r c e n t a g e of t o t a l A„,„ m a t e r i a l (7.2) a s s o c i a t e d w i t h SSI ( m i c r o -260 ' c o c c a l ) f r a c t i o n ( T a b l e X ) . I t i s p o s s i b l e t h a t t h e SSI and SS2 f r a c t i o n s r e l e a s e d from DNase I d i g e s t e d n u c l e i r e p ^ s e n t s u b s e t s o f t h e SSI f r a c t i o n r e l e a s e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i . 161 2.. C h a r a c t e r i z a t i o n of t h e DNA fragments a s s o c i a t e d w i t h  s a l t - e x t r a c t e d c h r o m a t i n f r a c t i o n s The s i z e o f the DNA fragments a s s o c i a t e d ' w i t h each s a l t - e x t r a c t e d f r a c t i o n cannot be determined by e l e c t r o p h o r e s i s on n o n d e n a t u r i n g p o l y a c r y l a m i d e g e l s as t h i s r e s u l t s i n a smear. To o b t a i n an approximate s i z e of t h e DNA fragments, B i o - G e l A-5m g e l e x c l u s i o n chromatography was used. The e l u t i o n p r o f i l e s o f A„,-.-absorbing m a t e r i a l a s s o c i a t e d w i t h t h e s a l t -260 e x t r a c t e d f r a c t i o n s , SSI, SS2 and SS4T (DNase I) a r e shown i n F i g u r e s 39, 40 and 41, r e s p e c t i v e l y . The DNA fragment s i z e a s s o c i a t e d w i t h each column f r a c t i o n has p r e v i o u s l y been c h a r a c t e r i z e d i n experiments i n which m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i were f u r t h e r f r a c t i o n a t e d on B i o - G e l A-5m columns ( S e c t i o n I b ( 4 ) ) . The A2gQ-absorbing m a t e r i a l a s s o c i a t e d w i t h SSI (DNase I) r e s o l v e s as t h r e e peaks ( F i g . 39). The f i r s t peak e l u t i n g a t o r near t h e v o i d volume i s termed t h e m u l t i m e r f r a c t i o n ; and c o n s i s t s of fragments g r e a t e r than t r i m e r i n l e n g t h . The second peak (monomer f r a c t i o n ) c o n s i s t s of mononucleosomal s i z e fragments and t h e t h i r d peak c o n s i s t s o f o l i g o -n u c l e o t i d e s . The mononucleosomal fragment i s t h e most abundant of t h e nucleosome a s s o c i a t e d DNA fragments. 162 i FRACTION NO. FIG. 39. B i o - G e l A-5m (90 X 1.5 cm) s e p a r a t i o n of nucleosomes o b t a i n e d from e x t r a c t i o n of DNase I d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0.1 M NaCl i n B u f f e r D. 163 1.2 r O 0.8 co C N 0.4 40 FRACTION NO. 80 FIG. 40. B i o - G e l A-5m (.90 X 1.5 cm) s e p a r a t i o n of nucleosomes o b t a i n e d from e x t r a c t i o n of DNase I d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0.2 M NaCl i n B u f f e r D. The d i g e s t e d n u c l e i had been p r e v i o u s l y e x t r a c t e d w i t h 0.1 M NaCl i n B u f f e r D. .164 40 80 FRACTION NO. FIG. 41. B i o - G e l A-5m (90 X 1.5 cm) s e p a r a t i o n of nucleosomes o b t a i n e d from e x t r a c t i o n of DNase I d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0.4 M NaCl B u f f e r D. 165 The SS2 (DNase I) m a t e r i a l a l s o r e s o l v e s i n t o t h r e e peaks ( F i g . 40). The m a j o r i t y of t h e m a t e r i a l e l u t e s w i t h t h e monomer f r a c t i o n (42 t o 51). Lower amounts of A„,„ a b s o r b i n g m a t e r i a l e l u t e w i t h the 260 m u l t i m e r and i n t e r m e d i a t e (30 to 41) f r a c t i o n s . The i n t e r m e d i a t e f r a c t i o n s c o n s i s t of dimer, some t r i m e r and v e r y l i t t l e monomer ( S e c t i o n I b ( 4 ) ) . The SS4T (DNase I) f r a c t i o n , which c o n t a i n s a l l of the n u c l e o s o m a l s u b f r a c t i o n s n o r m a l l y r e l e a s e d i n SSI, SS2 and SS4 (DNase I ) , was f r a c t i o n a t e d i n t o t h r e e peaks ( F i g . 41). The m a j o r i t y o f t h e m a t e r i a l would be c h a r a c t e r i s t i c o f t h e SS4 (DNase I) f r a c t i o n ( T a b l e XIX): - t h a t i s , about 80% of t h e m a t e r i a l i s a s s o c i a t e d w i t h t h e SS4 (DNase I ) . The p r o f i l e i s almost i d e n t i c a l t o t h e p r o f i l e o f SS4T ( m i c r o c o c c a l ) column f r a c t i o n a t e d m a t e r i a l ( F i g . 32). The m a j o r i t y of t h e A „ , - a b s o r b i n g m a t e r i a l e l u t e s w i t h the 260 i n t e r m e d i a t e f r a c t i o n . From t h e column e l u t i o n p o s i t i o n s , approximate s i z e s o f t h e n u c l e o s o m a l DNA fragments were o b t a i n e d . B o t h t h e SSI and SS2 (DNase I) f r a c t i o n s c o n t a i n DNA . fragments m a i n l y o f monomeric s i z e w h i l e t h e SS4T (DNase I) f r a c t i o n c o n t a i n e d m a i n l y fragments of dimer s i z e o r 166 l a r g e r . Thus, the r e s u l t s s uggest t h a t the f r a c t i o n o f c h r o m a t i n which i s most s e n s i t i v e to DNase I. d i g e s t i o n i s . e l u t e d a t t h e lowest: NaCl c o n c e n t r a t i o n s . The s i z e o f the s i n g l e - s t r a n d e d DNA fragments, a s s o c i a t e d with, t h e s a l t - e x t r a c t e d f r a c t i o n s , was examined on 15% d e n a t u r i n g p o l y a c r y l a m i d e g e l s ( F i g . 4 2 ) . F r a c t i o n s SSI, SS2, SS4 and SS6 (DNase.I) a l l p r e s e n t e d t h e 10 base DNA r e p e a t . V i s u a l i n s p e c t i o n o f t h e i n t e n s i t y o f each, band r e l a t i v e t o the 80 base band s u g g e s t e d each, f r a c t i o n c o n t a i n e d the same q u a n t i t y o f any p a r t i c u l a r band. However, one o b s e r v a b l e d i f f e r -ence was t h a t the SS4 and SS6 (DNase I) f r a c t i o n s c o n t a i n e d a g r e a t e r abundance o f fragments g r e a t e r t h a n 140 b a s e s . T h i s i s c o n s i s t e n t with, the o b s e r v a t i o n t h a t SSI and SS2 (DNase I) f r a c t i o n s c o n t a i n p r e d o m i n a n t l y mononucleosomes w h i l e the SS4 (DNas.e I) f r a c t i o n c o n t a i n s m a i n l y dinucleosomes and o t h e r p o l y -nucleosomes. The p a t t e r n of s i n g l e - s t r a n d e d fragments a s s o c i a t e d with, each f r a c t i o n d i d n o t v a r y a p p r e c i a b l y w i t h . t h e e x t e n t o f d i g e s t i o n I n the range, between 1.5% and 30% s o l u b i l i z a t i o n of the DNA. 167 H r I 1 HH ? _ ^ i 1 a b e d -50 CO Q> CO CO o c FIG. 42. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f s i n g l e - s t r a n d e d DNA fragments generated by DNase I d i g e s t i o n o f t r o u t t e s t i s n u c l e i and r e l e a s e d by s t e p w i s e i n c r e a s e s i n NaCl c o n c e n t r a t i o n s . The s a l t - e x t r a c t e d f r a c t i o n s were d i a l y z e d a g a i n s t 10. mM NH^HCO^ o v e r n i g h t and samples c o r r e s p o n d i n g t o 0.5 -^26Q w e r e l y o p h i l i z e d . The samples were r e d i s s o l v e d i n sample b u f f e r c o n t a i n i n g SDS and a p p l i e d to the g e l . The g e l was s t a i n e d w i t h e t h i d i u m bromide and photographed under u l t r a v i o l e t l i g h t . a - d, f r a c t i o n s s u c c e s s i v e l y r e l e a s e d by B u f f e r D c o n t a i n i n g 0.1 M - 0.6 M NaCl (SSI - SS6), r e s p e c t i v e l y . 168 C h a r a c t e r i z a t i o n o f t h e p r o t e i n s a s s o c i a t e d w i t h s a l t - e x t r a c t e d c h r o m a t i n f r a c t i o n s The p r o t e i n a s s o c i a t e d with. each, o f the s u p e r s n a t a n t f r a c t i o n s was a n a l y z e d on 15% p o l y a c r y l a m i d e SDS gels: ( F i g . 4 3). SSI (DNase I) contains, c o r e h i s t o n e s , HMG-T1 (27K, f o r m e r l y c l a s s i f i e d as HMG-T ( 1 5 9 ) ) , HMG-T2 (25K) , and HMG-T3 (22K). (.The nomen-c l a t u r e a s s i g n e d t o th e s e p r o t e i n s i s as d e s c r i b e d by B. S. B h u l l a r ( p e r s o n a l communication)). T h i s f r a c t i o n a l s o c o n t a i n s n o n h i s t o n e chromosomal p r o t e i n s i n the 30 - 90,000 m o l e c u l a r weight range. SS2 (DNase I) i s s i m i l a r t o SSI (DNase I) except t h a t the c o n t e n t s o f HMG-T2 and HMG-T3 a r e lower, and low l e v e l s , o f HI a r e obs e r v e d . The spectrum o f n o n h i s t o n e p r o t e i n s i n the 30 - 90,000 m o l e c u l a r weight range i s s i m i l a r to t h a t . from t h e SSI (DNase I) f r a c t i o n , but t h e r e a r e d i f f e r e n c e s i n the c o n t e n t o f some p r o t e i n s . SS4 and SS6 (DNase I) c o n t a i n e d t h e nuc l e o s o m a l h i s t o n e s , l a r g e amounts o f HI (HI b e i n g h i g h e s t i n SS4), two u n i d e n t i f i e d p r o t e i n s o f 28,000 and 29,000 apparent m o l e c u l a r weight but no HMG-T proteins.. F i g u r e 43 p r e s e n t s a sample (SS4 ( I I ) ) which c l e a r l y shows t h e '. two u n i d e n t i f i e d p r o t e i n s a s s o c i a t e d with- the SS4 (DNase I) f r a c t i o n . 169 a b c d e f FIG. 43. 15% PAGE SDS separation of proteins released from DNase I digested trout t e s t i s n u c l e i by successively higher NaCl concentrations. Samples were prepared as described i n the legend to F i g . 24. The gel was stained with Coomassie blue. a - d, f r a c t i o n s released from DNase I digested n u c l e i by Buffer D containing 0.1 M - 0.6 M NaCl (SSI - SS6), res p e c t i v e l y ; e, f r a c t i o n released by dig e s t i o n of n u c l e i by DNase I (SO (D)); f, f r a c t i o n released by dig e s t i o n of n u c l e i by micrococcal nuclease (SO (M)); and (g) f r a c t i o n released by Buffer D containing 0.4 M NaCl (SS4 (II)) a f t e r previous extractions of DNase I digested n u c l e i with Buffer D containing 0.1 M NaCl followed by extraction with Buffer D contain-ing 0.2 M NaCl. SO (D), SO (M) and SS4 (II) were a l l obtained from d i f f e r -ent experiments than those from which SSI - SS6 were derived. 170 The SO (DNase I) f r a c t i o n contains, low l e v e l s , o f t h e c o r e h i s t o n e s , and lower l e v e l s , o f p r o t e i n s found a s s o c i a t e d w i t h t h e SSI (DNase I) f r a c t i o n ( F i g . 4 3 ) . Thes:e p r o t e i n s may be r e l e a s e d from damaged n u c l e i . The p r o t e i n s , a s s o c i a t e d w i t h each, f r a c t i o n were examined a f t e r t h e n u c l e i had been d i g e s t e d t o v a r y i n g e x t e n t s w i t h DNase I'.(.1.5% t o 30% DNA s o l u b i l i z e d ) . Each s a l t - e x t r a c t e d f r a c t i o n c o n t a i n e d t h e same c h a r a c t e r i s t i c s e t o f p r o t e i n s r e g a r d l e s s o f t h e ex t e n t o f DNase I d i g e s t i o n . Thus, t h e e x t e n t o f n u c l e a r d i g e s t i o n does not a p p r e c i a b l y a f f e c t t h e p a t t e r n o f s o l u b i l i z e d p r o t e i n s r e l e a s e d i n t o each s a l t - e x t r a c t e d f r a c t i o n . A comparison o f t h e photographs o f t h e g e l s p r e s e n t e d i n F i g u r e s 24 and 43 demonstrates s i m i l a r -i t i e s and d i s s i m i l a r i t i e s i n t h e s a l t - e x t r a c t e d f r a c t i o n s r e l e a s e d from e i t h e r m i c r o c o c c a l n u c l e a s e o r DNase X d i g e s t e d n u c l e i . The SO ( m i c r o c o c c a l ) f r a c t i o n ( F i g . 43 and 24) con t a i n s . HMG-T1,. HMG-T2,: and HMG-T3 which, a r e not a s s o c i a t e d with; t h e SO (DNase I) f r a c t i o n but w i t h t h e SSI and SS2 (DNase I) f r a c t i o n s : ( F i g . 4 3 ) . Levy W. et a l . (162). have 171 r e p o r t e d s i m i l a r r e s u l t s . The p r e f e r e n t i a l s o l u b i l -i z a t i o n o f HMG-T by m i c r o c o c c a l n u c l e a s e but not DNase I d i g e s t i o n o f t r o u t t e s t i s n u c l e i suggested t o t h e a u t h o r s t h a t HMG-T was l o c a t e d i n t h e i n t e r n u c l e o s o m a l r e g i o n . In the. same experiments (162) Levy et a l . o b s e r v e d t h e s e l e c t i v e s o l u b i l i z a t i o n of H6 from DNase I d i g e s t e d n u c l e i . The s e l e c t i v e s o l u b i l i z a t i o n o f t h i s p r o t e i n has not been o b s e r v e d i n t h i s l a b o r a t o r y , and t h e d i s c r e p a n c y may be due t o Levy e t al... u s i n g t r o u t t e s t i s t h a t were a t a l a t e s t a g e o f m a t u r a t i o n . The SSI (DNase I) and SS2 (DNase I) f r a c t i o n s a r e q u a l i t a t i v e l y v e r y s i m i l a r t o t h e c o r r e s p o n d i n g SSI ( m i c r o c o c c a l ) and SS2 ( m i c r o c o c c a l ) f r a c t i o n s i n t h a t they c o n t a i n the f o u r n u c l e o s o m a l h i s t o n e s , low l e v e l s of HI and n o n h i s t o n e chromosomal p r o t e i n s i n t h e 30 -90,000 m o l e c u l a r weight range. The p r o t e i n s a s s o c i a t e d w i t h t h e SS4 (DNase 1) and SS6 (DNase I) f r a c t i o n s a r e a l s o s i m i l a r t o t h o s e a s s o c i a t e d w i t h t h e c o r r e s p o n d i n g SS4 ( m i c r o c o c c a l ) and SS6 ( m i c r o c o c -c a l ) f r a c t i o n s w i t h r e g a r d t o t h e i r c o n t e n t of nuc l e o s o m a l h i s t o n e s , HI and the u n i d e n t i f i e d p r o t e i n s of 28,000 and 29,000 apparent m o l e c u l a r weight. The r e s u l t s suggest t h a t the nu c l e o s o m a l s u b t r a c t i o n and chromosomal p r o t e i n s s o l u b i l i z e d i n t h e s a l t - e x t r a c t e d f r a c t i o n s from e i t h e r DNase I d i g e s t e d n u c l e i o r 172 m i c r o c o c c a l d i g e s t e d n u c l e i a r e v e r y a i m i l a r . 4. Quant i t at i o n o f t h e a c e t y l a t ed h i s t o n e sp e c i e s assoc. i a t ed with- t h e s a l t - e x t r a c t ed chr o m a t i n f r a c t i o n s A c i d - e x t r a c t e d h i s t o n e s from t h e d i f f e r e n t f r a c t i o n s were a n a l y z e d on a c i d - u r e a g e l s (194).. The h i s t o n e p a t t e r n s were examined^ f o r d i f f e r e n c e s , i n the c o n t e n t o f a c e t y l a t e d h i s t o n e s p e c i e s . However, q u a n t i t a t i o n o f t h e a c e t y l a t e d s p e c i e s , e s p e c i a l l y t h o s e o f H4, f o r f r a c t i o n s SSI and SS2 (DNase I) was i m p o s s i b l e as n o n h i s t o n e p r o t e i n s , c o m i g r a t e d w i t h t h e a c e t y l a t e d h i s t o n e s p e c i e s (not shown). The l e v e l s o f a c e t y l a t e d H4 s p e c i e s c o u l d be determined f o r f r a c t i o n s SS4 and SS6 (DNase I ) . F i g u r e 44 p r e s e n t s g e l scans o f SS4 (DNase.I) and SS6 (DNase I) a s s o c -i a t e d h i s t o n e s . r e s p e c t i v e l y . The l e v e l s , o f t h e a c e t y l a t e d H3 s p e c i e s appear v e r y h i g h but t h i s i s due t o t h e c o m i g r a t i o n o f a n o n h i s t o n e p r o t e i n d e s i g n a t e d 29K ( F i g . 33) w i t h t h e a c e t y l a t e d s p e c i e s of H3. V i s u a l i n s p e c t i o n o f t h e scans, suggest t h a t t h e SS.6 (DNase I ) f r a c t i o n c o n t a i n s lower l e v e l s , o f t h e a c e t y l a t e d H4 species, than does t h e SS4 (jDNase I), f r a c t i o n ( F i g . 44). The e x t e n t o f H4 a c e t y l a t i o n f o r t h e SS4 and SS6 (DNase I) f r a c t i o n s , was. c a l c u l a t e d as 173 O IT) FIG. 44. Gel scans, of his.tones, analyzed on acid-urea gels,. Hi s.t ones, were acid-extracted from nucleosomes. that had been released from DNase I digested n u c l e i by! stepwis.e,. increasing NaCl concentrations: (0.1, 0.2, 0-4 and 0.6. M NaCl y i e l d i n g SSI, SS2, SS4 and SS6, r e s p e c t i v e l y ) . Gels.were stained with Coomassie blue.and scanned i n a G i l f o r d spectrophotometer at 550 nm. the gel scans: of histones associated with SS4 and SS6 are. presented, unacetylated H4; A^, A^, and. A^, mono-, d i - , and t r i - a c e t y l a t e d H4, respectively. Only: 174 a r a t i o of t h e c o n t e n t of A^ and s p e c i e s d i v i d e d by t h e sum of A q and A^ s p e c i e s ( T a b l e XX). The e x t e n t o f H4 a c e t y l a t i o n a s s o c i a t e d w i t h t h e SS4 (DNase I) f r a c t i o n was s l i g h t l y h i g h e r than t h a t o f t h e SS6 (DNase I) f r a c t i o n . A l s o , t h e e x t e n t of H4 a c e t y l a t i o n f o r t h e r e s p e c t i v e f r a c t i o n s d i d not v a r y w i t h t h e e x t e n t o f d i g e s t i o n . I n t e r e s t i n g l y , t h e c o n t e n t o f a c e t y l a t e d H4 s p e c i e s a s s o c i a t e d w i t h t h e SS4 and SS6 (DNase I) f r a c t i o n s ( T a b l e XX) was s i m i l a r t o t h a t from SS4 and SS6 ( m i c r o c o c c a l ) f r a c t i o n s ( T a b l e X I ) . These r e s u l t s f u r t h e r suggest t h a t the n u c l e o s o m a l s u b f r a c t i o n s o l u b i l i z e d i n SS4 or SS6 from DNase I or m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i a r e v e r y s i m i l a r . 