UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies on the acetylation of trout testis histones Candido, Edward Peter Mario 1972

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STUDIES ON THE ACETYLATION OF TROUT TESTIS HISTONES by E. PETER M. CANDIDO B . S c , M c G i l l U n i v e r s i t y , 1968 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of B i o c h e m i s t r y F a c u l t y of Medicine We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard June, 1972 U n i v e r s i t y o f B r i t i s h Columbia In present ing t h i s thes is in p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the Un ivers i t y of B r i t i s h Columbia, I agree that 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 for reference and study. I fu r the r agree that permission for extensive copying o f th i s t h e s i s fo r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representa t i ves . It i s understood that copying or p u b l i c a t i o n of th i s t h e s i s fo r f i n a n c i a l gain sha l l not be allowed without my wr i t ten permiss ion . Department of The Un i ve rs i t y of B r i t i s h Columbia Vancouver 8, Canada -i -A b s t r a c t H i s t o n e s are b a s i c chromosomal p r o t e i n s which are complexed w i t h DNA i n e u k a r y o t i c organisms. During the s e x u a l maturation of the rainbow t r o u t (Salmo g a i r d n e r i i ) , there i s e x t e n s i v e a c e t y l a t i o n of the t e s t i s h i s t o n e s . Four of the f i v e major h i s t o n e f r a c t i o n s were found t o be a c e t y l a t e d , and the a c e t a t e i n c o r p o r a t e d was shown to be l a r g e l y p r e s e n t as e-N-acetyl groups a t t a c h e d to i n t e r n a l l y s y l r e s i d u e s i n these p r o t e i n s . P u r i f i e d h i s t o n e s were seen to e x h i b i t c o n s i d e r a b l e h e t e r o g e n e i t y when e l e c t r o p h o r e s e d on s t a r c h g e l s , and t h i s was shown t o be l a r g e l y due to the presence of a c e t y l a t e d components i n the p r e p a r a t i o n s . H i s t o n e I l b i was shown to c o n t a i n a s i n g l e major a c e t y l a t i o n s i t e a t l y s y l r e s i d u e 5; h i s t o n e IIb2 c o n t a i n s a c e t y l a t e d l y s y l r e s i d u e s a t p o s i t i o n s 5, 10, 13 and 18, h i s t o n e I I I a t p o s i t i o n s 9, 14, 18 and 23, and h i s t o n e IV a t p o s i t i o n s 5, 8, 12 and 16. A l l of these m o d i f i c a t i o n s are found i n the very b a s i c amino-terminal r e g i o n s of the p r o t e i n s , which may be DNA b i n d i n g s i t e s . I t i s p o s t u l a t e d t h a t the func-t i o n of these m o d i f i c a t i o n s i s to modulate the p o s i t i v e charge d e n s i t y of h i s t o n e s , and hence t h e i r b i n d i n g to DNA. The a c e t y l a t i o n of h i s t o n e s i n d i f f e r e n t t r o u t t e s t i s c e l l types was i n v e s t i g a t e d . A l l c e l l types i n c o r p o r a t e d a c e t a t e i n t o h i s t o n e s , but whereas h i s t o n e a c e t y l a t i o n was accompanied by s y n t h e s i s of these p r o t e i n s i n spermatocytes, the m o d i f i c a t i o n i n spermatid c e l l s o c c u r r e d i n the absence of s i g n i f i c a n t h i s t o n e s y n t h e s i s . I t i s suggested t h a t h i s t o n e a c e t y l a t i o n i n spermatids may be i n v o l v e d i n the process by which these p r o t e i n s are r e -p l a c e d by protamine, a s m a l l e r , a r g i n i n e - r i c h p r o t e i n , d u r i n g the maturation of t r o u t t e s t i s spermatids. Histone a c e t y l a t i o n i n c e l l types which are a c t i v e l y s y n t h e s i z i n g these p r o t e i n s may be i n -v o l v e d i n a c h i e v i n g the c o r r e c t b i n d i n g of newly s y n t h e s i z e d h i s t o n e s to DNA. An enzyme a c t i v i t y which t r a n s f e r s a c e t y l groups from a c e t y l coenzyme A to h i s t o n e s was i s o l a t e d from t r o u t t e s t i s chromatin, and some p r o p e r t i e s of t h i s p r e p a r a t i o n have been noted. ACKNOWLE DGMENT The author wishes t o express h i s a p p r e c i a t i o n t o P r o f e s s o r G.H. Dixon f o r h i s e x c e l l e n t guidance and encouragement d u r i n g the course o f t h i s work, t o Drs. B. Malchy, V. L i n g , D.T. Wigle, M. Sung, M. Sanders, and Mr. A. Louie f o r exchange of i d e a s , and to Mr. J . Durgo f o r ex p e r t t e c h n i c a l a s s i s t a n c e . The M e d i c a l Research C o u n c i l o f Canada i s thanked f o r p r o v i d i n g a F e l l o w s h i p t o the author f o r the p e r i o d 1968-72. -iv-DEDICATION ' my mother and father and my wife G l o r i a -v-TABLE OF CONTENTS ABSTRACT * ACKNOWLEDGMENT . . i i i DEDICATION iv LIST OF TABLES viii LIST OF FIGURES . ix INTRODUCTION . . . . . . . . 1 H i s t o n e s : Nomenclature and S t r u c t u r e . . . . . . . 1 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 o f His t o n e s . . . . . . 10 (i) A c e t y l a t i o n . . . . . . . . . . . . . . . 10 ( i i ) M e t h y l a t i o n 13 ( i i i ) P h o s p h o r y l a t i o n 15 I n t e r a c t i o n s o f His t o n e s w i t h DNA . . 18 The Process o f Spermatogenesis . . . 21 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . 25 I. Chemicals and A b b r e v i a t i o n s . . . . 25 (b) A b b r e v i a t i o n s . . 25 I I . I s o l a t i o n and P r e l i m i n a r y C h a r a c t e r i z a t i o n o f In V i v o A c e t y l a t e d T r o u t T e s t i s H i s t o n e s ... 26 (a) Source o f the Hi s t o n e s . . . . . . . . . . 26 (b) C e l l I n c u b a t i o n s 27 (c) I s o l a t i o n o f B a s i c P r o t e i n s . . . . . . . 27 (d) F r a c t i o n a t i o n o f Hist o n e 28 (e) A c i d H y d r o l y s i s o f t 1 ''C] - a c e t a t e L a b e l e d H i s t o n e s 30 (f) T r y p s i n - P r o n a s e D i g e s t i o n of t ^ C ] -a c e t a t e L a b e l e d H i s t o n e s . . . . 31 (g) S y n t h e s i s o f e-N- [* " c ] - a c e t y l l y s i n e . . . 31 (h) High V o l t a g e E l e c t r o p h o r e s i s and Paper Chromatography 32 (i) Amino A c i d A n a l y s i s 33 (j) S t a r c h G e l E l e c t r o p h o r e s i s 33 (k) Autoradiography of S t a r c h Gels . . . . . . 34 - v i -l l i . S t u d i e s on the S i t e s o f In V i v o A c e t y l a t i o n i n T r o u t T e s t i s H i s t o n e s 34 (a) Dowex 50 Chromatography of Histone IV T r y p t i c P e p t i d e s 35 (b) Chemical A c e t y l a t i o n o f Histones I l b j and IV 35 (c) Manual Edman Degradation o f Pe p t i d e s L a b e l e d w i t h [ l * C ] - a c e t a t e 36 (i) H i s t o n e IV 36 ( i i ) H i s t o n e I l b i . 36 (d) Amino T e r m i n a l Determination of Pe p t i d e s . 37 (i) H i s t o n e IV 37 ( i i ) H i s t o n e I l b i • 38 (e) Automated P r o t e i n Sequencing . . . . . . . 3 9 (i) Quadrol Program 39 ( i i ) DMAA Program • . 40 IV. A c e t y l a t i o n of His t o n e s i n D i f f e r e n t C e l l Types from Developing T r o u t T e s t i s 41 (a) T r o u t T e s t i s . . . . . . . . 41 (b) Incubations . . . . . . . . . . 41 (c) C e l l S e p a r a t i o n s 42 (d) S t a r c h Gel E l e c t r o p h o r e s i s o f B a s i c P r o t e i n s from Whole C e l l s 43 (e) R a d i o a c t i v i t y A n a l y s i s . 44 v ' (f) Pulse-chase S t u d i e s '•" 44 Vi Some P r o p e r t i e s o f the Hi s t o n e A c e t y l t r a n s -f e r a s e s from T r o u t T e s t i s 45 (a) Enzyme Assays . . . . . 45 (i) Assay of A c e t y l t r a n s f e r a s e ' A c t i v i t y i n I s o l a t e d N u c l e i 45 ( i i ) Assay of A c e t y l t r a n s f e r a s e A c t i v i t y i n S o l u b l e P r e p a r a t i o n s . . 4 7 (b) P r e p a r a t i o n o f N u c l e o h i s t o n e . . . . . . . 47 (c) E x t r a c t i o n and Ammonium S u l f a t e F r a c -t i o n a t i o n o f Hi s t o n e A c e t y l t r a n s f e r a s e A c t i v i t y from T r o u t T e s t i s N u c l e i . . . . 48 RESULTS Z. I s o l a t i o n and P r e l i m i n a r y C h a r a c t e r i z a t i o n o f In V i v o A c e t y l a t e d T r o u t T e s t i s H i s t o n e s . . . 50 (i) H i s t o n e I l b i 67 ( i i ) H istone I I b 2 . . 70 ( i i i ) H i s t o n e I I I . 71 (iv) H i s t o n e IV 72 - v i l -l i . S t u d i e s on the S i t e s o f In V i v o A c e t y l a t i o n i n T r o u t T e s t i s H i s t o n e s 74 (i) H i s t o n e IV 74 ( i i ) H istone I l b i . 8 7 ( i i i ) Histone I I I 94 (iv) H i s t o n e I I b 2 104 I I I . A c e t y l a t i o n of H i s t o n e s i n D i f f e r e n t C e l l Types From Developing T r o u t T e s t i s . . . . . . 112 IV. Some P r o p e r t i e s of 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 from T r o u t T e s t i s 127 DISCUSSION 142 REFERENCES 160 . • * • • -u^^^-LIST OF TABLES Page 1. Nomenclature and Some C h a r a c t e r i s t i c s of the P r i n -c i p a l Components of C a l f Thymus Hist o n e 2 2. E f f e c t of Cycloheximide on A c e t y l a t i o n of T r o u t T e s t i s Histones i n C e l l Suspensions 53 3. A c i d L a b i l i t y of t 1 ''C]-acetate i n T r o u t T e s t i s r 4. Amino A c i d Compositions of [*^c]-acetate Lab e l e d H i s t o n e IV P e p t i d e s . . . . . . . . . . . . . . . . . . 80 5. Edman Degradation o f P e p t i d e s T A l a ' and T A l b . . . . . 86 6. Amino A c i d Compositions of H i s t o n e I l b i P e p t i d e s L a b e l e d w i t h V ''C]-acetate 92 7. S e q u e n t i a l Degradation of T r o u t T e s t i s H i s t o n e I I I . . 100 8. S e q u e n t i a l Degradation of T r o u t T e s t i s H i s t o n e I I b 2 . . 1 0 6 9. R e l a t i v e I n c o r p o r a t i o n o f I1 "c]-acetate and [ 3H]-l y s i n e i n t o H i s t o n e s o f D i f f e r e n t C e l l Types From 10. Heat L a b i l i t y 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 A c t i v i t y i n T r o u t T e s t i s N u c l e i 129 11. E x t r a c t i o n 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 A c t i v i t y from T r o u t T e s t i s N u c l e i by NaCl . . . . . . . . . . . 133 12. Ammonium S u l f a t e P r e c i p i t a t i o n of Histone A c e t y l -t r a n s f e r a s e A c t i v i t y , and E f f e c t o f D i a l y s i s on Enzyme A c t i v i t y 134 13. Sequences Around the A c e t y l a t e d L y s y l Residues of T r o u t T e s t i s H i s t o n e s . . . . . . . . . . . . . . . . . 148 14. I1 kC]-acetate 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 per C e l l f o r D i f f e r e n t T r o u t T e s t i s C e l l Types . . . . . . „ „ , 158 -ix-LIST OF FIGURES Page 1. (a) Amino A c i d Sequence of C a l f Thymus Histone IV . . . 4 (b) Amino A c i d Sequence of C a l f Thymus Hist o n e I I b 2 . • 4 2. (a) Sequence of C a l f Thymus Histone I I I 7 (b) " P a r t i a l Sequence of H i s t o n e I l b i from T r o u t T e s t i s « 7 (c) P a r t i a l Sequence of H i s t o n e T from T r o u t T e s t i s . . 7 3. P a r t i a l Sequences of Two Histone I Components from Rabbit Thymus, and of One Component from C a l f 4. S e p a r a t i o n of t 1 ''C]-acetate L a b e l e d Histone from 5. Sephadex G-25 Chromatography of a T r y p s i n - P r o n a s e D i g e s t of Whole Hist o n e . . . . . . . . . . 5 5 6. P u r i f i c a t i o n of e - N - a c e t y l l y s i n e from a T r y p s i n -Pronase D i g e s t of Whole Hist o n e 56 (a) High V o l t a g e E l e c t r o p h o r e s i s a t pH 6.5 . . . . . . 56 (b) High V o l t a g e E l e c t r o p h o r e s i s a t pH 3.6 . . . . . . 56 (C) Paper Chromatography . . . . . . . . . . . . . . . 56 7. Amino A c i d A n a l y s i s of the i1 **C]-acetyl L a b e l e d Com-pound Obtained from a T r y p s i n - P r o n a s e D i g e s t of 8. A c e t y l a t i o n of T r o u t T e s t i s H i s t o n e s . F r a c t i o n a t i o n of Histones by Bio-Rex 70 Chromatography . . . . . . . 60 9. P u r i f i c a t i o n of H i s t o n e s I I I and IV . . . . . . . . . . 61 10. F r a c t i o n a t i o n of A c e t y l a t e d H i s t o n e s by Chromato-graphy on B i o - G e l P-10 63 11. S t a r c h Gel E l e c t r o p h o r e s i s of P u r i f i e d H i s t o n e s . . . . 65 12. S t a r c h Gel E l e c t r o p h o r e s i s of P u r i f i e d H i s t o n e s . . . . 66 13. A c e t y l a t e d Components o f T r o u t T e s t i s H i s t o n e s . . . . 6 8 -x-14. A c e t y l a t e d Components of T r o u t T e s t i s H i stones . . . . 69 15. S e p a r a t i o n o f [*^C]-labeled T r y p t i c - P e p t i d e s o f His t o n e IV on Dowex 50 75 16. High V o l t a g e E l e c t r o p h o r e t i c S e p a r a t i o n o f Hi s t o n e IV [ l k C ] - a c e t a t e Labeled T r y p t i O P e p t i d e s 76 17. Amino-Terminal Sequence of C a l f Thymus Hist o n e IV, Showing S i t e s o f A c e t y l a t i o n i n T r o u t T e s t i s H i s t o n e IV . . 78 18. (a) Chemical A c e t y l a t i o n o f In V i v o L a b e l e d [ I l fC]-a c e t a t e h i s t o n e IV 81 (b) T h e r m o l y s i n Cleavage o f P e p t i d e T A l 81 (c) P u r i f i c a t i o n o f P e p t i d e T A l a 81 19. I d e n t i f i c a t i o n o f Dansyl D e r i v a t i v e s from P e p t i d e s T A l b and T A l a ' . . . . . . . . . . . . . 84 20. Summary of S t r a t e g y Used i n E l u c i d a t i n g the S i t e s of A c e t y l a t i o n i n Hi s t o n e IV 88 21. P u r i f i c a t i o n o f a T r y p t i c P e p t i d e o f Hi s t o n e I l b i L a b e l e d w i t h [ ^ C ] - a c e t a t e . . . . . . . . . . . . . . 90 22. (a) High V o l t a g e E l e c t r o p h o r e s i s o f the I1 l*C] - a c e t a t e L a b e l e d T r y p t i c P e p t i d e from H i s t o n e I l b i 91 (b) T r y p t i c P e p t i d e Obtained from I l b i a f t e r Chemical A c e t y l a t i o n o f the In Vivo L a b e l e d P r o t e i n . . . . 91 23. Comparison o f the N H 2 - t e r m i n a l Regions of Hist o n e I l b i from T r o u t T e s t i s , and Histone IV from C a l f Thymus 93 24. Paper Chromatography of the [x * C ] - a c e t a t e Labeled P e p t i d e Obtained by T r y p t i c H y d r o l y s i s o f In V i v o l a b e l e d , C h e m i c a l l y A c e t y l a t e d I l b i . . . . . . . . . . 95 25. Edman Degradation o f the I1 l f C ] - a c e t a t e Labeled Pep-t i d e from In V i v o L a b e l e d , C h e m i c a l l y A c e t y l a t e d I l b i . 96 26. Sequence of the f i r s t 25 Residues o f T r o u t T e s t i s 27. Recovery o f A l a n i n e During the Automated Degradation of H i s t o n e I I I . . . . . . . . . . . . . 101 ( 28. (a) Automated Degradation o f [* kC]-acetate L a b e l e d H i s t o n e I I I Using the Quadrol Program 103 (b) Automated Degradation o f [* ''C]-acetate Labeled H i s t o n e I I I Using V o l a t i l e Reagents 103 29. Sequence o f the F i r s t 22 Residues o f T r o u t T e s t i s H i s t o n e I I b 2 . 105 30. Recovery o f A l a n i n e During the Automated Degradation of H i s t o n e I I b 2 107 31. Comparison of the Amino-Terminal Sequence of C a l f Thymus Hi s t o n e I I b 2 With That o f T r o u t T e s t i s I I b 2 . . 109 32. Automated Degradation o f [ x H C ] - a c e t a t e L a b e l e d H i s t o n e I I b 2 Using V o l a t i l e Reagents 110 33. I n c o r p o r a t i o n of [ l **C]-acetate i n t o D i f f e r e n t C e l l Types o f T r o u t T e s t i s 114 34. A c e t y l a t i o n o f Hi s t o n e s i n D i f f e r e n t T e s t i s C e l l Types . . 115 35. I n c o r p o r a t i o n o f [l*C]-acetate i n t o T r o u t T e s t i s 36. A c e t y l a t i o n o f His t o n e s i n T r o u t T e s t i s Spermatids . . 118 37. I n c o r p o r a t i o n o f [ x''C]-acetate and [ 3 H ] - l y s i n e I n t o D i f f e r e n t C e l l Types From T r o u t T e s t i s . . . . . . . . 120 38. S y n t h e s i s and A c e t y l a t i o n o f His t o n e s i n D i f f e r e n t C e l l Types from T r o u t T e s t i s 121 39. Turnover o f I 1''C]-acetyl Groups i n T r o u t T e s t i s H l S t O X l G S 0 9 e O 9 9 » » 9 9 9 0 9 9 9 « » « « O « » * X 2 4 40. I n c o r p o r a t i o n o f [ 3 H ] - a r g i n i n e i n t o T r o u t T e s t i s W i t h T l I T l G « o • 0 « a o o e o • e • « o o o o o • e o o X2 5 41. Uptake of [*''C]-acetate i n t o A c i d - S o l u b l e P r o t e i n s o f T r o u t T e s t i s N u c l e i . . . . . . . 128 42. Dependence o f the Histo n e A c e t y l t r a n s f e r a s e A c t i v i t y o f N u c l e o h i s t o n e on Mg + + Ions . . . . . . . . 130 43. Dependence o f the Histo n e A c e t y l t r a n s f e r a s e A c t i v i t y o f N u c l e o h i s t o n e on K+ Ions • . 131 -x-ii,-44. Dependence of Hi s t o n e A c e t y l t r a n s f e r a s e A c t i v i t y on Enzyme C o n c e n t r a t i o n 135 45. Dependence o f Histone A c e t y l t r a n s f e r a s e A c t i v i t y on H i s t o n e C o n c e n t r a t i o n 136 46. Time Course of Hi s t o n e A c e t y l t r a n s f e r a s e A c t i v i t y . . . 137 47. pH Dependence of the Hist o n e A c e t y l t r a n s f e r a s e A c t i v i t y from T r o u t T e s t i s Chromatin . 139 48. Sephadex G-200 Chromatography of Histone A c e t y l -t r a n s f e r a s e A c t i v i t y 140 49. Summary of the N- t e r m i n a l Sequences and S i t e s o f Mod-i f i c a t i o n i n Tr o u t T e s t i s H i s t o n e s IV, I l b i , I I I and H b 2 147 50. H e l i c a l Wheel o f Hi s t o n e IV 153 -1-I n t r o d u c t i o n  H i s t o n e s ; Nomenclature and S t r u c t u r e . Histones are very b a s i c p r o t e i n s which are found a s s o c i a t e d w i t h the DNA of e u k a r y o t i c organisms (1-3). From e a r l y s t u d i e s (2,4,5), i t had appeared t h a t c e r t a i n h i s t o n e f r a c t i o n s v a r i e d i n k i n d or amount from one t i s s u e t o another, and i t was suggested t h a t h i s t o n e s were s p e c i f i c gene r e p r e s s o r s ( 2 ) . However, sub-sequent work has demonstrated t h a t much of the apparent hetero-g e n e i t y of h i s t o n e s was a r t i f a c t u a l . I t was r e c o g n i z e d , f o r i n s t a n c e , t h a t p r o t e o l y s i s c o u l d generate a r t i f a c t s d u r i n g i s o -l a t i o n of the h i s t o n e s (6-8). Another source of h e t e r o g e n e i t y proved to be non-covalent a g g r e g a t i o n , and t h i s was e l i m i n a t e d by the use of urea (9-12), guanidinium c h l o r i d e (13) or a c i d (14) i n p u r i f i c a t i o n and f r a c t i o n a t i o n procedures. Furthermore, the presence of c y s t e i n e i n the a r g i n i n e - r i c h h i s t o n e I I I was shown to l e a d to c o v a l e n t a g g r e g a t i o n , w i t h r e s u l t i n g molecular com-p l e x i t y (15,16) of t h i s f r a c t i o n . I t i s now r e c o g n i z e d t h a t the number of d i f f e r e n t h i s t o n e s i s r e l a t i v e l y s m a l l , and t h a t the h i s t o n e complement v a r i e s l i t t l e from one t i s s u e t o another or from one s p e c i e s to another. Some p r o p e r t i e s of the major h i s t o n e c l a s s e s , and the two main systems of nomenclature i n c u r r e n t use are g i v e n i n T a b l e 1. Throughout t h i s t h e s i s , the nomenclature of Rasmussen e t a l . (17) w i l l gen-e r a l l y be f o l l o w e d . In a d d i t i o n to these h i s t o n e f r a c t i o n s , which are found i n the somatic c e l l s o f a l l animals and p l a n t s , there i s Table 1 Nomenclature and Some C h a r a c t e r i s t i c s o f the P r i n c i p a l Components of C a l f Thymus H i s t o n e * C l a s s F r a c t i o n Lys M o l e c u l a r Weight N - t e r m i n a l C - t e r m i n a l Rasmussen Johns and et al.(17) B u t l e r ( 1 8 ) Arg L y s i n e - r i c h l a f l b f l 20 (200) 20 (200) 21,000 (200) 21,000 (200) Ac-Ser (201) Ac-Ser (201) Lys (202) Lys (202) S l i g h t l y L y s i n e - r i c h I l b i f2a2 I I b 2 f2b 1.0-1.2 (178) 2.5 (31) 13,000-17,000 (30) 13,774 (31) Ac-Sef (178) Pro (31,32) Lys (178) Lys (31,32) A r g i n i n e - r i c h I I I f3 IV f 2 a l 0.7-0.8 (30) 0.8 (30) 15,324 (33) 11,282 (26) A l a (180) AcSer (26) A l a (180) Gly (26) * Reference numbers are given i n parentheses. -3-a moderately l y s i n e - r i c h , s e r i n e - r i c h h i s t o n e ( f r a c t i o n V or f2c) found i n the e r y t h r o c y t e s o f b i r d s (19) and of f i s h , r e p t i l e s and amphibians (20) , and a moderately l y s i n e - and a l a n i n e - r i c h h i s t o n e ( h i s t o n e T) found i n t r o u t t i s s u e s (21). I t should a l s o be noted t h a t the sperm c e l l n u c l e i of many animal s p e c i e s c o n t a i n s m a l l , h i g h l y b a s i c p r o t e i n s c a l l e d protamines (22,132), which may r e -p l a c e the h i s t o n e s normally found i n the somatic c e l l s . With the advent of r e l i a b l e methods f o r the l a r g e - s c a l e pur-i f i c a t i o n of i n d i v i d u a l h i s t o n e s (23,24) , d e t a i l e d s t r u c t u r a l s t u d i e s on these p r o t e i n s became f e a s i b l e . The f i r s t sequence of a h i s t o n e t o be r e p o r t e d was t h a t o f h i s t o n e IV from c a l f thymus, which was achieved independently by DeLange e t a l . (25,26) and by Ogawa e t a l . (27). The complete sequence i s g i v e n i n F i g . l a . Notable f e a t u r e s o f t h i s p r o t e i n are the presence of an a c e t y l a t e d l y s y l r e s i d u e a t p o s i t i o n 16 i n approximately 50% of the molecules, and a methylated l y s y l r e s i d u e a t p o s i t i o n 20 (present as mono-methyl and dimethyl l y s i n e i n a r a t i o o f 1:3 (27)). Although m e t h y l a t i o n of l y s y l r e s i d u e s occurs i n a number of p r o t e i n s (28), a c e t y l a t i o n of l y s y l r e s i d u e s i s not known to occur n a t u r a l l y i n any p r o t e i n s other than the h i s t o n e s . Another s t r i k i n g f e a t u r e o f the p r o t e i n i s the d i s t r i b u t i o n o f amino a c i d r e s i d u e s (26): the net charge of the amino-terminal r e g i o n ( r e s i d u e s 1-45) i s +16, w i t h nine of the b a s i c r e s i d u e s being p r e s e n t i n the f i r s t 20 r e s i d u e s ; i n c o n t r a s t t o t h i s , the net charge of the c a r b o x y - t e r m i n a l r e g i o n ( r e s i d u e s 46-102) i s on l y +3, and most o f the hydrophobic r e s i d u e s occur i n t h i s r e g i o n . FIG. 1 (a) 10 20 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 - G l y - A l a - L y s ( A c ) - A r g - H i s - A r g - L y s ( M e ) - V a l -30 40 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 - G l y - G l y - V a l - L y s - A r g - A r g -50 60 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 - L e u - G l u - A s n - V a l - I l e - A r g - A s p - A l a -70 80 90 Va 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 - M 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 -100 Gly-Arg-Thr-Leu-Tyr-Gly-Phe-Gly-Gly-COOH FIG. l a . Amino a c i d sequence o f c a l f thymus h i s t o n e IV, as deduced by DeLange e t a l . (26) 10 20 H 2 N - P r o - G l n - P r o - A l a - L y s - S e r - A l a - P r o - A l a - P r o - L y s - L y s - G l y - S e r - L y s - L y s - A l a - V a l - T h r - L y s - A l a - G l n - L y s -30 40 Ly s - A s p - G l y - L y s - L y s - A r g - L y s - A r g - S e r - A r g - L y s - G l u - S e r - T y r - S e r - V a l - T y r - V a l - T y r - L y s - V a l - L e u - L y s - G l n -50 60 70 Val-His-Pro-Asp-Thr-Gly-Ile-Ser-Ser-Lys-Ala-Met-Gly-Ile-Met-Asn-Ser-Phe-Val-Asn-Asp-Ile-Phe-Glu-80 90 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 - A s n - L y s - A r g - S e r - T h r - I l e - T h r - S e r - A r g - G l u - I l e - G l n -100 110 Th 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 - 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 -120 Lys-Tyr-Thr-Ser-Ser-Lys-COOH FIG. l b . Amino a c i d sequence o f c a l f thymus h i s t o n e I I b 2 , as deduced by Iwai e t a l . (31). L y s y l r e s i d u e 19 i n r e f . 