5. B i o - G e l A-0.5m column f r a c t i o n a t i o n o f s a l t - e x t r a c t e d  nucleosome f r a c t i o n s ( i ) Column f r a c t i o n a t i o n o f t h e chromosomal p r o t e i n s  a s s o c i a t e d w i t h t h e s a l t - e x t r a c t e d c h r o m a t i n  f r a c t i o n s The B i o - G e l A-0.5m column (40 x 1.5 cm) e q u i l i b r a t e d w i t h 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA, and 0.4 M NaCl was chosen t o s e p a r a t e unbound p r o t e i n s , from t h e nu c l e o s o m a l m a t e r i a l . SSI f r a c t i o n s from n u c l e i t h a t had been d i g e s t e d w i t h e i t h e r DNase I (4.5% o f DNA s o l u b i l i z e d ) ( F i g . 46) or m i c r o c o c c a l n u c l e a s e (5.7% of DNA 175 TABLE XX. Q u a n t i t a t i o n of A c e t y l a t e d S p e c i e s of H i s t o n e H4 i n S a l t - E x t r a c t e d Nucleosome F r a c t Ions R e l e a s e d from DNase I D i g e s t e d N u c l e i The SS4 and SS6 f r a c t i o n s were o b t a i n e d as d e s c r i b e d i n " M a t e r i a l s and Methods" from n u c l e i t h a t had been i n c u b a t e d w i t h DNase I f o r v a r i o u s t i m e s . The h i s t o n e s t h a t were prep a r e d by a c i d - e x t r a c t i o n of t h e s a l t - e x t r a c t e d nucleosome f r a c t i o n s were s e p a r a t e d on a c i d - u r e a g e l s . The g e l s were s t a i n e d w i t h Coomassie b l u e . and scanned a t 550 nm u s i n g a G i l f o r d s p e c t r o p h o t o m e t e r . P e r c e n t of T o t a l A Released i n to SO 260 F r a c t i o n : SS4 SS6 R a t i o o f H, A 2 + A 3 * 1.5 2.9 5.4 7.8 AVG. 0.38 0.32 0.36 0.34 0.35 A + A, o 1 0.26 0.27 0.25 0.24 AVG.0.26 * Determined from t h e A peak h e i g h t s of t h e scanned H4 s p e c i e s . 176 -2 0 4 0 FRACTION NO. FIG. 45. B i o - G e l A - 0 . 5 m C 4 0 X 1.5, cm) s e p a r a t i o n of nucleosomes o b t a i n e d 7M e!J?C t 1 0; « m i ^ r O C ° c u C a l n u c l e a s e d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0.1 M NaCl m B u f f e r D. The column e l u a n t was. 10 mM T r i s - H C l , P H 7 5 0.7.mM EDTA, 0.4 M Na C l . P ' 177 0.8 0.6 o CN ^ 0.4 0.2 • m • • • • • • • : / : / i • • • i i • i • 1 • • i i • 1 • • 1 • 1 • I t • • • i • ( i • • i i • I I • i • i • • • • • • • • > • : : :\ . ' i I » " i \ "* • - a' • i • • • • • f • • * 0 20 40 FRACTION NO. FIG. 46 B i o - G e l A-0.5 f f i(40 X 1.5 cm) s e p a r a t i o n o f nucleosomes o b t a i n e d from e x t r a c t i o n o f DNase I d i g e s t e d t r o u t t e s t i s n u c l e i w i t h 0 1 M NaCl o!^ M M l ! " C ° 1 U m n 6 l U a n t W 3 S 1 0 m M T r i s - H C 1 » P h 7.5, 0.7 mM EDTA, 178 s o l u b i l i z e d ) ( F i g . 45) were f u r t h e r f r a c t i o n -a t e d on t h i s : column. The SSI ( m i c r o c o c c a l ) f r a c t i o n r e s o l v e s as f o u r peaks. ( E i g . 45). A l t h o u g h t h e s i z e o f t h e DNA fragments was not a n a l y z e d on 3% n o n d e n a t u r i n g g e l s , the f i r s t (multimer f r a c t i o n ) and second (monomer f r a c t i o n ) peaks p r o b a b l y c o n t a i n d i n -ucleosomes p l u s l a r g e r o l i g o n u c l e o s o m e s : and mononucleosomes, r e s p e c t i v e l y . (The B i o - G e l A-0.5m column has an e x c l u s i o n l i m i t o f 500,000 m o l e c u l a r weight; t h e r e f o r e , d i nucleosomes would p r o b a b l y be e x c l u d e d ) . The t h i r d and f o u r t h peaks p r o b a b l y c o n t a i n o l i g o n u c l e o t i d e s and d i n u c l e o t i d e s , r e s p e c t i v e l y . The SSI (DNase I) f r a c t i o n r e s o l v e s as t h r e e peaks ( F i g . 4 6 ) . The f i r s t and second peaks c o r r e s p o n d t o t h e m u l t -imer and monomer f r a c t i o n s , r e s p e c t i v e l y , w h i l e the t h i r d peak p r o b a b l y c o n s i s t s of o l i g o -n u c l e o t i d e s . The p r o f i l e f o r t h e column f r a c t i o n a t e d SSI ( m i c r o c o c c a l ) and SSI (DNase I) f r a c t i o n s remained t h e same i f t h e B i o - G e l A-0.5m column was e q u i l i b r a t e d w i t h 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA, and 0.1 M NaCl (not shown). 179 P r o t e i n s , a s s o c i a t e d with: t h e d i f f e r e n t f r a c t i o n s , were a n a l y z e d on 15% p o l y a c r y l a m i d e -SDS gels:. F i g u r e 47 l a n e (a) presents, t h e SSI (DNase I) sample b e f o r e f r a c t i o n a t i o n on t h e column. T h i s sample c o n t a i n s t h e c o r e h i s t o n e s , HMG-T1, HMG-T2 and HMG-T3. There i s a l s o a m u l t i t u d e o f n o n h i s t o n e p r o t e i n s , i n the 30 -90,000 m o l e c u l a r weight range. The SSI (DNase I) was f r a c t i o n a t e d on a B i o - G e l A-0.5m column e q u i l i b r a t e d w i t h 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and e i t h e r 0.1 M NaCl o r 0.4 M N a C l . The nucleosomal f r a c t i o n ( F i g u r e 46, f r a c t i o n s 13 to 22) and the o l i g o n u c l e o t i d e f r a c t i o n ( F i g . 46, f r a c t i o n s 24 t o 36) were p o o l e d separately;-and d i a l y z e d o v e r n i g h t a g a i n s t 10 mM NH^HCO^ a t 4°C. An e q u i v a l e n t amount of sample (0.6 A^^Q u n i t s ) from each f r a c t i o n was examined on a 15% po l y a c r y l a m i d e - S D S g e l . The same spectrum of p r o t e i n s was i d e n t i f i e d i n t h e c o r r e s p o n d i n g n u c l e o s o m a l and o l i g o n u c l e o t i d e f r a c t i o n s r e g a r d -l e s s of t h e i o n i c s t r e n g t h o f t h e column b u f f e r (compare F i g . 47, l a n e (b) t o l a n e ( d ) , and l a n e Cc) to l a n e ( e ) ) . The nucleosome f r a c t i o n s c o n t a i n e d t h e c o r e h i s t o n e s and some n o n h i s t o n e p r o t e i n s i n 30 - 90,000 m o l e c u l a r weight range. 180 • II • i _ J — — HMG-T1. HMG-T&V FIG. 47. 15% PAGE SDS s e p a r a t i o n o f chromosomal p r o t e i n s a s s o c i a t e d w i t h column f r a c t i o n a t e d , 0.1 M NaCl e x t r a c t e d nucleosome f r a c t i o n s . DNase I d i g e s t e d t r o u t t e s t i s n u c l e i o r m i c r o c o c c a l n u c l e a s e d i g e s t e d t r o u t t e s t i s n u c l e i were e x t r a c t e d w i t h 0.1 M NaCl i n B u f f e r D ( S S I ) . The s a l t - e x t r a c t e d nucleosome f r a c t i o n s were f u r t h e r f r a c t i o n a t e d u s i n g a B i o - G e l A-0.5m column e q u i l i b r a t e d w i t h e i t h e r 0.4 M NaCl, 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA or 0.1 M NaCl, 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA. Nucleosomal (combined m u l t -imer and monomer f r a c t i o n s ) and o l i g o n u c l e o t i d e f r a c t i o n s f o r SSI (DNase I ) , and n u c l e o s o m a l , o l i g o n u c l e o t i d e and d i n u c l e o t i d e f r a c t i o n s f o r SSI ( m i c r o -c o c c a l ) were p o o l e d s e p a r a t e l y . The samples were p r e p a r e d f o r e l e c t r o p h o r e s i s as d e s c r i b e d i n t h e l e g e n d t o F i g . 24. a, SSI (DNase I) u n f r a c t i o n a t e d ; b and c, n u c l e o s o m a l and o l i g o n u c l e o t i d e f r a c t i o n s , r e s p e c t i v e l y , from SSI (DNase I) f r a c t i o n a t e d on a B i o - G e l A-0.5m column e q u i l i b r a t e d w i t h 0.1 M NaCl, 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA; d and e, n u c l e o s o m a l and o l i g o -n u c l e o t i d e f r a c t i o n s , r e s p e c t i v e l y , from SSI (DNase I) f r a c t i o n f r a c t i o n a t e d on t h e column e q u i l i b r a t e d w i t h 0.4 M NaCl, 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA; f , SSI ( m i c r o c o c c a l ) u n f r a c t i o n a t e d ; g, h and i , n u c l e o s o m a l , o l i g o -n u c l e o t i d e and d i n u c l e o t i d e f r a c t i o n s , r e s p e c t i v e l y , from SSI ( m i c r o c o c c a l ) f r a c t i o n f r a c t i o n a t e d on t h e column e q u i l i b r a t e d w i t h 0.1 M NaCl, 10 mM T r i s -H C l , pH 7.5, 0.7 mM EDTA; j , 1 and m, n u c l e o s o m a l , o l i g o n u c l e o t i d e and d i -n u c l e o t i d e f r a c t i o n s , r e s p e c t i v e l y , from SSI ( m i c r o c o c c a l ) f r a c t i o n f r a c t i o n -a t e d on t h e column e q u i l i b r a t e d w i t h 0.4 M NaCl, 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA; k, m i c r o c o c c a l n u c l e a s e . Lanes k, 1 and m were from a s e p a r a t e g e l than t h a t o f the o t h e r s . 181 The predominant p r o t e i n s a s s o c i a t e d with, t h e o l i g o n u c l e o t i d e f r a c t i o n s ; were the HMG p r o t e i n s . (HMG-Tl, HMG-T2 and HMG-.-T3)'. Low l e v e l s : o f o t h e r n o n h i s t o n e p r o t e i n s were a l s o c o n t a i n e d i n t h i s : f r a c t i o n . F i g u r e 47, l a n e ( f ) p r e s e n t s t h e SSI ( m i c r o c o c c a l ) sample b e f o r e column f r a c t i o n a t i o n . T h i s f r a c t i o n c o n t a i n s t h e c o r e h i s t o n e s and non-h i s t o n e p r o t e i n s i n c l u d i n g t h e HMG p r o t e i n s . The SSI ( m i c r o c o c c a l ) f r a c t i o n was f u r t h e r f r a c t -i o n a t e d on a B i o - G e l A-0.5m column e q u i l i b r a t e d w i t h 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and • e i t h e r 0.1 M NaCl o r 0.4 M NaCl ( F i g . 4 5 ) . The column f r a c t i o n a t e d samples were p o o l e d i n t o t h r e e f r a c t i o n s : t h e nucleosome f r a c t i o n (.14 to 22), t h e o l i g o n u c l e o t i d e f r a c t i o n (.24 t o 29) and t h e d i n u c l e o t i d e f r a c t i o n (31 to 3 8 ) . The spectrum o f p r o t e i n s a s s o c i a t e d w i t h each, f r a c t i o n d i d not a l t e r w i t h t h e i o n i c s t r e n g t h : of t h e column b u f f e r (compare F i g . 47, l a n e '(g) • t o l a n e ( j ) , l a n e (h). t o l a n e ( 1 ) , and l a n e ( i ) t o l a n e (m)). The nucleosome f r a c t i o n s c o n t a i n e d t h e c o r e h i s t o n e s : and low l e v e l s , of 182 n o n h i s t o n e p r o t e i n s i n the 30 - 90,000 m o l e c u l a r weight range. The o l i g o n u c l e o t i d e f r a c t i o n s c o n t a i n e d n o n h i s t o n e p r o t e i n s , i n c l u d i n g HMG-Tl, HMG-T2, and HMG-T3, and low l e v e l s of t h e c o r e h i s t o n e s . M i c r o c o c c a l n u c l e a s e was t h e most abundant p r o t e i n c o n t a i n e d i n t h e d i n u c l e o t i d e f r a c t i o n ( F i g . 47, compare l a n e ( i ) o r l a n e (m) t o l a n e ( k ) ) . The SSI (DNase I) nucleosome f r a c t i o n ( F i g . 47, l a n e (b)) c o n t a i n s a lower amount o f c o r e h i s t o n e s but h i g h e r amounts o f p r o t e i n s when compared t o t h e SSI ( m i c r o c o c c a l ) n u c l e o s o m a l f r a c t i o n ( F i g . 47, l a n e Cg)) per u n i t of a b s o r b i n g m a t e r i a l . T h i s may be due to t h e f a c t t h a t t h e SSI (DNase I) f r a c t i o n c o n t a i n s a g r e a t e r amount of nucleosomal m a t e r i a l e l u t i n g w i t h t h e mu l t i m e r f r a c t i o n r e l a t i v e t o t h e mono-mer f r a c t i o n than t h a t o f t h e SSI ( m i c r o c o c c a l ) f r a c t i o n (compare F i g . 45 t o F i g . 4 6 ) . I n t e r e s t i n g l y j t h e HMG p r o t e i n s do not remain bound to e i t h e r the DNase..I o r m i c r o c o c c a l n u c l e a s e produced n u c l e o s o m a l m a t e r i a l i n t h e p r e s e n c e of 0.1 M N a C l . A l t h o u g h t h e nucleosome f r a c t i o n s c o n t a i n nucleosomes w i t h l i n k e r DNA 183 (.1.e. dinucleosomes) t o which t h e HMG p r o t e i n s a r e s u p p o s e d l y bound (162), t h e HMG p r o t e i n s a r e .:. a p p a r e n t l y u n a b l e to b i n d to t h i s r e g i o n i n t h e p r e s e n c e of 0.1 M N a C l . As HMG p r o t e i n s a r e n o t r e l e a s e d from DNase I d i g e s t e d n u c l e i ( F i g . 4 3 ) , t h e y p r o b a b l y remain bound t o t h e l i n k e r DNA at i o n i c s t r e n g t h s l e s s t h a n 0.1. ( i i ) Q u a n t i t a t i o n of t h e a c e t y l a t e d h i s t o n e s p e c i e s  a s s o c i a t e d w i t h n u c l e o s o m a l s u b t r a c t i o n s DNase I d i g e s t e d n u c l e i were e x t r a c t e d w i t h B u f f e r D c o n t a i n i n g 0.1 M NaCl f o l l o w e d by e x t r a c t i o n w i t h B u f f e r D c o n t a i n i n g 0.4 M NaCl to y i e l d s u p e r n a t a n t f r a c t i o n s SSI and SS4T, r e s p e c t i v e l y . The SSI (DNase I) f r a c t i o n was f u r t h e r f r a c t i o n a t e d on a B i o - G e l A-0.5m column e q u i l i b r a t e d w i t h 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and 0.4 M N a C l . The column p r o f i l e o b t a i n e d was s i m i l a r t o t h e p r o f i l e shown i n F i g . 46. The h i s t o n e a s s o c i a t e d w i t h t h e nucleosomes ( f r a c t i o n s . 13 t o 22) from the column f r a c t i o n -a t e d SSI (DNase I) f r a c t i o n ^ and t h e h i s t o n e s a s s o c i a t e d with, t h e SS4T (DNase I) f r a c t i o n were a c i d - e x t r a c t e d , and s e p a r a t e d on an a c i d - u r e a g e l i n t h e f i r s t d i mension f o l l o w e d by e l e c t r o -184 p h o r e s i s on a 15% p o l y a c r y l a m i d e - SDS g e l i n the second d i m e n s i o n . As seen i n F i g u r e 48A, a l l h i s t o n e f r a c t i o n s a r e s e p a r a t e d from each o t h e r i n t h i s system. V i s u a l I n s p e c t i o n o f t h e photographed g e l suggests t h a t t h e l e v e l s of t h e a c e t y l a t e d H4 s p e c i e s , A„ and A,, r e l a t i v e t o A 2 3 o and A^ a r e h i g h e r f o r the SSI (.DNase I) f r a c t i o n ( F i g . 48A) t h a n f o r t h e SS4T (DNase I) f r a c t i o n ( F i g . 48B). H i s t o n e s H2A and H2B appear to be s i m i l a r f o r both f r a c t i o n s , but h i s t o n e H3 a s s o c i a t e d w i t h t h e SSI (DNase I) f r a c t i o n a p pears to c o n t a i n lower l e v e l s of a c e t y l a t e d s p e c i e s than t h e H3 a s s o c i a t e d w i t h the SS4T ('DNase I) f r a c t i o n . An u n i d e n t i f i e d component of s l i g h t l y .lower.: m o b i l i t y t han ;H4, d e s i g n a t e d 12K, m i g r a t e s between A^ and A^., and i s b a r e l y v i s i b l e i n t h e photograph o f t h i s g e l ( F i g . 48A and B) f o r b o t h f r a c t i o n s , SSI and SS4T (DNase I ) . Another f a i n t , u n i d e n t i f i e d component, 14K, i s o b s e r v e d m i g r a t i n g ahead of H2B f o r both, f r a c t i o n s . The 28K and 29K components a s s o c i a t e d w i t h the SS4T (DNas.e.I) f r a c t i o n can be seen m i g r a t i n g s l i g h t l y f a s t e r t h a n HI ( F i g . 48B).. Both: o f t h e s e components: c o m i g r a t e w i t h t h e a c e t y l a t e d s p e c i e s of H3 i n t h e f i r s t d i m e n s i o n . 185 FIG. 48. Two-dimensional PAGE separation of histones from salt-extracted nucleosome fractions. The f i r s t (horizontal) dimension consisted of an acid-urea gel (194) and the second (vertical) dimension consisted of the polyacrylamide SDS gel system of Laemmli (226) as modified by Weintraub et a l . (5). The prepara-tion of the f i r s t dimension gel for electrophoresis on the second dimension gel was as described in the legend to Fig. 33. (A) Histones from the nucleo-somal fraction of a SSI (DNase I) fraction fractionated on a Bio-Gel A-0.5m column as described in the legend to Fig. 46. (B) Histones from SS4T (DNase I) fraction obtained by extracting DNase I digested nuclei that had been previously extracted with Buffer D containing 0.1 M NaCl, with Buffer D con-taining 0.4 M NaCl. AQ, unacetylated H4; A]_, A2, A3 and A4, mono-, d i - , t r i - and tetra-acetylated H4, respectively. 186 Baaed on t h e p e r c e n t a g e o f A^^Q abs.prbing m a t e r i a l a s s o c i a t e d with, t h e SSI and SS2 (DNase I) f r a c t i o n s ( T a b l e X I X ) , t h e s e f r a c t i o n s a r e p o s s i b l y t h e s u b s e t s o f t h e SSI ( m i c r o c o c c a l ) f r a c t i o n (see S e c t i o n I I I b ( l ) ) . T h e r e f o r e , i n s t e a d o f i n i t i a l l y e x t r a c t i n g DNase I d i g e s t e d n u c l e i w i t h B u f f e r D c o n t a i n i n g 0.1 M NaCl, B u f f e r D c o n t a i n i n g 0.2 M NaCl was used t o y i e l d a s u p e r n a t a n t d e s i g n a t e d SS2T. The SS2T (DNase I) was f u r t h e r f r a c t i o n a t e d on a B i o - G e l A-0.5m column e q u i l i b r a t e d w i t h 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and 0.4 M NaCl ( F i g . 