31 has been transposed t o p o s i t i o n 16 (30). -5-On the b a s i s of these f i n d i n g s , i t was suggested t h a t the amino-t e r m i n a l r e g i o n might c o n t a i n the primary DNA-binding s i t e s , and t h a t any conformation which the h i s t o n e might have by i t s e l f (not superimposed by DNA) would most l i k e l y be i n the c a r b o x y - t e r m i n a l r e g i o n of the p r o t e i n . Probably the most s u r p r i s i n g c h a r a c t e r i s t i c of the h i s t o n e IV sequence i s i t s extreme e v o l u t i o n a r y conservatism, which became e v i d e n t when the sequence of pea h i s t o n e IV was completed (29). Pea h i s t o n e IV, l i k e the c a l f thymus p r o t e i n , c o n t a i n s 102 amino a c i d r e s i d u e s , and i t s sequence i s i d e n t i c a l t o t h a t of the bovine p r o t e i n , except f o r two c o n s e r v a t i v e replacements: r e s i d u e 60 i s v a l i n e i n the c a l f , but i s o l e u c i n e i n the pea, and r e s i d u e 77 i s l y s i n e i n the c a l f but a r g i n i n e i n the pea. A l s o , l y s y l r e s i d u e 20 i s not methylated i n the pea h i s t o n e , and i n a d d i t i o n t o a c e t y l a t i o n of l y s y l r e s i d u e 16, a t l e a s t one of l y s y l r e s i d u e s 5,8 or 12 was found t o be a c e t y l a t e d i n the pea. No other p r o t e i n i s known to have such constancy of sequence i n s p e c i e s as d i s t a n t l y r e l a t e d as peas and c a t t l e (131). T h i s suggests t h a t the f u n c t i o n of h i s t o n e IV imposes very s t r i c t l i m i t a t i o n s on i t s e n t i r e s t r u c t u r e , and t h a t mutations i n the h i s t o n e s t r u c t u r a l gene are e i t h e r suppressed by r e g u l a t o r y mech-anisms, or t h a t most mutations are l e t h a l and are never observed (30). The next h i s t o n e sequence t o be completed was t h a t of c a l f thymus IIb2, by Iwai e t a l . (31), and i t i s shown i n F i g . l b . Most of the sequence was a l s o deduced by H n i l i c a e t a l . (32). -6-The p o l y p e p t i d e c h a i n c o n s i s t s of 125 r e s i d u e s and, as i n the case of h i s t o n e IV, the amino-terminal r e g i o n (34 r e s i d u e s ) i s very b a s i c , w i t h a charge of +15; the middle p a r t of the molecule (45 r e s i d u e s ) has a charge of o n l y +1, and the c a r b o x y - t e r m i n a l r e g i o n (46 r e s i d u e s ) has a charge of +6. No evidence f o r the presence of a c e t y l l y s i n e or m e t h y l l y s i n e was o b t a i n e d . The sequence of c a l f thymus h i s t o n e I I I has r e c e n t l y been completed (33) , and i s i l l u s t r a t e d i n F i g . 2a. Again, the amino-t e r m i n a l r e g i o n i s the most b a s i c p a r t of the molecule. P a r t i a l e - a c e t y l a t i o n of l y s y l r e s i d u e s 14 and 23 and m e t h y l a t i o n of l y s y l r e s i d u e s 9 and 27 was found. The molecule c o n t a i n s two c y s t e i n e r e s i d u e s , a t p o s i t i o n s 96 and 110. S i n c e h i s t o n e I I I from c a l f thymus and pea bud have ve r y s i m i l a r p e p t i d e maps, i t seems t h a t t h i s h i s t o n e has a l s o remained very c o n s e r v a t i v e throughout e v o l u t i o n (34). Pea h i s t o n e I I I , however, c o n t a i n s o n l y a s i n g l e c y s t e i n e r e s i d u e , and i t i s t h e r e f o r e i n f e r r e d t h a t the h i g h e r o r d e r multimers which can form w i t h c a l f thymus I I I are not b i o l o g i c a l l y meaningful, and t h a t o n l y monomers and dimers are of p o s s i b l e b i o l o g i c a l importance (34). Sequence s t u d i e s on h i s t o n e s I and I l b i are not y e t complete. Most of the sequence of h i s t o n e I l b i from c a l f thymus has been done by Simson and P h i l l i p s (35) , and from t r o u t t e s t i s by B a i l e y and Dixon (36). The sequence of the amino-terminal 23 r e s i d u e s i s g i v e n i n F i g . 2b. T h i s r e g i o n of the molecule i s v e r y s i m i l a r i n t r o u t t e s t i s and i ) FIG. 2 (a) 10 20 H2N-Ala-Arg-Thx-Lys-Gln-Thr-Ala-Arg-Lys (CH3) 0-2-Ser-Thr-Gly-Gly-Lys (Ac) -Ala-Pro-Arg-Lys-Gln-Leu-30 40 Ala-Thr-Lys (Ac) -Ala-Ala-Arg-Lys (CH3) 0-2-Ser-Ala-Pro-Ala-Thr-Gly-Gly-Val-Lys-Lys-Pro-His-Arg-Tyr-50 60 Arg-Pro-Gly-Thr-Val- Ala-Leu- Arg-Glu-Ile-Arg-Arg-Tyr-Gln-Lys-Ser-Thr-Glu-Leu-Leu-Ile-Arg-Lys-Leu-70 80 Pro-Phe-Gln-Arg-Leu-Val-Arg-Glu-Ile-Ala-Gln-Asp-Phe-Lys-Thr-Asp-Leu-Arg-Phe-Gln-Ser-Ser-Ala-Val-90 100 110 Met-Ala-Leu-Gln-Glu-Ala-Cys-Glu-Ala-Tyr-Leu-Val-Gly-Leu-Phe-Glu-Asp-Thr-Asn-Leu-Cys-Ala-Ile-His-120 130 Ala-Lys-Arg-Val-Thr-Ile-Met-Pro-Lys-Asp-Ile-Gln-Leu-Ala-Arg-Arg-Ile-Arg-Gly-Glu-Arg-Ala-COOH FIG. 2a. Sequence of c a l f thymus histone' I I I , according to DeLange et a l . (33). (b) 10 20 Ac-Ser-Gly-Arg-Gly-Lys-Thr-Gly-Gly-Lys-Ala-Arg-Ala-Lys-Ala-Lys-Thr-Arg-Ser-Ser-Arg-Ala-Gly-Leu FIG. 2b. P a r t i a l sequence of histone I l b i from trout t e s t i s , elucidated by Bailey and Dixon (36). (c) Pro-Lys-Arg-Lys-Gly-Ala- (Thr) -Lys-Gly-Asp-Glu-Pro-Ala-Arg- (Arg) -Ser-Ala-Arg-Leu-Ser-?-Arg-Pro-Val-Pro-Lys-Pro-Ala-Ala FIG. 2c. P a r t i a l sequence of histone T from trout t e s t i s , according to Huntley and Dixon (42) . -8-1 10 RT-3; A c - S e r - G l u - A l a - P r o - A l a - G l u - T h r - A l a - A l a - P r o -RT-4: A c - S e r - G l u - A l a - P r o - A l a - G l u - T h r - A l a - A l a - P r o -CT-1: A c - S e r - G l u - A l a - P r o - A l a - G l u - T h r - A l a - A l a - P r o -20 A l a - P r o - A l a - G l u - L y s - S e r - P r o - A l a - L y s - - L y s -A l a - P r o - A l a - - L y s - S e r - P r o - A l a - L y s - T h r - P r o -A l a - P r o - A l a - P r o - L y s - S e r - P r o - A l a - L y s - T h r - P r o -30 L y s - L y s - A l a - A l a - L y s - L y s - P r o - G l y - A l a - G l y - A l a -V a l - L y s - A l a - A r g - L y s - L y s - L y s - S e r - A l a - G l y - A l a -V a l - L y s - A l a - A l a - L y s - L y s - L y s - L y s - P r o - A l a - G l y -40 A l a - L y s - A r g - L y s - A l a - A l a - G l y - P r o - P r o - V a l - S e r -* A l a - 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 - S e r -* A l a - A r g - A r g - L y s - A l a - S e r - G l y - P r o - P r o - V a l - S e r -50 G l u - L e u - I l e - T h r - L y s - A l a - V a l - A l a - A l a - S e r - L y s -G l u - L e u - I l e - T h r ~ L y s - A l a - V a l - A l a - A l a - S e r - L y s -G l u - L e u - I l e ~ T h r - L y s - A l a - V a l - A l a - A l a - S e r - L y s -60 Glu-Arg-Asn-Gly-Leu-Ser-Leu-Ala-Ala-Leu-Lys-Glu-Arg-Ser-Gly-Val-Ser-Leu-Ala-Ala-Leu-Lys-Glu-Arg-Ser-Gly-Val-Ser-Leu-Ala-Ala-Leu-Lys-70 L y s - A l a - L e u - A l a - A l a - G l y - G l y - T y r Lys- Ala-Leu- A l a - A l a - A l a - G l y - T y r L y s - A l a - L e u - A l a - A l a - A l a - G l y - T y r FIG. 3. P a r t i a l amino a c i d sequences of two h i s t o n e I components from r a b b i t thymus (RT-3, RT-4) and of one component from c a l f thymus (37). The s e r y l r e s i d u e s a t p o s i t i o n 37 ( a s t e r i s k s ) are s i t e s o f p h o s p h o r y l a t i o n (38,39). -9-c a l f thymus, except at r e s i d u e 6, where the g l u t a m i n y l r e s i d u e of the c a l f thymus p r o t e i n i s r e p l a c e d by a t h r e o n y l r e s i d u e i n t r o u t t e s t i s . The sequence of the f i r s t 73 r e s i d u e s of s e v e r a l h i s t o n e I s u b f r a c t i o n s has been done by R a i l and Cole (37). These are shown i n F i g . 3. The s e r y l r e s i d u e a t p o s i t i o n 38 has been shown to be the s i t e of p h o s p h o r y l a t i o n i n c a l f thymus (38) and i n r a b b i t thymus (39). In one of the r a b b i t thymus components (RT-3, F i g . 3) the s e r y l r e s i d u e has been r e p l a c e d by an a l a n y l r e s i d u e , thus p r e c l u d i n g p h o s p h o r y l a t i o n of t h i s s i t e . Cleavage of the h i s t o n e I molecule by v a r i o u s means has r e v e a l e d t h a t the most b a s i c p a r t of t h i s h i s t o n e i s the c a r b o x y - t e r m i n a l r e g i o n , and t h a t most hydrophobic and a c i d i c r e s i d u e s are p r e s e n t i n the amino-t e r m i n a l r e g i o n (40). Hence, the d i s t r i b u t i o n of r e s i d u e s i n h i s t o n e I i s d i f f e r e n t from t h a t of the o t h e r h i s t o n e s . The mul-t i p l i c i t y of the l y s i n e - r i c h h i s t o n e s suggests t h e r e i s a d i v e r s i t y i n f u n c t i o n w i t h i n t h i s group. S t u d i e s on the sequence of h i s t o n e I from t r o u t t e s t i s are a l s o c u r r e n t l y i n p r o g r e s s (41). A p a r t i a l amino-terminal sequence of h i s t o n e T from t r o u t t e s t i s has r e c e n t l y been e l u c i d a t e d (42) , and t h i s p r o t e i n i s seen to be d i s t i n c t from any of the o t h e r major h i s t o n e f r a c t i o n s ( F i g . 2c ) . Thus, one may expect t h a t the sequences of a l l the major h i s t o n e s from some sources w i l l be completed w i t h i n the next year or two. T h i s i n f o r m a t i o n w i l l be i n v a l u a b l e t o anyone attempting to deduce the b i o l o g i c a l r o l e s o f these chromosomal p r o t e i n s . -10-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 of H i s t o n e s . (i) A c e t y l a t i o n ; The occurrence of a c e t y l a t e d and methylated l y s y l r e s i d u e s i n some h i s t o n e f r a c t i o n s was mentioned above. The presence of e - N - a c e t y l l y s i n e i n h i s t o n e s was f i r s t noted independently by DeLange e t a l . (25,29) and by A l l f r e y e t a l . (43,44). I t had p r e v i o u s l y been found by P h i l l i p s (45) t h a t a c e t y l groups were pre s e n t a t the amino-terminal of s e v e r a l h i s t o n e f r a c t i o n s (Table 1 ) , but the above s t u d i e s by A l l f r e y e t a l . showed t h a t f o l l o w i n g the i n c u b a t i o n of c a l f thymus n u c l e i w i t h t 1 l*C]-acetate, most of the l a b e l was i n c o r p o r a t e d as e-N-acetyl groups. The r e a c t i o n was not i n h i b i t e d by c o n c e n t r a t i o n s of puromycin s u f f i c i e n t to b l o c k p r o t e i n s y n t h e s i s , and i t was concluded t h a t the r a d i o -a c t i v e groups were at t a c h e d t o the p r o t e i n a f t e r completion of the p o l y p e p t i d e c h a i n (46) . A c e t y l a t i o n of the amino-terminal of h i s t o n e s , however, was c o r r e l a t e d w i t h h i s t o n e s y n t h e s i s i n d i v i d i n g c e l l s (47,48) , and i t has been claime d t h a t N - a c e t y l s e r i n e may be i n v o l v e d i n the i n i t i a t i o n of h i s t o n e s y n t h e s i s (48). Huang and Bonner (49) f i r s t observed t h a t DNA-dependent RNA s y n t h e s i s by RNA polymerase on pea embryo DNA was i n h i b i t e d by h i s t o n e s . These o b s e r v a t i o n s l e d t o a renewed i n t e r e s t i n the i d e a of h i s t o n e s as s p e c i f i c gene r e p r e s s o r s (50), o r i g i n a l l y proposed by Stedman and Stedman (2). S i n c e the a c e t y l a t i o n of a l y s y l r e s i d u e e l i m i n a t e s i t s pos-i t i v e charge, and thus r e s u l t s i n a decrease i n the net p o s i t i v e charge of the p r o t e i n , a c e t y l a t i o n of h i s t o n e s might a f f e c t the -11-c a p a c i t y of these p r o t e i n s to i n h i b i t RNA s y n t h e s i s i n v i v o (46). Thus, A l l f r e y e t a l . found t h a t chemical a c e t y l a t i o n of h i s t o n e s i n v i t r o t o degrees which d i d not a f f e c t t h e i r a b i l i t y t o combine w i t h DNA lowered the i n h i b i t i o n of RNA polymerase a c t i v i t y on c a l f thymus DNA by 21-66% (46). An i n c r e a s e i n h i s t o n e a c e t y l a t i o n was observed i n lymphocytes which were s t i m u l a t e d t o grow and d i v i d e by the mit o g e n i c agent, phytohemagglutinin (51) , and i n r e g e n e r a t i n g r a t l i v e r (52). The i n c r e a s e i n h i s t o n e a c e t y l a t i o n appeared t o precede i n c r e a s e d RNA s y n t h e s i s i n these c e l l s . Polymorphonuclear l e u c o c y t e s , which do not d i v i d e , showed a decrease i n t h e i r r a t e s of RNA 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 when t r e a t e d w i t h phyto-hemagglutinin (51). In r e g e n e r a t i n g l i v e r , furthermore, the i n c r e a s e i n h i s t o n e a c e t y l a t i o n was a s s o c i a t e d w i t h a decreased r a t e of a c e t y l group turnover (52). C o r t i s o l treatment of adrenalectomized r a t s leads t o i n c r e a s e s i n the rate, of h i s t o n e a c e t y l a t i o n i n l i v e r (53,54), and e r y t h r o p o i e t i n s t i m u l a t e s h i s t o n e a c e t y l a t i o n and RNA s y n t h e s i s i n the sp l e e n c e l l s of polycythemic mice (55). Enzyme p r e p a r a t i o n s which t r a n s f e r a c e t y l groups from a c e t y l coenzyme A t o h i s t o n e s have been ob t a i n e d from pigeon l i v e r (56, 57) and r a t l i v e r (58-60). The r a t l i v e r enzymes are b e s t char-a c t e r i z e d ; they were o b t a i n e d from r a t l i v e r n u c l e i by s o n i c a t i o n i n the presence of 1M ammonium s u l f a t e (59). Two h i s t o n e a c e t y l -a t i n g enzymes have been i s o l a t e d i n t h i s way from r a t l i v e r and kidney, and t h r e e from r a t thymus (60). Enzymes from d i f f e r e n t organs showed d i f f e r i n g chromatographic p a t t e r n s on D E A E - c e l l u l o s e , -12-and d i f f e r e n t s p e c i f i c i t i e s f o r c a l f thymus h i s t o n e f r a c t i o n s f2a and f3 (60). An enzyme which c a t a l y z e s the d e a c e t y l a t i o n o f h i s -tones has been d e t e c t e d i n c a l f thymus e x t r a c t s (61) and i n t r o u t t e s t i s (62). The c a l f thymus enzyme showed g r e a t e r a c t i v i t y t o -wards h i s t o n e s a c e t y l a t e d i n vivo, versus h i s t o n e s l a b e l e d i n v i t r o w i t h i1 ^C]-acetic anhydride. A d i r e c t c a u s e - a n d - e f f e c t r e l a t i o n s h i p between h i s t o n e a c e t y l -a t i o n and i n c r e a s e d DNA template a c t i v i t y has not y e t been demon-s t r a t e d ; the r e s u l t s d i s c u s s e d above are onl y s u g g e s t i v e i n t h i s r e g a r d . The r e l a t i v e l y low m u l t i p l i c i t y o f h i s t o n e s probably r u l e s out the p o s s i b i l i t y o f t h e i r f u n c t i o n i n g as s p e c i f i c gene r e p r e s s o r s by themselves (63); however, they might s t i l l c o n c e i v -a b l y f u n c t i o n i n such a c a p a c i t y i f combined w i t h other macro-molecules such as non-histone chromosomal p r o t e i n s (64) or RNA molecules (65,66), which might d i r e c t h i s t o n e s t o s p e c i f i c s i t e s on DNA. A l t e r n a t i v e l y , non-histone chromosomal p r o t e i n s might a c t t o b l o c k h i s t o n e b i n d i n g t o s p e c i f i c areas o f DNA. The a c e t y l a t i o n of h i s t o n e s i s not meant to e x p l a i n the s p e c i f i c i t y o f gene a c t i v a t i o n , but r a t h e r i s assumed to be p a r t of a "loosening-up" p r o c e s s which makes the DNA template a v a i l -a b l e f o r the a c t i v a t i o n o f s p e c i f i c RNA s y n t h e s i s by ot h e r mech-anisms. The analogy g i v e n i s t h a t of p u l l i n g out a f i l e - d r a w e r b e f o r e s e a r c h i n g the f i l e s f o r a p a r t i c u l a r type of i n f o r m a t i o n (54). I n co n n e c t i o n w i t h t h i s h y p o t h e s i s , i t i s i n t e r e s t i n g t o note t h a t S h i h and Bonner (67) have found evidence f o r two types - 1 3 -of mechanism i n v o l v e d i n the i n h i b i t i o n of E. c o l i RNA polymerase a c t i v i t y by b a s i c p o l y p e p t i d e s . I f the DNA-basic p o l y p e p t i d e com-p l e x was i n a t i g h t l y aggregated s t a t e , i t was u n a v a i l a b l e f o r use as a template by the enzyme; i f the complex was i n a l o o s e , h i g h l y hydrated s t a t e , i t was a b l e to b i n d enzyme, but RNA s y n t h e s i s was s t i l l i n h i b i t e d . In the l a t t e r case, the b a s i c p o l y p e p t i d e s ( p o l y -l y s i n e , p o l y - a r g i n i n e or protamine) were b e l i e v e d to be i n t e r f e r i n g w i t h l o c a l unwinding of the DNA. As w i l l be d i s c u s s e d l a t e r i n t h i s t h e s i s , r e c e n t evidence suggests t h a t a major f u n c t i o n of h i s t o n e a c e t y l a t i o n i s to decrease the charge d e n s i t y of the p r o t e i n so t h a t i t can assume the c o r r e c t conformation on the DNA, p r i o r t o becoming f i r m l y bound t o i t (see " D i s c u s s i o n " ) . For reviews on h i s t o n e a c e t y l a t i o n , see r e f s . 3 5 , 5 4 and 6 8 . ( i i ) M e t h y l a t i o n : The presence of e-N-methyllysine i n h i s t o n e s was f i r s t r e -p o r t e d by Murray (69) , and the presence of methylated l y s y l r e s -idues a t s p e c i f i c p o s i t i o n s i n the a r g i n i n e - r i c h h i s t o n e s was d i s -cussed above. The presence of both monomethyl and d i m e t h y l l y s i n e i n c a l f thymus h i s t o n e s was noted by P a i k and Kim ( 7 0 ); enzymes which methylate h i s t o n e s u s i n g methionine as the methyl donor are found i n the n u c l e i o f c a l f thymus ( 4 6 , 7 1 ) and r a t l i v e r (72) c e l l s . The r e a c t i o n i s not i n h i b i t e d by puromycin, and i t has been con-c l u d e d t h a t the m o d i f i c a t i o n o c c u r s a f t e r s y n t h e s i s of the h i s t o n e ( 4 6 ) . -14-There appear to be s p e c i e s d i f f e r e n c e s i n the m e t h y l a t i o n of a r g i n i n e - r i c h h i s t o n e s ; thus, c a l f thymus h i s t o n e IV was found t o c o n t a i n a mixture of monomethyl- and d i m e t h y l l y s i n e a t p o s i t i o n 20, whereas no m e t h y l a t i o n a t t h i s p o s i t i o n was observed i n pea s e e d l i n g h i s t o n e IV (29). Pea s e e d l i n g chromatin incubated w i t h ,[ 3H]-methyl methionine r e s u l t e d i n the i n c o r p o r a t i o n o f l a b e l i n t o h i s t o n e s I I I and l i b , but not i n t o IV, a p a t t e r n s i m i l a r t o t h a t seen i n v i v o (73). The b i o l o g i c a l s i g n i f i c a n c e o f l y s i n e m e t h y l a t i o n i n h i s t o n e s remains obscure. S t u d i e s on the t i m i n g of h i s t o n e m e t h y l a t i o n f o l l o w i n g p a r t i a l hepatectomy have r e v e a l e d t h a t the peak i n methyl-a t i o n o f the a r g i n i n e - r i c h h i s t o n e s o c c u r s a t a time when the r a t e s o f h i s t o n e s y n t h e s i s and DNA s y n t h e s i s have a l r e a d y begun t o d e c l i n e (74). The m e t h y l a t i o n of the e-NH2 group of a l y s y l r e s i d u e r a i s e s the pK of t h a t group (28), and i t has been suggested t h a t methyl-a t i o n may be i n v o l v e d i n the condensation o f chromatin which occurs p r i o r to c e l l d i v i s i o n (74). The presence o f c j-N-methylarginine i n r a t l i v e r h i s t o n e s has r a l s o been r e p o r t e d (75), and when c a l f thymus n u c l e i are incubated w i t h S-adenosyl-L-methionine-(methyl 1 " c ) , a t r a n s f e r o f methyl groups t o a r g i n i n e r e s i d u e s o c c u r s (76). I n c u b a t i o n of duck r e t i c u l o c y t e s and e r y t h r o c y t e s i n the presence o f l a b e l e d methionine r e s u l t e d i n the 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 i t y as 3 - m e t h y l h i s t i d i n e i n h i s t o n e s f l and f 2 c (77), and t h i s d e r i v a t i v e has a l s o been de-t e c t e d i n s m a l l amounts i n h i s t o n e s from the l i v e r s of r a t s i n j e c --15-t e d w i t h l a b e l e d methionine (35). The s i g n i f i c a n c e of these mod-i f i c a t i o n s i s not known. P r o t e i n m e t h y l a t i o n has r e c e n t l y been reviewed by P a i k and Kim (28). ( i i i ) P h o s p h o r y l a t i o n : Histone p h o s p h o r y l a t i o n has been s t u d i e d e x t e n s i v e l y i n s e v e r a l systems, s i n c e Ord and Stocken (78) and K l e i n s m i t h e t a l . (79) r e p o r t e d the presence of phosphoserine i n h i s t o n e s . P h o s p h o r y l -a t i o n of a l l major h i s t o n e f r a c t i o n s (80,81), as w e l l as of p r o t -amine (80,82,83) takes p l a c e i n d e v e l o p i n g t r o u t t e s t i s ; i n ot h e r systems, the p h o s p h o r y l a t i o n of h i s t o n e I has been most s t u d i e d (84-88) although p h o s p h o r y l a t i o n o f h i s t o n e s I I b 2 and I I I (89) has a l s o been r e p o r t e d i n c a l f thymus. The a v i a n e r y t h r o c y t e h i s t o n e V a l s o seems to be phosp h o r y l a t e d (90). The p h o s p h o r y l a t i o n of a s e r y l r e s i d u e a t p o s i t i o n 37 i n c e r t a i n s u b f r a c t i o n s of h i s t o n e I from c a l f thymus and r a b b i t thymus was d i s c u s s e d above (38,39), and i s i n d i c a t e d i n F i g . 3. In t r o u t t e s t i s h i s t o n e s I l b i and IV, the amino-terminal N - a c e t y l -s e r y l r e s i d u e i s the s i t e o f p h o s p h o r y l a t i o n (81,91), and h i s t o n e I i s phosphorylated a t a Lys-Ser-Pro-Lys sequence i n the carboxy-t e r m i n a l r e g i o n of the molecule (81). In one of the components of t r o u t t e s t i s protamine, t h r e e s e r y l r e s i d u e s i n the sequence Arg-Ser-Ser-Ser-Arg-Pro were found t o be phosphorylated i n a f r a c -t i o n o f the molecules (83). Enzyme systems which phos p h o r y l a t e h i s t o n e s i n v i t r o u s i n g ATP have been d e s c r i b e d . Langan (92) o b t a i n e d an enzyme pre p a r a --16-t i o n from rat l i v e r which phosphorylated histones, and which was stimulated four-to s i x - f o l d by 3',5'-cyclic AMP. This stimulation could also be demonstrated i n vivo, by int r a p e r i t o n e a l i n j e c t i o n s of c y c l i c AMP and d i b u t y r y l c y c l i c AMP, and the same s i t e i n histone I was shown to be modified i n both the i n vivo and i n v i t r o cases (93). Siebert et a l . (94) found histone phosphokinase ac-t i v i t y both i n the cytoplasmic f r a c t i o n and on the chromatin of r a t l i v e r c e l l s . The cytoplasmic enzyme was stimulated f o u r - f o l d by 1 uM c y c l i c AMP, while the nuclear enzyme was only marginally activated, with histone I as substrate. A kinase which phosphoryl-ates protamine p r e f e r e n t i a l l y , and i s activated by c y c l i c AMP, has been p u r i f i e d from rainbow trout t e s t i s (95). Phosphorylation of histone I from d i f f e r e n t species and from d i f f e r e n t organs with t h i s protamine kinase, followed by t r y p t i c digestion, showed differences i n the phosphopeptide pattern of histone I both from d i f f e r e n t organs i n the same species and from the same organ i n d i f f e r e n t species (96). A phosphatase s p e c i f i c for histones and protamines has also been i s o l a t e d (97); two fractions with d i f f e r e n t s p e c i f i c i t i e s for histone I and protamine were separable, and the a c t i v i t y was detected i n a l l eukaryotic c e l l s examined, but not i n bacteria or blue-green algae. As i n the case of histone acetylation, attempts have been made to correlate phosphorylation with changes i n gene a c t i v i t y . Siebert et a l . (94) found a s i x - f o l d increase i n the a c t i v i t y of a histone phosphokinase from the n u c l e i of regenerating rat l i v e r s -17-22 hr a f t e r p a r t i a l hepatectomy; Sung e t a l . (98) showed t h a t f o l l o w i n g p a r t i a l hepatectomy, p h o s p h o r y l a t i o n of the amino-t e r m i n a l s e r y l r e s i d u e i n h i s t o n e l i b * c o u l d be demonstrated. An i n c r e a s e i n h i s t o n e phosphate content has a l s o been noted d u r i n g r e g e n e r a t i o n of the pancreas f o l l o w i n g e t h i o n i n e treatment (99). An i n c r e a s e d r a t e of turnover of h i s t o n e phosphate was observed d u r i n g the i n i t i a l p e r i o d o f RNA s y n t h e s i s i n lymphocytes s t i m -u l a t e d by phytohemagglutinin, and a net i n c r e a s e i n the phos-p h o r y l a t i o n of h i s t o n e I c o r r e l a t e d w i t h the p e r i o d of DNA syn-t h e s i s (100) . The e f f e c t s of v a r i o u s hormones on h i s t o n e p h o s p h o r y l a t i o n have a l s o been i n v e s t i g a t e d . Langan (101) found t h a t adminis-t r a t i o n of glucagon t o r a t s causes an i n c r e a s e i n the p h o s p h o r y l -a t i o n of a s p e c i f i c s e r y l r e s i d u e i n h i s t o n e I of l i v e r d u r i n g a one hour p e r i o d f o l l o w i n g a d m i n i s t r a t i o n of the hormone. Hydro-c o r t i s o n e and a d r e n o c o r t i c o t r o p h i c hormone had no e f f e c t , whereas i n s u l i n a d m i n i s t r a t i o n a l s o caused an i n c r e a s e i n p h o s p h o r y l a t i o n . The a d d i t i o n of i n s u l i n t o mouse mammary e p i t h e l i a l c e l l s i n organ c u l t u r e caused an e i g h t to t e n - f o l d i n c r e a s e i n n u c l e a r p r o t e i n - p h o s p h o r y l a t i o n , c o n c o m i t a n t l y w i t h a s t i m u l a t i o n o f RNA s y n t h e s i s , and p r i o r t o a s t i m u l a t i o n of h i s t o n e s y n t h e s i s (102). P r o l a c t i n s t i m u l a t e d p h o s p h o r y l a t i o n of c e r t a i n h i s t o n e f r a c t i o n s and of non-histone n u c l e a r p r o t e i n s a f t e r treatment of the e x p l a n t s w i t h i n s u l i n and h y d r o c o r t i s o n e , but not before (102). The e x t e n s i v e p h o s p h o r y l a t i o n of h i s t o n e s observed i n develop-i n g t r o u t t e s t i s l e d t o the i d e a t h a t these m o d i f i c a t i o n s might be -18-i n v o l v e d i n the detachment of h i s t o n e s from DNA d u r i n g the process of t h e i r replacement by protamine (80). However, r e c e n t e x p e r i -ments, i n which the p h o s p h o r y l a t i o n of h i s t o n e s i n d i f f e r e n t c e l l types was s t u d i e d (103), showed there was very l i t t l e p h osphoryla-t i o n of h i s t o n e s . i n spermatids, the c e l l type i n which the r e p l a c e -ment process occurs (104). Furthermore, the r a t e o f p h o s p h o r y l a t i o n was c o r r e l a t e d w i t h the r a t e of DNA s y n t h e s i s i n d i f f e r e n t c e l l t y p e s , and r e c e n t evidence suggests t h a t p h o s p h o r y l a t i o n o f h i s t o n e I l b i may be i n v o l v e d i n the c o r r e c t b i n d i n g o f t h i s p r o t e i n t o DNA (105). The p h o s p h o r y l a t i o n of protamine may p l a y a s i m i l a r r o l e , and the d e p h o s p h o r y l a t i o n o f protamine, which probably o c c u r s on the chromatin (106), may be i n v o l v e d i n the c o n t r a c t i o n of the spermatid nucleus (107) I n t e r a c t i o n s of Hi s t o n e s w i t h DNA. The major h i s t o n e f r a c t i o n s d i f f e r i n the ease w i t h which they may be d i s s o c i a t e d from n u c l e o h i s t o n e by i n c r e a s i n g s a l t con-c e n t r a t i o n s (108) or d e c r e a s i n g pH (109). Thus, the l y s i n e - r i c h h i s t o n e I i s r e l e a s e d i n the range 0.4-0.5M NaCl or pH 1.7, the ' s l i g h t l y l y s i n e - r i c h f r a c t i o n s I I b 2 and I l b i i n the range 0.8-1.2M NaCl or pH 1.0, and the a r g i n i n e - r i c h f r a c t i o n s I I I and IV i n the range 0.8-1.6M NaCl or pH 0.7-0.2. T h e r e f o r e , the b i n d i n g of h i s t o n e s t o DNA i s very t i g h t , and would not be r e a d i l y r e v e r s i b l e under p h y s i o l o g i c a l c o n d i t i o n s o f i o n i c s t r e n g t h and pH. In c o n t r a s t t o t h i s , histone-RNA complexes were found t o d i s s o c i a t e a t 0.15M NaCl (110),' s u g g e s t i n g t h a t perhaps the double-stranded -19-s t r u c t u r e of DNA i s r e q u i r e d f o r t i g h t b i n d i n g of the h i s t o n e s . In accord w i t h t h i s i d e a , A k i n r i m i s i e t a l . ( I l l ) found t h a t a t low s a l t c o n c e n t r a t i o n s (0.05-0.2M), protamines and i n d i v i d u a l h i s t o n e f r a c t i o n s e x h i b i t e d a g r e a t e r a f f i n i t y f o r n a t i v e than f o r denatured DNA. On the ot h e r hand, the f i n d i n g t h a t h i s t o n e -RNA complexes can be d i s s o c i a t e d by n a t i v e , denatured or i s o l a t e d s i n g l e - s t r a n d e d DNA i s d i f f i c u l t t o e x p l a i n (110) . Many s t u d i e s have been done on the conformation of h i s t o n e s i n s o l u t i o n by o p t i c a l r o t a t o r y d i s p e r s i o n (ORD), c i r c u l a r d i -chroism (CD), n u c l e a r magnetic resonance (NMR), and i n f r a r e d s pectroscopy (IR). In g e n e r a l , i t i s found t h a t h i s t o n e s i n aqueous s o l u t i o n s at low i o n i c s t r e n g t h are mainly i n a random c o i l conformation, but as the i o n i c s t r e n g t h i s i n c r e a s e d , or DNA or o r g a n i c s o l v e n t s are added, a s i g n i f i c a n t amount of ordered s t r u c t u r e appears (112-116). An important o b s e r v a t i o n i n t h i s r e g a rd i s the f a c t t h a t w h i l e most of the h i s t o n e f r a c t i o n s seem t o c o n t a i n a f a i r amount of ^ - h e l i c a l s t r u c t u r e a t h i g h i o n i c s t r e n g t h s , the l y s i n e - r i c h h i s t o n e s seem to form an i s o l a t e d ex-tended c h a i n conformation (112). T h i s i s i n keeping w i t h the o b s e r v a t i o n t h a t the l y s i n e - r i c h h i s t o n e i s by f a r the most e f f i c i e n t o f the h i s t o n e s i n p r e c i p i t a t i n g DNA, and wi t h the r e -s u l t i n g s u g g e s t i o n t h a t h i s t o n e I i s i n v o l v e d i n c r o s s - l i n k i n g DNA (117,118). B o u b l i k e t a l . (115) have estimated the p r o b a b i l i t y of °<>-helix formation f o r v a r i o u s r e g i o n s of h i s t o n e IV by u s i n g the h e l i c a l wheel r e p r e s e n t a t i o n of S c h i f f e r and Edmundson (119), -20-and Prothero's r u l e (120) . They concluded t h a t r e s i d u e s 1-36 and 91-102 are l e a s t l i k e l y t o form h e l i x , whereas r e s i d u e s 55-72 have the g r e a t e s t p o t e n t i a l f o r h e l i x f o r m a t i o n , w i t h r e s i d u e s 37-54 and 73-90 a l s o showing hi g h p o t e n t i a l . Zubay and Doty (121) f i r s t showed t h a t a p o l y p e p t i d e c h a i n i n the a l p h a - h e l i c a l conformation c o u l d be accommodated r e a d i l y i n the major groove of DNA, and Sung and Dixon (81) and S h i h and Bonner (122) have suggested t h a t the b a s i c amino-terminal of h i s t o n e IV binds to DNA i n t h i s manner. However, s t u d i e s u s i n g i n h i b i t o r s such as actinomycin D and phleomycin, which b i n d to DNA, have been e q u i v o c a l i n a s s i g n i n g b i n d i n g s i t e s to h i s t o n e s (123,124), and i t must be admitted t h a t s t r o n g evidence f o r s p e c i f i c b i n d i n g s i t e s o f h i s t o n e s on DNA i s s t i l l l a c k i n g (30). The conformation of DNA i n chromatin i s a l t e r e d as compared to f r e e DNA. X-ray d i f f r a c t i o n measurements suggest t h a t n u c l e o -h i s t o n e may have a s u p e r - h e l i c a l s t r u c t u r e (125-126), and there i s evidence t o suggest t h a t d i v a l e n t c a t i o n s (Ca or Mg ) may be i n v o l v e d i n the maintenance o f t h i s conformation (126). I t seems to be g e n e r a l l y agreed t h a t removal o f h i s t o n e I has l i t t l e e f f e c t on the conformation o f n u c l e o h i s t o n e , as measured by CD or ORD (116,127), but l i t t l e e l s e can be s a i d about s p e c i f i c s t r u c t u r a l r o l e s f o r i n d i v i d u a l h i s t o n e s i n chromatin. There i s evidence from s t u d i e s o f the b i n d i n g of p o l y l y s i n e to chromatin and from the a c t i o n of nucleases (128), as w e l l as from DNA m e l t i n g p o i n t s t u d i e s (122) t h a t not a l l r e g i o n s of the chromatin are complexed w i t h -21-h i s t o n e s . The l y s i n e - r i c h h i s t o n e s may b i n d p r e f e r e n t i a l l y t o regions of DNA r i c h i n A-T sequences, and the a r g i n i n e - r i c h h i s -tones to G-C sequences (129). C r i c k (203) has proposed t h a t e u k a r y o t i c DNA i s composed of r e g i o n s o f f i b r o u s DNA which codes f o r p r o t e i n s , separated by r e g i o n s of g l o b u l a r , c o n t r o l DNA. The g l o b u l a r DNA i s p o s t u l a t e d t o be r e q u i r e d f o r r e c o g n i t i o n p r o c e s s e s , and i t i s assumed t h a t the double h e l i x i s unwound b e f o r e r e c -o g n i t i o n . The f o r c e and energy needed f o r the unwinding i s supposed t o be p r o v i d e d by chromosomal p r o t e i n s , p r o b a b l y h i s t o n e s . The evidence f o r t h i s t h e o r y i s as y e t o n l y c i r c u m s t a n t i a l . For a comprehensive review of n u c l e o h i s t o n e s t r u c t u r a l s t u d i e s , see r e f . 35. The s t r u c t u r e of chromosomes has a l s o been reviewed r e c e n t l y (130). The Process of Spermatogenesis. During spermatogenesis, the germ c e l l s of the t e s t i s g i v e r i s e t o l a r g e numbers of spermatozoa, by a s e r i e s o f m i t o t i c and m e i o t i c d i v i s i o n s . F i r s t l y , as a r e s u l t of m i t o s i s of the d i p l o i d spermatogonia, l a r g e numbers of primary spermatocytes are pro-duced. These then undergo the f i r s t m e i o t i c d i v i s i o n t o produce secondary spermatocytes; the second m e i o t i c d i v i s i o n , i n v o l v i n g the secondary spermatocytes, g i v e s r i s e to the h a p l o i d spermatids. The spermatids, without d i v i d i n g f u r t h e r , undergo a s e r i e s of m o r p h o l o g i c a l changes known as spermiogenesis, which r e s u l t i n the f o r m a t i o n of the mature sperm. The t r a n s f o r m a t i o n of a spermatid i n t o a mature sperm c e l l i n v o l v e s a number of s t r i k i n g m o r p h o l o g i c a l changes. These i n --22-clude the condensation of the spermatid n u c l e u s , the s l o u g h i n g o f f of most of the cytoplasm, and the development of a p r o p u l s i v e t a i l . In the r a t , LeBlond and Clermont were ab l e t o d i v i d e the spermatids i n t o 19 stages of development (133). During t h e i r development, the spermatids of mammalian t e s t e s are i n t i m a t e l y . a s s o c i a t e d w i t h l a r g e c e l l s known as S e r t o l i or "nurse" c e l l s , the exact f u n c t i o n s of which are not c l e a r . The number of these c e l l s appears t o remain constant i n the t e s t i s , and they are known t o p h a g o c y t i z e the r e s i d u a l cytoplasm of the spermatids, and degenerating t e s t i s c e l l s of a l l types (134). Spermatogenesis i n f i s h f o l l o w s the g e n e r a l scheme o u t l i n e d above. However, whereas i n the mammal spermatozoa are u s u a l l y produced c o n t i n u o u s l y , so t h a t a l l c e l l types can be found i n the t e s t i s at a g i v e n time, i n f i s h t h e r e i s g e n e r a l l y a y e a r l y c y c l e of spermatogenesis. In Salmonid f i s h e s , t h i s c y c l e l a s t s s e v e r a l months, d u r i n g which time the t e s t i s grows from a t h r e a d r e p r e s e n t i n g perhaps 0.1% of the body weight to as much as 5% of the body weight (135,136). Perhaps because of t h i s s e asonal nature of spermatogenesis i n Salmonids, the development of the t e s t i s i s r e l a t i v e l y syn-chronous; the germ c e l l s are grouped t o g e t h e r i n nests or c y s t s , which are separated i n t o l o b u l e s by t h i n l a y e r s of c o n n e c t i v e t i s s u e (137). A l l o f the c e l l s i n a g i v e n c y s t are i n the same stage of m a t u r a t i o n . Fawcett e t a l . (138) r e p o r t e d the occurrence o f c y t o p l a s m i c b r i d g e s between groups of spermatocytes and be-tween spermatids i n many s p e c i e s , and suggested t h a t t h i s phen-omenon i s r e s p o n s i b l e f o r t h e i r synchronous d i f f e r e n t i a t i o n . No -23-c e l l s comparable t o the S e r t o l i c e l l s o f mammals have been ob-served i n Salmonids (139); the spermatids appear t o l i e f r e e l y i n the lumina o f the t e s t i s . Spermatogenesis i n v e r t e b r a t e s i s under the c o n t r o l of hor-mones from the p i t u i t a r y , the gonadotrophins (140). Thus, the development of the t e s t e s i n salmon and t r o u t can be induced by the i n j e c t i o n o f p i t u i t a r y e x t r a c t (141) or of crude gonado-t r o p h s p r e p a r a t i o n s (142). Two separate gonadotrophins, f o l l i c l e -s t i m u l a t i n g hormone (FSH) and l u t e i n i z i n g hormone (LH) can be de t e c t e d i n most v e r t e b r a t e s (143); however, evidence f o r the presence o f two hormones i n t e l e o s t f i s h e s i s not c o n c l u s i v e : FSH a c t i v i t y seems t o be very low, and the a c t i o n o f LH i n some s p e c i e s produces e f f e c t s u s u a l l y a s s o c i a t e d w i t h the two separate f r a c t i o n s (143). I n a d d i t i o n t o the m o r p h o l o g i c a l changes e v i d e n t d u r i n g spermiogenesis, s t r i k i n g b i o c h e m i c a l changes occur as w e l l . In t r o u t and salmon, these i n c l u d e a marked decrease i n the RNA content of maturing spermatids (144), t h e - c e s s a t i o n o f RNA syn-t h e s i s (145), and the appearance of s p e r m - s p e c i f i c chromosomal p r o t e i n s , the protamines (22,146). During t h i s time, the h i s t o n e s which were p r e s e n t on the chromatin d i s a p p e a r c o m p l e t e l y , t o be r e p l a c e d by the protamines. The m o d i f i c a t i o n o f t r o u t t e s t i s h i s t o n e s by p h o s p h o r y l a t i o n was d i s c u s s e d e a r l i e r . P r o t -amines are a l s o p h o s p h o r y l a t e d (147), soon a f t e r t h e i r s y n t h e s i s i n t he cytoplasm on a c l a s s of s m a l l polysomes (148). Dephos-p h o r y l a t i o n o f protamine o c c u r s on the chromatin (106), and may -24-be i n v o l v e d with the condensation of the spermatid nucleus (107) f The complete sequences of t h r e e protamine components from r a i n -bow t r o u t have been r e p o r t e d by Ando and Watanabe (149). The a v a i l a b i l i t y of l a r g e q u a n t i t i e s o f m a t e r i a l , the r e l a t i v e synchrony of development, the ease of p r e p a r a t i o n of chromatin (145) , and the phenomenon of the replacement p r o c e s s d e s c r i b e d above make the Salmonid t e s t i s , and p a r t i c u l a r l y t h a t of the rainbow t r o u t (Salmo g a i r d n e r i i ) a very f a v o u r a b l e system f o r the study o f chromosomal p r o t e i n s . T h i s t h e s i s r e p o r t s the r e s u l t s of s t u d i e s on the a c e t y l a t i o n o f h i s t o n e s i n d e v e l o p i n g t r o u t t e s t i s . I t i s shown t h a t the a c e t a t e i n c o r p o r a t e d i n t o h i s t o n e s i s l a r g e l y i n the form of e - N - a c e t y l groups a t t a c h e d to l y s y l r e s i d u e s i n these p r o t e i n s ; the d i s t r i b u t i o n of a c e t y l groups w i t h i n the v a r i o u s h i s t o n e f r a c t i o n s i s determined, and the s i t e s of the m o d i f i e d l y s y l r e s i d u e s are l o c a t e d f o r a l l f r a c t i o n s known to be a c e t y l a t e d i n t r o u t t e s t i s ; the p a r t i a l amino a c i d sequences o f two o f these h i s t o n e s have been deduced; the h e t e r o g e n e i t y of p u r i f i e d h i s t o n e f r a c t i o n s i s shown t o be due i n l a r g e p a r t to the presence Of a c e t y l a t e d components; the a c e t y l a t i o n of h i s t o n e s i n d i f f e r -ent c e l l types o f the t e s t i s has been i n v e s t i g a t e d , and some p r o p e r t i e s of the enzymes i n v o l v e d i n the a c e t y l a t i o n of h i s t o n e s are noted. i -25-M a t e r i a l s and Methods I. Chemicals and A b b r e v i a t i o n s (a) Chemicals. A l l chemicals o b t a i n e d commercially were of the h i g h e s t p u r i t y or reagent grade. For sequence s t u d i e s u s i n g the Beckman 890 sequencer, Sequencer Grade chemicals, s u p p l i e d by Beckman Instruments, Inc., or P i e r c e Chemical Co., were employed. R a d i o a c t i v e compounds: sodium 1- **C]-acetate (61 mCi/mmole) , ( a c e t y l - 1 - 1 ''C) -coenzyme A (59 mCi/mmole) , (acetic-1- 1''C) anhydride (121 mCi/mmole), and D,L-5- [ 3 H ] - a r g i n i n e (12 mCi/mmole), were o b t a i n e d from Amersham-Searle C o r p o r a t i o n ; L- [ 3 H ] - l y s i n e (3 mCi/mmole), from New England N u c l e a r C o r p o r a t i o n . Cycloheximide was o b t a i n e d from Calbiochem; Hanks' balanced s a l t s o l u t i o n , and a p e n i c i l l i n -s t reptomycin mixture, from B a l t i m o r e B i o l o g i c a l L a b o r a t o r y ; B i o -Gel P-10, B i o - G e l P-60, and Bio-Rex 70, from Bio-Rad L a b o r a t o r i e s ; NCS s o l u b i l i z e r , from Amersham-Searle C o r p o r a t i o n ; C a b - O - S i l , from Mann Research L a b o r a t o r i e s Inc.; 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; Nonidet P-40, from S h e l l O i l Company. (b) A b b r e v i a t i o n s . For the sake of c l a r i t y , non-standard a b b r e v i a t i o n s have been kept t o a minimum. When used, t h e i r meaning i s e x p l a i n e d i n the body of t e x t where they are f i r s t encountered. -26-DNS-C1 or d a n s y l c h l o r i d e : - 1, dimethylaminonaphthalene-5-sulfonyl c h l o r i d e - a f l u o r e s c e n t compound which r e a c t s w i t h f r e e amino groups a t a l k a l i n e pH to y i e l d a c i d - s t a b l e d e r i v a t i v e s ; used f o r N H 2 - t e r m i n a l d e t e r m i n a t i o n s of p r o t e i n s and pep-t i d e s (150). POPOP:- 1,4-Bis-(5-phenyloxazol-2-yl)-benzene PPO:- 2,5-diphenyloxazole c TCA - t u n g s t a t e : - a p r o t e i n p r e c i p i t a n t f i r s t d e s c r i b e d by Gardner e t a l . (151), c o n t a i n i n g 5% t r i c h l o r o a c e t i c a c i d and 0.25% sodium t u n g s t a t e a t pH 2. TMKS:- an i s o t o n i c medium c o n t a i n i n g T r i s - H C l (50 mM, pH 7.4), magnesium c h l o r i d e (1 mM), potassium c h l o r i d e (25 mM), and sucrose (0.25 M). TMK:- a b u f f e r c o n t a i n i n g the same i n g r e d i e n t s as TMKS, except t h a t i t l a c k s s u c r o s e . I I . I s o l a t i o n and P r e l i m i n a r y C h a r a c t e r i z a t i o n of In V i v o  A c e t y l a t e d T r o u t T e s t i s H i s t o n e s . (a) Source of the H i s t o n e s . Testes at v a r i o u s stages of spermatogenesis were o b t a i n e d d u r i n g the months of September to December from n a t u r a l l y maturing rainbow t r o u t (Salmo g a i r d n e r i i ) r a i s e d at the Sun V a l l e y T r o u t Farm, M i s s i o n , B r i t i s h Columbia. T e s t e s were removed from the t r o u t , packed i n i c e , and t r a n s p o r t e d to the l a b o r a t o r y w i t h i n approximately 2% hours. The t e s t e s were then r i n s e d i n c o l d run-n i n g tap water, and e i t h e r used f o r i n c u b a t i o n immediately o r -27-f r o z e n and s t o r e d a t -80°C. (b) C e l l I n c u b a t i o n s . T e s t e s were s c i s s o r - m i n c e d i n Hanks'-Tris medium (Hanks' medium (152) , -with NaHC0 3 r e p l a c e d by 5 mM T r i s , pH 7.4) , and a c e l l suspension was prepared by g e n t l e hand homogenization i n a P o t t e r - E l v e h j e m homogenizer w i t h a g l a s s - r e i n f o r c e d T e f l o n p e s t l e . The suspension was f i l t e r e d through f o u r l a y e r s of c h e e s e c l o t h , d i l u t e d approximately 1:3 (W/V) w i t h H a n k s ' - T r i s , and sodium a c e t a t e - 1 - [^C] was added to a f i n a l c o n c e n t r a t i o n of 3 uCi/ml. I n c u b a t i o n s were c a r r i e d out at 18° f o r 50-90 min on a g y r a t o r y water bath. In most experiments, i n c u b a t i o n s were done i n the presence of 0.2 mM cycloheximide t o prevent p r o t e i n s y n t h e s i s . A f t e r i n c u b a t i o n , c e l l 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 washed w i t h TMKS. R a d i o a c t i v i t y was determined on a N u c l e a r Chicago 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 counter a t an e f f i c i e n c y of 75-80% f o r lhC. (c) I s o l a t i o n of B a s i c P r o t e i n s Washed c e l l s were resuspended i n TMK, homogenized at moderate speed i n a P o t t e r - E l v e h j e m homogenizer w i t h a r o t o r -d r i v e n p e s t l e ( T r i - R Instruments, Inc., New York) and 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 (3,000 x g, 10 min). The p r o c e s s was repeated once more, the n u c l e a r p e l l e t was resuspended i n 0,05 M T r i s pH 7.4, and c e n t r i f u g e d a t 17,000 x g f o r 10 min. The r e s u l t -ant p e l l e t was used f o r e x t r a c t i o n of b a s i c p r o t e i n s . A l t e r n a t i v e l y , washed c e l l s were l y s e d by r e s u s p e n s i o n twice i n d i s t i l l e d water or i n TMKS c o n t a i n i n g 0.5% Nonidet P-40, a n o n i o n i c d e t e r g e n t -28-(Shell O i l ) . After each resuspension, the nuclear p e l l e t was c o l l e c t e d by centrifugation as before. When detergent was used, the p e l l e t was washed several times by hand homogenization i n 0.01 M T r i s , pH 7.4, p r i o r to extraction of basic proteins. Basic proteins were extracted from the p e l l e t s with at least four volumes of 0.4 N H2SO.» (0°, 30 min) , and p r e c i p i t a t e d with 4 volumes of 95% ethanol (-20° , 24 hr). The p r e c i p i t a t e was c o l l e c t e d by cen-t r i f u g a t i o n (17,000 x g, 20 min), washed with cold ethanol, and dried i n vacuo. Basic proteins were then converted to t h e i r acetate form by d i a l y s i s against 0.1 M acetic acid, and l y o p h i l i z e d . For the i s o l a t i o n of protamine along with the histones, d i a l y s i s must be avoided, since protamine molecules are small enough to penetrate the pores of the d i a l y s i s bag; thus, when protamine was required, basic proteins were converted from t h e i r sulfate to t h e i r chloride form by adsorption onto a small carboxymethyl Sephadex column (H + form), eluted with 0.2 N HCl, and l y o p h i l i z e d . (d) Fractionation of Histone. (i) T otal histone was separated from protamine by chromato-graphy on a Bio-Gel P-10 column (2.0 x 50 cm) e q u i l i b r a t e d and eluted with 0.2 M acetic acid as described by Ingles and Dixon (147). The fract i o n s comprising the histone region af t e r Bio-Gel chromato-graphy of t o t a l basic proteins were combined, l y o p h i l i z e d , and fractionated by column chromatography on Bio-Rex 70 (Bio-Rad; -29-e q u i v a l e n t to Amberlite CG-50) as d e s c r i b e d by Satake e t a l . (13). A 2.5 x 50 cm column was used f o r 50 mg of whole h i s t o n e . P r o t e i n s were e l u t e d w i t h a l i n e a r g r a d i e n t of 8 to 13% guanidinium c h l o r i d e (700 ml) f o l l o w e d by 40% guanidinium c h l o r i d e , i n 0.1 M phosphate b u f f e r pH 6.8. P r o t e i n c o n c e n t r a t i o n was determined by t u r b i d i t y measurement at 400 nm a f t e r p r e c i p i t a t i o n by 1.1 M t r i c h l o r o a c e t i c a c i d (13). For the assay of r a d i o a c t i v i t y , a l i q u o t s (0.05 ml) were mixed w i t h Bray's s c i n t i l l a t i o n f l u i d (5 ml) c o n t a i n i n g 2% Cab-O-Sil as recommended by Bonner e t a l . (24) and counted i n 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 counter. In l a t e r experiments, the i n i t i a l column chromatography on B i o - G e l P-10 was omitted; h i s t o n e s I I I and IV were separated from each o t h e r and from any protamine p r e s e n t by re-chromatography of the Bio-Rex I I I - I V f r a c t i o n on a B i o - G e l P-60 column (2 x 94 cm) i n 0.01 N HC1, as d e s c r i b e d by Fambrough and Bonner (153). The f r a c t i o n s comprising the I I I - I V peak from Bio-Rex were combined, d i a l y z e d a g a i n s t 0.1 M 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 0.01 N HC1. The h i s t o n e s were then a p p l i e d to the B i o - G e l P-60 column. Histones I l b i and I I b 2 were separated from each o t h e r by rechromatography o f the f r a c t i o n I I peak from Bio-Rex 70 on a long column of B i o - G e l P-10, as d e s c r i b e d below. ( i i ) H i s tones were f r a c t i o n a t e d on a B i o - G e l P-10 column by a m o d i f i c a t i o n (154) of the method d e s c r i b e d by Sung and Dixon (81). Whole h i s t o n e (100-200 mg, as the a c e t i c s a l t ) was d i s -s o l v e d (10-20 mg/ml) i n 0.1 M sodium bo r a t e b u f f e r , pH 8.9, -30-c o n t a i n i n g 4-6 M d e i o n i z e d urea and 0.02 M d i t h i o t h r e i t o l , t o reduce the d i s u l f i d e bonds i n h i s t o n e I I I . The s o l u t i o n was allowed to stand a t 25° f o r 90 minutes, a f t e r which iodoacetamide ( r e c r y s t a l l i z e d from acetone:chloroform, 1:1 by volume) was added . to a f i n a l c o n c e n t r a t i o n of about 0.08 M. A f t e r a f u r t h e r 60-90 minutes a t 25°, the s o l u t i o n was a c i d i f i e d w i t h N HCl and a p p l i e d d i r e c t l y t o the B i o - G e l P-10 column. The column, 3 x 320 cm, was prepared by c o n n e c t i n g two 160 cm columns i n s e r i e s , u t i l i z i n g connectors w i t h minimal dead space, and was e q u i l i b r a t e d w i t h 0.01 N HCl. The flow r a t e was 30-60 ml/hr, and 6 ml f r a c t i o n s were c o l l e c t e d . P r o t e i n c o n c e n t r a t i o n was monitored by absorbance a t 230 nm, and r a d i o a c t i v i t y was determined by mixing a l i q u o t s w i t h Bray's s c i n t i l l a t i o n f l u i d (155) and c o u n t i n g the samples i n 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 counter. A d d i t i o n a l p u r i f i c a t i o n of h i s t o n e s I l b i , I I b 2 and I I I , when