4 9 ) . The monomer f r a c t i o n (17 t o 21) was p o o l e d , and the h i s t o n e s were a c i d - e x t r a c t e d . H i s t o n e s from u n d i g e s t e d n u c l e i ( c o n t r o l ) were a l s o a c i d -e x t r a c t e d . H i s t o n e samples from t h e SS2T (DNase I) monomer f r a c t i o n , c o n t r o l and SSI ( m i c r o c o c c a l ) * were a n a l y z e d on a c i d - u r e a g e l s , and t h e h i s t o n e p a t t e r n s examined f o r d i f f e r e n c e s i n t h e e x t e n t of m o d i f i c a t i o n ( F i g . 5 0 ) . The l e v e l s o f t h e a c e t y l a t e d H4 s p e c i e s appear t o be s i m i l a r f o r t h e SS.2T (DNas.e I I and S.Sl. ( m i c r o c o c c a l ) f r a c t i o n s ( F i g . 50)_. A l s o , t h e c o n t e n t o f t h e a c e t y l a t e d H4 s p e c i e s a s s o c i a t e d with, t h e c o n t r o l sample i s lower than *The sample was p r e v i o u s l y p r e p a r e d f o r another experiment ( s e c t i o n I b ( 3 ) ) . 187 O <0 CM 2 0 FRACTION NO. 4 0 f r ™ l ± 0 ~ G e l t ° - 5 m ( 4 ° X 1 ' 5 m ) s e P ^ t i o n of nucleosomes obtained in B u S r " ^ * t r ° U t t e S t ± S ™ ^ w i t h 0.2 M NacT 188 O m in < FIG. 5Q. Gel scans, of hi s t o n e s . a n a l y z e d on a c i d - u r e a gels.. Histones. were a c i d - e x t r a c t e d from e i t h e r u n d i g e s t e d n u c l e i ( c l , nucleosomes; t h a t had been r e l e a s e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i . b.y B u f f e r D c o n t a i n -i n g 0.1 M NaCl (SSI (micrococcal),). o r mononucleosomes (SS2T (DNas;e 1)11 t h a t were p r e p a r e d by e x t r a c t i n g DNase I d i g e s t e d n u c l e i with: B u f f e r D c o n t a i n i n g 0.2 M NaCl and f r a c t i o n a t i n g t h e s o l u b i l i z e d f r a c t i o n on a B i o - G e l A-Q.5m column as: d e s c r i b e d i n t h e lege n d to F i g . 49. G e l s were s t a i n e d w i t h Coomassie b l u e and scanned i n a G i l f o r d s p e c t r o p h o t o m e t e r a t 550 nm. MN, m i c r o c o c c a l n u c l e a s e . A , u n a c e t y l a t e d H4; A^, A^, A^ and A^. d i - , t r i - and t e t r a - a c e t y l a t e a * H4, r e s p e c t i v e l y . mono-, 189 t h a t o f t h e SS2T (DNase I) and SSI ( m i c r o c o c c a l ) ; f r a c t i o n s . The e x t e n t o f H4 a c e t y l a t i o n f o r the d i f f e r e n t f r a c t i o n s was c a l c u l a t e d by d i v i d i n g t h e sum o f the A^ and s p e c i e s by t h e sum of t h e A q and A^ s p e c i e s ( T a b l e X X I ) . A g a i n , t h e c o n t e n t o f t h e H4 a c e t y l a t e d s p e c i e s i s s i m i l a r f o r t h e SS2T (DNase I) and SSI ( m i c r o c -c a l ) f r a c t i o n s , and t h e c o n t e n t o f t h e H4 a c e t y l a t e d s p e c i e s i s much h i g h e r f o r t h e s e f r a c t i o n s t h a n f o r t h e c o n t r o l sample. The t o t a l H4 p o p u l a t i o n ( c o n t r o l ) ( T a b l e XXI) has low l e v e l s o f the a c e t y l a t e d H.4 s p e c i e s s i m i l a r t o the SS6 (DNase I) f r a c t i o n ( T a b l e XX). The l e v e l s o f t h e a c e t y l a t e d H3 s p e c i e s a s s o c i a t e d w i t h t h e s e f r a c t i o n s appear t o d i f f e r . The h i g h l e v e l o f a c e t y l a t e d H3 s p e c i e s a s s o c i a t e d w i t h the c o n t r o l i s d e c e i v i n g as non-h i s t o n e p r o t e i n s , 28K and 29K, m i g r a t e i n t h i s r e g i o n . A l t h o u g h f r a c t i o n s . SS2T (DNase I) and SSI ( m i c r o c o c c a l ) do not c o n t a i n components 28K and 29K, the l e v e l o f a c e t y l a t e d H3 species, f o r f r a c t i o n SSI ( m i c r o c o c c a l l i s h i g h e r than t h a t o f f r a c t i o n SS.2T (DNase I) ( F i g . 50) . 190 TABLE XXI Q u a n t i t a t i o n o f A c e t y l a t e d S p e c i e s o f H i s t o n e H4 i n S a l t - E x t r a c t e d Nucleosome: F r a c t i o n s r e l e a s e d from e i t h e r M i c r o c o c c a l N u c l e a s e or DNase I D i g e s t e d N u c l e i H i s t o n e s p r e p a r e d from e i t h e r u n d i g e s t e d n u c l e i o r s a l t - e x t r a c t e d nucleosome f r a c t i o n s f o l l o w i n g m i c r o c o c c a l n u c l e a s e or DNase I d i g e s t i o n as d e s c r i b e d i n t h e l e g e n d t o F i g . 50. were s e p a r a t e d on a c i d - u r e a g e l s . The g e l s were s t a i n e d w i t h Coomassie blue, and scanned a t 550 nm u s i n g a G i l f o r d s p e c t r o p h o t o m e t e r . F r a c t i o n R a t i o of H4 A 2 + A 3 * A + A-o 1 c o n t r o l ( u n d i g e s t e d n u c l e i ) SSI ( m i c r o c o c c a l ) SS2T (DNase I) monomer 0.26 0.70 0.62 * Determined from t h e A 5 5 0 peak h e i g h t s o f t h e scanned H4 s p e c i e s . 191 To f u r t h e r r e s o l v e t h e p r o t e i n s , t h e samples, were e l e c t r o p h o r e s e s i n a second dimension on a 15% p o l y a c r y l a m i d e — SDS g e l ( F i g . 51). E x a m i n a t i o n of t h e SS2T (DNase I) f r a c t i o n and t h e SSI ( m i c r o c o c c a l ) f r a c t i o n s c o n f i r m s t h a t b o t h f r a c t i o n s c o n t a i n h i g h e r l e v e l s o f t h e a c e t y l a t e d H4 s p e c i e s than does t h e c o n t r o l which has t h e b u l k o f t h e H4 p r e s e n t i n t h e A q and forms. The l e v e l o f t h e a c e t y l a t e d H3 s p e c i e s a s s o c i a t e d w i t h t h e SS2T (DNase I) f r a c t i o n a p pears t o be lower than t h e l e v e l of t h e a c e t y l a t e d H3 s p e c i e s a s s o c i a t e d w i t h t h e SSI ( m i c r o c o c c a l ) o r c o n t r o l samples. A l s o , t h e t o t a l c o n t e n t of H3 appears t o be lower r e l a t i v e t o t h e o t h e r n u c l e o s o m a l h i s t o n e s f o r t h e SS2T (DNase I ) f r a c t i o n . Low l e v e l s of the a c e t y l a t e d H3 s p e c i e s were a l s o o b s e r v e d f o r t h e SSI (DNase I) f r a c t i o n ( F i g . 4 8 ) . S e v e r a l u n i d e n t i f i e d components a s s o c i a t e d w i t h t h e f r a c t i o n s were r e s o l v e d by t h i s two-d i m e n s i o n a l g e l system ( F i g . 51). The c o n t r o l sample c o n t a i n e d t h e 29K component n o r m a l l y a s s o c i a t e d with, t h e SS4 (DNase I or m i c r o c o c c a l ) f r a c t i o n . A l s o , a component t h a t m i g r a t e d s l i g h t l y f a s t e r than H2B and d e s i g n a t e d as 14K 1 9 2 11 f t ) MN n HZ-H2A / H2B - 1 4 K A 4 A 3 A 2 Aj A 0 ^ 1 4 K A 4 t t A 3 A 2 A i V - 1 4 K A 4 A 3 A 2 A, t» 1 K FIG. 51. Two-dimensional PAGE s e p a r a t i o n o f h i s t o n e s from s a l t e x t r a c t e d nucleosome f r a c t i o n s and from u n d i g e s t e d n u c l e i . The f i r s t ( h o r i z o n t a l ) dimension c o n s i s t e d o f an a c i d - u r e a g e l (194), and the second ( v e r t i c a l ) d i m e n s i o n c o n s i s t e d of the p o l y a c r y l a m i d e SDS g e l system o f Laemmli (226) as m o d i f i e d by Weintraub e t a l . (59) . The p r e p -a r a t i o n o f t h e f i r s t d i mension g e l f o r e l e c t r o p h o r e s i s on the second d i m e n s i o n g e l was as d e s c r i b e d i n the l e g e n d t o F i g . 33. (A) H i s t o n e s from the 0.1 M NaCl f r a c t i o n e x t r a c t e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i (SSI ( m i c r o c o c c a l ) ) ; (B) h i s t o n e s from u n d i g e s t e d n u c l e i ; (C) h i s t o n e s from mononucleosomes a s s o c i a t e d w i t h a f r a c t i o n p r e p a r e d by e x t r a c t i n g DNase I d i g e s t e d n u c l e i w i t h B u f f e r D c o n t a i n i n g 0.2 M NaCl as d e s c r i b e d i n t h e l e g e n d t o F i g . 49. Ag, u n a c e t y l a t e d H4; A-^ , k2, A3 and A4, mono-, d i - , t r i - and t e t r a - a c e t y l a t e d H4. MN, m i c r o c o c c a l n u c l e a s e . 193 was. seen. The 14K component was I d e n t i f i e d i n a l l t h r e e f r a c t i o n s . I n a d d i t i o n t o t h e 14K component, s e v e r a l o t h e r u n i d e n t i f i e d components were observed f o r both t h e SS2T (.DNase I) and SSI ( m i c r o c o c c a l ) f r a c t i o n s . . These components i n c l u d e d a p r o t e i n m i g r a t i n g ahead o f t h e s p e c i e s of H4 (11K) ., and s e v e r a l o t h e r p r o t e i n s p o s i t i o n e d between t h e A^ s p e c i e s of H4 and H2B. A l t h o u g h t h e n u c l e i were t r e a t e d w i t h t h e p r o t e a s e i n h i b i t o r PMSF, t h e p o s s i b i l i t y t h a t t h e s e components a r e p r o t e o l y t i c p r o d u c t s ( p o s s i b l y of H3) cannot be r u l e d o u t . 6. D e t e r m i n a t i o n of t h e degree of a c e t y l group i n c o r p o r a t i o n a s s o c i a t e d w i t h t h e s a l t - e x t r a c t e d  f r a c t i o n s The degree o f nucleosome a c e t y l group i n c o r p -o r a t i o n a s s o c i a t e d w i t h each f r a c t i o n i n a two hour p e r i o d i n v i t r o was determined by r a d i o a c t i v e l a b e l l i n g o f c e l l s u s p e n s i o n s . N u c l e i i s o l a t e d from t r o u t t e s t i s c e l l s l a b e l l e d w i t h [ l l*C] a c e t a t e were d i g e s t e d w i t h DNas.e I and s u b s e q u e n t l y t r e a t e d w i t h i n c r e a s i n g s a l t c o n c e n t r a t i o n s . . The h i s t o n e s from f r a c t i o n s , SSI, SS2 and SS4 (DNase I), were s e p a r a t e d on -15% po l y a c r y l a m i d e - S D S gels-, and t h e i r s p e c i f i c a c t i v i t i e s - i n terms o f cpm/Ac;c.nr were determined 194 ( T a b l e XXII)_. F o r H4 t h e degree of a c e t a t e i n c o r p o r a t i o n . i n c r e a s e d w i t h t h e I n c r e a s i n g NaCl c o n c e n t r a t i o n used i n t h e s e q u e n t i a l e x t r a c t i o n s . Thus, t h e l a b e l l i n g o f H4 a c e t y l groups does not c o r r e s p o n d to t h e l e v e l s o f H.4 a c e t y l a t i o n determined by scans o f s t a i n e d a c i d / u r e a g e l s o r two d i m e n s i o n a l g e l s . The r e a s o n s f o r t h i s a p p a r e n t d i s c r e p a n c y a r e not e n t i r e l y c l e a r . The s i m p l e s t e x p l a n a t i o n would be t h a t h i s t o n e H4 a s s o c i a t e d w i t h t h e SSI f r a c t i o n c o n s t i t u t e s a s u b p o p u l a t i o n o f m o l e c u l e s w h i c h under-goes a c e t y l group t u r n o v e r a t a r e l a t i v e l y s l b ^ r a t e ( r e f e r p. 107) . The combined r e s u l t s suggest t h a t low s a l t e x t r a c t i o n (0.1 M and 0.2 M NaCl) o f DNase I d i g e s t e d n u c l e i r e l e a s e s a p o p u l a t i o n o f nucleosomes t h a t c o n t a i n i n c r e a s e d l e v e l s o f t h e h i g h l y a c e t y l a t e d s p e c i e s o f H4, and, p o s s i b l y , low l e v e l s of t h e a c e t y l a t e d H3 s p e c i e s from n u c l e a s e s e n s i t i v e r e g i o n s of c h r o m a t i n . A l s o , t h e nucleosome s u b t r a c t i o n a s s o c i a t e d with: SSI and SS2 (DNase I.) f r a c t i o n s , p o s s i b l y c o n s t i t u t e s u b p o p u l a t i o n s o f t h e nucleosomes a s s o c i a t e d with: SSI ( m i c r o c o c c a l ) . Furthermore, t h e i n t e r - n u c l e o s o m a l l i n k e r DNA o f t h e DNase I s e n s i t i v e 195 TABLE X X I I S p e c i f i c A c t i v i t i e s of Histones. H3 and H4 i n S a l t - E x t r a c t e d  Nucleosome F r a c t i o n s H i s t o n e s l a b e l l e d w i t h [ l l fC] a c e t a t e were e x t r a c t e d from s a l t - e l u t e d nucleosome f r a c t i o n s o f DNase I d i g e s t e d n u c l e i . The h i s t o n e s were s e p a r a t e d on 15% p o l y a c r y l a m i d e SDS g e l s c o n t a i n i n g 0.6% N, N ' - d i a l -l y l t a r t a r d i a m i d e as c r o s s - l i n k e r . The g e l s l i c e s were scanned a t 550 nm a f t e r s t a i n i n g w i t h Coomassie blue., and counted a f t e r s o l u b i l i z a t i o n . S a l t - E x t r a c t e d S p e c i f i c A c t i v i t y o f H i s t o n e F r a c t i o n F r a c t i o n H3 H4 _ 3 _ 1!*C c p m / A 5 5 0 x 10 S S 1 1.09 1.55 s s 2 1.30 1.88 SS4 1.58 1 > 7 5 196 regions; o f c h r o m a t i n may c o n t a i n n o n h i s t o n e p r o t e i n s with: t h e predominant p r o t e i n s b e i n g HMG-T1, HMG-T2 and HMG-T3.' DNase I d i g e s t i o n o f mononucleosome E a r l y i n the c o u r s e o f t h i s p r o j e c t , t h e p o s s i b i l i t y t h a t mononucleosomes c o n t a i n i n g h i g h l e v e l s o f t h e a c e t y l a t e d h i s t o n e s p e c i e s might be more s e n s i t i v e t o DNase I a t t a c k was examined.• The [ 1' tC] - a c e t a t e and [ 3H] - l y s i n e l a b e l l e d mononucleosomes were d i g e s t e d w i t h DNase I . However, a t t h a t time i t was not r e a l i z e d t h a t [ 1 4 C ] a c e t a t e might not l a b e l a l l nucleosome sub-f r a c t i o n s e q u a l l y ( s e c t i o n I I I b ( 6 ) ) . L a b e l l e d mononucleosomes were d i g e s t e d w i t h DNase I to 50% a c i d s o l u b i l i t y and t h e d i g e s t p r o d u c t s f r a c t i o n a t e d on a B i o -Gel A-0.5m column e q u i l i b r a t e d w i t h 10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA and 6 M u r e a a t 4°C ( F i g . 52B). The 6 M u r e a was added t o produce a more " l o o s e l y " a s s o c i a t e d nucleosome, and i n t h i s way, perhaps, t h e more e x t e n s i v e l y d i g e s t e d nucleosomes might be more s u s c e p t i b l e t o d i s s o c i a t i o n . U n d i g e s t e d l a b e l l e d mono-nucleosomes were f r a c t i o n a t e d on a B i o - G e l A-0.5m column ( F i g . 52A). The u n d i g e s t e d mononucleosomes r e s o l v e d , as. one peak w i t h maximal A 2 g Q a b s o r b i n g m a t e r i a l and r a d i o a c t i v e c o u n t s e l u t i n g a t f r a c t i o n 35. The DNase I d i g e s t e d mononucleosomes, r e s o l v e d as two A^^Q - a b s o r b i n g peaks ( F i g . 52B). 197 1 .2 0.8 0.4 o ID 1.2 0.8 0.4 B u A d o :TV-\\\ 6 0 4 0 2 0 m b E a 6 0 ^ 4 0 2 0 12 8 b E Q. o 3 0 FRACTION NO. 6 0 FIG. 52. B i o - G e l A-0.5m column (90.x 1.0 cm) f r a c t i o n a t i o n bf .DNase I d i g e s t e d mononucleosomes. (A) Mononucleosomes (.10 A ^ Q ) , t h a t were i s o l a t e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i as d e s c r i b e d i n t h e le g e n d t o F i g . 6, l a b e l l e d w i t h [ 1 1 +C] a c e t a t e and I 3H] l y s i n e were a p p l i e d t o a B i o - G e l A-Q..5m column e q u i l i b r a t e d w i t h 10 mM T r i s - H C l , pH. 7,5, 0.7 mM EDTA and 6 M u r e a . (B) Mononucleosomes (. 20 A ^ ) , t h a t were p r e p a r e d as d e s c r i b e d as above, l a b e l l e d w i t h [ l t +C] a c e t a t e and [ 3H] l y s i n e were d i g e s t e d w i t h DNase I u n t i l 5Q% of t h e DNA was a c i d s o l u b l e . The d i g e s t s were a p p l i e d t o a Bio^-Gel A-Q.5m column e q u i l i b r a t e d with. 10 mM T r i s - H C l , pH. 7,5, 0.7 mM EDTA and 6 M u r e a . R a d i o a c t i v i t y was determined as d e s c r i b e d i n t h e l e g e n d t o F i g . 7. 198 The s p e c i f i c a c t i v i t y ( 1 1 +C cpm/A^g) of t h e nucleosome peak f r a c t i o n ( f r a c t i o n 35) was dete r m i n e d f o r each p r o f i l e ( T a b l e X X I I I ) . The s p e c i f i c a c t i v i t y o f t h e s u r v i v i n g nucleosomes from t h e d i g e s t i s much h i g h e r than t h a t of t h e u n d i g e s t e d nucleosomes. A l s o , t h e 3H cpm/A„,„ r a t i o remains 2oU c o n s t a n t f o r b o t h f r a c t i o n s w h i l e t h e lL{C cpm/A^^Q r a t i o d e c r e a s e s f o r t h e d i g e s t e d f r a c t i o n . These r e s u l t s suggest t h a t t h e c o n t e n t of h i s t o n e s , as measured by [ 3 H ] - l y s i n e label.' bound t o n u c l e o s o m a l DNA, remains c o n s t a n t but t h a t t h e n u c l e o -somes c o n t a i n i n g a c e t y l a t e d h i s t o n e s ([ l l + C ] - a c e t a t e ) a r e s e l e c t -i v e l y d i g e s t e d . E x a m i n a t i o n o f t h e column p r o f i l e o f t h e d i g e s t e d mononucleosomes shown i n F i g u r e 52B f u r t h e r s t r e n g t h e n s t h e s u g g e s t i o n . The h i s t o n e s e l u t i n g a f t e r t h e n u c l e o s o m a l peak c o n t a i n an enrichment o f a c e t y l a t e d h i s t o n e s : - t h a t i s , t h e 3H/ l l fC r a t i o d e c r e a s e s d r a m a t i c a l l y a f t e r the n u c l e o s o m a l peak. T h i s , experiment has been/repeated w i t h two other, p r e p a r - ; :... a t i o n s o f l a b e l l e d mononucleosomes, and each y i e l d e d i d e n t i c a l r e s u l t s . However, one p a r t i c u l a r p r e p a r a t i o n o f l a b e l l e d mononucleosomes d i d not demonstrate t h e i n c r e a s e i n t h e 3H/ 1 I +C r a t i o o f t h e n u c l e o s o m a l peak a f t e r DNase I d i g e s t i o n . The re a s o n f o r t h i s r e s u l t i s u n c l e a r . The h i s t o n e s e l u t i n g a f t e r t h e n u c l e o s o m a l peak i n F i g . 