necessary, was achieved by r e -chromatography of these f r a c t i o n s on the same column, and by column chromatography on Sephadex G-100 i n 0.01 N HCl. For l a r g e -s c a l e s e p a r a t i o n s , a l a r g e r P-10 column (4 x 360 cm) was used. (e) A c i d H y d r o l y s i s of [* **C]-acetate L a b e l e d H i s t o n e s . To t e s t f o r the a c i d l a b i l i t y o f the [ x''C]-acetate i n h i s t o n e s , a l i q u o t s of the l a b e l e d h i s t o n e c o n t a i n i n g measured amounts of r a d i o a c t i v i t y were d r i e d , r e d i s s o l v e d i n 6 N HCl and h y d r o l y z e d i n evacuated, s e a l e d tubes a t 110° f o r 19 hours. The h y d r o l y s a t e s were d r i e d i n vacuo over a d e s i c c a t o r , r e d i s s o l v e d i n d i s t i l l e d water, and counted i n 5 ml of Bray's s c i n t i l l a t i o n f l u i d (155). -31-(f) Trypsin-Pronase D i g e s t i o n of [*"C]-acetate Labeled H i s t o n e s . The sample of h i s t o n e (60 mg, c o n t a i n i n g 860,000 cpm [ l *C]-acetate) was i n c u b a t e d w i t h 1 mg of c r y s t a l l i n e p o r c i n e t r y p s i n (Novo I n d u s t r i ) i n 3.5 ml of 0.2 M NHi»HC0 3 pH 7.8, a t 40° f o r 7 h r s . The i n s o l u b l e m a t e r i a l was removed by c e n t r i f u g a t i o n and the s o l u b l e supernatant, c o n t a i n i n g the r a d i o a c t i v i t y , was l y o p h i -l i z e d . T h i s l y o p h i l i z e d d i g e s t mixture was then r e d i s s o l v e d i n 1.0 ml of 0.2 M NHi,HC0 3 , and d i g e s t e d f u r t h e r w i t h 1 mg of Pronase (B grade) at 40° f o r 9 h r s . A f t e r l y o p h i l i z a t i o n , the d i g e s t was a p p l i e d t o a G-25 column (1.3 x 130 cm) e q u i l i b r a t e d and e l u t e d w i t h 0.1 M N H i»HC0 3. (g) S y n t h e s i s of e-N- [* * * ( ; ] - a c e t y l l y s i n e . The s y n t h e s i s o f e - N - a c e t y l l y s i n e , l a b e l e d w i t h U C a t the c a r b o n y l carbon of the a c e t y l group, was c a r r i e d out a c c o r d i n g t o the method d e s c r i b e d by L e c l e r c and Benoiton (156). A c c o r d i n g to t h i s t e chnique, p - n i t r o p h e n y l a c e t a t e i s used as the a c y l a t i n g agent, and o n l y the e-amino group of l y s i n e i s a c y l a t e d a t pH 11. The a c y l a t i n g agent, p - n i t r o p h e n y l a c e t a t e l a b e l e d a t the a c e t y l group w i t h ^ C , was prepared e s s e n t i a l l y as d e s c r i b e d by Chattaway (157): 5 u l of u n l a b e l e d c a r r i e r a c e t i c anhydride was added t o a v i a l c o n t a i n i n g 10 y C i and 2.04 mg o f a c e t i c 1- t 1 1 1 C ] - a n h y d r i d e , and c o o l e d on i c e . To t h i s s o l u t i o n was added 1.2 ml of p - n i t r o p h e n o l s o l u t i o n i n a l k a l i , c o n t a i n i n g 7.36 mg of p - n i t r o p h e n o l and 2.4 mg NaOH/ml. A f t e r shaking r a p i d l y , a white p r e c i p i t a t e of p-n i t r o p h e n y l a c e t a t e formed, and 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 . -32-The p r e c i p i t a t e was resuspended i n water, and warmed to f a c i l i t a t e s o l u b i l i z a t i o n ; a few drops of ethyl acetate were also added to keep the ester i n solution. This solution was then added to a solution (10 ml) containing 0.075 mmoles of L-lysine hydrochloride at pH 11, and the resultant solution was s t i r r e d with the addition of 2 N NaOH to maintain the pH at 11.0, as measured by a pH meter. After 45 minutes the pH ceased to drop, and the reaction was judged completed. The solution was extracted three times with ethyl ether, and l y o p h i l i z e d . The residue was redissolved i n water, and aliquots were compared with authentic unlabeled e-N-acetyllysine (kindly provided by Dr. L. Benoiton, University of Ottawa) by high voltage electrophoresis. A single radioactive component was present i n the preparation, with a mobility i d e n t i c a l to that of authentic e-N-acetyllysine under conditions (pH 1.9) which c l e a r l y separated the <^-and e-acetyl d e r i v a t i v e s . (h) High Voltage Electrophoresis and Paper Chromatography. High voltage electrophoresis of peptides and amino acids was c a r r i e d out on Whatman 3MM paper at pH 6.5 i n a toluene-cooled tank, or at pH 3.6 or 1.9 i n varsol-cooled tanks, for 40-60 minutes. The voltage gradient was usually 50-60 V/cm. The pH 6.5 buffer consisted of pyridine/acetic acid/water, 100:4:900; the pH 3.6 buffer was pyridine/acetic acid/water 1:10:89, and the pH 1.9 buffer was 2% formic acid:8% acetic acid. For electrophoresis of -33-a r a d i o a c t i v e compound at s e v e r a l d i f f e r e n t pH v a l u e s , the area of paper c o n t a i n i n g the d e s i r e d component ( l o c a t e d by autoradiography) was c u t out, sewn i n t o a f r e s h sheet of paper, and e l e c t r o p h o r e s i s a t a d i f f e r e n t pH or paper chromatography was then c a r r i e d o ut. Paper chromatography was c a r r i e d out i n a descending f a s h i o n , u s i n g a s o l v e n t system c o n s i s t i n g of b u t a n o l / a c e t i c acid/water/ p y r i d i n e , 15:3:12:10 by volume (158), f o r 12-14 hours on Whatman 3MM paper. (i) Amino A c i d A n a l y s i s . P e p t i d e s and p r o t e i n s were hy d r o l y z e d at 110° i n 6 N HC1 (0.2 ml) i n evacuated, s e a l e d tubes f o r 16-24 hours. A f t e r removal o f the HC1 by r a p i d e v a c u a t i o n over NaOH and HaSOif, amino a c i d a n a l y s e s were performed on a Beckman model 120C amino a c i d a n a l y z e r u s i n g a s i n g l e column system as d e s c r i b e d by Devenyi (159). (j) S t a r c h G e l E l e c t r o p h o r e s i s . Urea-aluminum l a c t a t e s t a r c h g e l s were prepared as des-c r i b e d by Sung and Smithies (160), except t h a t the amount of s t a r c h used was 80-90 g (equal p a r t s of s t a r c h from Connaught L a b o r a t o r i e s , Toronto, Canada, and E l e c t r o s t a r c h Co., 0. H i l l e r , Madison, Wis., U.S.A.). Samples of h i s t o n e were a p p l i e d t o the g e l i n d i s t i l l e d water or g e l b u f f e r . 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 6 V/cm (180 V acro s s the g e l , 35 ma) w i t h water c o o l e d g e l t r a y s , i n the c o l d room. The g e l s were t r i s e c t e d h o r i z o n t a l l y , and the bottom and middle s l i c e s used f o r s t a i n i n g . S t a i n i n g was done i n 1% Amido B l a c k f o r 10 min, and g e l s were d e s t a i n e d i n 2% a c e t i c a c i d . -34-(k) Autoradiography of S t a r c h G e l s . For the autoradiography o f t 1 **C]-acetate l a b e l e d h i s t o n e s , the method of F a i r b a n k s e t a l . f o r d r y i n g p o l y a c r y l a m i d e d i s c g e l s (161) was adapted t o s t a r c h g e l s . A home-made s u c t i o n f i l t r a t i o n apparatus s i m i l a r t o t h a t d e s c r i b e d by the above authors was used, except t h a t no heat was a p p l i e d t o the d r y i n g g e l . The d e s t a i n e d g e l s l i c e was p l a c e d on a sheet of Whatman 3MM paper, which was s e t upon a t h i c k l a y e r (approximately 1") of paper towels on the f i l t r a t i o n apparatus. The top of the g e l was covered w i t h Saran ' wrap, and the vacuum a p p l i e d . Two t o th r e e days were r e q u i r e d f o r thorough d r y i n g o f the g e l , w i t h s e v e r a l changes of the paper towels. The d r i e d g e l s adhere s t r o n g l y t o the f i l t e r paper, and can be kept f l a t by s t a p l i n g the paper backing t o a p i e c e of c o r -rugated cardboard. The X-ray f i l m (Kodak Blue Brand M e d i c a l X-ray f i l m ) i s then a p p l i e d d i r e c t l y t o the dry g e l f o r autoradiography. In o r d e r t o prevent c r a c k i n g o f the d r i e d g e l , 1% g l y c e r o l was sometimes i n c l u d e d i n the f i n a l g e l d e s t a i n i n g wash. I I I . S t u d i e s on the S i t e s o f In V i v o A c e t y l a t i o n i n T r o u t T e s t i s  H i s t o n e s . P r e p a r a t i o n of t 1 ^ C ] - a c e t a t e l a b e l e d h i s t o n e s , p u r i f i c a t i o n of i n d i v i d u a l h i s t o n e f r a c t i o n 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 and paper chromatography of p e p t i d e s , and amino a c i d a n a l y s e s were c a r r i e d out as d e s c r i b e d above i n P a r t I I . -35-(a) Dowex 50 Chromatography of Histone IV T r y p t i c P e p t i d e s Approximately 60 mg of c a r r i e r h i s t o n e IV was added t o 10.5 mg of i1 ^ C]-acetate l a b e l e d h i s t o n e IV, c o n t a i n i n g 700,000 cpm. The p r o t e i n was d i s s o l v e d i n 0.1 M N H i»HC0 3, pH 7.8, and d i g e s t e d w i t h 2 mg of CP t r y p s i n (Novo I n d u s t r i ) f o r 3 hours a t 37°. The mixture was then l y o p h i l i z e d , r e d i s s o l v e d i n a few mis. of d i s -t i l l e d water, 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 . The supernatant s o l u t i o n was a c i d i f i e d t o approximately pH 1 w i t h HCl, and a p p l i e d t o a j a c k e t e d 0.9 x 100 cm column of Dowex 50 x 2 r e s i n , maintained a t 38°. P e p t i d e s were e l u t e d from the column w i t h a g r a d i e n t of p y r i d i n i u m a c e t a t e from pH 3.1 (0.2 M, 333 ml) to pH 5.0 (2.0 M, 666 ml) a c c o r d i n g t o Schroeder (162). A l i q u o t s of 0.05 ml were removed from each f r a c t i o n , d r i e d , and counted i n 5 ml of Bray's s c i n t i l l a t i o n f l u i d (155). (b) Chemical A c e t y l a t i o n o f Histones I l b i and IV The procedure was e s s e n t i a l l y t h a t d e s c r i b e d by Riordan and V a l l e e (163). The i n v i v o , [* **C]-acetate l a b e l e d h i s t o n e was mixed w i t h c a r r i e r and d i s s o l v e d i n d i s t i l l e d water. An equal volume of s a t u r a t e d sodium a c e t a t e was added, produ c i n g a f i n a l pH o f 7.5. The s o l u t i o n was c o o l e d on i c e , and i c e c o l d a c e t i c anhydride (Reagent grade) was added i n 5-10 u l a l i q u o t s , a t i n t e r v a l s of 10-15 min, w i t h continuous s t i r r i n g , f o r 1 hr; a f u r t h e r 10 u l of a c e t i c anhydride was then added, and s t i r r i n g c o n t i n u e d f o r 1 h r . The pH was p e r i o d i c a l l y a d j u s t e d t o 7.5 with N NaOH. The suspension was then d i a l y z e d a g a i n s t 0.6 M a c e t i c a c i d , and l y o p h i l i z e d . -36-(c) Manual Edman Degradation o f P e p t i d e s L a b e l e d With  t l **€]-acetate, (i) H i s t o n e IV: The procedure was based on t h a t d e s c r i b e d by Edman (164) , as m o d i f i e d by Hew (165). The p e p t i d e (0.09-0.12 umole) was d i s -s o l v e d i n 100 u l of 50% aqueous p y r i d i n e (V/V, p y r i d i n e r e d i s t i l l e d over s o l i d n i n h y d r i n ) , and 100 u l of p h e n y l i s o t h i o c y a n a t e (PITC) s o l u t i o n (100 u l PITC i n 1 ml r e d i s t i l l e d N - e t h y l morpholine) was added. The tube was f l u s h e d w i t h N 2 * and c o u p l i n g was c a r r i e d out a t 37° f o r 2h hours. The s o l u t i o n was d r i e d down over NaOH and P 2 O 5 i n a heated vacuum d e s i c c a t o r (50°), and 200 u l of an-hydrous t r i f l u o r o a c e t i c a c i d ( r e d i s t i l l e d ) was added. The tube was again f l u s h e d w i t h N 2, and c y c l i z a t i o n c a r r i e d out f o r 1 hr a t 37°. The contents o f the tube were d r i e d down as b e f o r e , 0.5 ml H 20 was added t o the dry r e s i d u e , and the s o l u t i o n was e x t r a c -t e d t h r e e times w i t h reagent grade e t h y l a c e t a t e . The e t h y l a c e t a t e phase c o n t a i n i n g the t h i a z o l i n o n e d e r i v a t i v e o f the amino a c i d was d r i e d down and counted i n 10 ml of T o l u e n e - T r i t o n X-100 s c i n t i l l a t i o n f l u i d (166). The aqueous phase was a l s o d r i e d down, and another Edman c y c l e c a r r i e d out. Before each Edman st e p , an a l i q u o t o f the p e p t i d e (0.01 umole) was removed f o r d a n s y l a t i o n a c c o r d i n g t o Gray (150) . ( i i ) H istone I l b i : The procedure was based on t h a t o f Edman (164) w i t h the f o l l o w i n g m o d i f i c a t i o n . The p e p t i d e was d i s s o l v e d i n e i t h e r 150 -37-or 300 y l of 5% N-ethylmorpholine (twice d i s t i l l e d ) pH 9.3, and 150 or 300 y l of 5% phenylisothiocyanate solution i n r e d i s t i l l e d pyridine was added. The tube was flushed with N 2, and coupling was c a r r i e d out at 40° for 3 hours. The solution was dried down over NaOH and H 2 SOi , i n a heated vacuum desiccator (60°) , and 200 or 300 u l of anhydrous t r i f l u o r o a c e t i c acid ( r e d i s t i l l e d ) was added. The tube was again flushed with N 2 and incubated at 40° for 1 hour. The contents of the tube were dried down as before,-200 y l of deionized water was added, and the solution was extracted twice with 1.5 ml of n-butyl acetate (reagent grade). The butyl acetate phases were combined, dried down, and counted i n 10 ml of Bray's s c i n t i l l a t i o n f l u i d (155). An aliquot of the peptide was then removed from the aqueous phase for dansylation (150), and the cycle was repeated. (d) Amino Terminal Determination of Peptides After each manual Edman degradation, the NH 2-terminal of the r e s i d u a l peptide was usually determined by the 'dansylation' technique of Gray (150). (i) Histone IV: The peptide, i n a 9 x 75 mm glass tube, was dissolved i n 25 y l of 0.1 M NaHCOa, dried i n vacuo over NaOH and H 2 SOi» (50°), and redissolved i n 25 y l of H 20. To the peptide solution was then added 25 y l of D N S-Cl ('dansyl chloride', 1-dimethyl-aminonaphthalene-5-sulfonyl chloride) solution i n acetone (2.5 mg/ml). The r e s u l t i n g solution was incubated at 37° f o r 2 hrs, -38-and d r i e d i n vacuo. The d a n s y l d e r i v a t i v e o f the N H 2 - t e r m i n a l amino a c i d was then l i b e r a t e d by h y d r o l y s i s of the p e p t i d e i n 6 N HC1 f o r 16 hrs at 110° i n an evacuated, s e a l e d tube. A f t e r d r y i n g down i n vacuo, the sample was taken up i n 10 u l of 2 M NHi.0H. Dansyl d e r i v a t i v e s were i d e n t i f i e d by t h i n l a y e r chromato-graphy on g l a s s p l a t e s coated w i t h s i l i c a g e l . These were prepared by spreading a s l u r r y (25 g i n 50 ml) of s i l i c a g e l G (a c c o r d i n g t o S t a h l , E.; Merck A.G., Darmstadt) i n a 0.25 mm l a y e r (Desaga spreader) on 20 x 20 cm g l a s s p l a t e s , and the p l a t e s were a c t i v a t e d at 120° i n an oven f o r 30 minutes b e f o r e use. The s o l v e n t system c o n s i s t e d o f B e n z e n e / P y r i d i n e / A c e t i c A c i d 80:20:2 as d e s c r i b e d by Morse and Horecker (167). Chromatography was done a t room temp-e r a t u r e . ( i i ) H i s t o n e I l b i : I n t h i s case, the r e c e n t l y developed micro d a n s y l a t i o n method d e s c r i b e d by H a r t l e y (168) was used. The d a n s y l a t i o n r e a c t i o n was c a r r i e d out e s s e n t i a l l y as d e s c r i b e d above f o r h i s t o n e IV, except t h a t a 5 x 60 mm tube, and volumes of 10 u l i n s t e a d of 25 u l , were used. The amount of p e p t i d e was u s u a l l y 10 nanomoles i n both cases. A f t e r a c i d h y d r o l y s i s i n 50 u l of 6 N HC1 as d e s c r i b e d above, the sample was d r i e d i n vacuo, taken up i n 5 u l of e t h a n o l / a c e t i c a c i d 3:2, and approximately one t h i r d of the sample was s p o t t e d i n one corner of a 5 x 5 cm polyamide sheet (Polyamide Layer, Cheng Chin T r a d i n g Co., Hankow S t . , T a i p e i , Taiwan; d i s t r i b u t e d i n the U.S.A. by G a l l a r d - S c h l e s i n g e r Chemical Mfg. Corp., C a r l e P l a c e , N.Y.). The sample was run alone on one -39-s i d e of the d o u b l e - s i d e d sheet, and on the other s i d e was mixed w i t h a s e l e c t e d marker mixture. The sample was f i r s t run i n S o l v e n t System 1 (1.5% f o r m i c a c i d ) , d r i e d , and examined under u l t r a v i o l e t l i g h t . The sample was then run i n S o l v e n t System 2 (Benzene/Acetic A c i d 9:1) a t r i g h t angles t o the f i r s t d i r e c t i o n , d r i e d and examined. F i n a l l y , the sample was run i n S o l v e n t System 3 ( E t h y l Acetate/Methanol/ A c e t i c A c i d 20:1:1) i n the same d i r e c t i o n as S o l v e n t 1. By running the s o l v e n t f r o n t s e x a c t l y 3 cm from the o r i g i n i n each system, the m o b i l i t i e s of the v a r i o u s standards had c o n s t a n t v a l u e s , and unknowns c o u l d be i d e n t i f i e d by superimposing t h e i r chromatograms on t r a c i n g s of the standard chromatograms, thus e l i m i n a t i n g the need f o r s p o t t i n g standards w i t h each unknown. Using t h i s method, 1 nanomole of DNS-amino a c i d i s r e a d i l y i d e n t i f i e d . (e) Automated P r o t e i n Sequencing For s t u d i e s on the amino-terminal sequences and s i t e s of a c e t y l a t i o n i n t r o u t t e s t i s h i s t o n e s I I b 2 and I I I , a Beckman 890 automatic p r o t e i n sequencer was used (169). The sequencer cup was loaded with 0.5-1.2 umole of p r o t e i n i n 0.6 ml of d i s t i l l e d water. Automated Edman degr a d a t i o n s were then c a r r i e d out by the instrument, a c c o r d i n g to one of the two f o l l o w i n g programs: (i) Quadrol program (169): T h i s program p r o v i d e d s e q u e n t i a l a d d i t i o n s o f p h e n y l i s o t h i o c y a n a t e and Quadrol b u f f e r , washing w i t h benzene and e t h y l a c e t a t e , double cleavage w i t h h e p t a f l u o r o b u t y r i c -40-a c i d , and e x t r a c t i o n of the t h i a z o l i n o n e d e r i v a t i v e w i t h b u t y l c h l o r i d e , ( i i ) DMAA program (170): In t h i s program d i m e t h y l -a l l y l a m i n e (DMAA) t r i f l u o r o a c e t i c a c i d i n s t e a d of Quadrol was used to b u f f e r the p h e n y l i s o t h i o c y a n a t e / and the e t h y l a c e t a t e e x t r a c t i o n was omitted. Other p a r t s of the program were s i m i l a r to those of the Quadrol program. The 2 - a n i l i n o - 5 - t h i a z o l i n o n e d e r i v a t i v e s of the amino a c i d s -from the sequencer were conv e r t e d to t h e i r more s t a b l e 3-phenyl-2 - t h i o h y d a n t o i n (PTH) d e r i v a t i v e s , as d e s c r i b e d by Edman and Begg (169) , except t h a t the 1 N HCl c o n t a i n e d ImM ethane t h i o l . T h i s c o n s i s t e d i n h e a t i n g the tubes c o n t a i n i n g the d e r i v a t i v e s i n the IN HCl s o l u t i o n a t 80° f o r 10 min i n a water bath. The tubes were f l u s h e d w i t h N 2 and capped p r i o r to t h i s o p e r a t i o n . The PTH d e r i v a t i v e s were then e x t r a c t e d from the HCl phases w i t h e t h y l a c e t a t e (2 x 0.7 ml, Reagent Grade), and d r i e d under a stream of N 2. A r g i n i n e was e i t h e r d e t e c t e d a f t e r a c i d h y d r o l y s i s of the HCl phase f o l l o w i n g the c o n v e r s i o n to i t s PTH d e r i v a t i v e (169), or e l s e PTH a r g i n i n e was f i r s t e x t r a c t e d from the HCl phase w i t h e t h y l a c e t a t e a f t e r n e u t r a l i z a t i o n of the HCl w i t h 1M disodium hydrogen phosphate. Amino a c i d s were i d e n t i f i e d on a Beckman 120C amino a c i d a n a l y z e r a f t e r h y d r o l y s i s of t h e i r PTH d e r i v a t i v e s i n 6N HCl c o n t a i n i n g 0.01M mercaptoethanol at 140°C f o r 24 hrs i n evacuated, s e a l e d tubes (171). Threonine was i d e n t i f i e d as -41-g l y c i n e a f t e r b a s i c h y d r o l y s i s i n oxygen-free 0.1M NaOH a t 110°C f o r 13 h r s , as d e s c r i b e d by A f r i c a and Carpenter (172). For the d e t e r m i n a t i o n o f r a d i o a c t i v i t y , a l i q u o t s o f the 2-a n i l i n o - 5 - t h i a z o l i n o n e d e r i v a t i v e s i n e t h y l a c e t a t e were t r a n s -f e r r e d t o s c i n t i l l a t i o n v i a l s , d r i e d , and counted i n 5 ml of Bray's s c i n t i l l a t i o n f l u i d (155). IV. A c e t y l a t i o n of Histones i n D i f f e r e n t C e l l Types from  Developing T r o u t T e s t i s . (a) T r o u t T e s t i s For c e l l s e p a r a t i o n s , t e s t e s were o b t a i n e d from rainbow t r o u t which were kept i n a q u a r i a , and induced t o mature by t w i c e -weekly i n j e c t i o n s of salmon p i t u i t a r y e x t r a c t , as d e s c r i b e d p r e -v i o u s l y (141). These f i s h mature i n approximately 10 weeks, as opposed t o the 5 or 6 months r e q u i r e d f o r n a t u r a l l y maturing f i s h . The t e s t e s i n the hormonally induced f i s h a l s o develop more synchronously than those o f n a t u r a l l y maturing f i s h , and the d i f f e r e n t c e l l types are more c l e a r l y r e s o l v e d i n c e l l sep-a r a t i o n experiments (103). A l s o , by choosing t e s t e s a t a p p r o p r i a t e e -1 stages of development, almost homogeneous p o p u l a t i o n s of p a r t i c u l a r c e l l t y p e s , e.g. spermatids, can be ob t a i n e d . (b) I n c u b a t i o n s T e s t e s were s c i s s o r - m i n c e d i n 2 volumes of TMKS con-t a i n i n g 0.1% glu c o s e , and a c e l l suspension was prepared by g e n t l e hand homogenization 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 . The suspension was f i l t e r e d through c h e e s e c l o t h , and a -42-0.5 ml a l i q u o t , c o n t a i n i n g 1-7 x 10 8 c e l l s , was used f o r the i n -c u b a t i o n . Sodium 1- t 1 **C]-acetate was added t o a f i n a l c o n c e n t r a t i o n of 330 uCi/ml, and L- [ 3 H ] - l y s i n e , when p r e s e n t , was at a co n c e n t r a -t i o n of 130 uCi/ml. When o n l y t 1''CJ-acetate was used, c y c l o h e x i m i d e was added t o the i n c u b a t i o n mixture, t o a f i n a l c o n c e n t r a t i o n of O.lmM. Inc u b a t i o n was c a r r i e d out a t 16° f o r 90 min on a g y r a t o r y water bath. At the end of the i n c u b a t i o n , the c e l l suspension was d i l u t e d to 25 ml wi t h phosphate-buffered s a l i n e (PBS, c o n t a i n s NaCl, 0.14M; K C l , 2.7mM; N a 2 H P O i , , 8mM; KH 2PO.», 1.5mM; C a C l 2 , 0.9mM; MgCl 2 , 0.5mM; pH 7.4) and the c e l l 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 at 1900 x g f o r 10 min. The c e l l s were then resuspended i n 5-10 ml of PBS, and a s u i t a b l e a l i q u o t c o n t a i n i n g approximately 7-9 x 10 7 c e l l s was taken f o r sedime n t a t i o n on a serum albumin g r a d i e n t . (c) C e l l S e p a r a t i o n s S e p a r a t i o n o f c e l l s by v e l o c i t y s edimentation a t u n i t g r a v i t y was c a r r i e d out as d e s c r i b e d by Lam e t a l . (173), except t h a t the bovine serum albumin (BSA, Calbiochem, F r a c t i o n V, grade B) i n PBS s o l u t i o n was t i t r a t e d t o pH 7.2 wi t h IN NaOH and f i l t e r e d through a g l a s s f i b e r f i l t e r (AP-20-047-00, M i l l i p o r e F i l t e r Corp., Bedford, Mass.). The g r a d i e n t v e s s e l was loaded w i t h 20 ml o f phosphate-buffered s a l i n e f o l l o w e d by 7-9 x 10 7 c e l l s i n 15 ml of 0.5% BSA i n the same s o l u t i o n . The 15 ml l a y e r of c e l l s was separa t e d from the 600 ml 1-3% l i n e a r BSA g r a d i e n t by 5 ml of -43-0.75% BSA. A f t e r a sedimentation time of 255 or 555 min a t 4°, approximately 85 f r a c t i o n s of 7 ml volume were c o l l e c t e d at the r a t e of one f r a c t i o n every 30 sees. The t o t a l sedimentation time was t h e r e f o r e 300 or 600 min. The l a s t f r a c t i o n corresponds to the top of the g r a d i e n t . C e l l s were counted on a hemocytometer. (d) S t a r c h Gel E l e c t r o p h o r e s i s of B a s i c P r o t e i n s from Whole  C e l l s The procedure of L o u i e and Dixon (103) was used. C e l l s from a p p r o p r i a t e p a r t s o f the g r a d i e n t were f i l t e r e d by s u c t i o n onto g l a s s f i b e r f i l t e r s ( M i l l i p o r e AP-20-025-00) over a 6 x 11 mm r e c t a n g u l a r area u s i n g a T e f l o n template, washed w i t h PBS, and f i x e d w i t h 95% e t h a n o l . For the e x t r a c t i o n of b a s i c p r o t e i n s , the areas of the f i l t e r s c o n t a i n i n g the c e l l s were cu t out, moistened w i t h 95% e t h a n o l , and p l a c e d d i r e c t l y i n urea-aluminum l a c t a t e s t a r c h g e l s l o t s c o n t a i n i n g 0.4 N HCl. E x t r a c t i o n was allowed t o proceed f o r approximately 15 min, d u r i n g which time the f i l t e r s were a g i t a t e d p e r i o d i c a l l y i n the s l o t s by means of f i n e f o r c e p s . The g e l s l o t s were then s e a l e d w i t h petrolatum, 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 above i n P a r t I I . A f t e r e l e c t r o p h o r e s i s , the g e l s were t r i s e c t e d h o r i z o n t a l l y . The bottom s l a b was s t a i n e d by the s e n s i t i v e cobalt-Amido B l a c k p r o -cedure and d e s t a i n e d with 0.5M H2SCK , as d e s c r i b e d by Sung and S m i t h i e s (160), and the middle s l a b was used f o r r a d i o a c t i v i t y a n a l y s i s , as d e s c r i b e d below. -44-(e) R a d i o a c t i v i t y A n a l y s i s T o t a l r a d i o a c t i v i t y i n c o r p o r a t e d i n t o the n u c l e i of the c e l l s i n the g r a d i e n t f r a c t i o n s was determined by f i l t e r i n g an a l i q u o t of the BSA c e l l suspension through a g l a s s f i b e r f i l t e r ( M i l l i p o r e AP-20-025-00), washing wi t h PBS, 5% t r i c h l o r o a c e t i c a c i d -0.25% sodium t u n g s t a t e pH 2.0 (151), 95% e t h a n o l , and d r y i n g i n an oven at 50°, as d e s c r i b e d by Louie and Dixon (103). The f i l t e r s were then counted i n 5 ml of t o l u e n e s c i n t i l l a t i o n f l u i d ( toluene c o n t a i n i n g 0.01% POPOP and 0.4% PPO) i n a Nuclear Chicago U n i l u x I I r counter. For the a n a l y s i s of r a d i o a c t i v i t y i n c o r p o r a t e d i n t o h i s t o n e s , the procedure of L o u i e and Dixon (103) was f o l l o w e d : the r e l e v a n t area of the middle s l a b of the s t a r c h g e l was c u t i n t o 2 mm s l i c e s , which were incubated i n capped s c i n t i l l a t i o n v i a l s w i t h 0.5 ml NCS Reagent (Amersham-Searle) f o r 15 hr at room temperature. Toluene s c i n t i l l a t i o n f l u i d (5 ml) was then added, and the v i a l s i n c u b a t e d f o r a f u r t h e r 3 hr at 45°, b e f o r e c o o l i n g and c o u n t i n g . (f) Pulse-chase S t u d i e s For the measurement of a c e t a t e turnover i n h i s t o n e s , 1 x 10 8 t e s t i s c e l l s from a pre-spermatid stage f i s h were i n -cubated w i t h 100 u C i of L- [ 3 H ] - a r g i n i n e or L- [ 3 H ] - l y s i n e (New England N u c l e a r , 10 mCi/mmole and 3mCi/mmole, r e s p e c t i v e l y ) , 200-300 u C i of [l^C]-acetate (Amersham-Searle, 61 mCi/mmole), and 100 u n i t s of p e n i c i l l i n - s t r e p t o m y c i n mixture (Baltimore B i o l o g i c a l ) , i n a t o t a l volume of 0.7 ml. The medium c o n s i s t e d of TMKS-0.1% glu c o s e -45-c o n t a i n i n g 10-20% Waymouth's medium (174) with v i t a m i n s a c c o r d i n g t o E a g l e (175), the sodium b i c a r b o n a t e b u f f e r r e p l a c e d by lOmM T r i s - H C l , pH 7.4 and l a c k i n g e i t h e r l y s i n e or a r g i n i n e , as r e -q u i r e d . A f t e r i n c u b a t i o n at 16° f o r 90 min on a g y r a t o r y water bath, the c e l l suspension was d i l u t e d t o 15 ml with i c e c o l d TMKS-. 0.1% g l u c o s e , and c e n t r i f u g e d a t 1900 x g f o r 10 min. The c e l l s were then resuspended by g e n t l e hand homogeniza-t i o n with a P o t t e r - E l v e h j e m homogenizer, i n 20 ml of f r e s h TMKS-0.1% glucose c o n t a i n i n g 10% Waymouth's medium and 100 u n i t s / m l o f p e n i c i l l i n - s t r e p t o m y c i n or a l t e r n a t i v e l y i n the same medium c o n t a i n i n g 0.05M T r i s - a c e t a t e i n s t e a d of T r i s - H C l , and ImM a r g i n i n e o r l y s i n e , t o ensure r a p i d d i l u t i o n o f the r a d i o a c t i v e compounds. The c e l l s were in c u b a t e d a t 16° as b e f o r e , and samples c o n t a i n i n g 5 x 10 6 c e l l s were removed a t i n t e r v a l s and c o l l e c t e d on g l a s s f i b e r f i l t e r s . The c e l l s on the f i l t e r s were washed w i t h TMKS-0.1% gl u c o s e , and f i x e d w i t h e t h a n o l . R a d i o a c t i v i t y i n the h i s t o n e s was then determined 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 and c o u n t i n g as d e s c r i b e d above. V. Some P r o p e r t i e s of the His t o n e A c e t y l t r a n s f e r a s e s from  T r o u t T e s t i s , (a) Enzyme Assays (i) Assay of A c e t y l t r a n s f e r a s e A c t i v i t y i n I s o l a t e d N u c l e i : N u c l e i were prepared from f r e s h o r f r o z e n t i s s u e by homogenization i n a Waring blendor f o r 1-2 min, at h i g h speed, i n TMK pH 7.4 or 8.0. A l l o p e r a t i o n s were performed a t 0-4°. N u c l e i -46-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 at 3,000 x g f o r 10 min, and resuspended by hand homogenization 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 . 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 as b e f o r e , and the hand homogenization step was repeated ( i n some experiments, a motor-driven p e s t l e was used to resuspend the n u c l e i ) . T h i s procedure y i e l d s an opaque white suspension of n u c l e i , i n which the chromatin remains i n a condensed s t a t e , due t o the presence of the magnesium io n s i n the TMK. For the assay of h i s t o n e a c e t y l a t i n g a c t i v i t y , an a l i q u o t ( u s u a l l y 100 ul) of a n u c l e a r suspension (1:2, W/V) i n TMK, prepared as d e s c r i b e d above, was i n c u b a t e d w i t h 40,000-80,000 cpm of 1- t 1 ' ' C l - a c e t y l Coenzyme A (59 mCi/mmole, Amersham-Searle Corp.) a t room temperature f o r 15 minutes. The r e a c t i o n was stopped by the a d d i t i o n of 5 u l of 12 N HCl t o the 100 u l i n c u -b a t i o n mixture, r e s u l t i n g i n a f i n a l c o n c e n t r a t i o n of 0.6 N HCl; t h i s a l s o served t o e x t r a c t the a c i d - s o l u b l e p r o t e i n s from the n u c l e i . The i n s o l u b l e m a t e r i a l was removed by c e n t r i f u g a t i o n (3,000 x g, 10 min), and the supernatants were p i p e t t e d onto f i l t e r paper d i s c s (Whatman 3MM 2.4 cm F i l t e r Paper C i r c l e s ) . The f i l t e r d i s c s h e l d a maximum of approximately 110 u l of s o l u t i o n . To p r e c i p i t a t e the p r o t e i n s , and e l i m i n a t e contaminating r a d i o a c t i v e compounds, the f i l t e r d i s c s were t r e a t e d as f o l l o w s : a f t e r a p p l i c a t i o n of the samples, the d i s c s were immersed i n i c e - c o l d 5% TCA-tungstate, and allowed to stand f o r 15-30 minutes; they were then b o i l e d i n the TCA-tungstate f o r 2 minutes, the s o l u t i o n was poured o f f , and they were b o i l e d a g a i n i n f r e s h TCA-tungstate -47-(at l e a s t 5 ml per f i l t e r d i s c was used i n each wash) f o r a f u r t h e r 2 minutes. The d i s c s were then r i n s e d once wi t h i c e - c o l d TCA-t u n g s t a t e , twice with 95% e t h a n o l (to remove the TCA), twice w i t h e t h e r , and d r i e d under a stream o f warm a i r . The d i s c s were then counted i n 2-5 ml o f Bray's s c i n t i l l a t i o n f l u i d (155) i n 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 counter. ( i i ) Assay o f A c e t y l t r a n s f e r a s e A c t i v i t y i n S o l u b l e Prep- a r a t i o n s : For the assay of a c e t y l t r a n s f e r a s e a c t i v i t y i n e x t r a c t s of n u c l e i , ammonium s u l f a t e f r a c t i o n s , and column f r a c t i o n s , whole h i s t o n e was added as s u b s t r a t e . Whole h i s t o n e was prepared as d e s c r i b e d i n " M a t e r i a l s and Methods", P a r t I I . I t was u s u a l l y d i s s o l v e d i n TMK (5-10 mg/ml) immediately before use. V a r i a b l e amounts of h i s t o n e were in c u b a t e d w i t h the enzyme p r e p a r a t i o n and 50,000-80,000 cpm of 1- [ x " C ] - a c e t y l CoA, i n a f i n a l volume of 105 y l , a t room temperature f o r 15 minutes, or a t 19° i n a shaking water bath, f o r longer p e r i o d s . The r e a c t i o n was stopped w i t h 5 y l of 12N HCl and the mixtures were p i p e t t e d onto f i l t e r d i s c s and t r e a t e d as d e s c r i b e d above. I f any p r e c i p i t a t e was p r e s e n t , i t was f i r s t removed by c e n t r i f u g a t i o n , and the supernatants were p i p e t t e d onto f i l t e r d i s c s . (b) P r e p a r a t i o n of N u c l e o h i s t o n e N u c l e i were prepared i n TMK, as d e s c r i b e d above. They were then resuspended and homogenized 2 or 3 times i n 0.01M T r i s , pH 8.0, i n a P o t t e r - E l v e h j e m homogenizer wi t h a T e f l o n p e s t l e , -48-and the chromatin was co l l e c t e d each time by centrifugation (17,000 x g, 15 min). This procedure causes the chromatin to expand, and become viscous and translucent. At t h i s stage, shearing of the chromatin r e s u l t s i n s o l u b i l i z a t i o n of the nucleo-histone; the nucleoprotamine, being more compact, i s not so rea d i l y sheared, remains insoluble, and can be separated by cen-t r i f u g a t i o n at 17,000 x g for 10 minutes (145). In t h i s case, shearing was accomplished by sonication of 10 ml aliquots of the chromatin i n a s o n i f i e r (Model W185D, Ultrasonics, Inc., Plainview, L.I., N.Y.) at setting 8 (75 Watts) for l%-2 min, during which time the tube containing the chromatin was immersed i n an ice bath. The sonicate was centrifuged at 17,000 x g for 10 minutes, and the supernatant, containing the nucleohistone, was pipetted o f f and used immediately for assays of acetyltransferase a c t i v i t y . (c) Extraction and Ammonium Sulfate Fractionation of Histone  Acetyltransferase A c t i v i t y from Trout T e s t i s Nuclei For the extraction of histone acetyltransferase a c t i v i t y from t e s t i s n u c l e i , a nuclear suspension was prepared i n TMK, as described above. The nuc l e i were then c o l l e c t e d by c e n t r i f -ugation (3,000 x g, 10 min) and resuspended i n TMK containing 0.4 M NaCl, using a Potter-Elvehjem homogenizer. The suspension was allowed to stand on ice for 15-30 min, with occasional s t i r r i n g , and the nuc l e i were then removed by centrifugation as before. The supernatant was then brought to 30% saturation with ammonium su l f a t e by the gradual addition of the s o l i d , with -49-c o n s t a n t s t i r r i n g on i c e . 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 by c e n t r i f u g a t i o n (17,000 x g, 10 min), the supernatant was brought t o 60% s a t u r a t i o n w i t h ammonium s u l f a t e , and the p r e c i p i t a t e was c o l l e c t e d as b e f o r e . O c c a s i o n a l l y , a s i n g l e f r a c t i o n p r e c i p i t a t e d by 70% s a t u r a t i o n w i t h ammonium s u l f a t e was used. The p r e c i p i -t a t e s were r e d i s s o l v e d i n a s m a l l volume of TMK or TMK-0.4 M NaCl, and d i a l y z e d a g a i n s t the same b u f f e r . I n s o l u b l e m a t e r i a l was then removed by c e n t r i f u g a t i o n , and the supernatants were used f o r enzyme experiments. -50-R e s u l t s I . I s o l a t i o n and P r e l i m i n a r y C h a r a c t e r i z a t i o n of In V i v o  A c e t y l a t e d T r o u t T e s t i s H i s t o n e s . I t was f i r s t observed by L i n g and Dixon (176) t h a t the i n c u -b a t i o n of t r o u t t e s t i s c e l l s i n suspension with sodium 1- [ l **C] - a c e t a t e r e s u l t e d i n c o n s i d e r a b l e l a b e l i n g of the h i s t o n e s . I t had a l r e a d y been shown t h a t the h i s t o n e s o f c a l f thymus c e l l s c o u l d be l a b e l e d i n a s i m i l a r manner, and most of the r a d i o a c t i v e a c e t a t e c o u l d be shown to be p r e s e n t as e - N - a c e t y l l y s i n e a f t e r t o t a l enzymatic d i g e s t i o n of l a b e l e d h i s t o n e s (26,43,44). During the sequence work on c a l f thymus h i s t o n e IV, a s i n g l e s p e c i f i c l y s y l r e s i d u e was found to be a c e t y l a t e d i n approximately 50% of the molecules (26). T h i s work has been d i s c u s s e d i n more d e t a i l above, under " I n t r o -d u c t i o n " . As p r e l i m i n a r y steps i n the study o f h i s t o n e a c e t y l a t i o n i n t r o u t t e s t i s c e l l s , i t was necessary to examine the nature of the a c e t y l groups i n c o r p o r a t e d i n t o the h i s t o n e s , and the d i s t r i b u t i o n o f these groups w i t h i n the v a r i o u s h i s t o n e f r a c t i o n s . S t u d i e s on these two aspects of h i s t o n e a c e t y l a t i o n w i l l be d e s c r i b e d i n t h i s s e c t i o n . F i g . 4 shows the p r o f i l e from a B i o - G e l P-10 column (2.5 x 50 cm) of t 1''C]-acetate l a b e l e d b a s i c p r o t e i n s e x t r a c t e d from t r o u t t e s t i s n u c l e i o b t a i n e d from n a t u r a l l y maturing rainbow t r o u t d u r i n g the month of October. I t i s seen t h a t the h i s t o n e s are e x t e n s i v e l y l a b e l e d w i t h t 1''C]-acetate, whereas t h e r e i s no d e t e c -t a b l e r a d i o a c t i v i t y i n the protamine r e g i o n . T h i s r e s u l t showed t h a t the h i s t o n e s were being s p e c i f i c a l l y l a b e l e d w i t h a c e t a t e , and t h a t the i n c o r p o r a t i o n was probably not due to random i n c o r -p o r a t i o n of [ l **C]-acetate i n t o the amino a c i d "backbone" of these p r o t e i n s . The l a t t e r p o s s i b i l i t y was a l s o u n l i k e l y i n view o f the f a c t t h a t a c e t y l a t i o n of h i s t o n e s c o n t i n u e d when p r o t e i n s y n t h e s i s was i n h i b i t e d by 0.1 mM cycloheximide (Table 2). I t i s e v i d e n t , furthermore, t h a t the peak o f t 1 **C]-acetate l a b e l i s skewed towards the low molecular weight s i d e of the absorbance peak f o r the h i s t o n e s . I t w i l l be shown below t h a t h i s t o n e I , which has the h i g h e s t molecular weight of a l l the h i s t o n e s (177), i n c o r p o r a t e s no a c e t a t e i n t o e-amino groups; t h i s h i s t o n e migrates f a s t e r than the o t h e r s d u r i n g g e l f i l t r a t i o n chromatography, and even on a s h o r t column such as t h a t i l l u s t r a t e d i n F i g . 4, i t occupies the l e a d i n g edge of the h i s t o n e peak, hence, the r a d i o a c t i v i t y p a t t e r n i s skewed toward the r i g h t (low molecular weight) s i d e of the h i s t o n e peak. I f the t 1 **C]-acetate i n c o r p o r a t e d i n t o h i s t o n e s i s p r e s e n t as a c e t y l groups r a t h e r than as p a r t of the carbon s k e l e t o n of c e r t a i n amino a c i d s , the r a d i o a c t i v i t y should be rendered v o l a t i l e f o l -lowing a c i d h y d r o l y s i s of the p r o t e i n . Table 3 i l l u s t r a t e s the r e s u l t s of an experiment i n which a measured a l i q u o t of a [ X I*C]-a c e t a t e l a b e l e d h i s t o n e p r e p a r a t i o n was s u b j e c t e d to a c i d h y d r o l y s i s . I t i s seen t h a t a f t e r the a c i d h y d r o l y s a t e has been d r i e d i n vacuo, the r e s i d u e c o n t a i n s no r a d i o a c t i v i t y , i n d i c a t i n g t h a t the l a b e l has been l o s t , presumably as a c e t i c a c i d . Hence, the [ x''C]-acetate l a b e l must have been p r e s e n t i n the form of a c e t y l groups i n the h i s t o n e s . -52.-FRACTION NO., 7 ML. FIG. 4. S e p a r a t i o n of [*^C]-acetate l a b e l e d h i s t o n e from p r o t a -mine. T o t a l b a s i c p r o t e i n s , e x t r a c t e d from chromatin a f t e r a p e r i o d o f i n c u b a t i o n of t e s t i s c e l l s w i t h t 1 ''C] - a c e t 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", were loaded onto a column of B i o - G e l P-10 i n 1.5 ml of 0.2 M a c e t i c a c i d . The column (2 x 50 cm) was then e l u t e d w i t h the same s o l u t i o n . -53-Table 2 E f f e c t of cycloheximide on a c e t y l a t i o n of t r o u t t e s t i s h i s t o n e s i n c e l l suspensions* Cycloheximide (0 .1 mM) (-) (+) 1 [lkC]-acetate 28,252 36 ,000 cpm/mg h i s t o n e * A c e l l suspension was prepared from lOg of t e s t i s and 25 ml of Hanks' medium, as d e s c r i b e d i n " M a t e r i a l s and Methods", and d i v i d e d i n t o two p a r t s . Both suspensions - one w i t h , and one without c y c l o h e x i m i d e . were p r e i n c u b a t e d at 20° f o r 30 min., 416 u C i of sodium l-> [***C]-acetate was then added t o each, and i n c u b a t i o n c a r r i e d out f o r 5 hr. Ta b l e 3 A c i d l a b i l i t y of t 1 **C]-acetate i n t r o u t t e s t i s h i s t o n e s * (a) Before h y d r o l y s i s 6,433 cpm i n whole h i s t o n e sample (b) A f t e r h y d r o l y s i s 32 * See " M a t e r i a l s and Methods" f o r d e t a i l s . Samples were hy-d r o l y z e d i n 6N HCl a t 110° f o r 19 h r . , d r i e d i n vacuo, and counted. -54-I t has been shown by Gershey e t a l . (43) t h a t the r a d i o a c t i v i t y i n [lkC]-acetate l a b e l e d c a l f thymus h i s t o n e s can be r e c o v e r e d l a r g e l y as e - N - a c e t y l l y s i n e a f t e r d i g e s t i o n of the h i s t o n e s w i t h t r y p s i n , f o l l o w e d by pronase. We attempted to i s o l a t e e - N - a c e t y l l y s i n e from t r o u t t e s t i s h i s t o n e s i n the same manner, and F i g . 5 shows the p r o f i l e on Sephadex G-25 of a t r y p s i n - p r o n a s e d i g e s t of [* **C]-acetate l a b e l e d t r o u t t e s t i s h i s t o n e s . Most o f the r a d i o a c t i v i t y chrom-atographs as a s i n g l e peak w i t h a slower-running shoulder, a p r o f i l e v e ry s i m i l a r t o t h a t o b t a i n e d by Gershey e t a l . (43) from c a l f thymus h i s t o n e s . The r a d i o a c t i v e f r a c t i o n s were pooled as shown, l y o p h i l i z e d , and a l i q u o t s were taken f o r a n a l y s i s by h i g h v o l t a g e e l e c t r o p h o r e s i s . F i g . 6 A shows the autoradiogram o b t a i n e d from the h i g h v o l t -age e l e c t r o p h o r e t o g r a m a t pH 6.5 of a t r y p s i n - p r o n a s e d i g e s t of I 1'•C]-acetate l a b e l e d whole h i s t o n e , a f t e r i n i t i a l chromatography on Sephadex G-25. Most of the r a d i o a c t i v i t y i s seen t o migrate w i t h a marker of a u t h e n t i c e - N - a c e t y l l y s i n e , prepared as d e s c r i b e d under " M a t e r i a l s and Methods". There i s a l s o some r a d i o a c t i v i t y m i g r a t i n g somewhat f a s t e r towards the cathode, which probably r e p r e s e n t s l a b e l i n i n c o m p l e t e l y d i g e s t e d t r y p t i c p e p t i d e s , which would a l l end with a COOH-terminal a r g i n i n e or l y s i n e , and hence would have a net p o s i t i v e charge a t pH 6.5. The m o b i l i t y of the main r a d i o a c t i v e component o f the d i g e s t was f u r t h e r compared w i t h the e - N - a c e t y l l y s i n e marker by e l e c t r o p h o r e s i s at pH 3.6, and paper chromatography i n a s o l v e n t system c o n s i s t i n g of n - b u t a n ol/ a c e t i c a c i d / w a t e r / p y r i d i n e , 15/3/12/10 by volume, and the correspond--55-i i FRACTION NO., 2.0 ML FIG. 5. Sephadex G-25 chromatography of a t r y p s i n - p r o n a s e d i g e s t of whole h i s t o n e . A sample of whole h i s t o n e (60 mg, c o n t a i n i n g 860,000 cpm t 1 **C]-acetate) was d i g e s t e d w i t h 1 mg of t r y p s i n i n 3.5 ml of 0.2 M NJUHC0 3, pH 7.8, a t 40° f o r 7 h r s . I n s o l u b l e m a t e r i a l was removed by c e n t r i f u g a t i o n and the supernatant, c o n t a i n i n g the r a d i o a c t i v i t y , was l y o p h i l i z e d . The l y o p h i l i z e d p e p t i d e s were then d i g e s t e d w i t h 1 mg of Pronase i n the same b u f f e r , a t 40° f o r 9 h r s . A f t e r l y o p h i l i z a t i o n , the d i g e s t was a p p l i e d t o a G-25 column (1.3 x 130), e q u i l i b r a t e d and e l u t e d w i t h 0.1 M NHi,HC0 3 . • t CO e-DNP LYS $>»• i i t-Ac Lys DIGEST pH6.5 A DIGEST ' «-Ac lys pH 3.6 B r DIGEST . ( A c Ly» FIG. 6. P u r i f i c a t i o n of e - N - a c e t y l l y s i n e from a t r y p s i n - P r o n a s e d i g e s t of whole h i s t o n e . A, autoradiogram: h i g h v o l t a g e e l e c t r o -p h o r e s i s of the d i g e s t a t pH 6.5. B , autoradiogram: h i g h v o l t a g e e l e c t r o p h o r e s i s at pH 3.6 of the main r a d i o a c t i v e component from pH 6.5. C, autoradiogram: chromatography i n b u t a n o l / a c e t i c a c i d / w a t e r / p y r i d i n e 15:3:12:10, of the r a d i o a c t i v e compound obtained a f t e r e l e c t r o p h o r e s i s a t pH 3.6. A marker of e - N - ( a c e t y l - 1 - 1 l*C) l y s i n e i s shown w i t h each d i g e s t sample; e-DNP l y s i n e i s a n e u t r a l marker. i n g autoradiograms are shown i n F i g s . 6 B and 6 C, r e s p e c t i v e l y . The r a d i o a c t i v e compound again migrates w i t h the marker, w i t h o n l y a s l i g h t r e t a r d a t i o n i n m o b i l i t y , due probably to a s l i g h t over-l o a d i n g of the d i g e s t sample. A f t e r p u r i f i c a t i o n by e l e c t r o p h o r e s i s and paper chromatography, the r a d i o a c t i v e compound from the t r y p s i n - p r o n a s e d i g e s t of t r o u t t e s t i s h i s t o n e s was f u r t h e r compared with standard e -N-acetyl l y s i n e by a n a l y s i s on a Beckman 120C amino a c i d a n a l y z e r , u s i n g a b u f f e r system which r e s o l v e s a l l of the amino a c i d s commonly found i n a c i d h y d r o l y s a t e s of p r o t e i n s (see " M a t e r i a l s and Methods"). F i g . 7 A shows the e l u t i o n diagram of a standard e -N-acetyl l y s i n e marker; F i g . 7 B shows the e l u t i o n diagram of the unknown compound from the d i g e s t , and F i g . 7 C i s an e l u t i o n diagram ob t a i n e d a f t e r the a d d i t i o n of approximately 0.02 ymoles of the e -N-acetyl l y s i n e standard to an a l i q u o t of the d i g e s t i d e n t i c a l to t h a t used i n the chromatogram i l l u s t r a t e d i n F i g . 7 B. Comparison of F i g s . 7 B and 7 C shows t h a t although the d i g e s t mixture c o n t a i n s s m a l l amounts of p r o l i n e and v a l i n e , as w e l l as ammonia, the major component ^from the d i g e s t emerges from the a n a l y z e r column i n e x a c t l y the same p l a c e as e - N - a c e t y l l y s i n e . No other amino a c i d commonly found i n p r o t e i n s emerges at t h i s p o s i t i o n ( e l u t i o n time 62 min) from the a n a l y z e r column. The above s t u d i e s suggested t h a t most, i f not a l l of the r a d i o a c t i v e a c e t a t e i n c o r p o r a t e d i n t o t r o u t t e s t i s h i s t o n e s i s p r e s e n t as e - N - a c e t y l groups a t t a c h e d to l y s y l r e s i d u e s i n these -58-FIG. 7. Amino a c i d a n a l y s i s of the [ ^ C ] - a c e t y l l a b e l e d com-pound ob t a i n e d from a t r y p s i n - P r o n a s e d i g e s t of whole h i s t o n e a f t e r p u r i f i c a t i o n by h i g h v o l t a g e e l e c t r o p h o r e s i s and chrom-atography (see F i g . 6). A, standard e - N - a c e t y l l y s i n e ; B, pur-i f i e d [ C ] - a c e t y l l a b e l e d compound from t r y p s i n - P r o n a s e d i g e s t C, p u r i f i e d compound from d i g e s t w i t h 0.02 umoles o f added e - N - a c e t y l l y s i n e standard. The s i n g l e column system o f DeVenyi (159) was used. -59-p r o t e i n s . Subsequent s t u d i e s , t o be d e s c r i b e d i n s e c t i o n I I below, f u r t h e r supported t h i s assumption. D e t a i l e d s t u d i e s of the l a b e l -i n g p a t t e r n s of i n d i v i d u a l h i s t o n e s were t h e r e f o r e undertaken. In e a r l y experiments, a p r e l i m i n a r y f r a c t i o n a t i o n of t o t a l h i s t o n e s was achieved by chromatography on columns of Bio-Rex 70, a weak c a t i o n exchange r e s i n , a c c o r d i n g to the procedure f i r s t d e s c r i b e d by Satake e t a l . (13). T h i s procedure separates whole h i s t o n e i n t o t h r e e f r a c t i o n s , as shown i n F i g . 8, and when the h i s t o n e s have been p r e v i o u s l y l a b e l e d w i t h i1 **C]-acetate, most o f the r a d i o a c t i v i t y i s seen t o emerge wit h the peak c o n t a i n i n g the a r g i n i n e - r i c h h i s t o n e s I I I and IV. There i s a l s o a peak of r a d i o a c t i v i t y a s s o c i a t e d w i t h the h i s t o n e I I peak, but very l i t t l e w ith the h i s t o n e I peak. In order to examine the d i s t r i b u t i o n of r a d i o a c t i v i t y i n the a r g i n i n e -r i c h f r a c t i o n , the p r o t e i n i n the I I I - I V peak from the Bio-Rex 70 column was p o o l e d as shown, d i a l y z e d a g a i n s t 0.2M a c e t i c a c i d , l y o p h i l i z e d , and a p p l i e d to a column of B i o - G e l P-60 (2 x 94 cm) e l u t e d w i t h 0.01N HCl. T h i s technique separates h i s t o n e I I I from h i s t o n e IV (153), and F i g . 