52B were p o s s i b i l y bound t o DNA fragments r a t h e r than f r e e i n s o l u t i o n . N - e t h y l [ 3H] maleimide (NEM) was used t o measure 199 TABLE .XXIII S p e c i f i c A c t i v i t y o f Hlatones; A s s o c i a t e d with: Mononucleosomes  B e f o r e and A f t e r DNase I D i g e s t i o n Mononucleosomes and DNase I d i g e s t e d mononucleosomes l a b e l l e d with, b o t h [ l l fC] a c e t a t e and [ 3H] l y s i n e were f r a c t i o n a t e d on B i o - G e l A-0.5m columns, and t h e r a d i o a c t i v i t y o f t h e peak f r a c t i o n s ( f r a c t i o n 35) d e t e r m i n -ed a s d e s c r i b e d i n t h e l e g e n d t o F i g . 52. F r a c t i o n 35 S p e c i f i c A c t i v i t y o f F r a c t i o n R a t i o 3H cpm/A o c n 260 C ° P m / A 2 60 3 r c P m / l l t c c P m U n d i g e s t e d mononucleosomes 32,688 7,912 4 1 DNase I d i g e s t e d mononucleosomes 33,959 5,106 6.6 200 t h e degree o f a c c e s s i b i l i t y o f t h e t h i o l group i n the H3 m o l e c u l e (191). Mononucleosomes were d i g e s t e d by DNase I t o 50% a c i d s o l u b i l i t y f o l l o w e d by r e a c t i o n w i t h NEM i n t h e p r e s e n c e o f e i t h e r a low i o n i c s t r e n g t h . b u f f e r (10 mM T r i s - H C l , pH 7.5, 0.7 mM EDTA), 6 M u r e a , 0.5 M NaCl or a c o m b i n a t i o n of 6 M u r e a and 0.5 M N a C l . U n d i g e s t e d nucleosomes ( c o n t r o l ) were t r e a t e d i n a s i m i l a r manner ( T a b l e XXIV). The r e a c t -i v i t y o f t h e H3 t h i o l groups o f t h e c o n t r o l and d i g e s t e d nucleosomes i n e i t h e r T r i s / E D T A , 6 M u r e a o r 0.5 M N a C l was ex t r e m e l y low. Maximal r e a c t i v i t y o f t h e H3 t h i o l groups f o r b o t h t h e c o n t r o l and nucleosomes was o b t a i n e d i n 6 M u r e a and 0.5 M N a C l . Thus, t h e H3 s u l f h y d r y l groups a r e f u l l y exposed i n t h e p r e s e n c e of 0.5 M NaCl and 6 M u r e a , but t h e t h i o l groups a r e . p r o t e c t e d by h i s t o n e - h i s t o n e i n t e r a c t i o n s and/or histone-DNA i n t e r a c t i o n s i n t h e p r e s e n c e of T r i s / E D T A , 6 M u r e a , or 0.5 M N a C l . As t h e t h i o l groups of t h e d i g e s t e d n u c l e o -somes d i d not e x h i b i t a g r e a t e r r e a c t i v i t y t o NEM i n t h e p r e s e n c e o f 6 M u r e a than t h a t o f t h e c o n t r o l , the h i s t o n e s t r a i l i n g a f t e r t h e n u c l e o s o m a l peak ( F i g . 52B) a r e p r o b a b l y bound to DNA: - t h a t i s , DNase I d i g e s t i o n o f nucleosomes.. f o l l o w e d by exposure o f t h e d i g e s t e d nucleosomes t o 6 M urea;-., does not l e a d t o t h e r e l e a s e o f f r e e h i s t o n e s . Simpson (178) has r e p o r t e d t h a t nucleosome c o r e p a r t i c l e s c o n t a i n i n g h i g h l e v e l s ; of the a c e t y l a t e d H3 and H4 s p e c i e s a r e d i g e s t e d by DNase I at a r a t e e q u i v a l e n t t o t h a t o f c o r e 201" TABLE XX TV N - E t h y l [ 3H] Maleimide R e a c t i v i t y , of the:'Cyst e i n y l Residue  of H i s t o n e H3 A s s o c i a t e d w i t h E i t h e r Mononucleosomes or DNase I D i g e s t e d Mononucleosomes i n V a r i o u s  D e n a t u r i n g S o l u t i o n s Mononucleosomes were p r e p a r e d as d e s c r i b e d i n t h e l e g e n d t o F i g . 6j, and d i g e s t e d w i t h DNase I as d e s c r i b e d i n " M a t e r i a l s and Methods". R e a c t i o n of t h e nucleosomes ( d i g e s t e d or u n d i g e s t e d ) w i t h N - e t h y l [ 3H] maleimide was as d e s c r i b e d . i n " M a t e r i a l s and Methods". U n d i g e s t e d DNase I D i g e s t e d S o l u t i o n Mononucleosomes Mononucleosomes 3H cpm T r i s / E D T A 173 186 6 M u r e a 356 132 0.5 M NaCl 222 221 6 M u r e a and 0.5 M NaCl 3,104 2,854 202 p a r t i c l e s , c o n t a i n i n g much, lower l e v e l s o f ..the a c e t y l a t e d H3 and H4 s p e c i e s . Thus, t h e c o n t e n t s o f a c e t y l a t e d h i s t o n e s d i d not a l t e r t h e r a t e of d i g e s t i o n o f t h e c o r e p a r t i c l e . How-ever, t h e DNA s i t e , 60 n u c l e o t i d e s from t h e 5' end of t h e nucleosome c o r e p a r t i c l e , was more s u s c e p t i b l e t o DNase I d i g e s t i o n f o r t h o s e p a r t i c l e s c o n t a i n i n g t h e i n c r e a s e d l e v e l s o f t h e a c e t y l a t e d H3 and H4 s p e c i e s . The e v i d e n c e s u g g e s t s t h a t a c e t y l a t i o n o f t h e a m i n o - t e r m i n a l p o s i t i o n o f t h e h i s t o n e s r e s u l t s i n l o s s o f p r o t e c t i o n from DNase I a t t a c k a t t h i s DNA s i t e . The p r e s e n c e o f a p e r t u r b i n g agent such as ure a might p o s s i b l y e n a b l e t h e more e x t e n s i v e l y n i c k e d nucleosomes c o n t a i n -i n g e n r i c h e d l e v e l s o f the a c e t y l a t e d h i s t o n e s p e c i e s t o r e a d i l y d e n a t u r e . P r e l i m i n a r y r e s u l t s p r o v i d e some support f o r t h i s p o s s i b i l i t y . I d e a l l y , t h e experiment s h o u l d be r e p e a t e d u s i n g a c i d - u r e a g e l s t o examine t h e c o n t e n t o f t h e a c e t y l a t e d h i s t o n e s p e c i e s i n s t e a d o f u s i n g r a d i o a c t i v e l a b e l s . Summary A l t h o u g h DNase I d i g e s t s b o t h i n t e r - and i n t r a n u c l e o s o m a l DNA a t a p p r o x i m a t e l y e q u i v a l e n t r a t e s , nucleosomes can s t i l l be o b t a i n e d from t h e DNase I d i g e s t e d n u c l e i . When the s i n g l e -s t r a n d e d DNA fragments a s s o c i a t e d w i t h t h e mononucleosomal o r p o l y n u c l e o s o m a l f r a c t i o n were examined on d e n a t u r i n g p o l y a c r y l -amide g e l s , both, nucleosome f r a c t i o n s were found t o c o n t a i n fragments t h a t were m u l t i p l e s , o f t e n bases a p a r t . In a d d i t i o n , t h e p o l y n u c l e o s o m a l f r a c t i o n s c o n t a i n e d fragments g r e a t e r t h a n 203 140 bases.. The c o n t e n t o f a c e t y l a t e d h i s t o n e s p e c i e s was s i m i l a r f o r b o t h mono- and polynucleosomes. although, the c o n t e n t o f t h e a c e t y l a t e d H3 s p e c i e s appeared low f o r t h e mononucleosome f r a c t i o n . Both f r a c t i o n s c o n t a i n e d low l e v e l s o f t h e a c e t y l -a t e d H4 s p e c i e s ( F i g . 38). T h i s r e s u l t i s e x p e c t e d as t h e nucleosomes t h a t a r e r e l e a s e d i n t o s o l u t i o n by l y s i n g m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i w i t h EDTA,.. a r e r e p r e s e n t -a t i v e o f t h e nucleosomes a s s o c i a t e d with, t h e b u l k o f c h r o m a t i n ( F i g . 4 4 ) : - t h a t i s , t h e m a j o r i t y o f nucleosomes a s s o c i a t e d w i t h c h r o m a t i n c o n t a i n low l e v e l s o f t h e h i g h l y a c e t y l a t e d H4 s p e c i e s ( A 2 and A 3 ) ( T a b l e XIX). F r a c t i o n a t i o n o f t h e nucleosome p o p u l a t i o n i n t o sub-f r a c t i o n s was made p o s s i b l e by e x t r a c t i n g DNase I d i g e s t e d n u c l e i w i t h s e q u e n t i a l l y i n c r e a s i n g s a l t c o n c e n t r a t i o n s . R e s u l t s from t h e s e experiments suggested t h a t the i n t e r -n u c l e o s o m a l l i n k e r DNA o f DNase I s e n s i t i v e r e g i o n s o f c h r o m a t i n may c o n t a i n HMG-T1, HMG-T2, HMG-T3 and low l e v e l s of o t h e r n o n h i s t o n e proteins.. A l s o , t h e nucleosomes i n t h e s e r e g i o n s c o n t a i n h i g h l y a c e t y l a t e d H4 and p o s s i b l y low. l e v e l s of t h e a c e t y l a t e d H3 species:. In a d d i t i o n t o the s e l e c t i v e e x c i s i o n of t h e nucleosomes: c o n t a i n i n g h i g h l y a c e t y l a t e d H4, p r e l i m i n a r y e v i d e n c e s u g g e s t s t h a t nucleosomes c o n t a i n i n g h i g h l y a c e t y l a t e d 204 h i s t o n e s a r e more s u s c e p t i b l e t o i n t e r n a l DNase I a t t a c k t h a n nucleosomes c o n t a i n i n g low l e v e l s o f the a c e t y l a t e d h i s t o n e s p e c i e s . Weintraub and Groudine (133) have demonstrated t h a t t r a n s c r i p t i o n a l l y competent genes e x h i b i t an i n c r e a s e d s u s c e p t i b i l t y t o DNase I . The r e s u l t s may be i n t e r p r e t e d as i n d i c a t i n g an a l t e r a t i o n i n nucleosome s t r u c t u r e o r pac k a g i n g a t t r a n s c r i p t i o n a l l y competent l o c i . H i s t o n e a c e t y l a t i o n (178 - 182) and HMG p r o t e i n s (137) have been i m p l i c a t e d i n p r o d u c i n g a DNase I s e n s i t i v e c h r o m a t i n s t r u c t u r e . The r e s u l t s suggest t h a t h i s t o n e a c e t y l a t i o n , p r i n c i p a l l y o f H4 : and n o n h i s t o n e p r o t e i n s , such, as HMG-T1', HMG-T2"and HMG-T3'might be i n v o l v e d i n m a i n t a i n i n g t h e DNase I s e n s i t i v e c o n f o r m a t i o n of c h r o m a t i n . 204a, PART C - EFFECT OF SODIUM N-BUTYRATE ON HISTONE ACETYLATION 205. RESULTS Sodium b u t y r a t e i n m i l l i m o l a r c o n c e n t r a t i o n s has been r e p o r t e d to have a wide v a r i e t y o f m o r p h o l o g i c a l and p h y s i o l o g i c a l e f f e c t s on mammalian c e l l s grown i n t i s s u e c u l t u r e . Furthermore, i t appears t h a t most of t h e o b s e r v e d changes a r e r e a d i l y r e v e r s i b l e once t h i s s h o r t c h a i n f a t t y a c i d has been removed from t h e c e l l c u l t u r e medium. ( F o r a r e c e n t r e v i e w of t h i s s u b j e c t , see P r a s a d and S i n h a ( 2 3 1 ) ) . Of a l l t h e r e p o r t e d e f f e c t s of b u t y r a t e on c u l t u r e d c e l l s , p erhaps t h e most i n t e r e s t i n g have been t h o s e l i n k i n g low c o n c e n t r a t i o n s o f t h i s f a t t y a c i d t o t h e i n d u c t i o n o f F r i e n d e r y t h r o l e u k e m i c c e l l s , c a u s i n g t h e s e c e l l s to d i f f e r e n t i a t e i n t o n o n d i v i d i n g , h e m o g l o b i n - s y n t h e s i z i n g o r t h o c h r o m a t o p h i l i c n o r m o b l a s t - l i k e c e l l s (232). D u r i n g t h i s b u t y r a t e i n d u c t i o n , t h e h i s t o n e s , p a r t i c u l a r l y H3 and H4, become h i g h l y a c e t y l a t e d (175) i n p a r a l l e l w i t h a d e c r e a s e i n t h e r a t e of DNA r e p l i c a t i o n (233). The re a s o n s why b u t y r a t e causes i n d u c t i o n o f c e l l u l a r d i f f e r e n t i a t i o n a r e unknown. S i n c e , however, a c o n s i d e r a b l e body of e x p e r i m e n t a l e v i d e n c e has suggested t h a t a c e t y l a t i o n o f h i s t o n e s may have an important r o l e i n e i t h e r t h e c o n t r o l o f c h r o m a t i n t r a n s c r i p t i o n a l a c t i v i t y (234) o r c h r o m a t i n assembly ( 3 2 ) , i t was of c o n s i d e r a b l e i n t e r e s t t o e l u c i d a t e the b i o c h e m i c a l mechanisms u n d e r l y i n g t h e s e e f f e c t s of b u t y r a t e . The f o l l o w i n g s e t of experiments was done i n c o l l a b o r a t i o n w i t h Dr. Reeves who m a i n t a i n e d t h e c e l l l i n e s (except t h e hamster BHK-21 c e l l l i n e which was m a i n t a i n e d by Dr. R i c h a r d s o n ) , t r e a t e d t h e c e l l l i n e s w i t h b u t y r a t e 206 (ex c e p t t h e hamster BHK-21 c e l l l i n e ) and l a b e l l e d t h e t i s s u e c u l t u r e s and w i t h Dr. E. P. M. Candido who i s o l a t e d t h e h i s t o n e s from a l l t h e c e l l l i n e s except t h e hamster BHK-21 c e l l l i n e . B o t h Dr. R. Reeves and Dr. E. P. M. Candido performed the a s s a y s of h i s t o n e a c e t y l a s e and d e a c e t y l a s e a c t i v i t y a s s o c i a t e d w i t h e r y t h r o l e u k e m i c c e l l s . I . E f f e c t s of B u t y r a t e on E r y t h r o l e u k e m i c C e l l H i s t o n e s F i g u r e 53 shows t h e p r o f i l e s o f h i s t o n e s e x t r a c t e d from F r i e n d . e r y t h r o l e u k e m i c c e l l s ( s u b l i n e FrC18 o b t a i n e d from clone' 745A) and s e p a r a t e d on a c i d - u r e a g e l s by e l e c t r o p h o r e s i s . I t i s e v i d e n t from t h i s p hotograph t h a t i n e r y t h r o l e u k e m i c c e l l s t h a t have been exposed to 5 mM b u t y r a t e f o r 24 h ( g e l s l o t B) , t h e h i s t o n e s , p a r t i c u l a r l y H3 and H4, have become v e r y h i g h l y a c e t y l a t e d compared w i t h t h e h i s t o n e s from c o n t r o l c e l l s n ot exposed to b u t y r a t e ( g e l s l o t A ) . The mono-, d i - and t r i a c e t y l a t e d s p e c i e s of H4 a r e p a r t i c u l a r l y n o t i c e a b l e i n t h i s photograph, but i t can a l s o be seen t h a t H3 has i n c r e a s e d i t s p o p u l a t i o n o f a c e t y l a t e d s p e c i e s r e l a t i v e t o t h e c o n t r o l c e l l s . T h i s p o i n t i s v > . ' . " ' more c l e a r l y e v i d e n t i n t h e o p t i c a l scans shown i n F i g u r e 54. I n „ c o n t r a s t to t h e i n c r e a s e d a c e t y l a t i o n o f H3 and H4, H2A and H2B do not seem t o e x h i b i t i n c r e a s e d l e v e l s of a c e t y l a t i o n i n b u t y r a t e - t r e a t e d F r i e n d c e l l s . These: f i n d i n g s are: i n agreement w i t h those: r e p o r t e d by Ingram's l a b o r a t o r y . ( 1 7 5 , 233). In a d d i t i o n , two f u r t h e r p o i n t s can be made from F i g u r e s 53 and 54. The f i r s t i s t h a t i n e r y t h r o l e u k e m i c c e l l s exposed f o r 24 h o r more to 2% d i m e t h y l s u l f o x i d e (DMSO), a known i n d u c e r of e r y t h r o i d 207 A B C FIG. 53. Histone patterns of Friend erythroleukemic cel l s . (A) Electrophoretic pattern of histones from control, unindu?.ed cells; (B) Histone pattern from ce l l s induced with butyrate (5 mM for 24 h. ); (C) Histone pattern from cells induced with DMSO (2% for 96 h ). Chromat and histone preparation were as described in "Materials and Methods" (Part C Section III). Electrophoresis was carried out in 15% acid-urea gels (194). The gel was stained with Coomassie blue. 208 FIG. O p t i c a l scans o f h i s t o n e s on a c i d - u r e a g e l s (A) H i s t o n e s from uninduced F r i e n d e r y t h r o l e u k e m i c c e l l s ; (B) h i s t o n e s from F r i e n d c e l l s i n d u c e d w i t h b u t y r a t e (5 mM f o r 24,h~p); (0) h i s t o n e s from t r o u t t e s t i s c e l l s f o r comparison. The p o s i t i o n s of.H4 and H3 a r e i n d i c a t e d . Peak 1 i s u n a c e t y l a t e d H4, and peaks 2 to 4 r e p r e s e n t mono-, d i - and t r i a c e t y l a t e d H4, r e s p e c t i v e l y . The g e l s were s t a i n e d w i t h Coomassie b l u e and scanned a t 550 nm i n a G i l f o r d s p e c t r o p h o t o m e t e r . 209 d i f f e r e n t i a t i o n f o r t h e s e c e l l s (.19.8), no i n c r e a s e i n t h e amount of h i s t o n e a c e t y l a t i o n i s observed ( F i g . 53, g e l C) r e l a t i v e t o u n t r e a t e d c o n t r o l c e l l s . The second p o i n t t o be made i s e v i d e n t from t h e g e l scan o f t r o u t t e s t i s h i s t o n e s shown i n F i g u r e 54C. When t h e scan i s compared w i t h t h a t of h i s t o n e s i s o l a t e d from b u t y r a t e - t r e a t e d F r i e n d c e l l s shown above i t i n F i g u r e 54B, i t i s r a t h e r s t r i k i n g t h a t the amount o f a c e t y l a t i o n found i n t r o u t t e s t i s H3 and H4 h i s t o n e s i s analogous t o t h a t found i n t h e same h i s t o n e s from b u t y r a t e - t r e a t e d c e l l s . The p o s s i b l e s i g n i f i c a n c e of t h i s o b s e r v a t i o n i s t r e a t e d f u r t h e r i n the D i s c u s s i o n . I I . E f f e c t s of B u t y r a t e on Other C e l l L i n e s To i n v e s t i g a t e t h e e f f e c t o f b u t y r a t e t r e a t m e n t on h i s t o n e a c e t y l a t i o n i n o t h e r c u l t u r e c e l l t y p e s , a number of permanent v e r t e b r a t e c e l l l i n e s w i t h r a d i c a l l y d i f f e r e n t m o r p h o l o g i e s and growth c h a r a c t e r i s t i c s were s t u d i e d . The c e l l l i n e s t h a t M e r e t e s t e d f o r r e s p o n s e to b u t y r a t e treatment were: 3T3 c e l l s , a s t r o n g l y c o n t a c t -i n h i b i t e d c e l l l i n e o f embryonic mouse f i b r o b l a s t o r i g i n (199) ; BHK-21 c e l l s , a f i b r o b l a s t - l i k e c e l l l i n e t h a t i s p a r t i a l l y c o n t a c t -i n h i b i t e d , d e r i v e d from the c u l t u r e of k i d n e y c e l l s from baby S y r i a n hamsters (200); IRC8 c e l l s . , a lymphoid leukemia c e l l l i n e o b t a i n e d from F i s h e r rats, ( l i n e 334) t h a t can grow e i t h e r i n s u s p e n s i o n c u l t u r e o r as. a s c i t e s : tumors: when i n j e c t e d i n t o i s o l o g o u s h o s t s (201); and X58 c e l l s , p a r t i a l l y c o n t a c t - i n h i b i t e d e p i t h e l o i d c e l l s o b t a i n e d from w i l d - t y p e embryos o f the amphibian Xeridpus l a e v i s (202). F i g u r e s 210 55 and 56 show t h e r e s u l t s o f ex p e r i m e n t s i n wh i c h h i s t o n e s i s o l a t e d from u n t r e a t e d c o n t r o l c u l t u r e s o f t h e s e c e l l l i n e s a r e compared w i t h h i s t o n e s i s o l a t e d from p a r a l l e l c u l t u r e s t r e a t e d f o r about 24 h w i t h 5 mM sodium'.butyrate. From t h e photograph o f t h e s t a i n e d h i s t o n e s s e p a r a t e d by e l e c t r o p h o r e s i s on a c i d - u r e a g e l s shown i n F i g u r e 55, i t i s app a r e n t t h a t a l l of t h e t i s s u e c u l t u r e c e l l l i n e s t e s t e d , r e g a r d l e s s o f t h e i r morphology o r growth c h a r a c t e r i s t i c s , responded to b u t y r a t e t r e a t m e n t by a c c u m u l a t i n g i n c r e a s e d l e v e l s o f a c e t y l a t e d h i s t o n e s H3 and H4. I t s h o u l d be no t e d , however, t h a t t h e amount o f b u t y r a t e - i n d u c e d h i s t o n e a c e t y l a t i o n v a r i e d from c e l l l i n e .to c e l l l i n e . Thus, t h e embryonic amphibian c e l l l i n e from Xenopus. (X58) showed t h e low e s t r e s p o n s e t o b u t y r a t e w i t h r e s p e c t t o h i s t o n e a c e t y l a t i o n , whereas the r a t c e l l l i n e IRC8 not o n l y e x t e n s i v e l y a c e t y l a t e d h i s t o n e s H3 and H4 but a l s o appeared t o have a c e t y l a t e d h i s t o n e s H2A and H2B ( F i g u r e 55, g e l s 3 and 4; F i g u r e 56, second p a n e l ) . A q u a n t i t a t i v . e , c o m p a r i s o n w i t h u n t r e a t e d c o n t r o l i c e l l h i s t o n e s r e v e a l s t h a t a f t e r 24 h a t 5 mM b u t y r a t e t r e a t m e n t , a maximum of 20 - 25% of h i s t o n e H4 i s l e f t u n a c e t y l a t e d i n t h e t r e a t e d IRC8 c e l l s , t h e r e m a i n i n g 75 - 80% of t h e H4 b e i n g i n t h e mono-, d i - , t r i - and perhaps t e t r a a c e t y l a t e d forms. A s i m i l a r amount of h i s t o n e H4 i s c o n v e r t e d t o a c e t y l a t e d forms i n b u t y r a t e - t r e a t e d F r i e n d e r y t h r o l e u k e m i c c e l l s ( F i g u r e 53, s l o t B; F i g u r e 54B; ( 1 7 5 ) ) . These r e s u l t s c o n f i r m and extend t h o s e o b t a i n e d w i t h human ca n c e r c e l l s ( H e l a c e l l s ; 175) and w i t h c h i c k embryo" f i b r o b l a s t c e l l ' s , ( 2 3 3 ) - . and i n d i c a t e t h a t t h e i n d u c t i o n o f h i s t o n e a c e t y l a t i o n by b u t y r a t e 211 X58 IRC8 3T3 BHK C BA C BA C BA C BA FIG. 55. E f f e c t o f b u t y r a t e on h i s t o n e a c e t y l a t i o n i n v a r i o u s c e l l t y p e s . C e l l s (see " M a t e r i a l s and Methods" ( P a r t C)) were exposed to 5 mM b u t y r a t e i n c u l t u r e f o r 24 h. Chromatin was prep a r e d and h i s t o n e s were e x t r a c t e d .as d e s c r i b e d i n " M a t e r i a l s and Methods". A l l samples were a n a l y z e d on a s i n g l e a c i d - u r e a 15% a c r y l a m i d e s l a b g e l (194). (C) C o n t r o l c e l l s grown i n t h e absence of b u t y r a t e ; (BA) c e l l s t r e a t e d w i t h 5 mM b u t y r a t e f o r 24 h; (X58) Xenopus l a e v i s embryonic c e l l s ; (IRC8) r a t a s c i t e s c e l l s ; (3T3) mouse f i b r o b l a s t s ; (BHK) baby hamster kidney c e l l s . 2 1 2 FIG. 56. O p t i c a l scans of histones from various c e l l , types analyzed on acid-urea acrylamide gels. Abbreviations are as i n the legend to Figure 55. For each pair , the top scan i s of histones from c o n t r o l c e l l s . Arrows indicate acetylated components which are increased i n amount i n c e l l s exposed to 5 mM butyrate for 24 h i , (lower scan). The gels were stained with Coomassie blue and scanned at 550 nm i n a G i l f o r d spectrophotometer. 213 t r e a t m e n t o f t i s s u e c u l t u r e c e l l s i s a g e n e r a l phenomenon. I t i s n o t dependent upon t h e degree o f c o n t a c t i n h i b i t i o n shown by the c e l l s , t h e r a t e of growth o r t h e growth c h a r a c t e r i s t i c s o f t h e c e l l s , n o r i s i t a f u n c t i o n o f t h e v e r t e b r a t e c l a s s from which t h e c e l l s a r e d e r i v e d . G i v e n t h e u b i q u i t y and c o n s e r v a t i o n o f t h e c o r e , s t r u c t u r e o f , t h e nucleosome and the g e n e r a l i t y of a c e t y l a t i o n of t h e c o r e h i s t o n e s H2A, H2B, H3:.and H4 (32, 227, 234), i t seems p r o b a b l e t h a t t h e mode of a c t i o n o f b u t y r a t e on h i s t o n e a c e t y l a t i o n i s s i m i l a r i n a l l v e r t e b r a t e c e l l t y p e s a f f e c t e d . I I I . H i s t o n e A c e t y l a s e A c t i v i t y i n T r o u t T e s t i s C e l l S u s p e n s i o n s and i n E r y t h r o l e u k e m i c C e l l L y s a t e s To examine t h e e f f e c t o f b u t y r a t e on h i s t o n e a c e t y l a s e a c t i v i t y , t h e r a t e s o f i n c o r p o r a t i o n o f r a d i o a c t i v e a c e t a t e i n t o h i s t o n e s i n e i t h e r t r o u t t e s t i s c e l l s u s p e n s i o n s , whole e r y t h r o l e u k e m i c c e l l homogenates o r i s o l a t e d e r y t h r o l e u k e m i c c e l l n u c l e i were measured. T r o u t t e s t i s c e l l s were l a b e l l e d w i t h [ 1 1 +C] a c e t a t e i n t h e p r e s e n c e o r absence o f 1 mM b u t y r a t e . A f t e r v a r i o u s l a b e l l i n g t i m e s , t h e h i s t o n e s from t h e c o n t r o l and b u t y r a t e - t r e a t e d c e l l s were i s o l a t e d and counted f o r r a d i o a c t i v i t y . E x a m i n a t i o n o f t h e r e s u l t s shown i n F i g u r e 57 s u g g e s t s b o t h t h e c o n t r o l and b u t y r a t e - t r e a t e d t r o u t t e s t i s c e l l s i n c o r p o r a t e d t h e [^C] a c e t a t e l a b e l i n t o h i s t o n e a t e q u i v a l e n t r a t e s . In o t h e r experiments, e i t h e r c o n t r o l e r y t h r o l e u k e m i c c e l l s o r c e l l s t r e a t e d f o r 24 h p r e v i o u s l y w i t h 5 mM b u t y r a t e were homo-21\ CO O 20 10 • • _.. - - * * * • • *• # # • 0 *' # 0 / y / / ° • •.•••*' • 3 4 8 12 TIME(hours) FIG. 57. H i s t o n e a c e t y l a s e a c t i v i t y i n s u s p e n s i o n s of t r o u t t e s t i s c e l l C e l l s u s p e n s i o n s from e i t h e r c o n t r o l c e l l s ( • - • ) or b u t y r a t e t r e a t e d c e l l s ( o - o) were assayed u s i n g [ 1 4 C ] a c e t a t e as s u b s t r a t e as d e s c r i b e d i n " M a t e r i a l s and Methods" (See P a r t C S e c t i o n V ) . 215 g e n i z e d i n an a p p r o p r i a t e b u f f e r s o l u t i o n , and [ 3H] - a c e t y l - C o A was. added t o the d i s r u p t e d c e l l s : . A f t e r v a r i o u s , l a b e l l i n g p e r i o d s , t h e h i s t o n e s were i s o l a t e d from t h e s e m i x t u r e s and counted f o r r a d i o -a c t i v i t y . The r e s u l t s of one such experiment a r e shown i n F i g u r e 58. From t h i s graph, i t i s apparent t h a t b o t h c o n t r o l c e l l s and b u t y r a t e -t r e a t e d c e l l s a c e t y l a t e h i s t o n e s a t t h e same r a t e s when they a r e d i s r u p t e d and s u p p l i e d w i t h r a d i o a c t i v e a c e t y l - C o A . S i m i l a r r e s u l t s were o b t a i n e d f o r t h e i n c o r p o r a t i o n of a c e t a t e i n t o i s o l a t e d n u c l e i f rom t h e s e two c e l l p o p u l a t i o n s ( d a t a not shown). Thus, b u t y r a t e does not a c t i v a t e h i s t o n e a c e t y l a s e s . The same c o n c l u s i o n was r e ached by Hagopian et a l . (233) from s t u d i e s of i h v i v o r a t e s of a c e t a t e i n c o r p o r a t i o n . IV. H i s t o n e D e a c e t y l a s e A c t i v i t y i n C e l l L y s a t e s G iven the r e s u l t s of t h e above exper i m e n t s , t h e p o s s i b i l i t y t h a t b u t y r a t e i n m i l l i m o l a r c o n c e n t r a t i o n s might i n h i b i t t h e a c t i v i t y o f h i s t o n e d e a c e t y l a s e s i n c r u d e e x t r a c t s o f e r y t h r o l e u k e m i c c e l l s was examined. The experiments i n v o l v e d t h e a d d i t i o n of [ 3H] - a c e t a t e l a b e l l e d h i s t o n e s ( o b t a i n e d from i n v i v o l a b e l l e d e r y t h r o l e u k e m i c c e l l s ) t o c r u d e l y s a t e s of e i t h e r c o n t r o l c e l l s or b u t y r a t e - i n d u c e d c e l l s . . , and m o n i t o r i n g t h e r e l e a s e of I 3H] - a c e t i c a c i d a f t e r a p e r i o d of i n c u b a t i o n a t 22°C. The r e s u l t s : o f such an experiment a r e shown i n F i g u r e 59.. Crude c e l l l y s a t e s . from e i t h e r c o n t r o l c e l l s , or c e l l s ; p r e v i o u s l y grown i n 5 mM b u t y r a t e i s p r e s e n t In t h e l y s a t e s . T h i s 216 CO I o Q_ U < 1 L < I z 10 20 TIME , min 30 FIG. 58. H i s t o n e a c e t y l a s e a c t i v i t y i n c e l l - f r e e e x t r a c t s o f F r i e n d e r y t h r o l e u k e m i c c e l l s . C e l l l y s a t e s from e i t h e r c o n t r o l c e l l s ( • - • ) or b u t y r a t e - i n d u c e d c e l l s ( » • ) were assayed u s i n g [ 3 H ] - a c e t y l - C o A as s u b s t r a t e (see " M a t e r i a l s and Methods" P a r t C S e c t i o n V ) . 217 CO I Q. U u 3 < LU U T 1 Z co C O N T R O L b I N D U C E D e a II FIG. 59. H i s t o n e deacfetylase a c t i v i t y i n c e l l - f r e e e x t r a c t s of F r i e n d e r y t h r o l e u k e m i c c e l l s . C e l l l y s a t e s from e i t h e r c o n t r o l or b u t y r a t e - i n d u c e d c e l l s were' i n c u b a t e d w i t h 50,000 cpm of [ 3H] a c e t a t e , i n v i v o l a b e l l e d h i s t o n e . R e a c t i o n volumes were 140 y l , and i n c u b a t i o n was f o r 4 h •• at 22 C. [ 3H] a c e t i c a c i d r e l e a s e was measured by e t h y l a c e t a t e e x t r a c t i o n . , as d e s c r i b e d i n " M a t e r i a l s and Methods". (a and d) B o i l e d l y s a t e s of c o n t r o l and induced c e l l s , r e s p e c t i v e l y ; (b and e) t o t a l d e a c e t y l a s e a c t i v i t y i n l y s a t e s o f c o n t r o l and induced c e l l s , r e s p e c t i v e l y ; (c and f ) d e a c e t y l a s e a c t i v i t y i n t h e p r e s e n c e of 5 mM b u t y r a t e f o r c o n t r o l and ind u c e d c e l l l y s a t e s , r e s p e c t i v e l y . 218 c l e a r l y i n d i c a t e s , t h a t growth, o f e r y t h r o l e u k e m i c c e l l s , i n b u t y r a t e does not r e s u l t i n t h e l o s s of d e a c e t y l a t i n g enzymes:. Rather, t h e b u t y r a t e must somehow i n h i b i t t h e e x i s t i n g enzymes; t h i s , i s c o n f i r m e d by t h e f i n d i n g t h a t 5 mM b u t y r a t e added t o l y s a t e s o f e i t h e r c o n t r o l o r b u t y r a t e - g r o w n c e l l s g r e a t l y i n h i b i t s h i s t o n e d e a c e t y l a t i o n . T h i s i s a l s o shown i n F i g u r e 59. The e x t e n t of i n h i b i t i o n o f h i s t o n e d e a c e t y l a t i o n by b u t y r a t e i s v i r t u a l l y i d e n t i c a l i n t h e two l y s a t e s : — t h a t i s , 82% i n h i b i t i o n f o r c o n t r o l c e l l s and 83% f o r b u tyrate-grown c e l l s . DISCUSSION The experiments t h a t have been d e s c r i b e d demonstrate t h a t m i l l i -m olar c o n c e n t r a t i o n s o f b u t y r a t e i n h i b i t h i s t o n e d e a c e t y l a s e a c t i v i t i e s i n a r e v e r s i b l e manner, and t h a t i t i s p r o b a b l y t h i s i n h i b i t i o n which, l e a d s t o h y p e r a c e t y l a t i o n o f h i s t o n e s i n . v i v o d u r i n g b u t y r a t e t r e a t m e n t o f v e r t e b r a t e c e l l s i n c u l t u r e . The r e v e r s i b l e n a t u r e o f t h i s i n h i b i t i o n a g r e e s w i t h t h e f i n d i n g s o f o t h e r i n v e s t i g a t o r s t h a t t h e e f f e c t s o f b u t y r a t e on h i s t o n e a c e t y l a t i o n a r e r e v e r s i b l e i n v i v o (185, 233). S e v e r a l r e c e n t r e p o r t s a g r e e w i t h t h e c o n c l u s i o n t h a t b u t y r a t e e x e r t s i t s e f f e c t s by i n h i b i t i o n o f t h e h i s t o n e deacetylaseCs) (.176, 177, 185). In a d d i t i o n , Cousens. e t a l . (185) have r e c e n t l y demonstrated t h a t b u t y r a t e i s , a n o n c o m p e t i t i v e i n h i b i t o r of h i s t o n e d e a c e t y l a s e , and t h e authors, suggest t h a t b u t y r a t e may be a c t i n g as a t i g h t b i n d i n g d e t e r g e n t In i n h i b i t i n g t h e d e a c e t y l a s e ( s ) . 219 The o n l y o t h e r r e p o r t e d s i t u a t i o n i n which, b u t y r a t e a p p a r e n t l y acts, t o i n h i b i t enzyme a c t i v i t i e s , d i r e c t l y i s i n t h e case o f l i v e r c e l l s , where low c o n c e n t r a t i o n s : of t h i s ; f a t t y a c i d i n h i b i t t h e g l u c o s e - p h o s p h o r y -l a t i n g enzymes,, g l u c o k i n a s e and hexokinas.e, when added t o c e l l homogenates (231). In t h i s c a s e , i t i s not known whether the e f f e c t on t h e enzymes, i s r e v e r s i b l e , a l t h o u g h g i v e n t h e g e n e r a l r e v e r s i b i l i t y o f most b u t y r a t e e f f e c t s on c u l t u r e d c e l l s (231), t h i s seems t o be p r o b a b l e . Thus., t h e f i n d i n g s r e p o r t e d h e r e f o r t h e r e v e r s i b l e i n h i b i t i o n o f d e a c e t y l a s e a c t i v i t y by b u t y r a t e may be i n d i c a t i v e o f a much l a r g e r c l a s s o f r e v e r s i b l e enzyme i n h i b i t i o n s brought about i n c u l t u r e d v e r t e b r a t e c e l l s by low c o n c e n t r a t i o n s o f t h i s f a t t y a c i d . B u t y r a t e causes an a c c u m u l a t i o n o f a c e t y l a t e d h i s t o n e s i n t h e c h r o m a t i n of a l l v e r t e b r a t e c e l l t y p e s examined, but t h e e x t e n t of t h i s e f f e c t v a r i e s w i t h d i f f e r e n t c e l l t y p e s . L a r g e i n c r e a s e s i n a c e t y l a t e d H3 and H4 o c c u r i n F r i e n d e r y t h r o l e u k e m i c c e l l s , 3T3 c e l l s and IRC8 c e l l s ; moderate i n c r e a s e s a r e observed i n BHK c e l l s , and. t h e lowest r e s p o n s e was o b s e r v e d i n Xenopus X58 c e l l s . The r a t lymphoid c e l l l i n e IRC8 gave t h e most d r a m a t i c o v e r a l l response., i n which a c e t y l a t e d s p e c i e s p r o b a b l y c o r r e s p o n d i n g t o H2A and H2B a l s o accumulated. Whether t h e s e i n d i v i d u a l r e s p o n s e s t o b u t y r a t e a r e r e l a t e d t o t h e t y p e s o f h i s t o n e m e t a bolism n o r m a l l y o c c u r r i n g i n t h e s e c e l l t y p e s i s unknown. I t is. i n t e r e s t i n g t o n o t e t h a t t h e h i s t o n e s . of c o n t r o l IRC8 c e l l s show, a prominent band i n the 220 p o s i t i o n o f t r i a c e t y l a t e d H4,, as seen i n F i g u r e 56. I t i s tempting to s p e c u l a t e t h a t t h i s may be r e l a t e d to t h e e x t e n s i v e h y p e r a c e t y l a t i o n i n t h e s e c e l l s i n r e s p o n s e to b u t y r a t e . I f h i s t o n e a c e t y l a t i o n w i t h i n a nucleosome c o r e p a r t i c l e were c o o p e r a t i v e : — t h a t i s , i f m o d i f i c a t i o n of H4 t o t h e t r i a c e t y l l e v e l promoted a c e t y l a t i o n o f H2A and H2B — t h i s might account f o r t h e o b served r e s u l t s . The d i f f e r e n t i a l e f f e c t of b u t y r a t e on d e a c e t y l a t i o n i n d i c a t e s t h a t t h e r e i s no s i m p l e feedback c o n t r o l on h i s t o n e a c e t y l a t i o n : — t h a t i s , an i n c r e a s e i n t h e l e v e l of a