9 a shows t h a t both p r o t e i n s are l a b e l e d w i t h t 1'*C]-acetate. For the d e t a i l e d chemical s t u d i e s t o be des-c r i b e d i n S e c t i o n I I below, these p r o t e i n s were f u r t h e r p u r i f i e d by re-chromatography on the same column ( F i g s . 9 b and 9 c ) . The [ l ""C]-acetate l a b e l e d h i s t o n e I I peak o b t a i n e d from Bio-Rex chrom-atography was f u r t h e r f r a c t i o n a t e d to o b t a i n pure p r e p a r a t i o n s of h i s t o n e s I l b i and I I b 2 , by chromatography on a long B i o - G e l P-10 -60-F R A C T I O N N O . - 5 m l FIG. 8. Acetylation of trout t e s t i s histones. Basic pro-teins extracted from three separate incubation mixtures were pooled, applied to a 2.5 x 50 cm Bio-Rex 70 column, and eluted with a l i n e a r gradient of 8 to 13% guanidinium chloride (700 ml) followed by 40% guanidinium chloride. Radioactivity was determined on aliquots i n a Unilux l i q u i d s c i n t i l l a t i o n counter using Bray's f l u i d containing Cab-O-Sil (24). Protein concentration was determined by t u r b i d i t y , at 400 nm following p r e c i p i t a t i o n by 1.1 M t r i c h l o r o a c e t i c acid. -61-1000 500 2 SO 70 40 60 SO F R A C T I O N N O . . 3.2 m l 20 40 60 80 F R A C T I O N N O . , 3.0 ml FIG. 9. P u r i f i c a t i o n o f h i s t o n e s III and IV. a, F r a c t i o n s comprising the III-IV peak from Bio-Rex 70 chromatography of whole h i s t o n e s ( F i g . 8) were pool e d , d i a l y z e d a g a i n s t two 2 & changes o f 0.2 M a c e t i c a c i d , and l y o p h i l i z e d . The d r i e d p r o t e i n was r e d i s s o l v e d i n 4 ml of 0.01 N HCl, a p p l i e d t o a column o f B i o - G e l P-60, and e l u t e d w i t h 0.01 N HCl. b and c, the peaks c o r r e s p o n d i n g t o h i s t o n e s III and IV i n F i g . 9a were po o l e d as shown, l y o p h i l i z e d , and r e r u n on the same column. -62-column, as d e s c r i b e d below. T h i s l a t t e r technique was developed by Dr. Michael Sung i n t h i s l a b o r a t o r y (81), and proved t o be a very convenient method f o r the f r a c t i o n a t i o n of whole h i s t o n e s as w e l l . I f a mixture of whole h i s t o n e s , l a b e l e d with [x l*C]-acetate d u r i n g a c e l l i n c u b a t i o n , i s chromatographed on a long column (3 x 320 cm) of B i o - G e l P-10 e l u t e d w i t h 0.01N HCl (see " M a t e r i a l s and Methods"), a good s e p a r a t i o n of a l l the major h i s t o n e s can be obt a i n e d . In order t o ensure the s e p a r a t i o n of h i s t o n e I I I from I l b i and I I b 2 , i t i s f i r s t n e cessary t o reduce any d i s u l f i d e bonds p r e s e n t i n h i s t o n e I I I by treatment o f the whole h i s t o n e w i t h d i t h i o t h r e i t o l or mercaptoethanol f o l l o w e d by iodoacetamide t o bl o c k the s u l f h y d r y l groups of the c y s t e i n e r e s i d u e s (16). The h i s t o n e I I I i s then converted from the dimer form to the monomer, and a l l of the major h i s t o n e s can be r e s o l v e d by P-10 chromato-graphy, as i l l u s t r a t e d i n F i g . 10. I t i s seen from t h i s e l u t i o n diagram t h a t a l l major h i s t o n e s from t r o u t t e s t i s , except h i s t o n e I , i n c o r p o r a t e r a d i o a c t i v i t y d u r i n g a p e r i o d of i n c u b a t i o n w i t h [* ''C]-acetate i n c e l l suspen-s i o n . S i n c e the amino t e r m i n i o f h i s t o n e s I , I l b i , and IV are bl o c k e d by a c e t y l groups (45,178) which are probably added d u r i n g s y n t h e s i s of these p r o t e i n s (47,48) , the l a c k o f l a b e l i n h i s t o n e I and the presence of a c e t y l groups i n h i s t o n e s I I b 2 and I I I , which have f r e e amino t e r m i n i , show t h a t the [l ^C]-acetate i n c o r p o r a t e d i n t o h i s t o n e s i n these experiments i s not p r e s e n t as amino-terminal °<-N-acetyl groups, but i n s t e a d must be p r e s e n t as e- N - a c e t y l FIG. 10. F r a c t i o n a t i o n of a c e t y l a t e d h i s t o n e s . H i s t o n e s were e x t r a c t e d from 15g of t e s t i s c e l l s a f t e r a 60 minute per-i o d of l a b e l i n g w i t h [ C ] - a c e t a t e . B a s i c p r o t e i n s were then d i s s o l v e d i n 0.1 M sodium borate b u f f e r , f o r r e d u c t i o n and a l k y l a 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". The r e -duced and a l k y l a t e d h i s t o n e s were a p p l i e d to' a 3 x 320 cm B i o - G e l P-10 column e q u i l i b r a t e d and e l u t e d w i t h 0.01 N HCl. The flow r a t e was approximately 50 ml/hr., and the v o i d volume o f the column was 600 ml. R a d i o a c t i v i t y was determined by mixing 200 y l a l i q u o t s of the column f r a c t i o n s w i t h 5 ml o f Bray's s c i n t i l l a t i o n f l u i d 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 counter. P r o t e i n c o n c e n t r a t i o n was determined by absorbance a t 230 nm. -64-groups on i n t e r n a l l y s y l r e s i d u e s . The l a c k of a c e t y l a t i o n of h i s t o n e I has been noted i n c a l f thymus as w e l l (44) , and i s s t r i k i n g i n view of the f a c t t h a t t h i s p r o t e i n c o n t a i n s the h i g h e s t amount of l y s i n e of a l l the h i s t o n e s (178). I t i s worth n o t i n g here t h a t the a c e t y l a t e d components of each h i s t o n e emerge from the P-10 column s l i g h t l y ahead of the main p r o t e i n peak. T h i s i s probably due to the presence of a s m a l l number of a n i o n i c groups i n the B i o - G e l matrix, which c o n f e r upon i t weak i o n exchange p r o p e r t i e s and the a c e t y l a t e d h i s t o n e s , being l e s s b a s i c , are e l u t e d e a r l i e r . The phosphorylated forms of the h i s t o n e s behave i n a s i m i l a r manner (154). When the v a r i o u s t r o u t t e s t i s h i s t o n e s , p u r i f i e d by i o n -exchange on Bio-Rex 70 f o l l o w e d by B i o - G e l chromatography, or by d i r e c t chromatography on long columns of B i o - G e l P-10 as d e s c r i b e d above, are analyzed by e l e c t r o p h o r e s i s on s t a r c h g e l s , they are seen to be heterogeneous. F i g . 11 i l l u s t r a t e s the p a t t e r n s o b t a i n e d w i t h [l **C]-acetate l a b e l e d h i s t o n e s I l b i , H b 2 , I I I and IV f o l l o w i n g e l e c t r o p h o r e s i s on a urea-aluminum l a c t a t e s t a r c h g e l and Araido B l a c k s t a i n i n g . These f r a c t i o n s were p u r i f i e d by i n i t i a l i o n exchange chromatography on Bio-Rex 70 f o l l o w e d by g e l e x c l u s i o n chromatography of the h i s t o n e I I and I I I - I V peaks on B i o - G e l columns as d e s c r i b e d above. In F i g . 12, the p a t t e r n s o f the same f r a c t i o n s , p u r i f i e d t h i s time by d i r e c t chromatography o f whole h i s t o n e s on B i o - G e l P-10, are i l l u s t r a t e d (the samples used here were taken from the p o s i t i o n s -65-IIB in IIB, 2 M i . FIG. 11. S t a r c h g e l e l e c t r o p h o r e s i s o f p u r i f i e d h i s t o n e s . F r a c t i o n s were p u r i f i e d by Bio-Rex 70 chromatography f o l l o w e d by chromatography on B i o - G e l P-10 and P-60 ( F i g s . 8 and 9). E l e c t r o p h o r e s i s was f o r 16 hrs a t 6 vo l t s / c m . S t a i n i n g was done i n 1% Amido Black f o r 10 min, and d e s t a i n i n g i n 2% a c e t i c a c i d . - 6 6 -FIG. 12. S t a r c h g e l e l e c t r o p h o r e s i s o f p u r i f i e d h i s t o n e s . A l i q u o t s of each l a b e l e d h i s t o n e f r a c t i o n (arrows) from the P-10 column of F i g . 10, p. 63, were a p p l i e d t o 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 16 hrs at 6 v o l t s / cm. S t a i n i n g was done i n 1% Amido B l a c k f o r 10 min, and de-s t a i n i n g i n 2% a c e t i c a c i d . Each sample was run i n d u p l i c a t e . -67-marked by arrows i n the column p r o f i l e of F i g . 10) . The reasons f o r the g r e a t d e a l of h e t e r o g e n e i t y observed i n 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 s remained l a r g e l y obscure u n t i l a method was d e v i s e d to o b t a i n a u t o r a d i o g r a p h i c images of [ l l fC]-l a b e l e d h i s t o n e samples on s t a r c h g e l s . T h i s technique i n v o l v e s p r i o r d r y i n g of the s t a r c h g e l s l a b , as d e s c r i b e d i n " M a t e r i a l s and Methods", and i s an e x t e n s i o n of the method d e s c r i b e d by F a i r b a n k s e t a l . f o r the autoradiography of [ l**C]-labeled p r o t e i n s on p o l y a c r y l a m i d e d i s c g e l s (161). F i g u r e s 13 and 14 i l l u s t r a t e the same p r o t e i n p a t t e r n s as F i g s . 11 and 12, r e s p e c t i v e l y , except t h a t beside each Amido Black s t a i n e d p r o t e i n p a t t e r n , the c o r r e s p o n d i n g autoradiogram produced by the presence of the t 1 kC]~ a c e t a t e l a b e l i s shown. The nature of the v a r i o u s p r o t e i n bands i n each h i s t o n e w i l l now be d i s c u s s e d i n d e t a i l . (i) H i s t o n e I l b i : In F i g . 13, the h i s t o n e I l b i sample has been r e s o l v e d i n t o t h r e e bands - a f a s t e r - r u n n i n g d o u b l e t (A 0 & Ai) and a slower, s i n g l e band (P). The autoradiogram of the p r o t e i n shows t h a t o n l y the second band, A i , i s l a b e l e d w i t h [x ^C]-acetate. The band marked "P" i n the f i g u r e has been shown by Dr. M. Sung to i n c o r p o r a t e 3 2 P d u r i n g c e l l i n c u b a t i o n s w i t h r a d i o a c t i v e i n o r g a n i c phosphate and t h i s band moves t o the p o s i t i o n of band A 0 upon treatment w i t h a l k a l i n e phosphatase (154). These data are a l l con-s i s t e n t w i t h the i d e a t h a t band Ao r e p r e s e n t s unmodified h i s t o n e I l b i , band A i r e p r e s e n t s a c e t y l a t i o n a t a s i n g l e l y s y l r e s i d u e i n lib, llb2 III iv FIG. 13. Acetylated components of trout t e s t i s histones: autoradiography of [x **C]-acetate labeled, p u r i f i e d histone fractions following starch gel electrophoresis. Histones were p u r i f i e d by Bio-Rex chromatography, followed by chromatography on Bio-Gel P-10 and P-60. The protein s t a i n i s shown on the l e f t , and the corresponding autoradiogram on the r i g h t of each sample. A0-A2?-llb2 : 0 l i i l Af Ar III IV A1-A3 A + P " P,A,-P,A„ P.AaV P,A4 FIG. 14. Acetylated components of trout t e s t i s histones. Aliquots of each labeled histone f r a c t i o n (arrows) from the P-10 column of Fi g . 10, p. 63, containing 300-500 ug of pro-t e i n and 10-20,000 cpm of [ x^C]-acetate were l y o p h i l i z e d , redissolved i n d i s t i l l e d water and applied to a urea-aluminum lactate starch gel. Electrophoresis was for 16 hours at 6 volts/cm. The middle s l i c e of the gel was dried a f t e r s t a i n -ing i n 1% Amido Black for 10 min and destaining i n 2% acetic acid. The dried gel s l i c e was exposed to the X-ray f i l m for three weeks. For each histone f r a c t i o n , the s t r i p on the l e f t represents the protein staining pattern, and that on the r i g h t , i t s corresponding autoradiogram. -70-I l b i , and band P r e p r e s e n t s p h o s p h o r y l a t i o n of I l b i a t a s i n g l e s i t e . The s i t e of p h o s p h o r y l a t i o n of t r o u t t e s t i s I l b i has been d e s c r i b e d (81). S t u d i e s on the s i t e o f a c e t y l a t i o n of I l b i , t o be d e s c r i b e d i n S e c t i o n I I , c o n f i r m the presence of a s i n g l e major a c e t y l a t e d l y s y l r e s i d u e i n t h i s p r o t e i n . In F i g . 14, the p a t t e r n of I l b i i s s i m i l a r t o t h a t j u s t d e s c r i b e d , except t h a t another band i s v i s i b l e behind A;, and may r e p r e s e n t another a c e t y l a t e d s p e c i e s . The f a s t e s t - r u n n i n g band i n t h i s sample i s an uniden-t i f i e d contaminant. A comparison of the two samples of h i s t o n e I l b i i n F i g s . 13 and 14 shows t h a t the a c e t y l a t e d component, A i , c o n s t i t u t e s a somewhat l a r g e r p r o p o r t i o n o f the t o t a l p r o t e i n i n the sample i l l -u s t r a t e d i n F i g . 13, as compared to t h a t i n F i g . 14. T h i s i s be-cause the p u r i f i c a t i o n o f the I l b i sample i n F i g . 13 i n v o l v e d p r e l i m i n a r y f r a c t i o n a t i o n by i o n exchange on a Bio-Rex 70 column. The a c e t y l a t e d (and phosphorylated) components, being s l i g h t l y l e s s b a s i c , tend t o emerge from the column e a r l i e r than the bulk of the h i s t o n e I I , as shown by the l o c a t i o n of t h e 1 [l **C]-acetate peak i n F i g . 8, p. 60. Hence, when the f r a c t i o n s are pooled on the b a s i s of the I 1''C]-acetate p r o f i l e , the r e s u l t i n g p r o t e i n i s en-r i c h e d i n the a c e t y l a t e d s p e c i e s . The same s i t u a t i o n holds f o r the components of h i s t o n e IIb2, t o be d i s c u s s e d below. ( i i ) H istone IIb2: In F i g . 13, two d i s t i n c t bands are v i s i b l e i n h i s t o n e I I b 2 . The second of these (marked "Ai") i s an a c e t y l a t e d s p e c i e s , as shown by the autoradiogram. -71-In F i g . 14, the g e l sample has been d e l i b e r a t e l y overloaded i n order t o p r o v i d e enough r a d i o a c t i v i t y f o r autoradiography; hence, the p r o t e i n bands are not ve r y d i s t i n c t . However, f o u r bands can be d i s t i n g u i s h e d , and f. 1 ^ C ]-acetate l a b e l can be d e t e c t e d over com-ponents A i and A 2 i n the autoradiogram. The band marked Ao c o n t a i n s no l a b e l , and t h e r e f o r e presumably r e p r e s e n t s the unmodified h i s -tone. Thus i t seems, from t h i s data, t h a t h i s t o n e I I b 2 can be a c e t y l a t e d a t two, and p o s s i b l y t h r e e d i f f e r e n t s i t e s i n the mol-e c u l e . ( i i i ) Histone I I I : In F i g . 13, most of the h i s t o n e I I I i s p r e s e n t i n i t s dimer (oxidized) form, which runs, on s t a r c h g e l s , behind a l l of the o t h e r h i s t o n e s . The autoradiogram shows t h a t the I1 kC]-acetate i s l o c a t e d e n t i r e l y over t h i s component. The band l a b e l e d " I I I m " may r e p r e s e n t some reduced h i s t o n e I I I . The f a s t e s t - r u n n i n g component i n t h i s sample i s probably some con-t a m i n a t i n g h i s t o n e I I b 2 , s i n c e t h i s band was s t a i n e d blue by the s e n s i t i v e Cobalt-Amido Black s t a i n of Sung and Smithies (160). By t h i s technique (see " M a t e r i a l s and Methods", S e c t i o n I I I ) h i s t o n e s I l b i , I I I and IV, being r e l a t i v e l y r i c h i n a r g i n i n e , appear b l a c k , whereas h i s t o n e s I and I I b 2 y i e l d a blue c o l o r a f t e r d e s t a i n i n g . I n F i g . 14, the h i s t o n e I I I i s pr e s e n t as the monomer (reduced) form, s i n c e the h i s t o n e s were reduced and a l k y l a t e d p r i o r t o the B i o - G e l P-10 chromatography, as p r e v i o u s l y d e s c r i b e d . Again, the sample has been overloaded f o r purposes of autoradiography. T y p i -c a l l y , reduced h i s t o n e I I I runs as a group of t h r e e bands, c l o s e l y -72-spaced, and of almost equal i n t e n s i t y . I t i s seen from the auto-radiogram t h a t the two slowest-running bands (Ai and A 2) are d i s -t i n c t l y l a b e l e d w i t h t 1 **C]-acetate. I t i s c l e a r , furthermore, t h a t the f a s t e s t s t a i n e d component (Ao) i s not l a b e l e d w i t h [ l ^ C ] - a c e t a t e , and hence r e p r e s e n t s unmodified h i s t o n e I I I . The g e l p a t t e r n of t h i s h i s t o n e , then, p r e d i c t s the e x i s t e n c e of two major s i t e s of a c e t y l a t i o n i n the molecule. (iv) Histone IV: The p a t t e r n of h i s t o n e IV on urea-aluminum l a c t a t e s t a r c h g e l s i s the most complex of a l l . In F i g . 13, f i v e s t a i n e d p r o t e i n bands are c l e a r l y v i s i b l e ; two other bands were v i s i b l e behind Ai» on the o r i g i n a l g e l , but are not d i s c e r n i b l e i n the photograph. I t i s e v i d e n t from the autoradiogram of t h i s sample t h a t bands A i to Ai. are l a b e l e d w i t h [l **C]-acetate, and these have been i n t e r p r e t e d as a c e t y l a t e d d e r i v a t i v e s of the unmodified h i s t o n e , r e p r e s e n t e d by band Ao. Thus, A i r e p r e s e n t s a c e t y l a t i o n of h i s t o n e IV at a s i n g l e l y s y l r e s i d u e , A 2 r e p r e s e n t s h i s t o n e IV m o d i f i e d at two s i t e s , e t c . There i s a f a i n t t r a c e of l a b e l i n g over band A 0, and t h i s c o u l d be due to i n c o r p o r a t i o n of [l kC]-acetate i n t o the amino t e r m i n a l of the p r o t e i n , which i s normally a c e t y l -a ted i n t h i s h i s t o n e (45). In F i g . 14, a t o t a l of nine components are v i s i b l e i n the h i s t o n e IV sample. Bands Ai-Ai* are agai n c l e a r l y l a b e l e d w i t h I1 " C ] - a c e t a t e . The bands marked " P i A i " t o "PiAi»" d i s a p p e a r upon treatment o f h i s t o n e IV w i t h a l k a l i n e phos-phatase, and i n c o r p o r a t e 3 2 P ; the band l a b e l e d "A^+Pi" d i m i n i s h e s i n i n t e n s i t y a f t e r a l k a l i n e phosphatase treatment, and i n some experiments has been r e s o l v e d i n t o two separate bands (81), -73-making a t o t a l of 10 h i s t o n e IV components; furthermore, h i s t o n e IV from t r o u t t e s t i s has been shown to be phosphorylated a t a s i n g l e s e r y l r e s i d u e i n the molecule (91). A l l of the above f a c t s are c o n s i s t e n t with the f o l l o w i n g i n t e r p r e t a t i o n : bands A i - A i f r e p r e s e n t a c e t y l a t i o n of unmodified h i s t o n e IV (band Ao) a t one, two, t h r e e , or f o u r l y s y l r e s i d u e s , r e s p e c t i v e l y . The band l a b e l e d " P i " , which can be r e s o l v e d s e p a r a t e l y from A^ on some g e l s , r e p r e s e n t s p h o s p h o r y l a t i o n of unmodified h i s t o n e IV (band Ao), w h i l e bands P i A i to PiAi» r e p r e s e n t p h o s p h o r y l a t i o n of bands Ai to A i , r e s p e c t i v e l y , at a s i n g l e s i t e . Hence, a p o p u l a t i o n of h i s t o n e IV molecules can be separated, on the b a s i s of t h e i r charge, i n t o 10 separate components. I t i s p o s s i b l e t h a t the more e x t e n s i v e l y m o d i f i e d components might emerge from a c a t i o n exchange column e a r l i e r than the l e s s m o d i f i e d s p e c i e s , and t h i s may e x p l a i n the s m a l l e r number of v i s i b l e components i n h i s t o n e IV prepared by Bio-Rex 70 chromatography ( F i g . 13). There i s evidence which suggests t h a t these more h i g h l y m o d i f i e d s p e c i e s are p r e s e n t i n l a r g e r amounts i n t e s t e s o b t a i n e d from f i s h induced to mature by i n j e c t i o n s of salmon p i t u i t a r y e x t r a c t , as opposed to t e s t e s o b t a i n e d from n a t u r a l l y maturing f i s h (179). In the s t u d i e s d e s c r i b e d here, the l a t t e r souce of m a t e r i a l was used. The a c e t y l a t i o n of t r o u t t e s t i s h i s t o n e s i n hormonally induced t e s t i s w i l l be d i s c u s s e d i n S e c t i o n I I I . -74-I I . Studies on the Sites of In Vivo Acetylation i n Trout T e s t i s  Histones. (i) Histone IV; As a r e s u l t of the studies described i n Section I above, we became interested i n obtaining more detailed knowledge of the s i t e s of acetylation i n trout t e s t i s histones. Since histone IV was one of the most extensively modified of these proteins, and since the amino acid sequence of t h i s protein from c a l f thymus and pea seedlings had recently become available as a re s u l t of the work of DeLange et a l . (26,29) , we began our det a i l e d s t r u c t u r a l studies on t h i s histone. As a preliminary step i n these studies, I1''C]-acetate labeled histone IV, obtained from lab e l i n g experiments c a r r i e d out with t e s t i s c e l l suspensions, was digested with t r y p s i n and the radio-active peptides were examined. F i g . 15 shows the e l u t i o n pattern of t r y p t i c acetylated peptides on Dowex 50. Three major peaks of r a d i o a c t i v i t y were obtained. When aliquots of each peak were sub-jected to paper electrophoresis at pH 6.5, a t o t a l of nine d i s t i n c t peptides could be seen by autoradiography, as shown i n F i g . 16. The section of paper corresponding to each labeled spot was cut out, sewn into a fresh sheet of Whatman No. 3MM paper, and e l e c t r o -phoresed at pH 1.9, to see whether further f r a c t i o n a t i o n could be obtained. A l l of the peptides ran as single components at pH 1.9 except T l a , which contained minor amounts of a s l i g h t l y f a s t e r moving peptide along with the major component, which was designated T l a ' . -75-F R A C T I O N N O . - 4 m l FIG. 15. Separation of [* l*c}-acetate labeled, t r y p t i c peptides of histone IV. Seventy mg of p u r i f i e d histone IV, contain-ing 700,000 cpm of [* "*Cj-acetate, was digested with 2 mg of CP tr y p s i n for 3 hrs at 37°, i n 0.1 M NH^HC03 pH 7.8. The mix-ture was l y o p h i l i z e d , redissolved i n d i s t i l l e d water, c l a r i -f i e d by centrifugation, a c i d i f i e d to pH 1, and applied to a 0.9 x 100 cm jacketed column of Dowex 50 x 2 r e s i n maintained at 38°. The ..column was eluted with a f l a t convex gradient of pyridinium acetate from pH 3.1 (0.2 M) to pH 5.0 (2.0 M) as described by Schroeder et a l . (162). A constant flow rate of 40 ml/hr was maintained with a Milton-Roy pump. Aliquots (0.05 ml) of each f r a c t i o n were removed, dried i n s c i n t i l l a t i o n v i a l s , and counted i n 5 ml of Bray's s c i n t i l l a t i o n f l u i d (155). -76-t c-> T2 n C . b a -c _ b-a -7 3 O C-DNP LYSINE FIG. 16. Autoradiography of high voltage electrophoric sep-aration of histone IV, £ cl-acetate labeled t r y p t i c peptides. The fractions from each of the radioactive Dowex peaks from F i g . 15 were pooled, l y o p h i l i z e d , redissolved i n a small volume of d i s t i l l e d water, and aliquots were spotted on Whatman 3MM paper. Electrophoresis was ca r r i e d out at 56 V/cm for 60 min, at pH 6.5 i n a toluene-cooled tank. The paper was then removed, dried, and exposed to a sheet of Kodak Blue Brand Medical X-ray f i l m for several days. -77-In s e c t i o n I above, evidence was pres e n t e d which suggested the presence of f o u r a c e t y l a t i o n s i t e s i n h i s t o n e IV. The f i n d i n g o f 9 o r 10 t r y p t i c p e p t i d e s i n a d i g e s t o f t h i s p r o t e i n was, t h e r e -f o r e , a p u z z l i n g r e s u l t a t f i r s t . In the course o f t h e i r sequence work on h i s t o n e IV from c a l f thymus,.DeLange e t a l . (26) found t h a t l y s y l r e s i d u e 16 was approximately 50% a c e t y l a t e d . Comparison o f t h i s sequence w i t h t h a t o f pea s e e d l i n g h i s t o n e IV r e v e a l e d t h a t , i n a d d i t i o n t o l y s y l r e s i d u e 16, a t l e a s t one of l y s y l r e s i d u e s 5,8, o r 12 was p a r t i a l l y a c e t y l a t e d i n the pea (29). The complex p a t t e r n o f a c e t y l a t e d t r y p t i c p e p t i d e s o b t a i n e d from t r o u t t e s t i s h i s t o n e IV c o u l d be e x p l a i n e d , then, i f i t was assumed t h a t a l l f o u r o f the suspected a c e t y l a t i o n s i t e s were p r e s e n t i n the amino-t e r m i n a l end of the molecule, i . e . i f l y s y l r e s i d u e s 5,8,12 and 16 were a l l m o d i f i e d t o some ex t e n t i n t r o u t t e s t i s h i s t o n e IV, and i f i t was assumed, furthermore, t h a t m o d i f i c a t i o n a t each of these f o u r r e s i d u e s c o u l d occur independently o f the o t h e r s . S i n c e t r y p s i n w i l l c l e a v e on the c a r b o x y - t e r m i n a l s i d e o f a l y s y l o r a r g i n y l r e s -i d u e , but not at an a c e t y l a t e d l y s y l r e s i d u e , a c e t y l a t i o n o f the f o u r l y s y l r e s i d u e s i n v a r i o u s combinations c o u l d g i v e r i s e t o a complex mixture of t r y p t i c p e p t i d e s . Examination of the amino-t e r m i n a l sequence of h i s t o n e IV ( F i g . 