c e t y l a t e d h i s t o n e s does n o t l e a d to a d e c r e a s e i n t h e a c t i v i t y o f a c e t y l a t i n g enzymes. I t i s p r o b a b l e t h a t t h e two p r o c e s s e s a r e c o n t r o l l e d i n d e p e n d e n t l y and a r e c o u p l e d to o t h e r key n u c l e a r e v e n t s . Hagopian e t a l . (233) have found t h a t i n h i b i t i o n o f DNA s y n t h e s i s and t h e a c c u m u l a t i o n of a c e t y l a t e d H4 f o l l o w s i m i l a r t i m e c o u r s e s i n b u t y r a t e -t r e a t e d H e l a c e l l s , and they have suggested t h a t t h e two p r o c e s s e s may be l i n k e d . Whereas i n h i b i t i o n o f DNA s y n t h e s i s may be a n e c e s s a r y c o n d i t i o n f o r t h e a c c u m u l a t i o n of a c e t y l a t e d h i s t o n e s , however, i t c l e a r l y i s not s u f f i c i e n t - s i n c e t h e i n h i b i t i o n o f DNA s y n t h e s i s by o t h e r means has v e r y l i t t l e e f f e c t on h i s t o n e a c e t y l a t i o n (227) . The c o n v e r s e , however, may be t r u e : — t h a t i s , h i s t o n e a c e t y l a t i o n may l e a d to d e c r e a s e d DNA s y n t h e s i s , as suggested by Hagopian e t a l . (233). T h i s p o s s i b i l i t y i s more d i f f i c u l t t o t e s t ; s i n c e l e v e l s o f h i s t o n e a c e t y l a t i o n a r e not r e a d i l y m a n i p u l a t a b l e o t h e r t h a n by b u t y r a t e . I t i s i n t e r e s t i n g t o n o t e t h a t Z l a n t a n o v a and S w e t l y (240) have r e p o r t e d t h a t t h e h i s t o n e s from e r y t h r o l e u k e m i c mouse 221 s p l e e n c e l l s ( F r i e n d c e l l s ) t r e a t e d w i t h n - b u t y r a t e a r e s y n t h e s i z e d a t a time when DNA s y n t h e s i s i s b l o c k e d , i n d i c a t i n g t h a t t h e s y n t h e s i s o f h i s t o n e s has been u n c o u p l e d from t h a t o f DNA i n t h e t r e a t e d c e l l s . A l s o i n t h i s c o n t e x t , i t i s i n t e r e s t i n g to n o t e t h a t t r o u t t e s t i s h i s t o n e s n o r m a l l y c o n t a i n h i g h l e v e l s o f a c e t y l a t e d h i s t o n e s as seen i n F i g u r e 54. The predominant c e l l t y p e s i n t h i s t i s s u e have g e n e r a t i o n t i m e s o f 3 - 7 days (10) .. and t h e r e f o r e may spend a s h o r t e r p r o p o r t i o n o f t h e i r c y c l e i n S phase. Thus t h e h i g h e r l e v e l s o f h i s t o n e a c e t y l a t i o n may c o r r e s p o n d w i t h the l o n g p e r i o d s d u r i n g which DNA s y n t h e s i s i s q u i e s c e n t i n t h e s e c e l l s . H i s t o n e a c e t y l a t i o n i s a complex p r o c e s s , and a t l e a s t two d i f f e r e n t phases can be d i s t i n g u i s h e d : a c e t y l a t i o n of de novo s y n t h e s i z e d h i s t o n e s and a c e t y l a t i o n o f " o l d " o r preformed h i s t o n e s . L o u i e and Dixon (10) showed t h a t n e w l y - s y n t h e s i z e d H4 i s r a p i d l y a c e t y l a t e d i n c e l l s from d e v e l o p i n g t r o u t t e s t i s , and R u i z - C a r i l l o e t a l . (234) and J a c k s o n e t a l . (227) have shown t h a t n e w l y - s y n t h e s i z e d h i s t o n e s a r e r a p i d l y a c e t y l a t e d and d e a c e t y l a t e d i n duck e r y t h r o b l a s t s and HTC c e l l s , r e s p e c t i v e l y . These r e s u l t s argue f o r a r o l e o f h i s t o n e a c e t y l a t i o n and d e a c e t y l a t i o n i n the assembly o f new c h r o m a t i n . On t h e o t h e r hand, t r o u t s p e r m a t i d c e l l s , which a r e c o m p l e t e l y i n a c t i v e i n DNA s y n t h e s i s , a c e t y l a t e h i s t o n e s a t s i g r i i f . i g a n t r a t e s a t a time when t h e protamine! replacement p r o c e s s i s o c c u r r i n g ( 1 1 ) ; mature a v i a n e r y t h r o c y t e s , a l s o i n a c t i v e i n DNA s y n t h e s i s , s i m i l a r l y c a r r y out e x t e n s i v e h i s t o n e a c e t y l a t i o n (235), and a " l a t e " phase of H4 a c e t y l a -tion.) i n v o l v i n g preformed m o l e c u l e s o c c u r s i n duck e r y t h r o b l a s t s (234) . Thus, h i s t o n e a c e t y l a t i o n may a l s o be needed f o r post-S phase events i n t h e 222 c e l l c y c l e which, r e q u i r e changes, i n nucleosome c o n f o r m a t i o n , nucleosome s l i d i n g or h i s t o n e removal. The a v a i l a b i l i t y o f newly s y n t h e s i z e d and preformed h i s t o n e s has been shown to be d i f f e r e n t ; w i t h t h e newly s y n t h e s i z e d h i s t o n e s b e i n g more a v a i l a b l e t o t h e h i s t o n e a c e t y l t r a n s f e r a s e ( . s ) (.177). In a d d i t i o n , the a c c e s s i b i l i t y of t h e preformed h i s t o n e s t o t h e h i s t o n e a c e t y l t r a n s f e r a s e ( . s ) d i f f e r s markedly, p r o b a b l y , depending on t h e nucleosome environment i n which i t i s l o c a t e d (185). At t h i s t i m e , i t i s no t known i f b u t y r a t e a f f e c t s d e a c e t y l a t i o n o f b o t h de nOvo s y n t h e s i z e d and preformed h i s t o n e s o r o n l y one o f t h e s e c l a s s e s . In view o f t h e magnitude of t h e b u t y r a t e r e s p o n s e and. of t h e i n v i t r o d e a c e t y l a s e r e s u l t s , however, i t seems p r o b a b l e t h a t b o t h p r o c e s s e s a r e a f f e c t e d . In summary, t h e r e s u l t s r e p o r t e d h e r e i n d i c a t e t h a t b u t y r a t e t r e a t m e n t o f a wide v a r i e t y o f v e r t e b r a t e t i s s u e , c u l t u r e c e l l s l e a d s t o a d i r e c t i n h i b i t i o n o f h i s t o n e d e a c e t y l a s e enzyme a c t i v i t y . . . and t h a t t h i s e f f e c t i s r e a d i l y r e v e r s i b l e a f t e r removal of the f a t t y a c i d . T h i s r e v e r s i b l e e f f e c t o f b u t y r a t e on t h e d e a c e t y l a s e . enzymes o c c u r s b o t h i n v i v o and i n v i t r o . , and s h o u l d t h e r e f o r e p r o v i d e a u s e f u l t o o l f o r t h e f u r t h e r i n v e s t i g a t i o n o f t h e r o l e s t h a t h i s t o n e a c e t y l a t i o n may have i n n u c l e a r m e t a b o l i s m and chro m a t i n s t r u c t u r e . 223 CONCLUDING REMARKS The nuclease, sensitive regions of chromatin may be either newly replicated regions (148) or transcriptionally active regions (120, 133, 202), Seale (148) has reported that newly synthesized DNA was digested at a greater rate than the bulk of the DNA in Hela c e l l s . Alternatively, transcriptionally active regions are excised preferentially by nucleases since recent reports (139, 121, 122) demonstrate that active genes are cleaved into mononucleosomes more rapidly than is bulk chromatin by micrococcal nuclease or DNase II. In the present study, experiments have been described that suggest micrococcal nuclease selectively excises a population of mono-nucleosomes containing increased levels of newly synthesized histones. The possibility that micrococcal nuclease selectively excises nucleosomes containing newly synthesized DNA from chromatin was d i f f i c u l t to determine,, since the preferential micrococcal nuclease digestion of A/T rich regions may have produced mononucleosomes containing greater amounts of thymidine residues per DNA fragment length than that of polynucleosomes. Ideally, the experiment should be repeated by labelling trout testis c e l l s for a brief period with [3H] thymidine. As nucleosomes associated with nuclease sensitive regions of chromatin can be selectively solubilized by extracting micrococcal nuclease digested nuclei with, low concentrations of NaCl (0.1 M or 0.2 M} , the specific activity (.3K cpm/A2gQ) of the low. salt extracted fraction may be compared to the high, salt (0.4 and 0.6 M NaCl) extracted fraction which, represents: nucleosomes associated with, the bulk of chromatin (.190). Thus, the nuclease sensitivity of newly replicated chromatin regions 224 may be examined. Both m i c r o c o c c a l n u c l e a s e and d e o x y r i b o n u c l e a s e I I have been used s u c c e s s i v e l y t o f r a c t i o n a t e c h r o m a t i n i n t o t r a n s c r i p t i o n a l l y a c t i v e and i n a c t i v e r e g i o n s . (142, 122, 139). B o t h enzymes p r e f e r e n t i a l l y c l e a v e a c t i v e r e g i o n s by d i g e s t i o n between n u c l e o s o m a l c o r e p a r t i c l e s . I n t h e p r e s e n t s t u d y , t h e n u c l e i were d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e f o l l o w e d by t h e f o l l o w i n g m a n i p u l a t i o n s : 1. s e q u e n t i a l e x t r a c t i o n o f t h e n u c l e i w i t h i n c r e a s i n g c o n c e n t r a t i o n s of NaCl.(0.1, 0.2, 0.4 and 0.6 M N a C l ) , o r 2. l y s i s of t h e n u c l e i w i t h EDTA f o l l o w e d by the a d d i t i o n of NaCl ( f i n a l c o n c e n t r a t i o n , 0.1 M) to t h e s o l u b i l i z e d d i g e s t p r o d u c t s . The r e s u l t s i n d i c a t e t h a t the 0.1 M N a C l - s o l u b l e nucleosome s u b t r a c t i o n c o n t a i n s i n c r e a s e d l e v e l s o f h i g h l y a c e t y l a t e d H4 and t h a t the nucleosomes a r e a s s o c i a t e d w i t h n u c l e a s e s e n s i t i v e r e g i o n s o f c h r o m a t i n . Furthermore, the r e s u l t s suggest t h a t t h e i n t e r n u c l e o s o m a l l i n k e r DNA o f t h e n u c l e a s e s e n s i t i v e r e g i o n s of c h r o m a t i n may c o n t a i n i n c r e a s e d l e v e l s of HMG-Tl, HMG-T2 and HMG-T3 and p o s s i b i l y l e s s e r amounts of o t h e r n o n h i s t o n e p r o t e i n s . Levy W. and Dixon (142) have r e p o r t e d t h a t the 0.1 M NaCl s o l u b l e nucleosome s u b t r a c t i o n o b t a i n e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d t r o u t t e s t i s n u c l e i was e n r i c h e d 1 0 - f o l d f o r DNA sequences complementary to p o l y a d e n y l a t e d RNA. R e c e n t l y , Levy e t a l . ( 1 7 4 ) have r e p o r t e d a l s o t h a t nucleosomes a s s o c i a t e d w i t h n u c l e a s e s e n s i t i v e r e g i o n s o f c h r o m a t i n c o n t a i n h i g h l e v e l s of t h e a c e t y l a t e d H4 s p e c i e s . Thus, t h e r e s u l t s from t h e 225 p r e s e n t s t u d y a r e i n agreement w i t h t h o s e from Levy e t a l . (_174)_ and support t h e g e n e r a l Idea t h a t nucleosomes a s s o c i a t e d with: t r a n s c r i p t i o n a l l y competent c h r o m a t i n c o n t a i n h i g h l y a c e t y l a t e d H4. By d i g e s t i n g c h r o m a t i n w i t h d e o x y r i b o n u c l e a s e I I f o l l o w e d by f r a c t i o n a t i o n o f the p r o d u c t s w i t h 2 mM Mg , i t has been shown t h a t h i g h l y a c e t y l a t e d H 4 . i s a s s o c i a t e d w i t h a t r a n s c r i p t i o n a l l y a c t i v e c h r o m a t i n f r a c t i o n . The r e s u l t s suggest t h a t the 0.1 M NaCl - s o l u b l e nucleosome s u b f r a c t i o n o b t a i n e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i i s p r o b a b l y t h e same f r a c t i o n as o b t a i n e d from DNase I I d i g e s t e d c h r o m a t i n . I t i s i n t e r e s t i n g t o n o t e t h a t the p e r c e n t a g e o f t h e t o t a l DNA r e m a i n i n g s o l u b l e a f t e r d e o x y r i b o n u c l e a s e I I d i g e s t i o n and 2 mM Mg f r a c t i o n a t i o n o f c h r o m a t i n ( a p p r o x i m a t e l y 7%) c o r r e s p o n d s t o t h e p e r c e n t a g e of t o t a l DNA a s s o c i a t e d w i t h t h e 0.1 M NaCl - s o l u b l e f r a c t i o n o b t a i n e d from m i c r o c o c c a l n u c l e a s e d i g e s t e d n u c l e i . D e o x y r i b o n u c l e a s e I, an enzyme which d i g e s t s both, i n t r a - and i n t e r n u c l e o s o m a l DNA, s e l e c t i v e l y d e s t r o y s a c t i v e genes. Weintraub and Groudine (133) have suggested t h a t t h e nucleosomes a s s o c i a t e d w i t h t r a n s c r i p t i o n a l l y competent c h r o m a t i n r e g i o n s a r e i n an a l t e r e d c o n f o r m a t i o n . R e s u l t s from t h e p r e s e n t s t u d y suggest t h a t b o t h h i s t o n e a c e t y l a t i o n and n o n h i s t o n e chromosomal p r o t e i n s may be i n v o l v e d i n t h e maintenance of the a l t e r e d s t r u c t u r e . DNase I d i g e s t e d n u c l e i were e x t r a c t e d w i t h s e q u e n t i a l l y i n c r e a s -i n g c o n c e n t r a t i o n s o f NaCl ("0.1, 0.2, 0.4 and 0.6 M). The r e s u l t s i n d i c a t e t h a t low s a l t e l u t i o n (0.1 o r 0.2 M NaCl) of DNase I d i g e s t e d n u c l e i r e l e a s e s 226 a p o p u l a t i o n o f nucleosomes c o n t a i n i n g i n c r e a s e d levels., o f h i g h l y a c e t y l a t e d H4 and, perhaps, d e c r e a s e d l e v e l s o f a c e t y l a t e d H3 species;. In a d d i t i o n , t h e i n t e r n u c l e o s o m a l l i n k e r DNA o f n u c l e a s e s e n s i t i v e r e g i o n s o f c h r o m a t i n may c o n t a i n HMG-T1, HMG-T2 and HMG.-T3 and many o t h e r n o n h i s t o n e chromosomal p r o t e i n s . The r e s u l t s suggest t h a t the-low s a l t e l u t e d nucleosome s u b f r a c t i o n i s a s s o c i a t e d w i t h c h r o m a t i n t h a t i s i n a t r a n s c r i p t i o n a l l y competent, d e o x y r i b o n u c l e a s e I s e n s i t i v e s t a t e . T h e r e f o r e , t h e accumulated r e s u l t s suggest t h a t nucleosomes c o n t a i n i n g h i g h l y a c e t y l a t e d H4 a r e . a s s o c i a t e d w i t h t r a n s c r i p t i o n a l l y competent c h r o m a t i n r e g i o n s . Furthermore, t h e i n t e r n u c l e o s o m a l l i n k e r DNA a s s o c i a t e d w i t h t h e s e r e g i o n s seems t o c o n t a i n l i t t l e , i f any, h i s t o n e H i and h i g h l e v e l s o f HMG-T1, HMG-T2, HMG-T3 and o t h e r n o n h i s t o n e chromosomal p r o t e i n s . The b u l k o f c h r o m a t i n , which i s t r a n s c r i p t i o n a l l y i n a c t i v e , i s a s s o c i a t e d , w i t h nucleosomes c o n t a i n i n g low l e v e l s of a c e t y l a t e d H4 ( i . e . u n a c e t y l a t e d and mo n o a c e t y l a t e d H4 a r e t h e major s p e c i e s ) , and t h e i n t e r -n u c l e o s o m a l l i n k e r DNA i s a s s o c i a t e d m a i n l y w i t h h i s t o n e HI. The t r a n s c r i p t i o n a l l y i n a c t i v e n u c l e o s o m a l f r a c t i o n i s a l s o d i s t i n g u i s h e d by the p r e s e n c e o f two n o n h i s t o n e chromosomal p r o t e i n s d e s i g n a t e d as 28K and 29K. U n l i k e t h e 0.1 NaCl s o l u b i l i z e d nucleosome s u b f r a c t i o n e l u t e d from m i c r o c o c c a l n u c l e a s e or DNase. I d i g e s t e d n u c l e i , t h e 2 mM Mg - s o l u b l e f r a c t i o n o b t a i n e d from DNase I I d i g e s t e d c h r o m a t i n c o n t a i n e d h i s t o n e H i and the u n i d e n t i f i e d p r o t e i n d e s i g n a t e d 29.K which, a r e both, n o r m a l l y a s s o c i a t e d w i t h t r a n s c r i p t i o n a l l y i n a c t i v e c h r o m a t i n . One 227 e x p l a n a t i o n f o r t h e above r e s u l t may b.e i n e f f i c i e n c y o f t h e e x t r a c t i o n p r o c e d u r e used. The e x t r a c t i o n p r o c e d u r e i n v o l v e d e t h a n o l p r e c i p i t a t i o n of an a c i d e x t r a c t which, In some i n s t a n c e s , s e l e c t i v e l y p r e c i p i t a t e d t h e l y s i n e - r i c h h i s t o n e s . S i n c e t h e d i s c o v e r y o f Riggs et a l . (.175) t h a t the a d d i t i o n o f low l e v e l s o f t h e s h o r t c h a i n f a t t y a c i d , sodium n - b u t y r a t e , t o H e l a or F r i e n d e r y t h r o l e u k a e m i a c e l l s r e s u l t s i n h y p e r a c e t y l a t i o n o f h i s t o n e s H3 and H4, sodium n - b u t y r a t e t r e a t m e n t o f c e l l s has been a u s e f u l t o o l f o r f u r t h e r i n v e s t i g a t i o n o f the r o l e s t h a t h i s t o n e a c e t y l a t i o n may have i n c h r o m a t i n s t r u c t u r e . C o n c u r r e n t l y w i t h o t h e r l a b o r a t o r i e s (.176, 177, 185), i t was found t h a t sodium n - b u t y r a t e i s an i n h i b i t o r o f h i s t o n e deacetylase(.s) and t h a t i t i s due t o t h e i n h i b i t i o n of t h i s enzyme(s) t h a t the a c e t y l a t e d H3 and H4 s p e c i e s accumulate. By u s i n g b u t y r a t e t o e l e v a t e t h e l e v e l s o f a c e t y l a t e d h i s t o n e s , Simpson (178), M a t h i s et a l . (179) and V i d a l i e t a l . (182) have r e p o r t e d t h a t c h r o m a t i n c o n t a i n i n g h y p e r a c e t y l a t e d h i s t o n e s i s d i g e s t e d by DNase I a t a g r e a t e r r a t e than c h r o m a t i n c o n t a i n i n g normal l e v e l s o f t h e a c e t y l a t e d h i s t o n e s . A l s o , a l t h o u g h t h e r a t e o f m i c r o c o c c a l n u c l e a s e d i g e s t i o n o f n u c l e i from sodium n - b u t y r a t e t r e a t e d H e l a c e l l s was i d e n t i c a l t o t h e r a t e o f d i g e s t i o n o f n u c l e i from u n t r e a t e d c e l l s (.178, 179), t h e nucleosomes c o n t a i n i n g t h e h i g h e s t l e v e l s o f a c e t y l a t e d h i s t o n e s were e x c i s e d from t h e t r e a t e d c h r o m a t i n p r e f e r e n t i a l l y (.178). Shewmaker et a l . (181) have r e p o r t e d t h a t t r e a t m e n t .of c h r o m a t i n with, a c e t y l a d e n y l a t e r e s u l t s i n c h e m i c a l h y p e r a c e t y l a t i o n o f t h e h i s t o n e s . C a l f thymus c h r o m a t i n t r e a t e d i n such a manner had an i n c r e a s e d s e n s i t i v i t y t o 228 -DNase I r e l a t i v e to t h a t o f u n t r e a t e d c h r o m a t i n . The p r e f e r e n t i a l DNase I d i g e s t i o n o f c h r o m a t i n r e g i o n s c o n t a i n i n g n a t u r a l l y r - o c c u r r I n g h y p e r a c e t y l a t e d h i s t o n e s has a l s o been demonstrated (180-183). Thus, the consensus of t h e above r e p o r t s i s t h a t h i s t o n e a c e t y l a t i o n might c o n v e r t c h r o m a t i n i n t o a t r a n s c r i p t i o n a l l y a c t i v e , d e o x y r i b o n u c l e a s e I s e n s i t i v e s t a t e . Recent experiments by Reeves and C s e r j e s i (184) have demonstrated t h a t sodium n - b u t y r a t e treatment o f F r i e n d e r y t h r o l e u k e m i c c e l l s l e a d s t o the s y n t h e s i s o f new RNA t r a n s c r i p t s and accompanying s y n t h e s i s of new n u c l e a r and c y t o p l a s m i c p r o t e i n s . These r e s u l t s p r o v i d e s t r o n g e v i d e n c e t h a t h i s t o n e a c e t y l a t i o n i s p r o b a b l y a p a r t of t h e r e q u i r e d b i o c h e m i c a l mechanisms f o r t h e e x p r e s s i o n o f a gene. In agreement w i t h t h e above r e c e n t r e p o r t s , t h e p r e s e n t r e s u l t s i n d i c a t e t h a t h i g h l y a c e t y l a t e d 114 i s i n v o l v e d i n m a i n t a i n i n g a r e g i o n o f c h r o m a t i n i n a t r a n s c r i p t i o n a l l y competent form. The r o l e o f H4 a c e t y l -a t i o n i n t r a n s c r i p t i o n a l l y competent c h r o m a t i n may be to re d u c e i n t e r a c t i o n s between c o r e p a r t i c l e s as suggested by W h i t l o c k and S t e i n ( 7 7 ) , Simpson (178) and P o s p e l o v e t a l . (236) and thus r e n d e r t h e extended c h r o m a t i n r e g i o n a c c e s s i b l e t o RNA polymerases. C o n v e r s e l y , u n a c e t y l a t e d H4 may be i n v o l v e d i n m a i n t a i n i n g h i g h e r l e v e l s o f compaction o f t h e c h r o m a t i n . I t would be i n t e r e s t i n g t o f u r t h e r examine t h e l e v e l s of c h r o m a t i n compaction f o r r e g i o n s a s s o c i a t e d w i t h e i t h e r h i g h o r low l e v e l s of the a c e t y l a t e d H4 s p e c i e s . A l t e n b u r g e r et a l . (115) have r e p o r t e d t h a t 229 d e o x y r i b o n u c l e a s e I I may r e c o g n i z e d i f f e r e n t c o n f o r m a t i o n s t a t e s , o f c h r o m a t i n . DNase I I d i g e s t i o n o f chr o m a t i n leads, to. a c l e a v a g e p a t t e r n w i t h a 2 0 0 - h u c l e o t i d e p a i r p e r i o d i c i t y . However, i f c h r o m a t i n was d i g e s t e d i n t h e p r e s e n c e o f 150 mM NaCl o r 1 mM C a C l ^ , a 1 0 0 - n u c l e o t i d e p a i r r e p e a t p a t t e r n appeared. The l a t t e r i o n i c c o n d i t i o n s a r e known t o c o n t r a c t c h r o m a t i n . Thus, i f c h r o m a t i n c o n t a i n i n g h i g h l y a c e t y l a t e d H4 i s i n an extended form, t h e low s a l t e x t r a c t e d f r a c t i o n (0.1 M NaCl) e l u t e d from DNase I I d i g e s t e d n u c l e i c o n t a i n i n g h i g h l y a c e t y l a t e d H4 s h o u l d p r e s e n t the 2 0 0 - n u c l e o t i d e p a i r r e p e a t p a t t e r n , w h i l e t h e h i g h s a l t e x t r a c t e d f r a c t i o n (0.4 M NaCl) c o n t a i n i n g low l e v e l s of t h e a c e t y l a t e d H4 s p e c i e s s h o u l d p r e s e n t t h e 1 0 0 - n u c l e o t i d e p a i r r e p e a t p a t t e r n . In a d d i t i o n t o t h e p r e s e n t r e s u l t s which suggest t h a t t h e n u c l e a s e s DNase I, DNase I I and m i c r o c o c c a l n u c l e a s e r e c o g n i z e t h e extended form of c h r o m a t i n c o n t a i n i n g h i g h l y a c e t y l a t e d H4, Gross and Simpkins (237) have r e p o r t e d t h a t t h e r e i s an a l t e r e d a c c e s s i b i l i t y o f h i s t o n e H4 t o e n z y m a t i c a l l y - or c h e m i c a l l y - c a t a l y z e d i o d i n a t i o n of c h r o m a t i n . I f i o d i n a t i o n o f c h r o m a t i n i s c a t a l y z e d t h r o u g h t h e use o f t h e l a r g e m o l e c u l a r weight enzyme (80,000 M.W.).;. l a c t o p e r o x i d a s e , t h e mono- and d i a c e t y l a t e d forms o f H4 a r e p r e d o m i n a n t l y l a b e l l e d . However, i f the i o d i n a t i o n r e a c t i o n i s c a t a l y z e d by c h l o r a m i n e T, t h e mono-, d i - , and u n a c e t y l a t e d H4 s p e c i e s a r e l a b e l l e d t o a p p r o x i m a t e l y t h e same e x t e n t . Thus, t h e a c e t y l a t e d s p e c i e s o f H.4 a r e p o s s i b l y a s s o c i a t e d with: c h r o m a t i n t h a t e x i s t s i n an extended form. Through, t h e a i d of h i s t o n e a c e t y l a t i o n , t h e t r a n s c r i p t i o n a l l y 230 competent c h r o m a t i n t h a t i s i n an extended s t a t e would be a c c e s s i b l e to RNA polymerases, r e g u l a t o r s and n u c l e a s e . DNase I I and m i c r o c o c c a l n u c l e a s e would d i g e s t i n t e r n u c l e o s o m a l DNA, and t h i s would r e l e a s e nucleosomes c o n t a i n i n g the t r a n s c r i p t i o n a l l y competent DNA sequences. However, DNase I would d i g e s t b o t h i n t e r - and i n t r a n u c l e o s o m a l DNA, and t h i s would r e s u l t i n t h e s e l e c t i v e d e s t r u c t i o n o f t r a n s c r i p t i o n a l l y competent gene s e q u e n c e s : — t h a t i s , DNase I would d i g e s t t h e DNA t o such an e x t e n t t h a t t h e d i g e s t e d DNA p r o d u c t s would be u n a b l e to h y b r i d i z e t o a s p e c i f i c cDNA probe. A c e t y l groups a r e i n c o r p o r a t e d i n t o b o t h newly s y n t h e s i z e d h i s t o n e s (10, 238) and p r e - e x i s t i n g , " o l d " h i s t o n e s . L o u i e and Dixon (10) have suggested t h a t t h e r o l e of H4 a c e t y l a t i o n i n assembly may be to reduce t h e p o s i t i v e charge d e n s i t y i n t h e N - t e r m i n a l p o r t i o n of t h e m o l e c u l e and a l l o w p r o p e r histone-DNA b i n d i n g . Woodland (238) has r e p o r t e d t h a t a l t h o u g h H4 i s a c e t y l a t e d b e f o r e assembly, H3 i s not a c e t y l a t e d b e f o r e assembly. Thus, H3 and H4 do not behave i n a s i m i l a r manner w i t h r e g a r d to t h e i r p a t t e r n of a c e t y l a t i o n b e f o r e assembly. The n u c l e a s e s e n s i t i v e f r a c t i o n s of c h r o m a t i n which have been shown i n t h e s e s t u d i e s t o c o n t a i n e l e v a t e d l e v e l s o f a c e t y l a t e d H4 c o u l d a l s o i n c l u d e newly r e p l i c a t e d c h r o m a t i n . T h i s i s a p o s s i b i l i t y because newly r e p l i c a t e d c h r o m a t i n as w e l l as t r a n s c r i p t i o n a l l y competent c h r o m a t i n has been found to be n u c l e a s e s e n s i t i v e (148). Thus, h i s t o n e a c e t y l a t i o n may a l s o be i n v o l v e d i n c h r o m a t i n r e p l i c a t i o n : — t h a t i s , t h e extended form of c h r o m a t i n m a i n t a i n e d by h i s t o n e a c e t y l a t i o n would be a c c e s s i b l e t o 231 DNA polymerases. I t Is p u z z l i n g t h a t t h e H i g h l y a c e t y l a t e d H4 a s s o c i a t e d with: extended c h r o m a t i n undergoes a c e t y l a t i o n a t a slow: r a t e : — t h a t i s , th e extended c h r o m a t i n form s h o u l d be h i g h l y a c c e s s i b l e t o h i s t o n e a c e t y l t r a n s f e r a s e s , and d e a c e t y l a s e s . R e c e n t l y , Reeves and Candido C23) have p r e s e n t e d i n t e r e s t i n g e v i d e n c e o f a d e a c e t y l a s e i n h i b i t o r a s s o c i a t e d w i t h DNase I s e n s i t i v e c h r o m a t i n . The a s s o c i a t i o n o f such an i n h i b i t o r w i t h t r a n s c r i p t i o n a l l y competent c h r o m a t i n would e x p l a i n the slow r a t e o f a c e t y l group t u r n o v e r , and t h e i n h i b i t i o n o f t h e d e a c e t y l a s e s , perhaps one s p e c i f i c f o r H4, would l e a d t o a l o c a l i z e d r e g i o n o f c h r o m a t i n c o n t a i n i n g h y p e r a c e t y l a t e d H4 m o l e c u l e s . A l t h o u g h h i s t o n e a c e t y l a t i o n i s i m p l i c a t e d i n i n d u c i n g and m a i n t a i n i n g a t r a n s c r i p t i o n a l l y f u n c t i o n a l s t a t e i n c h r o m a t i n , i t i s d o u b t f u l t h a t t h i s r e a c t i o n a l o n e i s i n v o l v e d i n t h e s e p r o c e s s e s . Non-h i s t o n e p r o t e i n s have a l s o been suggested as r e g u l a t o r s o f g e n e t i c a c t i v i t y . HMG-1, HMG-2, HMG-14 and HMG-17 i n mammals and HMG-T i n t r o u t a r e suggested t o be a s s o c i a t e d w i t h t r a n s c r i p t i o n a l l y a c t i v e r e g i o n s o f ch r o m a t i n (.137, 161, 162). HMG-Tl, HMG-T2, HMG-T3 and o t h e r n o n h i s t o n e p r o t e i n s (H6, u b i q u i t i n (.167))., i n c o n j u n c t i o n w i t h h i s t o n e may p l a y an impo r t a n t r o l e i n m a i n t a i n i n g a r e g i o n o f t r o u t t e s t i s c h r o m a t i n i n an extended form, a s t r u c t u r e p r o b a b l y n e c e s s a r y f o r t h e f u n c t i o n i n g o f an a c t i v e l y t r a n s c r i b i n g gene. -232 BIBLIOGRAPHY 1. Romberg, R.D. (1974) S c i e n c e 184, 868-871 2. A l l f r e y , V.G. (1977) I n : Chromatin and Chromosome S t r u c t u r e , Eds. H.J. L i and R. A. E c k h a r d t . , Academic P r e s s , New York: pp. 167-191 3. H a y a s h i , H., I w a i , K . , Johnson, J.D. and Bonner, J . (1977) J . Biochem. 82^, 503 4. S t r i c k l a n d , M., S t r i c k l a n d , W.N., Brandt , W.F. and Von H o l t , C. (1977) E u r . J . Biochem. 77_, 263-276 5. S t r i c k l a n d , W.N., S t r i c k l a n d , M., Brandt , W.F. and Von H o l t , C. (1977) Eur. J . Biochem. 77_, 277-286 6. Dixon, G.H., Candido, E.P.M., Honda, B.M., L o u i e , A . J . , Macleod A.R. and Sung, M.T. (1975) CIBA F o u n d a t i o n Symposium 28: The S t r u c t u r e and F u n c t i o n o f Chromatin, Ed. Wolstenholme, D. ( C h u r c h i l l and L i v i n g s t o n e , E d i n b u r g h and London), pp. 229-258 7. ' Moore, M., J a c k s o n , V., S e a l y , L. and C h a l k l e y , R. (1979) B i o c h i m . B i o p h y s . A c t a 561, 248-260 8. J a c k s o n , V., S h i r e s , A., C h a l k e l y , R. and Granner, D.K. (1975) J . B i o l . Chem. 250, 4856-4863 9. Keruabon, A., P a r e l l o , J . and Mery, J . (1979) FEBS L e t t . 98, 152-156 10. L o u i e , A . J . and Dixon, G.H. (1972) P r o c . N a t l . Acad. S c i . U.S.A. 69_, 1975-1979 11. Candido, E.P.M. and Dixon, G.H. (1972) J . B i o l . Chem 247, 5506-5510 12. Honda, B.M., Candido, E.P.M. and Dixon, G.H. (1975) J . B i o l . Chem. 250, 8686-8689 13. Honda, B.M., Dix o n , G.H. and Candido, E.P.M. (1975) J . B i o l . Chem. 250, 8681-8685 14. Duerre, J.A. and Ch a k r a b a r t y , S. (1975) J . B i o l . Chem. 250 8457-8461 15. Duerre, J.A., Wallwork, J . C , Quick, D.P. and F o r d , K.M. (1977) J . B i o l . Chem. 252, 5981-5985 16. Delange, R.J. Famborough, D.M., Smith, E.L. and Bonner, J . (1969) J . B i o l . Chem. 244, 319-334 .233 B r a n d t , W.F., S t r i c k l a n d , W.N., Morgan, M. and Von H o l t , C. (1974) FEBS L e t t . 40, 167-172 Thomas, G., Lange, H.W. and Hempel, K. (1975) E u r . J . Biochem. 51, 609-615 Delange, R.J., Hooper, J.A. and Smith, E.L. (1973) J . B i o l . Chem. 248, 3261-3274 oper, J.A., Smith, E.L., Sommer, K.R. and C h a l k l e y , R. (1973) J . B i o l . Chem. 248, 3275-3275 Delange, R.J., Famborough, D.M., Smith, E.L. and Bonner, J . (1969) J . B i o l . Chem. 244, 5669-5679 P a t t h y , L., Smith, E.L. and Johnson, J . (1973) J . B i o l . Chem. 248, 6834-6840 Lee, H.W., P a i k , W.K. and Borun, T.W. (1973) J . B i o l . Chem. 248, 4194-4199 Greenway, P.J. and L e v i n e , D. (1974) B i o c h i m . B i o p h y s . A c t a 350 57/. OOI r J > Wallwork, J . C , Quick, D.P. and Duerre, J.A. (1977) J . B i o l . Chem. 252, 5977-5980 G u r l e y , L.R., W a l l e r s , R.A. and Tobey, R.A. (1975) J . B i o l . Chem. 250, 3936-3944 Marks, D.B., P a i k , W.K. and Borun, T.W. (1973) J . B i o l . Chem. 248, 5660-5667 Bradbury, E.M., I n g l i s , R.J. and Matthews, H.R. (1974) Nature 247, 257-261 Bradbury, E.M., I n g l i s , R.J., Matthews, H.R. and S a r n e r , N. (1973) Eur. J . Biochem. 33, 131-139 B a l h o r n , R., B a l h o r n , M. and C h a l k l e y , R. (1972) Dev. B i o l o g y 29 199-293 . — ' G u r l e y , L.R., D'Anna, J.A., Barham, S.S., Deaven, L.L. and Tobey, R.A. (1978) Eur. J . Biochem. 84, 1-15 L o u i e , A . J . Candido, E.P.M. and Dixon, G.H. (1974) Cold S p r i n g Harbor Symp. Quant. B i o l . 3_8, 803-819 Hohmann, P., Tobey, R.A. and G u r l e y , L.R. (1976) J . B i o l . Chem. 251, 3685-3692 234 B l u m e n f e l d , M., O r f , J.W., S i n a , B . J . , K r e b e r , R.A. C a l l a h a n , M.A., M u l l i n s , J . I . and Snyder, L.A. (1975) P r o c . N a t l . Acad. S c i . U.S.A. 75_, 866-870 Langan, T.A. (1969)' P r o c . N a t l . Acad. S c i . U.S.A. 64, 1276-1283 Langan, T.A. (1976) Fed. P r o c . 3_5, 1623 Shoemaker, C.B. and C h a l k l e y , R. (1978) J . B i o l . Chem. 253, 5802-5807 M a s a r a c c h i a , R.A., Kemp, B.E. and Walsh, D.A. (1977) J . B i o l . Chem. 252, 7109-7117 Lake, R.S., G o i d l , J.A. and Salzman, N.P. (1972) Exp. C e l l R e s e a r c h 73, 113-121 Smith, D.L; Chen, C C . , Bruegger, B.B., H o l t z . , S.L., H a l p e r , R.M. and Smith, R.A. (1974) B i o c h e m i s t r y 13, 3785-3789 Smith, D.L., Chen, C C , Bruegger, B.B., H o l t z , S.L., H a l p e r , R.M. and Smith, R.A. (1974) B i o c h e m i s t r y 13, 3780-3784 Chen, C C , Bruegger, B.B., Ke r n , C.W., L i n , Y . C , H a l p e r n , R.M. and Smith, R.A. (1977) B i o c h e m i s t r y 16, 4852-4855 Smith, J.A. and S t o c k e n , L.A. (1973) Biochem. B i o p h y s . Res. Commun. 54, 297-300 Veda, K., Omachi, A., K a w a i c h i , M. and M a y a i s h i , 0. (1975) P r o c . N a t l . Acad. S c i . U.S.A. 72, 205-209 P o i r i e r , G . C , Savard, P., R a j o t t e , D., M o r i s s e t , J . and L o r d , A. (1978) Can. J . B i o c . 56, 784-790 L o r i m e r I I I , W.S., Stone, P.R. and K i d w e l l , W.R. (1977) Exp. C e l l Res. 106, 261-266 G i r i , C P . , West, M.H.P. and Smulson, M. (1978) B i o c h e m i s t r y 17, 3495-3500 Dixon, G.H. (1976) L i f e S c i e n c e s R e s e a r c h R e p o r t s V o l . 4: O r g a n i z a t i o n a n d . E x p r e s s i o n o f Chromosomes, V.G. A l l f r e y , E.K.F. Bautz, B . J . McCarthy, R.T. Schimke, A. T i s s i e r e s , Eds: . pp. 197-207 H a y a i s h i , 0. and Veda, K. (1977) Ann. Rev. Biochem. 46, 95-116 P r o c . Seminar on P o l y (ADP-ribose) and A D P - r i b o s y l a t i o n of P r o t e i n (1975) J . Biochem. JJ_, 1-11 • ...235 51. M u l l i n s , D.W., G i r i , C P . and Smulson, M. (1977) B i o c h e m i s t r y 16, 506-513 52. G i r i , C P . , West, M.H.P. Ramirez, M.L. and Smulson, M. (1978) B i o c h e m i s t r y 17, 3501-3504 53. Wong, N.C.W., P o i r i e r , G.G. and Dixon, G.H. (1977) E u r . J . Biochem. 7_7, H - 2 1 54. N o l l , M. (1974) Nature 251, 249-251 55. Hewish, D.R. and Burgoyne, L.A. (1973) Biochem, B i o p h y s . Res. Commun. 