17) shows t h a t random a c e t y l -a t i o n o f l y s y l r e s i d u e s 5,8,12 and 16 would g i v e r i s e t o 10 p o s s i b l e t r y p t i c p e p t i d e s . These would a l l have a charge of +1, and m o b i l -i t i e s a t pH 6.5 s i m i l a r t o those a c t u a l l y observed. A f t e r e l e c t r o p h o r e s i s a t pH 1.9, each p e p t i d e was c u t out, e l u t e d from the paper w i t h 0.01 N NHi»OH, and s u b j e c t e d t o amino -78-TAIa TAlb Ac Ac Ac Ac Me Ac-Ser-Gly-Arg-Gly-Lys-Gly-Gly-Lys-Gly-Leu-Gly-Lys-Gly-Gly-Ala-Lys-Arg-His-Arg-Lys-Val-5 8 1 2 1 6 TAl FIG. 17. NH2-terminal sequence of c a l f thymus h i s t o n e IV a c c o r d i n g t o DeLange e t a l . (26). The l y s y l r e s i d u e s found t o be a c e t y l a t e d i n t r o u t t e s t i s h i s t o n e IV are i n d i c a t e d . P e p t i d e T A l was o b t a i n e d from a t r y p t i c d i g e s t of i n v i v o l a b e l e d [* "cj-acetate h i s t o n e IV which had been c h e m i c a l l y a c e t y l a t e d w i t h a c e t i c anhydride, t o b l o c k a l l l y s y l r e s i -dues not p r e v i o u s l y a c e t y l a t e d d u r i n g the c e l l i n c u b a t i o n . P e p t i d e s T A l a and TA l b were o b t a i n e d by cleavage of T A l w i t h t h e r m o l y s i n .(see pp. 79-84 f o r e x p e r i m e n t a l d e t a i l s ) . - 7 9 -a c i d a n a l y s i s . T a b l e 4 shows the r e s u l t s of the amino a c i d a n a l y s i s of T la'' and T l b . The composition of T l a / i n d i c a t e s t h a t i t must correspond to r e s i d u e s 4-17 i n the h i s t o n e IV sequence of DeLange e t a l . (26) ( F i g . 17). The m o b i l i t y of the p e p t i d e suggests t h a t i t has a charge of o n l y +1, and s i n c e i t c o n t a i n s f o u r l y s y l r e s -idues (at 5,8,12 and 16) and one a r g i n y l r e s i d u e , the e-amino groups must a l l be blocked by a c e t y l groups. The a n a l y s i s o f T l b c o r -responds t o the composition of r e s i d u e s 6-17 of the sequence, and suggests t h a t l y s y l r e s i d u e s 8,12 and 16 are blocked. A n a l y s i s of some o f the o t h e r t 1 ^ C ] - a c e t a t e l a b e l e d p e p t i d e s shown i n F i g u r e 16 r e v e a l e d t h a t they were mixtures of s e v e r a l components. Others were o b t a i n e d i n i n s u f f i c i e n t amounts f o r a c c u r a t e a n a l y s i s . To o b t a i n more c o n c l u s i v e evidence f o r the a c e t y l a t i o n of l y s y l r e s i d u e s 5,8,12 and 16, the f o l l o w i n g experiment was c a r r i e d out: A sample of i n v i v o , 1 [l **C]-acetate l a b e l e d h i s t o n e IV^ was mixed w i t h c a r r i e r and c h e m i c a l l y a c e t y l a t e d w i t h u n l a b -e l e d a c e t i c anhydride, so as t o a c e t y l a t e the e-amino group of every l y s y l r e s i d u e i n the h i s t o n e . The p r o t e i n was then d i g e s t e d w i t h t r y p s i n . I f o n l y l y s y l r e s i d u e s 5,8,12 and 16 are l a b e l e d w i t h I1 kC]-acetate d u r i n g the i n v i v o i n c u b a t i o n , and i f the sub-sequent chemical a c e t y l a t i o n i s complete, then the t r y p t i c d i g e s t should c o n t a i n o n l y one l a b e l e d p e p t i d e , c o r r e s p o n d i n g t o r e s i d u e s 4-17, i . e . T l a ' T h i s experiment a l s o has the advantage of p r o -v i d i n g y i e l d s f o r the t e t r a - a c e t y l p e p t i d e of up to 100%, and thus e l i m i n a t e s the need f o r l a r g e amounts of m a t e r i a l . -80-Table 4 Amino acid compositions of [l l*C]-acetate labeled histone IV peptides Amino Acid T l a Tib TAl TA2 TAla' TAlb TAlb(-) 4 Lysine 3.96(4) 2.90(3) 3.72(4) 3.06 1.78(2) 1.81(2) 1.08(1) Arginine 0.94(1) 0.90(1) 1.04(1) 2.00 0.91(1) 0.90(1) Glycine 7.20(7) 6.00(6) 6.84(7) 6.00 4.00(4) 3.16(3) 1.38(1) Alanine 1.91(1) 1.13(1) 1.08(1) 0.93 1.03(1) 1.00(1) Leucine 1.02(1) 1.20(1) 0.96(1) 1.06 0.94(1) -H i s t i d i n e - - - 0.53 - - -Valine - - 0.47 - -• -81-T A 2 «-* JJ> O «-©N* IYSINI • r i g i n 4 T T A l b -» T A l o -«-ONr LYS PH6.5 pH 6.5 pH 1.9 b c origin FIG. 18. (a) Chemical acetylation of i n vivo labeled f^cf-acetate histone IV. A t o t a l of 18 mg p u r i f i e d histone IV con-tai n i n g 180,000 cpm £ a c e t a t e was chemically acetylated with acetic anhydride, as described i n Materials and Methods. The dialyzed and l y o p h i l i z e d protein was redissolved i n 2.5 ml of 0.1 M NIUHC0 3 pH 7.8, and digested for 4 hrs at 37° with 0.4 mg CP Trypsin. The solution was l y o p h i l i z e d , and an aliquot applied to a sheet of Whatman 3MM paper for electrophoresis at pH 6.5, followed by autoradiography. (b) Thermolysin cleavage of peptide TAl. Peptide TAl shown i n Figure (a) above was cut out, sewn int o a clean sheet of Whatman 3MM paper, and electrophoresed at pH 1.9 (50 V/cm, 50 min). The location of the peptide was determined by auto-radiography, and i t was then eluted from the paper with 1M a c e t i c acid and l y o p h i l i z e d . An aliquot was set aside f o r amino acid analysis. The r e s t of TAl was then digested with 25 ug of thermolysin i n 0.6 ml of 0.1 M N-ethylmorpholine-acetic acid, 0.002 M calcium chloride, at 37° for 3 hr. Digest products were then separated by high voltage electrophoresis at pH 6.5. (c) P u r i f i c a t i o n of peptide TAla. Peptide TAla shown i n Figure (b) above was cut out and electrophoresed at pH 1.9. The peptide designated by the arrow (TAla' ) was cut out and eluted for analysis and Edman degradation. -82-F i g u r e 18 (a) shows the s e p a r a t i o n of the l a b e l e d t r y p t i c p e p t i d e s o f c h e m i c a l l y a c e t y l a t e d h i s t o n e IV. A s i n g l e major p e p t i d e i s o b t a i n e d ( T A l ) , w i t h a m o b i l i t y s i m i l a r t o t h a t of T l a . There i s a l s o a more b a s i c p e p t i d e (TA2), which i s p r e s e n t i n r e l a t i v e l y s m a l l amounts (5-6%). Each o f these p e p t i d e s was r e r u n a t pH 1.9, and each remained as a s i n g l e homogeneous spot. A n a l y s i s of an a l i q u o t o f T A l (Table 4) showed t h a t i t was indeed the p e p t i d e c o n t a i n i n g r e s i d u e s 4-17 of the molecule. A n a l y s i s of TA2 (Table 4) suggested t h a t i t might c o n s i s t o f a p i e c e from" the N H 2 - t e r m i n a l r e g i o n , contaminated w i t h one or more other p e p t i d e s . Thus, although the occurrence o f an a c e t y l a t i o n s i t e o u t s i d e of the N H 2 - t e r m i n a l r e g i o n of h i s t o n e IV cannot be r u l e d out e n t i r e l y , the p o s s i b i l i t y seems u n l i k e l y from these d a t a . P e p t i d e T A l was then d i g e s t e d w i t h t h e r m o l y s i n , i n order t o o b t a i n s m a l l e r p e p t i d e s which would be more amenable t o sequen-c i n g . T h e r m o l y s i n would be expected t o c l e a v e a t the N H 2 - t e r m i n a l s i d e o f the l e u c y l r e s i d u e a t p o s i t i o n 10, thus y i e l d i n g two p e p t i d e s . F i g u r e 18 (b) shows the s e p a r a t i o n of the t h e r m o l y s i n d i g e s t products by paper e l e c t r o p h o r e s i s a t pH 6.5. The fas t e r - m o v i n g b a s i c p e p t i d e TAlb corresponds, as expected, t o the sequence Leu-Gly-Lys ( A c ) - G l y - G l y - A l a - L y s (Ac)-Arg, as shown by the a n a l y s i s i n T a b l e 4. Pe p t i d e T A l a , which moves w i t h the n e u t r a l marker e-DNP-l y s i n e , s hould correspond t o the sequence Gly-Lys (Ac)-Gly-Gly-Lys (Ac)-Gly. E l u t i o n and a n a l y s i s of T A l a suggested t h a t i t was not -83-completely pure, and hence i t was rerun a t pH 1.9. S e v e r a l com-ponents were then separated (Figure 1 8 ( c ) ) , and a n a l y s i s o f the major one (TAla', arrow) showed i t t o be the expected p e p t i d e (Table 4). The o t h e r p e p t i d e s may be the r e s u l t of cleavage by t h e r m o l y s i n a t o t h e r r e s i d u e s i n T A l . Cleavage at a G l y - G l y bond has been r e p o r t e d by Sung and Dixon (81), f o r t h i s p r e p a r a t i o n of t h e r m o l y s i n . A l s o , the s i d e c h a i n o f an a c e t y l l y s y l r e s i d u e i s q u i t e hydrophobic, and t h e r m o l y s i n might c o n c e i v a b l y be a b l e to c l e a v e a t the N H 2 - t e r m i n a l s i d e of t h a t r e s i d u e . The t h e r m o l y s i n cleavage products of T A l a l s o i n c l u d e a f a i n t spot running toward the anode (F i g u r e 1 8 ( b ) ) . A n a l y s i s showed i t t o c o n t a i n o n l y g l y c i n e and l y s i n e , i n a r a t i o o f 2:1; however, i n o r d e r f o r i t to be a c i d i c i t would have t o have both i t s N H 2 - t e r m i n a l , and the e-NH2 group of the l y s y l r e s i d u e ( s ) b l o c k e d . The o r i g i n of t h i s spot remains unknown. In o r d e r t o prove t h a t both l y s y l r e s i d u e s 5 and 8 i n TAla', and r e s i d u e s 12 and 16 i n T A l b were l a b e l e d w i t h [* "*C)-acetate, the p e p t i d e s were s u b j e c t e d to Edman d e g r a d a 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 f t e r each d e g r a d a t i o n , a sample of the p e p t i d e was removed f o r N H 2 - t e r m i n a l d e t e r m i n a t i o n by the 'dansyl* method of Gray (150). The DNS-amino a c i d s were i d e n t i f i e d by t h i n l a y e r chromatography i n B e n z e n e / P y r i d i n e / A c e t i c a c i d (80:20:2), as d e s c r i b e d by Morse and Horecker (167), and the r e s u l t s are shown i n F i g . 19. The d e r i v a t i v e of e - a c e t y l l y s i n e (which becomes <X-DNS-lysine as a r e s u l t of the a c i d h y d r o l y s i s of the d a n s y l a t e d peptide) does not migrate i n t h i s s o l v e n t system, but s i n c e the -84-D N S - N H , 3 UQ c o p 0 © o o 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 1. DNS-Gly standard 2. TAlb derivative #1 3. DNS-Leu standard 4. TAlb derivative #2 5. DNS-Gly standard 6. TAlb derivative #3 7. cV-DNS-e-Ac-Lys standard 8. TAlb derivative #4 9. DNS-Gly standard 10. TAlb derivative #5 11. di-DNS-Lys standard ; 12. DNS-Arg standard 13. DNS-Ala standard 14. o< -DNS-e-Ac-Lys standard 1. DNS-Gly standard 2. TAla' derivative #1 3. <X -DNS-e-Ac-Lys standard 4. TAla' derivative #2 5. DNS-Gly standard 6. TAla' derivative #3 7. DNS-Gly standard 8. di-DNS Lys standard TAla' FIG. 19. I d e n t i f i c a t i o n of dansyl derivatives of amino acids from [ l**C]-acetate labeled histone IV peptides. Samples were dissolved i n 2 M NH^OH and applied to 20 x 20 cm glass plates coated with s i l i c a gel G. Chromatography was c a r r i e d out at room temperature i n Benzene/Pyridine/Acetic Acid 80:20:2 (167). - 8 5 -d e r i v a t i v e s o f a l l the other amino a c i d s found i n these p e p t i d e s do migrate (except the d e r i v a t i v e o f a r g i n i n e , which i s COOH-t e r m i n a l i n p e p t i d e TAlb) i t s i d e n t i t y can be deduced from t h i s f a c t , as w e l l as from the amino a c i d a n a l y s i s data, and the r a d i o -a c t i v i t y a n a l y s i s o f the Edman d e g r a d a t i o n (see below). T a b l e 5 shows the r e s u l t s o f the r a d i o a c t i v i t y a n a l y s i s of the d e r i v a t i v e s o b t a i n e d by Edman d e g r a d a t i o n o f T A l a and T A l b . For TAla', a s i g n i f i c a n t amount of r a d i o a c t i v i t y was removed from the p e p t i d e o n l y i n the second Edman st e p , i . e . c o i n c i d e n t w i t h the removal of a c e t y l l y s y l r e s i d u e 5 . T h i s corresponds t o a s p e c i f i c a c t i v i t y o f approximately 1 9 , 8 7 5 cpm/umole f o r t h i s r e s i d u e . The r a d i o a c t i v i t y remaining i n the p e p t i d e a f t e r two Edman steps must be p r e s e n t i n a c e t y l l y s y l r e s i d u e 8 . T h i s corresponds t o approximately 7 1 , 7 0 0 cpm/umole. The r a d i o a c t i v i t y removed i n step one probably r e p r e s e n t s l i m i t e d s o l u b i l i t y of the p e p t i d e i n the e t h y l a c e t a t e phase. The N H 2 - t e r m i n a l r e s i d u e of the degraded p e p t i d e a f t e r two Edman c y c l e s was i d e n t i f i e d as g l y c i n e by the d a n s y l a t i o n procedure, which f u r t h e r confirms the i d e n t i t y o f TAla'. I n the case o f T A l b , f o u r Edman steps were performed. S i g -n i f i c a n t r a d i o a c t i v i t y was removed o n l y at the t h i r d s t e p , c o i n -c i d e n t w i t h the expected removal of a c e t y l l y s y l r e s i d u e 1 2 . I t s s p e c i f i c a c t i v i t y was 5 7 , 8 1 1 cpm/umole. A f t e r f o u r s t e p s , t h e r e remained a t o t a l of 6 , 1 4 2 cpm i n the p e p t i d e . T h i s must be p r e s e n t i n a c e t y l l y s y l r e s i d u e 1 6 , and r e p r e s e n t s approximately 6 8 , 2 4 4 -86-T a b l e 5 Edman Degradation of P e p t i d e s T A l a ' and TA l b P e p t i d e Edman Net cpm e x t r a c - Amino A c i d ymoles Net Step t e d i n t o e t h y l i d e n t i f i e d by counted cpm/ a c e t a t e phase D a n s y l a t i o n ymol'e T A l a ' 1 200 Gly -2 795 L y s a 0.04 19,875 - Gly Remaining i n aqueous phase a f t e r Step 2: 2,151 cpm ymoles counted: 0.03 Net cpm/ymole remaining i n aqueous phase: 71,700 TAlb 1 294 Leu 2 359 Gly 3 5,203 L y s a 0.09 t 57,811 4 291 Gly • - Gly Remaining i n aqueous phase a f t e r Step 4: 6,142 ymoles counted: 0.09 Net cpm/ymole remaining i n aqueous phase: 68,244 a T h i s d a n s y l d e r i v a t i v e ( oC - d a n s y l Lys) does not move from the o r i g i n i n the s o l v e n t system used, but i t s i d e n t i t y can be deduced, s i n c e a l l o t h e r r e s i d u e s i n the p e p t i d e are accounted f o r . cpm/umole. The N H 2 - t e r m i n a l of TAlb a f t e r removal o f f o u r amino a c i d s was i d e n t i f i e d as g l y c i n e , and amino a c i d a n a l y s i s of t h i s degraded p e p t i d e showed the presence of one r e s i d u e each of g l y c i n e , a l a n i n e , l y s i n e , and a r g i n i n e (Table 4, p. 80, T A l b ( - ) 4 ) . T h i s again confirms the i d e n t i t y o f T A l b . These s t u d i e s demonstrate t h a t the f o u r l y s y l r e s i d u e s i n the N H 2 - t e r m i n a l r e g i o n o f h i s t o n e IV are a c e t y l a t e d i n v i v o , i n d e v e l o p i n g t r o u t t e s t i s . L y s y l r e s i d u e 5 i s a c e t y l a t e d t o about one t h i r d the e x t e n t of the other t h r e e l y s y l r e s i d u e s a t p o s i t i o n s 8,12 and 16. T h i s r a t i o r e f l e c t s the e x t e n t of a c e t y l a t i o n o f the f o u r s i t e s at a p a r t i c u l a r moment i n time, and might be d i f -f e r e n t i f t 1''C]-acetate i n c o r p o r a t i o n were allowed t o take p l a c e f o r l o n g e r or s h o r t e r p e r i o d s . A summary of the s t r a t e g y used i s shown i n F i g . 20. I t had been assumed throughout t h i s work t h a t the N H 2 - t e r m i n a l sequence of t r o u t t e s t i s h i s t o n e IV i s e s s e n t i a l l y i d e n t i c a l t o t h a t of c a l f thymus h i s t o n e IV. The experiments d e s c r i b e d above show t h a t t h i s assumption was v a l i d . S i n c e the h i s t o n e IV of two organisms as d i s t a n t l y r e l a t e d as pea and c a l f d i f f e r e d o n l y by two r e s i d u e s i n the COOH-terminal p o r t i o n of the molecule (29), t h i s r e s u l t i s not s u r p r i s i n g . ( i i ) H i s t o n e I l b i : I t was shown i n S e c t i o n I t h a t t r o u t t e s t i s I l b i c o u l d be a c e t y l a t e d , and t h a t t h e r e was p r o b a b l y a s i n g l e major s i t e of a c e t y l a t i o n i n t h i s p r o t e i n . 'Since the amino-t e r m i n a l of h i s t o n e s I l b i and IV both b e g i n w i t h the sequence Ac-S e r - G l y - A r g , w i t h the s e r y l r e s i d u e being the s i t e o f p h o s p h o r y l a t i o n -88-Sites of In Vivo Acetylation in Trout Testis Histone IV. 14 , ? c , ? ? ? (Ac) (Ac) (Ac) (Ac) Me Ac-SER.QLY.ARO .GLY.LYS.GLY.GLY. LYS.GLY.LEU .GLY. LYS.GLY.GLY. ALA.LYS.ARG.HIS. ARG.LYS. I Chemical acetylation ol intact Histone IV I Ac Ac V Ac Ac j AC-SER.OLY.ARGIGLY.LYS.QLY.OLY.LYS.GLY.LEU.GLY.LYS.GLY.GLY.ALA.LYS.ARG HIS.ARG.LYS. Ta1 Trypsin Al Trypsin OLY. c Ac I Ac .LYS.QLY.OLY:LYS.QLY LEU.GLY.LYS QLY LEU.G .LYS.GLY.GLY.ALA.LYS.ARG .L$S. Tala Thermolysin Ac Ac I Ac Ac QLY.LYS.QLY.OLY.LYS.GLY LEU.GLY.LYS.GLY.GLY. ALA.LYS. ARQ Talb LYS 5 Step2m19,87Scpm. LYS 12 Step 3a57,S11cpm. LYS 8 Lett In aqueous m71,TOO cpm. LYS 16 Left in aqueous a 68,244 cpm. FIG. 20. Summary of the s t r a t e g y used i n e l u c i d a t i n g the s i t e s of a c e t y l a t i o n i n h i s t o n e IV. Histone IV, l a b e l e d i n c e l l sus-p e n s i o n with [ x ^ C ] - a c e t a t e , was p u r i f i e d and c h e m i c a l l y a c e t y l a t e d w i t h n o n - r a d i o a c t i v e a c e t i c anhydride. The p r o t e i n was then d i -gested w i t h t r y p s i n , and the p u r i f i e d t r y p t i c p e p t i d e was c l e a v e d w i t h t h e r m o l y s i n . The t h e r m o l y s i n p e p t i d e s were then s u b j e c t e d t o Edman de g r a d a t i o n . The r a d i o a c t i v i t y d e t e c t e d a t v a r i o u s stages o f the d e g r a d a t i o n i s g i v e n , i n cpm/ymole. See a l s o T a b l e 5, p. 86. -89-i n both molecules (178), i t was of i n t e r e s t t o see whether the major s i t e of a c e t y l a t i o n i n I l b i was a l s o i n the amino-terminal r e g i o n . A t r y p t i c d i g e s t o f p u r i f i e d [x hC]-acetate l a b e l e d I l b i r e v e a l e d the presence of a s i n g l e major r a d i o a c t i v e p e p t i d e upon Sephadex G-25 chromatography or h i g h v o l t a g e e l e c t r o p h o r e s i s at pH 6.5 ( F i g . 21 and 2 2 ( a ) ) . A n a l y s i s of the p e p t i d e a f t e r f u r t h e r pur-'i i f i c a t i o n by e l e c t r o p h o r e s i s at pH 3.6 and paper chromatography (see " M a t e r i a l s and Methods") gave the composition shown i n T a b l e 6 ( I l b i A ) . The composition f i t s t h a t expected f o r a t r y p t i c p e p t i d e c o n s i s t i n g of r e s i d u e s 4-9 of the t r o u t t e s t i s I l b i sequence as determined by B a i l e y and Dixon (36)/ w i t h an e - a c e t y l group at l y s y l r e s i d u e 5 ( F i g . 22). In a d d i t i o n , NH2-terminal d e t e r m i n a t i o n on t h i s p e p t i d e by the d a n s y l method r e v e a l e d the presence of g l y c i n e , as expected. However, i n s u f f i c i e n t m a t e r i a l was a v a i l a b l e f o r f u r t h e r sequence work, and we t h e r e f o r e r e s o r t e d t o the s t r a t e g y used s u c c e s s f u l l y on h i s t o n e IV, i . e . chemical a c e t y l a t i o n of i n v i v o l a b e l e d t 1 1 1 C ] - a c e t a t e I l b i , f o l l o w e d by t r y p t i c d i g e s t i o n . As F i g . 23 shows, chemical a c e t y l a t i o n of both l y s y l r e s i d u e 9, and of any remaining unmodified l y s i n e at p o s i t i o n 5 should a l l o w t r y p t i c cleavage o n l y a t a r g i n y l r e s i d u e s 3 and 11, thus prod u c i n g a p e p t i d e c o n t a i n i n g r e s i d u e s 4-11 of the I l b i m olecule, i n good y i e l d . F i g u r e 22(b) shows the autoradiogram of a pH 6.5 h i g h v o l t a g e e l e c t r o p h o r e t i c s e p a r a t i o n of a t r y p t i c h y d r o l y s a t e o b t a i n e d from I l b i a f t e r l a b e l i n g i n v i v o w i t h [ ^ C ] -a c e t a t e , and chemical m o d i f i c a t i o n i n v i t r o w i t h n o n - r a d i o a c t i v e -90-4 0 0 £ 3 0 0 C L <D 2 0 0 D < i U 1 0 0 Void Vol. 1 0 2 0 3 0 4 0 5 0 Fraction No., 2-4ml. 6 0 7 0 FIG. 21. P u r i f i c a t i o n of a t r y p t i c p e p t i d e of h i s t o n e I l b i l a b e l e d w i t h [ : **C]-acetate. A t o t a l o f 43 mg of h i s t o n e l i b ! c o n t a i n i n g 48,000 cpm of [* **C]-acetate was d i g e s t e d w i t h 0.5 mg of c r y s t a l l i n e p o r c i n e t r y p s i n (Novo I n d u s t r i , Copenhagen) f o r 2% hours at 40°, i n 0.1 M N H i»HC0 3-NaOH, pH 8.3. The mixture was l y o p h i l i z e d , r e d i s s o l v e d i n 2 ml of 0.2 M a c e t i c a c i d , c l a r -i f i e d by c e n t r i f u g a t i o n , and a p p l i e d t o a 1.3 x 137 cm column of Sephadex G-25 ( f i n e ) e q u i l i b r a t e d w i t h 0.2 M a c e t i c a c i d . R a d i o a c t i v i t y was determined on a l i q u o t s of the column f r a c -t i o n s d i s s o l v e d i n Bray's s c i n t i l l a t i o n f l u i d . J*° e-DNP LYS a • (~W a b ORIGIN j i pH 6.5 | FIG. 22a, Autoradiogram - High v o l t a g e e l e c t r o p h o r e s i s o f the t 1 ^ C ] - a c e t a t e l a b e l e d t r y p t i c p e p t i d e from h i s t o n e I l b i . A sample of [x - a c e t a t e l a b e l e d I l b i from the column shown i n F i g . 21, p. 90, was d i g e s t e d w i t h t r y p s i n and an a l i q u o t was a p p l i e d t o a sheet o f Whatman 3MM paper f o r h i g h v o l t a g e e l e c -t r o p h o r e s i s a t pH 6.5. A f t e r e l e c t r o p h o r e s i s the paper was d r i e d , and the r a d i o a c t i v e p e p t i d e d e t e c t e d by autoradiography, b, t r y p t i c p e p t i d e o b t a i n e d from h i s t o n e I l b i a f t e r chemical a c e t y l a t i o n of the i n v i v o , [l **C] - a c e t a t e l a b e l e d p r o t e i n . Approx i m a t e l y 33 mg of I l b i c o n t a i n i n g 300,000 cpm I1 **C]-acetate was c h e m i c a l l y a c e t y l a t e d w i t h a c e t i c anhydride. The d i a l y z e d and l y o p h i l i z e d p r o t e i n was r e d i s s o l v e d i n 3 ml of 0.2 M NH1 .HCO3 pH 7.9, and d i g e s t e d w i t h 0.5 mg o f c r y s t a l l i n e p o r c i n e t r y p s i n a t 40° f o r 4 hours. The d i g e s t mixture was then s u b j e c t e d t o h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 6.5. -92-T a b l e 6 Amino a c i d compositions of h i s t o n e I l b i p e p t i d e s l a b e l e d w i t h [* 1 1C]-acetate I l b i A I l b i B Amino a c i d umoles r a t i o s umoles r a t i o s L y s i n e 0.021 1.90(2) 0.043 2.15(2) A r g i n i n e 0.020 1.00(1) G l y c i n e 0.034 3.09(3) 0.053 2.65(3) A l a n i n e 0.021 1.05(1) Threonine 0.010 0.91(1) 0.021 1.05(1) \ -93-Ac Ac Ac Ac IV Ac-Ser-Gly-Arg-Gly-Lys- -Gly-Gly-Lys-Gly-Leu-Gly-Lys-Gly-Gly-Ala-Lys-Ac lib Ac-Ser-Gly-Arg-Gly-Lys-Thr-Gly-Gly-Lys-Ala-Arg-Ala-Lys -Ala-Lys-Thr-Arg-1 1 s -nin- 10 15 5 -Gln-llbjA llbjB FIG.. 23. Comparison of the N H 2 - t e r m i n a l r e g i o n s of h i s t o n e I I b x from t r o u t t e s t i s , and h i s t o n e IV from c a l f thymus (26). I n c a l f thymus I l b j , the t h r e o n y l r e s i d u e at p o s i t i o n 6 i s r e p l a c e d by a g l u t a m i n y l r e s i d u e (35). The l y s y l r e s i d u e found t o be a c e t y l a t e d i n t r o u t t e s t i s I l b i i s i n d i c a t e d ( p o s i t i o n 5). Pe p t i d e I l b i A was o b t a i n e d from a t r y p t i c d i g e s t of I l b i : l a b e l e d i n v i v o w i t h 1 ^ C - a c e t a t e ; I l b i B was o b t a i n e d from i n v i v o l a b e l e d I l b i by chemical a c e t y l a t i o n w i t h a c e t i c anhydride and t r y p t i c c l e a v a g e . -94-a c e t i c anhydride. A s i n g l e major r a d i o a c t i v e p e p t i d e i s p r e s e n t , w i t h a m o b i l i t y s l i g h t l y l e s s than t h a t of I l b i A. A f t e r f u r t h e r p u r i f i c a t i o n by h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 3.6 and by paper chromatography 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 . 24), t h i s p e p t i d e gave the a n a l y s i s shown i n Table 6 ( I l b i B ) . I t s composition i s t h a t expected f o r r e s i d u e s 4-11 of t r o u t t e s t i s I l b i , and sequencing of the f i r s t f o u r r e s i d u e s by the dansyl-Edman procedure f u r t h e r confirmed the i d e n t i t y o f the p e p t i d e ( F i g . 25). Edman de g r a d a t i o n of the p e p t i d e r e s u l t e d i n l o s s o f the r a d i o a c t i v i t y i n the second c y c l e , c o n c u r r e n t l y w i t h the removal of l y s y l r e s i d u e 5 ( F i g . 25); thus l y s y l r e s i d u e 5 i s the major s i t e of a c e t y l a t i o n i n t r o u t t e s t i s h i s t o n e I l b i . F i g u r e 23, p. 93, shows the l o c a t i o n o f p e p t i d e s l i b ! A and I l b i B . i n r e l a t i o n t o the r e s t o f the N H 2 - t e r m i n a l sequence of t r o u t t e s t i s l i b , . During the p u r i f i c a t i o n of p e p t i d e I l b i B by paper chromatography, another, very weakly l a b e l e d r a d i o a c t i v e p e p t i d e was d e t e c t e d ( i n d i c a t e d by the dashes i n F i g . 