52, 504-510 56. Thomas, J.P. and Romberg, R.D. (1975) P r o c . N a t l . Acad. S c i . U.S.A. 7_2, 2626-2630 57. S t e i n , A., B i n a - S t e i n , M. and Simpson, R.T. (1977) P r o c . N a t l . Acad. S c i . U.S.A. 74-, 2780-2784 58. Chung, S., H i l l , W.E. and Doty, P. (1978) P r o c . N a t l . Acad. S c i . U.S.A. 75, 1680-1684 59. Weintraub, H., P a l t e r , K. and Van Len t e , F. (1975) C e l l _6, 85-110 60. Thomas, J.P. and B u t t l e r , P.J.G. (1977) J . M o l . B i o l . 116, 769-781 61. Campbell, A.M. and C o t t e r , R.I. (1976) FEBS L e t t . _70, 209-211 62. E i c k b u s h , T.H. and M o u d r i a n a k i s , E«N. (1978) B i o c h e m i s t r y 17, 4955-4963 63. L i , H.J. (1975) N u c l e i c A c i d s Res.. 2, 1275-1289 64. Bonner, W.M. and P o l l a r d , H.B. (1975) Biochem. B i o p h y s . Res. Commun. 64, 28.2-288 65. D'Anna, J r . , J.A. and I s e n b e r g , I . (1974) Biochem. B i o p h y s . Res. Commun. 6_1_, 343-347 66. Kawashima, S. and Imahori, K. (1979) J . Biochem. (Japan) 8_5, 197-202 67. H a r d i s o n , R . C , Z e i t l e r , D.P., Murphy, J.M. and C h a l k l e y , R. (1977) C e l l 12, 417-427 68. Bonner, W.M. (1978) N u c l e i c A c i d s Res. _5» 71-85 69. M a r t i n s o n , H.G., and McCarthy, B . J . (1976) B i o c h e m i s t r y 15, 2002-2007 70. M a r t i n s o n , H.G. and McCarthy, B . J . (1976) B i o c h e m i s t r y 15_, 4126-4131 Bradbury, E.M., Moss, T., Hayashi, H., Hjelm, H., Sucu, P., Stephens, R.M., Baldwin, J.P. and Crane-Robinson, C. (1977) Cold Spring Harbor Symp. Quant. Biol. 4_2, 277-286 Weintraub, H. and Van Lente, F. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4249-4253 Bohm, L., Hayashi, H., Cary, P.D., Moss, T., Crane-Robinson, C. and Bradbury, E.M. (1977) Eur. J . Biochem. 77_, 487-493 Finch, J.T., Noll, M. and Romberg, R.D. (1975) Proc. Natl. Acad Sci. U.S.A. 7_2_, 3320-3322 Finch, J.T., Lutter, L.C., Rhodes, S.D., Brown, R.S., Rushton, B Levitt, M. and Rlug, A. (1977) Nature 269, 29-36 Weintraub, H., Worcel, A. and A l b e r t s , B. (1976) C e l l % 409-417 W h i t l o c k , J r . , J.P. and S t e i n , A. (1978) J . B i o l . Chem. 253, 3857-3861 L i l l e y , D.M.J, and Tatchell, R. (1977) Nucleic Acids Res. 4, 2039-2055 ~ B i n a - S t e i n , M. and Simpson, R.T. (1977) C e l l 11, 609-618 Camepini-Oterd, R.D., Sollner-Webb, B. and F e l s e n f e l d , G. (1976) C e l l 8_, 333-347 N o l l , M. (1976) C e l l 8_, 349-356 M o r r i s , N,R, (1976) C e l l 8_, 357-363 Weintraub, H. (1978) N u c l e i c A c i d s Res. j>, 1179-1187 R i l l , R.L., N e l s o n , D.A., O o s t e r h o f , D.R. and H o z l e r , J.C. (1977) N u c l e i c A c i d s Res. 4_, 771-788 G o t t e s f e l d , J.M. and Me l t o n , D.A. (1978) Nature 273, 317-319 Compton, J . L . B e l l a r d , M. and Chambon, P. (1976) P r o c . N a t l . Acad. S c i . U.S.A. 7_3, 4382-4386 Thomas, J.O. and Thompson, R.J. (1977) C e l l 1_0, 633-640 R e i c h l i n e , L.D. and Wassarman, P.M. (1977) B i o c h i m . B i o p h y s . A c t a 475, 139-151 M o r r i s , N.R., (1976) C e l l 9, 627-632 Bakayev, V.V., Bakayeva, T.G. and Varshavsky, A . J . (1977) C e l l 11 619-629 — 237 Wilhelm, M.L. Mazen, A. and Wilhelm, F.X. (1977) FEB'.S L e t t . 7_9, 404 '' Chambon, P. (1977) C o l d S p r i n g Harbor Symp. Quant. B i o l . 42, 1209-1234 Campbell, A.M. and C o t t e r , R.I. (1977) N u c l e i A c i d s Res. 4_, 3877-3886 S p a d a f o r a y C , Oudet, P. and Chambon, P. (1978). N u c l e i c A c i d s Res. 5_, 3479-3489 N o l l , M. (1974) N u c l e i c A c i d s Res. 1, 1573-1578 Sollner-Webb, B., M e l c h i o r , J r . , W. and F e l s e n f e l d , G. (1978) C e l l 14, 611-627 W h i t l o c k , J r . J.P. (1977) J . B i o l . Chem. 252, 7635-7639 SOllner-Webb, B. and F e l s e n f e l d , G. (1977) C e l l 10, 537-547 W h i t l o c k , J r . , J.P., R u s h i z k y , G.W. and Simpson, R.T. (1977) J . B i o l . Chem. 252, 3003-3006 T r i f o n o v , E.N. and B e t t e c k e r , T. (1979) B i o c h e m i s t r y 18_, 454-456 L u t t e r , L.C. (1979) N u c l e i c A c i d s Res. b_, 41-57 Smith, J.A. and Stocken, L.A. (1975) Biochem. J . 147, 523-529 Hjelm, R.P., K r e a l e , G.G., Suau, P., Baldwin, J.P., Bradbury, E.M. and I b e l , K. (1977) C e l l 10, 139-151 Pardon, J . F . , W o r c e s t e r , D.L., Wooley, J . C , T a t c h e l l , K., Van Holde, K.E. and R i c h a r d s , B.M. (1975) N u c l e i c A c i d s Res. _2, 2163-2172 Pardon, J.F., Worcester, D.L.,'Wooley, J . C , C o t t e r , R.I., L i l l e y , D.M.F. and R i c h a r d s , B.M. (1977) N u c l e i c A c i d s Res 9_, 3199-3214 Dubochet, J . and N o l l , M. (.1978) S c i e n c e 202, 280-286 Schmitz, K.S. and Shaw, B.R. (1977) S c i e n c e 197, 661-662 B u s t i n , M., G o l d b l a t t , D. and S p e r l i n g , R. (1976) C e l l 7, 297-304 ~ Varashavsky, A.J.., Nedospasov, S.A., Schmatchenko, U.V., Bakageu, V.V., Chumackov, P.M. and G e o r g i e v , C P . (1977) N u c l e i A c i d s Res. k_, 3303-3325 ' 238 F i n c h , J.T. and K l u g , A. (1976) P r o c . N a t l . Acad. S c i . U.S.A. 73, 1897-1901 C a r p e n t e r , B.G., Baldwin,. J.P., Bradbury, E.M. and I b e l , K. (1976) N u c l e i c A c i d s Res. _3, 1739-1746 Renz, M., N e h l s , P. and H o z i e r , J . (1977) P r o c . N a t l . Acad. S c i . U.S.A. 74., 1879-1883 Gaubatz, J.W. and C h a l k l e y , R. (1977) N u c l e i c A c i d s Res. 4_, 3281-3301 Thoma, F. and R o l l e r , T. (1977) C e l l 12, 101-107 A l t e n b u r g e r , W., Horz, W. and Zachau, H.G. (1976) Nature 264, 517-522 St e i n m e t z , M., S t r e e c k , R.E. and Zachau, H.G. (1978) Eur. J . Biochem. 82, 615-628 C h r i s t i a n s e n , G. and G r i f f i t h , J . (1977) N u c l e i c A c i d s Res. _4, 1837-1851 Lacy, E. and A x e l , R. (1975) P r o c . N a t l . Acad. S c i . U.S.A 72_, 3978-3982 Kuo, M.T., Sahascrabuddhe, C.G. and Saunders, G.F. (1976) P r o c . N a t l . Acad. S c i . U.S.A. (1976) 721, 1572-1576 G a r e l , A. and A x e l , R. (1976) P r o c . N a t l . Acad. S c i . U.S.A. 13_, 3966-3970 B e l l a r d , M., Gannon, F. and Chambon, P. (1977) C o l d S p r i n g Harbor Symp. Quant. B i o l . 42, 779-791 Bloom, K.S. and Anderson, J.N. (1978) C e l l 15, 141-150 Johnson, E.M., A l l f r e y , V.G. Bradbury, E.M. and Matthews, H.R. (1978) P r o c . N a t l . Acad. S c i . U.S.A. 75, 1116-1120 Reeves, R. (1977) Eur. J . Biochem. 7_5, 545-560 M a t h i s , D'.J. and Gorovsky, M.A. (.1976) B i o c h e m i s t r y 15, 750-755 Reeves, R. (1977) C o l d S p r i n g Harbor Symp; Quant. B i o l . 42, 709-722 Scheer, U. (1978) C e l l 13, 535-549 T r e n d e l e n b u r g , M.F. and Gordon, J.B. (1978) Nature 276, 292-294 239 Gurdon, J.B. and Brown, D.D. (1978) Dev. B i o l . 67_, 346-356 Meneguzzi, G., P i g n a t t i , P.F., B a r b a n t i - B r o a d a n o , G. and M i l a n e s i , G. (1978) P r o c . N a t l . Acad. S c i . U.S.A. 75, 1126-1130 H a l l , M.R. (1977) Biochem; B i o p h y s . Res. Commun. 76, 698-704 Green, M..H. and Brooks, T.L. (1977) N u c l e i c A c i d s Res. 4_, 4279-4289 Weintraub, H. and Groudine, M. (1976) S c i e n c e 193, 848-856 G a r e l , A., Z o l a n , M. and A x e l , R. (1977) P r o c . N a t l . Acad. S c i . U.S.A. 74, 4867-4871 F l i n t , S.J. and Weintraub, H.M. (1977) C e l l 12, 783-794 Levy, W., B and Dixon, G.H. (1977) N u c l e i c A c i d s Res. 4_, 883-898 Weisbrod, S. and Weintraub, H. (1979) P r o c . N a t l . Acad. S c i . U.S.A. 76, 630-634 G o t t e s f e l d , J.M. (1977) In Methods i n C e l l B i o l o g y . ,; G. S t e i n , ed. (New York: Academic P r e s s ) . 16, 421-436 G o t t e s f e l d , J.M. and B u t l e r , P.J.G. (1977) N u c l e i c A c i d s . Res. 4, 3155-3173 G o t t e s f e l d , J.M. Murphy, R.F. and Bonner (1975) P r o c . N a t l . Acad. S c i . U.S.A. 72_, 4404-4408 t G o t t e s f e l d , J.M. G a r r a r d , W.T., B a g i , G., W i l s o n , R.F. and Bonner, J . (1974) P r o c . N a t l . Acad. S c i . U.S.A. 71, 2193-2197 Levy W., and B. and Dixon, G.H. (1978) N u c l e i c A c i d s Res. 5_, 4155-4163 Levy W., B., Conner, W. and Dixon, G.H. (1978) J . B i o l . Chem. 254, 609-620 Levy, W., B. and Dixon, G.H. (1979) P r o c . N a t l . Acad. S c i . U.S.A. ( i n p r e s s ) McKnight, S.L. and M i l l e r , J r . , O.L. (1977) C e l l 12, 795-804 S e a l e , R.L. (1976) C e l l 9/ 423-429 Hewish, D. (1977) N u c l e i c A c i d s . Res. 4_, 1881-1890 240 S e a l e , R.L. (1978) P r o c . N a t l . Acad. S c i . U.S.A. 75, 2717-2721 Weintraub, H. (.1976) C e l l 9_» 419-422 C r e m i s i , C , C h e s t i e r , A. and Y a n i v , M. (1977) C e l l 12, 947-951 Tan, K.B. (1977) P r o c . N a t l . Acad. S c i . U.S.A. 74, 2805-2809 Russev, G. and Tsanev, R. (1979) Eur. J . Biochem. 93, 123-128 L e f f a k , I.M. G r a i n g e r , R. and Weintraub, H. (1977) C e l l 12, 837-845 Laskey, R.A., M i l l s , A.D. and M o r r i s , N.R. (1977) C e l l 10, 237-243 Laskey, R.A., Honda, B.M., M i l l s , A.D. and F i n c h , A . J . (1978) Nature 275, 416-420 Germond, J . E . and B r u t l a g , D. (1979) J . Supramol. S t r u c t . Suppl.3_, 72 S t e r n e r , R., B o f f a , L.C. and V i d a l i , G. (1978) J . B i o l . Chem. 253, 3830-3836 Goodwin, G.H., Woodhead, L. and Johns, E.W. (1977) FEBS L e t t . 7_3, 85-88 Watson, D . C , P e t e r s , E.H. and Dixon, G.H. (1977) Eur. J . Biochem. 7*4, 53-60 Levy W., B., Wong, N.A.C.W., Watson, D . C , P e t e r s , E.H. and Dixon, G.H. (1977) C o l d S p r i n g Harbor Symp. Quant. B i o l . 42, 793-802 Levy W., B. and Dixon, G.H. (1978) Can. J . Biochem. 56_, 480-491 Levy W., B., Wong, N.C.W. and Dixon, G.H. (1977) P r o c . N a t l . Acad. S c i . U.S.A. 74, 2810-2814 Mathew, C.G.P., Goodwin, G.H. and Johns, E.W. (1979) N u c l e i c A c i d s . Res. 6_, V i d a l i , G., B o f f a , L.C. and A l l f r e y , V.G. (1977) C e l l 12, 409-415 Goodwin, G.H. and Johns, E.W. (1978) B i o c h i m . . B i o p h y s . A c t a . 519, 279-284 Goldknopf, I.L., F r e n c h , M.F., Musso, R. and Busch, H. (1977) P r o c . N a t l . Acad. S c i . U.S.A. 74, 5492-5495 Watson, D.C. Levy W., B. and Dixon, G.H. (1978) Nature 276, 196-198 24i,-,: Walker, J.M. H a s t i n g s , J.R.B. and Johns, E.W. (1977) Eur. J . Biochem. 76_, 461-468 L i b b y , P.R. (1972) Biochem. J . 130, 663-669 L i b b y , P.R. (.1973) Biochem. J . 134_, 907-912 S c h a f f h a u s e n , B.S. and Benjamin, T.L. (1976) P r o c . N a t l . Acad. S c i . U.S.A. 73, 1092-1096 Gorovsky, M.A., G l o v e r , C., Johmann, C.A., K e e v e r t , J.B., M a t h i s , D.J. and Sumvelson, M. (1977) C o l d S p r i n g Harbor Symp. Quant. B i o l . 42, 493-503 Levy W., B., G j e r s e t , R.A. and McCarthy, B.J. (1977) Biochim. B i o p h y s . A c t a 475, 168-175 Levy W.B., Watson, D.C. and Dixon, G.H. (1979) N u c l e i A c i d s . Res 6, 259-274 Riggs, M.G., W h i t t a k e r , R.G., Neumann, J.R. and Ingram, V.M. (1977) Nature 268, 462-464 B o f f a , L.C., V i d a l i , G., Mann, R.S. and A l l f r e y , V.G. (1978) J . B i o l . Chem. 253, 3364-3366 S e a l y , L. and C h a l k l e y , R. (1978) C e l l 14, 115-121 Simpson, R.T. (1978) C e l l 13, 691-699 M a t h i s , D.J., Oudet, P., Wasylyk, B. and Chambon, P. (1978) N u c l e i c A c i d s . Res. 5_> 3523-3547 Wong, L.C. and A l b e r t s , B.M. (1977) Fed. P r o c e e d . 36, 784 Shewmaker, C.K., Cohen, B.N. and Wagner, T.E. (1978) Biochem. B i o p h y s . Res. Commun. 84, 342-349 V i d a l i , G., B o f f a , L . C , Bradbury, E.M. and A l l f r e y , V.G. (1978) P r o c . N a t l . Acad. S c i . U.S.A. 75_, 2239-2243 Ne l s o n , D.A., P e r r y , W.M. and C h a l k l e y , R. (1978) Biochem. B i o p h y s . Res. Commun. 82_, 356-363 Reeves, R. and C s e r j e s i , P. (.1979) J . B i o l . Chem. ( i n p r e s s ) Cousens, L.S., G a l l w i t z , D. and A l b e r t s , B. (.1979) J . B i o l . Chem. 254, 1716-1723 Schmidt, P.J., M i t c h e l l , B.S., Smith, M. and T s u y u k i , H. (1965) Gen. Comp. E n d o c r i n o l . 5_, 197-206 242 Waymouth, C. (1956) J . Nat. Cancer I n s t . 17, 315-325 Honda, B.M., B a i l l i e , D.L. and Candido, E.P.M. (1974) FEBS L e t t . 48, 156-159 Shaw, B.R., Herman, T.M., K o v a c i c , R.T., Beaudreau, G.S., and Van Holde, K.E. (1976) P r o c . N a t l . Acad. S c i . U.S.A. 73_, 505-509 Sanders, M.M. (1978) J . C e l l B i o l . 79., 97-109 Wong, N.T.N, and Candido, E.P.M. (1978) J . B i o l . Chem. 253, 8263-8268 Bonner, J . , C h a l k l e y , G.R., Dahmus, M., Fambrough, D., F u j i m u r a , F., Huang, R.C., Huberman, J . Jensen, R., Marushige, K., Ohlenbusch, H., O l v e r a , B. and Widholm, J . (1968) Methods i n Enzymol. 12B, 22-23 . Sung, M. and S m i t h i e s , 0.(1969) Biopolymers 1, 39-58 Panyim, S. and C h a l k l e y , R. (1969) A r c h . Biochem. B i o p h y s . 130, 337-346 O ' F a r r e l l , P.H. (1975) J . B i o l . Chem. 250, 4007-4021 Weiner, A.M., P i a t t , P. and Weber, K. (1972) J . B i o l . Chem. 247, 3252-3251 L o e n i n g , U.E. (1967) Biochem. J . 102, 251-257. F r i e n d , C , P r e i s l e r , H.D. and Scher, W. (1974) C u r r . T o p i c s Dev. B i o l . 8_, 81-101 Todaro, G. and Green, G. (1963) J . C e l l B i o l . 17, 299-313 Macpherson, I.A. and S t o k e r , M.G.P. (1962) V i r o l o g y 16, 147-151 Dunning, W.J. and C u r t i s , M.R. (1957) J . Nat. Cancer. I n s t . 19, 845-853 Reeves, R. and Jones, A. (1976) Nature 260, 495-500 P a u l , J . (1970) C e l l and T i s s u e C u l t u r e ( E d i n b u r g h : E. and S. L i v i n g s t o n e ) Marushige, K. and Bonner, J . (1966) J . Mol. B i o l . 15, 160-174 Bray, G.A. (1960) A n a l . Biochem. 1, 279-285 L o u i e , A . J . (1972) ph. D. T h e s i s , Univ. o f B r i t i s h Columbia •• 243 207. Candido, E.P.M. (1972) Ph. D. T h e s i s , Univ. o f B r i t i s h Columbia 208. Honda, B.M., B a i l l i e , D.L. and Candido, E.P.M. (1975) J . B i o l . Chem. 250, 4643-4647 209. Simpson, R.T. and W h i t l o c k , J r . , J.P. (1976) N u c l e i c A c i d s . Res. 3/ 117-127 210. W h i t l o c k , J r . , J.P. and Simpson, R.T. (1976) B i o c h e m i s t r y 15, 3307-3313 211. A x e l , R., Cedar, H. and F e l s e n f e l d , G. (1975) B i o c h e m i s t r y 14, 2489-2495 212. McKnight, S.L., B u s t i n , M. and M i l l e r , J r . , O.L. (1977) C o l d S p r i n g Harbor Symp. Quant. B i o l . 42, 741-754 213. Foe, V.E., W i l k i n s o n , L.E. and L a i r d , C D . (1976) C e l l 9_, 131-146 214. Candido, E.P.M. and Dixon, G.H. (1972) J . B i o l . Chem. 247, 3868-3873 215. Candido, E.P.M. and Dixon, G.H. (1971) J . B i o l . Chem. 246, 3182-3188 216. Sung, M. and--Dixon, G.H. (1970) P r o c . N a t l . Acad. S c i . U.S.A. 67, 1616-1623 217. N o l l , M. and Romberg, R.D. (1977) J . Mol. B i o l . 109, 393-404 218. A x e l , R., M e l c h i o r , J r . , W., Sollner-Webb, B. and F e l s e n f e l d , G. (1974) P r o c . N a t l . Acad. S c i . U.S.A. 71, 4101-4105 219. Varshavsky, A . J . , Bakayev, V.V. and G e o r g i e v , C P . (1976) N u c l e i A c i d s . Res. 3_> 477-492 220. Ohlenbusch, H.H., O l i v e r a , B.M., Tuan, D. and Davidson, N. (1967) J . M o l . B i o l . 25, 299-315 221. B r a s c h , K. (1976) E x p t l . C e l l Res. 101, 396-410 222. Moore, M., Jackson, V., S e a l y , L. and C h a l k l e y , R. (1979) Biochim. B i o p h y s . A c t a 561, 248-260 223. Sanders, M.M. and Hsu, J.T. (1977) B i o c h e m i s t r y 16, 1690-1695 224. Goodwin, G.H., Sanders, C. and Johns, E.W. (1973) Eur. J . Biochem. 38, 14-19 225. Marushige, K. and Bonner, J . (1971) P r o c . N a t l . Acad. S c i . U.S.A. 68, 2941-2944 244 226. Laemmli, U.K. (1970) Nature 227, 680-685 227. Jackson, V., S h i r e s , A., C h a l k l e y , R. and Granner, D. (1975) J . B i o l . Chem. 250, 4856-4863 228. G o t t e s f e l d , J . M. and P a r t i n g t o n , G.A. (1977) C e l l 12, 953-962 229. W a l l a c e , R.B., Dube, S.K. and Bonner, J . (1977) S c i e n c e 198, 1166-1168 230. N o l l , M. (1977) J . Mol. B i o l . 116, 49-71 231. P r a s a d , K.N. and S i n h a , P.K. (1976) I n V i t r o 12, 125-132 232. Leder, A. and L e d e r , P. (1975) C e l l 5., 319-322 233. Hagopian, H.K., Ri g g s , M.G., Swartz, L.A. and Ingram, V.M. (1977) C e l l 12, 855-860 234. R u i z - C a r i l l o , A., Wangh, L . J . and A l l f r e y , V.G. (1975) S c i e n c e 190, 117-128 235. Sanders, L.A., S c h e c h t e r , H.M. and McCarty, K.S. (1973) B i o c h e m i s t r y 12_, 783-791 236. P o s p e l o v , V.A., S v e t l i k o v a , S.B. and Vord'ev, V . I . (1979) FEBS L e t t . 99, 123-128 237. Gross, D.S. and Simpkins, H. (1979) Fed. Pr o c e e d . 38_, 947 238. Woodland, H.R. (1979) Dev. B i o l . 68, 360-370 239. Reeves, R. and Candido, E.P.M. (1979) Biochem. B i o p h y s . Res. Commun. 89_, 571-579 240. Z l a t a n o v a , J . and Swetly, P. (1978) Nature 276, 276-277 241. Macleod, A.R., Wong, N.C.W. and Dixon, G.H. (1977) Eur. J . Biochem. 78, 281-291 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0094862/manifest

Comment

Related Items