24). A n a l y s i s suggested i t might a l s o o r i g i n a t e from the N H 2 - t e r m i n a l r e g i o n of I l b i , but complete p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of t h i s p e p t i d e was not achieved. Thus i t i s p o s s i b l e t h a t o t h e r l y s y l r e s i d u e s i n the molecule can accept a c e t y l groups, and t h i s p e p t i d e may a r i s e from the minor a c e t y l a t e d s p e c i e s l a b e l e d A 2 i n F i g . 14, p. 69. ( i i i ) H i s t o n e I I I : In r e c e n t y e a r s , the f i e l d of p r o t e i n sequencing has undergone a t e c h n i c a l r e v o l u t i o n , due t o the advent ORIGIN f t FIG. 24. Paper chromatography of [* **C]-acetate l a b e l e d p e p t i d e obtained by t r y p t i c h y d r o l y s i s of i n v i v o l a b e l e d , c h e m i c a l l y a c e t y l a t e d I l b i . The p e p t i d e was i n i t i a l l y p u r i f i e d by h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 3.6. Chromatography was c a r r i e d out i n b u t a n o l / a c e t i c a c i d / w a t e r / p y r i d i n e , 15:3:12:10 by volume, f o r 14 h r s . -96-RESIDUE NUMBER FIG. 25. Edman d e g r a d a t i o n o f the [*^C]-acetate t r y p t i c pep-t i d e from i n v i v o l a b e l e d , c h e m i c a l l y a c e t y l a t e d h i s t o n e I l b i . The p e p t i d e (0.300 umole) was d i s s o l v e d i n 300 u l of 5% N - e t h y l raorpholine pH 9.3, t o which was then added 300 u l of 5% ph e n y l -i s o t h i o c y a n a t e s o l u t i o n i n r e d i s t i l l e d p y r i d i n e . Edman degrada-t i o n was then c a r r i e d out f o r 6 c y c l e s , as d e s c r i b e d i n " M a t e r i a l s and Methods". A f t e r each c y c l e , the n - b u t y l a c e t a t e phases con-t a i n i n g the t h i a z o l i n o n e d e r i v a t i v e of the removed amino a c i d were pooled, d r i e d i n vacuo, and counted i n 10 ml of Bray's s c i n t i l -l a t i o n f l u i d . In a separate experiment (not shown), a l i q u o t s o f the p e p t i d e were taken a f t e r each of f o u r Edman c y c l e s , and NH 2-t e r m i n a l d e t e r m i n a t i o n s were c a r r i e d out by the d a n s y l method of Gray (150). Residues marked w i t h an a s t e r i s k were i d e n t i f i e d i n t h i s manner. The p o s i t i o n s of the oth e r f o u r r e s i d u e s are d e r i v e d from the sequence o f B a i l e y and Dixon (36) and i n the above ex-periment from the amino a c i d a n a l y s i s o f the r e s i d u a l p e p t i d e a f t e r f o u r Edman c y c l e s , and the known enzymatic s p e c i f i c i t y o f t r y p s i n . -97-of the automatic p r o t e i n sequencer, developed by Edman and Begg (169) . T h i s instrument permits one t o c a r r y out a s e r i e s of Edman degradations s e q u e n t i a l l y from the amino-terminal end o f a p r o t e i n or p e p t i d e a u t o m a t i c a l l y , and under c a r e f u l l y c o n t r o l l e d c o n d i t i o n s . In some cases, such as w i t h sperm whale myoglobin, the sequence of as many as 60 amino a c i d r e s i d u e s has been determined i n t h i s way (169). In h i s t o n e s I l b i and IV, the amino-terminal r e s i d u e i s N-a c e t y l s e r i n e , so t h a t automatic s e q u e n t i a l Edman d e g r a d a t i o n i s i m p o s s i b l e , but i n the case o f h i s t o n e s I I I and I I b 2 , the amino t e r m i n i c o n s i s t o f unblocked a l a n y l and p r o l y l r e s i d u e s , r e s p e c -t i v e l y (178) . During the course o f t h e i r sequence work on c a l f thymus h i s t o n e I I I , DeLange e t a l . d e t e c t e d the presence o f e - N - a c e t y l -l y s i n e a t p o s i t i o n s 14 and 23 (33) (see F i g . 2, p. 7). The sequence of the f i r s t 26 r e s i d u e s o f c a l f thymus h i s t o n e I I I was a l s o subsequently o b t a i n e d by Olson e t a l . (181), u s i n g an automatic p r o t e i n sequencer; i n t h i s study, however, no a c e t y l a t e d l y s y l r e s i d u e s were d e t e c t e d . We were i n t e r e s t e d i n deter m i n i n g the s i t e s of a c e t y l a t i o n i n t r o u t t e s t i s h i s t o n e I I I . A n a l y s i s o f p u r i f i e d t r o u t t e s t i s I I I on urea-aluminum l a c t a t e s t a r c h g e l s suggested the presence o f at l e a s t two major m o d i f i c a t i o n s i t e s ( F i g . 14, p. 69) i n t h i s h i s t o n e . i f the a c e t y l a t e d l y s y l r e s i d u e s i n t r o u t t e s t i s h i s t o n e I I I were p r e s e n t i n the amino-terminal end o f the molecule, i t -98-seemed p o s s i b l e t h a t they might be l o c a t e d by the automatic s e q u e n t i a l Edman de g r a d a t i o n of h i s t o n e I I I l a b e l e d i n v i v o w i t h [* **C]-acetate. The p o s i t i o n of the a c e t y l a t e d r e s i d u e s c o u l d be d e t e c t e d by r a d i o -a c t i v i t y a n a l y s i s of the amino a c i d d e r i v a t i v e s o b t a i n e d from the sequencer. T h i s technique proved to be f e a s i b l e , and i n the course of t h i s work, the sequence of the f i r s t 25 r e s i d u e s of t r o u t t e s t i s h i s t o n e I I I was a l s o determined. The sequence of the f i r s t 25 r e s i d u e s of t r o u t t e s t i s h i s t o n e I I I i s shown i n F i g u r e 26, and the y i e l d of the amino a c i d at each p o s i t i o n , i n T a b l e 7. The sequence seems to be e n t i r e l y i d e n t i c a l to the sequence r e p o r t e d f o r c a l f thymus h i s t o n e I I I by DeLange et a l . (33). The s e r y l r e s i d u e a t p o s i t i o n 10 was not d e t e c t e d , s i n c e i t i s d e s t r o y e d d u r i n g h y d r o l y s i s o f i t s p h e n y l t h i o h y d a n t o i n (PTH) d e r i v a t i v e , and amides were not determined; i n c a l f thymus I I I , r e s i d u e 5 i s glutamine, as i s a l s o r e s i d u e 19 (33). An average r e p e t i t i v e y i e l d of 93.7% f o r each d e g r a d a t i v e step was c a l c u l a t e d from the s l o p e of a s e m i - l o g a r i t h m i c p l o t of the y i e l d of a l a n y l r e s i d u e s v e r s u s the number of d e g r a d a t i o n s ( F i g . 27). The r e p e t i t i v e y i e l d , Y, i s g i v e n by the e q u a t i o n 1 (B) l o g Y = r = — ^ r — l o g -TTTT- where N_ and N w are N f i-N A (A) A B the numbers o f degradations r e q u i r e d to remove r e s i d u e s A and B, r e s p e c t i v e l y , and (A) and (B) are the r e l a t i v e y i e l d s of the two r e s i d u e s . The s l o p e of the l i n e i n F i g . 27 y i e l d s the v a l u e of -99-Ac IK Ala-Arg-Thf-Lys-Glx-Thr-Ala-Arg-Lys^Ser^hir-Gly-Gly-Lys-Ala-Pro -Arg-Lys-Glx-Leu -Ala-Thr-Lys-Ala -Ala 14 18 23 FIG. 26. Sequence of the f i r s t 25 residues, of t r o u t t e s t i s h i s t o n e I I I . The h i s t o n e (0.7-1.2 umole) was s u b j e c t e d t o a s e r i e s o f automated Edman degradations i n a Beckman automatic p r o t e i n sequencer, u s i n g a program s i m i l a r t o t h a t d e s c r i b e d by Edman and Begg (169). Amino a c i d s were i d e n t i f i e d on a Beckman 120C amino a c i d a n a l y z e r , a f t e r a c i d i c or b a s i c h y d r o l -y s i s of t h e i r p h e n y l t h i o h y d a n t o i n d e r i v a t i v e s , as d e s c r i b e d i n " M a t e r i a l s and Methods". The l y s y l r e s i d u e s found t o be p a r t i a l l y a c e t y l a t e d are i n d i c a t e d . -100-Table 7 Sequential Degradation of Trout Te s t i s Histone I I I ^ Residue I d e n t i f i c a t i o n Free Amino Acid Re- % Y i e l d Number covered, nanomoles (t 1 Ala 430 100.0 2 Arg 51 11.9 3 Thr (as Gly) 88 20.5 4 Lys 179 41.6 5 Glx 148 34.4 6 Thr (as Gly) 290 10.4 7 Ala 67.0 8 Arg 11 2.6 9 Lys 56 13.0 10 (Ser) — — 11 Thr (as Gly) 24 5.6 12 Gly 83 19.2 13 Gly 115 26.8 14 Lys 65 15.2 15 ' Ala 170 40.0 16 Pro 33 7.6 17 Arg 12 2.8 18 Lys 40 9.4 19 Glx 58 13.5 20 Leu 70 16.3 21 Ala 100 23.0 22 Thr (as Gly) 19 4.4 23 Lys 29 6.7 24 Ala 91 21.0 25 Ala 98 23.0 ^ The amount of protein used was approximately 0.5 umole. -101-4 8 12 16 20 24 NO. OF DEGRADATIONS FIG. 27. Recovery of a l a n i n e a t v a r i o u s steps of the automated d e g r a d a t i o n of h i s t o n e I I I . The l o g a r i t h m of the r e l a t i v e y i e l d o f a l a n i n e a t d i f f e r e n t steps i s p l o t t e d a g a i n s t the number o f degradations performed. The N H 2 - t e r m i n a l a l a n i n e r e p r e s e n t s 100%. P o i n t s which f a l l w e l l o f f the l i n e ( • ) r e p r e s e n t background l e v e l s o f a l a n i n e , whereas those on the l i n e ( © ) i n d i c a t e the presence of a l a n i n e a t the c o r r e s p o n d i n g p o s i t i o n i n the sequence. -102-The y i e l d s of the residues, as l i s t e d i n Table 7, vary a great deal depending on the nature of the amino acid d e r i v a t i v e . The o v e r a l l recovery of a given amino acid depends on the s t a b i l i t y of i t s anilinothiazolinone d e r i v a t i v e , the e f f i c i e n c y of conversion of t h i s derivative to the corresponding phenylthiohydantoin, the s o l -u b i l i t y of the phenylthiohydantoin i n the ethyl acetate which i s used for extracting i t , and the e f f i c i e n c y of conversion of the PTH derivative to the free amino acid by hydrolysis. Hence, the y i e l d s of threonine (recovered as glycine a f t e r basic hydrolysis (172)) are low because of the i n s t a b i l i t y of i t s PTH and t h i a -zolinone derivatives (182), and the y i e l d s of arginine are low be-cause of the lower s o l u b i l i t y of i t s PTH derivative i n ethyl acetate. However, the y i e l d of the amino acid assigned to a given p o s i t i o n was always well above i t s background l e v e l , so that i t s i d e n t i f i c a t i o n was unambiguous within the sequences presented. (This i s evident from the data presented i n F i g . 27.) Figures 28a and 28b show plots of r a d i o a c t i v i t y recovered versus the number of degradations performed, using both the se-quencer program of Edman and Begg (169), (Fig. 28a), and the program described by N i a l l et a l . (170), which used v o l a t i l e reagents (Fig. 28b) (see "Materials and Methods"). For each f i g u r e , background r a d i o a c t i v i t y was subtracted, and a l l values above t h i s background were then corrected to allow for a r e p e t i t i v e y i e l d of 93.7% for each degradation, using the equation given above. Two prominent peaks of r a d i o a c t i v i t y are seen, corresponding -103-i _ 16 12 8 Uj 4 Q CO %24 \ § 2 0 Q. O ,16 a 2 7 2 LU i i i i i i i i a zzr • \ \ I M ' 1 ' 1 1 i 1 1 1 — i 1 1 — ^ t -\ \ _i i_iJ i i t-4 8 12 16 20 24 28 NO. OF DEGRADATIONS FIG, 28. (a), Automated degradation of [* **C]-acetate labeled trout t e s t i s histone I I I , using a program s i m i l a r to that of Edman and Begg (169). Approximately 12 mg of c a r r i e r histone III was added to 5 mg of t 1 ^ C]-acetate labeled material con-tai n i n g 190,000 cpm, and the sample was subjected to a series of automated Edman degradations i n the Beckman 890 protein sequencer. (b) , automated degradation of t 1 ^ C ^ a cetate labeled histone I I I , using v o l a t i l e reagents (170). The sample contained 9 mg of protein, and 168,000 cpm of [**C]-acetate. Radioactivity recovered at each degradative step has . been corrected on the basis of a r e p e t i t i v e y i e l d of 93.7%. -104-to l y s y l residues 14 and 23. In addition, there i s l a b e l associated with l y s y l residues 9 and 18 i n both experiments, but none with l y s y l residue 4. Thus, besides the two s i t e s of acetylation so far observed i n c a l f thymus histone I I I , two other l y s y l residues i n the amino-terminal region seem to be acetylated, to a much lesser extent. In c a l f thymus histone I I I , l y s y l residue 9 i s p a r t i a l l y methylated (33). I t i s not clear at the moment whether a given l y s y l residue can be p a r t i a l l y modified both by methylation and acetylat i o n , or whether our observation of a low degree of acetylation at p o s i t i o n 9 i n trout t e s t i s histone III represents a species difference. (iv) Histone I I b 2 : The amino acid sequence of c a l f thymus histone I I b 2 has been deduced by Iwai et a l . (31), but no acetyla-t i o n of t h i s histone has been reported for c a l f thymus (30) . I t was shown i n Section I, however, that trout t e s t i s histone I I b 2 i s acetylated, and that at l e a s t two such modified components can be detected on starch gels. We therefore undertook a study of the amino-terminal end of t 1 **C]-acetate labeled I I b 2 using the Beckman protein sequencer, as described above for histone I I I . Figure 29 shows the sequence of the f i r s t 22 residues of trout t e s t i s histone I I b 2 , as determined by automated degradation, and the y i e l d of each residue i s l i s t e d i n Table 8. The r e p e t i t i v e y i e l d , calculated from the y i e l d of alanyl residues (Fig. 30), was 91%. The lower y i e l d i n t h i s case i s probably due to the presence of four p r o l y l residues i n the sequence, since i t i s -105-* c Ac Ac Hb2 Pro-Glx-Pro-Ala-Lys-(Ser)-Ala-Pro-Lys-Lys-Gly-(SerhLys 5 TO 13 Ac ~ Lys-Ala-VaLThr-Lys$fo>Ala-Gly-Lys-18 FIG. 29. Sequence of the f i r s t 22 r e s i d u e s o f t r o u t t e s t i s h i s t o n e I I b 2 . The l y s y l r e s i d u e s found t o be p a r t i a l l y a c e t y l -ated are i n d i c a t e d . The amount o f p r o t e i n used was 0.50 umole. -106-Tabl e 8 S e q u e n t i a l Degradation of T r o u t T e s t i s Histone I I b 2 Residue I d e n t i f i c a t i o n Free Amino A c i d Re- % Y i e l d Number covered, nanomoles 1 Pro 195 51.0 2 Glx 382 100.0 3 Pro 244 63.9 4 A l a 346 90.6 5 Lys 189 49. 5 6 (Ser) — — 7 A l a 285 74.6 8 Pro 78 20.4 9 Lys 117 30.6 10 Lys 118 30.9 11 Gly 140 36.6 12 (Ser) — — 13 Lys 87 22.9 14 Lys 90 23.5 15 A l a 123 32.2 16 V a l 124 32.5 17 Thr (as Gly) 16 4.2 18 Lys 50 13.1 19 (Ser or Thr) — — 20 A l a 77 20.2 21 Gly 47 12.4 22 Lys 39 10.3 The amount of p r o t e i n used was 0.50 umole. C a l c u l a t e d r e l a t i v e t o the gl u t a m i c a c i d r e c o v e r e d at step 2 r a t h e r than t o the p r o l i n e a t step 1; t h i s a voids the appear-ance o f y i e l d s i n excess of 100%, s i n c e p r o l i n e i s r o u t i n e l y r e c o v e r e d i n lower y i e l d than, f o r i n s t a n c e , glutamic a c i d or a l a n i n e . -107-FIG. 30. Recovery of a l a n i n e a t v a r i o u s s t e p s o f the auto-mated de g r a d a t i o n of h i s t o n e I I b 2 from t r o u t t e s t i s . The l o g -a r i t h m of the r e l a t i v e y i e l d o f a l a n i n e a t d i f f e r e n t steps i s p l o t t e d a g a i n s t the number of degradations performed. The a l a n y l r e s i d u e a t p o s i t i o n 4 r e p r e s e n t s 100%. P o i n t s which f a l l o f f the l i n e ( E ) r e p r e s e n t background l e v e l s of a l a n i n e , whereas those on the l i n e ( © ) i n d i c a t e the presence of a l a n i n e a t the c o r r e s p o n d i n g p o s i t i o n i n the sequence. -108-known t h a t the c o u p l i n g e f f i c i e n c y i n the Edman r e a c t i o n i s lower f o r t h i s r e s i d u e than f o r o t h e r amino a c i d r e s i d u e s . In the experiments on h i s t o n e I I b 2 , we attempted t o i n c r e a s e the e x t e n t of c o u p l i n g a t the amino-terminal p r o l y l r e s i d u e by r e p e a t i n g the c o u p l i n g step a second time i n the f i r s t d e g r a d a t i v e c y c l e . T h i s i s p r obably a u s e f u l p r e c a u t i o n to take i n the case of other very b a s i c p r o t e i n s which are h i g h i n l y s i n e c o n t e n t , s i n c e the e-amino groups of l y s y l r e s i d u e s compete wit h the amino t e r m i n a l of the p r o t e i n f o r the p h e n y l i s o t h i o c y a n a t e reagent, u n t i l they become f u l l y b locked by i t . Thus, i n the case of h i s t o n e I I b 2 , t h e r e are approximately 20 e-amino groups (31) which can r e a c t w i t h the Edman reagent i n the f i r s t c o u p l i n g s t e p , and t h i s c o m p e t i t i o n f o r reagent c o u l d decrease the y i e l d of the f i r s t r e s i d u e because o f incomplete c o u p l i n g i f the reagent i s not i n s u f f i c i e n t l y g r e a t excess; t h i s i s p a r t i c u l a r l y l i k e l y t o occur when r e l a t i v e l y l a r g e amounts of p r o t e i n are used f o r sequencing. The amino-terminal sequence of h i s t o n e I I b 2 from t r o u t t e s t i s d i f f e r s from t h a t r e p o r t e d f o r c a l f thymus (31,32). The two sequences are compared.in F i g . 31, and have been arranged so as t o show maximum homology. In c a l f thymus I I b 2 , the sequence of r e s i d u e s 7-10 i s A l a - P r o - A l a - P r o , whereas i n t r o u t t e s t i s I I b 2 one o f the A l a - P r o p a i r s has been d e l e t e d . The sequences are otherwise a p p a r e n t l y i i d e n t i c a l up t o r e s i d u e 20 o f c a l f thymus I I b 2 . Residue 19 i n t r o u t t e s t i s I I b 2 i s not a l a n i n e , as expected from the c a l f thymus sequence of I war e t a l . (31) ( a l l o w i n g f o r the d e l e t i o n i n the l a t t e r sequence), but i n s t e a d seems to be s e r i n e or t h r e o n i n e . ^ 1 0 20 (a) P r o - G l n - P r o - A l a - L y s - S e r - A l a - p r o - A l a - P r o - L y s - L y s - G l y - S e r - L y s - L y s - A l a - V a l - T h r - L y s - A l a - G l n -(b) Pro-Glx-Pro-Ala-Lys-(Ser}-Ala-Pro ^Lys-Lys-Gly-^er)-Lys-Lys-Ala-Val-Thr-Lys-^^j-Ala-24 (a) Lys-Lys (b) Gly-Lys FIG. 31. Comparison of the amino-terminal sequence of c a l f thymus h i s t o n e I I b 2 (a) (30,31) w i t h t h a t o f t r o u t t e s t i s I I b 2 ( b ) . The sequences have been arranged so as t o show maximum homology. H 1 o VO -110-8 82 16 20 24 . OF DEGRADATIONS FIG. 32. Automated d e g r a d a t i o n of [* **C]-acetate l a b e l e d t r o u t t e s t i s h i s t o n e I I b 2 , u s i n g the program of N i a l l e t a l . (170). The sample c o n t a i n e d approximately 0.25 umole of p r o t e i n and 180,000 cpm [l " c ] - a c e t a t e . R a d i o a c t i v i t y r e c o v e r e d a t each step has _ been c o r r e c t e d on the b a s i s of a r e p e t i t i v e y i e l d o f 91%. -111-The sequence f o l l o w i n g t h i s r e s i d u e i s then A l a - G l y - L y s i n t r o u t t e s t i s , and Gln-Lys-Lys- i n c a l f thymus I I b 2 (31). r F i g u r e 32 shows the p l o t of [x **C]-acetate r e l e a s e d d u r i n g the automated d e g r a d a t i o n of i n v i v o l a b e l e d h i s t o n e I I b 2 . The r a d i o -a c t i v i t y p r e s e n t i n the f i r s t t h r e e r e s i d u e s i s probably due t o washout of f l a k e s of p r o t e i n from the r e a c t i o n cup, s i n c e i n s o l -u b l e m a t e r i a l was p r e s e n t i n these f r a c t i o n s . There are two major peaks of r a d i o a c t i v i t y c o r r e s p o n d i n g to l y s y l r e s i d u e s 10 and 13 of the t r o u t t e s t i s sequence. There i s a l s o a l e s s e r amount of l a b e l p r e s e n t at l y s y l r e s i d u e s 5 and 18. I t i s i n t e r e s t i n g t o note t h a t w h i l e l y s y l r e s i d u e 10 i s exten-s i v e l y l a b e l e d , l y s y l r e s i d u e 9 c o n t a i n s no s i g n i f i c a n t amount of r a d i o a c t i v i t y . I t i s apparent from these r e s u l t s t h a t t h e r e are f o u r l y s y l r e s i d u e s i n the amino-terminal r e g i o n of t r o u t t e s t i s h i s t o n e I I b 2 which can be a c e t y l a t e d i n v i v o , a s i t u a t i o n analogous t o t h a t found f o r t r o u t t e s t i s h i s t o n e s I I I and IV. -112-I I I . A c e t y l a t i o n of H i s t o n e s i n D i f f e r e n t C e l l Types from Developing  T r o u t T e s t i s . As was mentioned i n the I n t r o d u c t i o n , d e v e l o p i n g t r o u t t e s t i s c o n t a i n s a number of d i f f e r e n t c e l l t y p e s : spermatogonial c e l l s , primary and secondary spermatocytes, and s e v e r a l c l a s s e s of spermatids. A t the spermatid stage, the h i s t o n e s g r a d u a l l y d i s -appear from the chromatin, and are r e p l a c e d by protamine. We have shown t h a t f o u r of the f i v e major h i s t o n e s are e x t e n s i v e l y and s p e c i f i c a l l y a c e t y l a t e d i n d e v e l o p i n g t r o u t t e s t i s . Thus, i t was of i n t e r e s t t o see whether a c e t y l a t i o n of t r o u t t e s t i s h i s t o n e s was a s s o c i a t e d w i t h any p a r t i c u l a r c e l l t y p e. The s e p a r a t i o n of d i f f e r e n t c l a s s e s of t e s t i s c e l l s i n s m a l l amounts was made p o s s i b l e by the s e d i m e n t a t i o n technique of M i l l e r and P h i l l i p s (183). A c c o r d i n g to t h i s method, a suspension o f c e l l s i s l a y e r e d on top of a volume of i s o t o n i c b u f f e r i n a c y l i n d r i c a l s edimentation chamber, and allowed t o sediment a t u n i t g r a v i t y f o r s e v e r a l hours. The c e l l s sediment as narrow bands, the l a r g e r c e l l s sedimenting more r a p i d l y ( S t o k e 1 s Law). T h i s method i s p a r t i c u l a r l y w e l l s u i t e d t o the study o f spermato-g e n e s i s , s i n c e the t r a n s i t i o n s from spermatogonial c e l l s t o spermatocytes and spermatids are accompanied by l a r g e changes i n volume. T h i s technique has been a p p l i e d t o the study of spermatogenesis i n the mouse by Lara e t a l . (173). In t h i s l a b -o r a t o r y , Mr. A . J . L o u i e has c h a r a c t e r i z e d the v a r i o u s c e l l s from t r o u t t e s t i s which are s e p a r a b l e by t h i s method (107); they are r e f e r r e d to on the b a s i s of t h e i r sedimentation v e l o c i t y , i n -113-mm/hr. C e l l s sedimenting at 3.5 and 2.8 mm/hr are d i p l o i d , and probably c o n s i s t of primary spermatocytes, and A and B stem c e l l s , r e s p e c t i v e l y ; e a r l y spermatids c o n t a i n i n g h i s t o n e s sediment a t 1.5 mm/hr; l a t e spermatids, i n which the t r a n s i t i o n from h i s t o n e s t o protamines takes p l a c e , sediment at 1.0 mm/hr (107), and mature sperm at 0.6 mm/hr. Mr. Lo u i e has developed methods f o r e x t r a c t i n g the b a s i c p r o t e i n s from the s m a l l numbers of c e l l s o b t a i n e d from d i f f e r e n t r e g i o n s o f these g r a d i e n t s (107), and these p r o t e i n s can then be separated and v i s u a l i z e d by e l e c t r o p h o r e s i s on urea-aluminum l a c t a t e s t a r c h g e l s and s t a i n i n g by the s e n s i t i v e c o b a l t -Amido Black procedure o f Sung and Smithi e s (160). F i g u r e 33 shows the p r o f i l e a f t e r 5 hours sedi m e n t a t i o n of t e s t i s c e l l s o b t a i n e d from a f i s h 44 days a f t e r the s t a r t of i n j e c t i o n s w i t h p i t u i t a r y e x t r a c t . Numbers above the arrows r e f e r to the sedimen t a t i o n v e l o c i t y o f the c e l l s i n d i c a t e d , i n mm/hr. I t i s seen t h a t a l l c e l l types i n c o r p o r a t e [ l''C]-acetate i n t o n u c l e a r m a t e r i a l . However, not a l l of t h i s i n c o r p o r a t i o n rep-r e s e n t s a c e t y l a t i o n o f h i s t o n e s ; some of the l a b e l i s probably p r e s e n t i n the deoxyribose r e s i d u e s o f DNA, and i n the p u r i n e and p y r i m i d i n e bases, e t c . C e l l s from v a r i o u s p a r t s of the g r a d i e n t were t h e r e f o r e pooled as shown, and the ex t e n t of t 1''C]-acetate i n c o r p o r a t i o n i n t o the h i s t o n e s was measured by e x t r a c t i o n o f the a c i d - s o l u b l e n u c l e a r p r o t e i n s f o l l o w e d 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 and r a d i o a c t i v i t y a n a l y s i s , 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 u r e 34 shows the p r o f i l e s on s t a r c h g e l s of -114-T 1 ~T 1 I T r-C -1 FRACTION NO. (7A ML) FIG. 33. I n c o r p o r a t i o n of [l **C]-acetate i n t o d i f f e r e n t c e l l types o f t r o u t t e s t i s . A suspension of t r o u t t e s t i s c e l l s was prepared from a f i s h 44 days a f t e r the s t a r t o f i n j e c t i o n s w i t h salmon p i t u i t a r y e x t r a c t , and incubated w i t h sodium l - t ^ C ] -a c e t 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". The l a b e l e d c e l l s were then f r a c t i o n a t e d by v e l o c i t y s e d i m e n t a t i o n (5 hr) at u n i t g r a v i t y i n a 1-3% g r a d i e n t of bovine serum albumin (173). A f t e r c o l l e c t i o n o f the f r a c t i o n s from the g r a d i e n t , c e l l numbers were counted u s i n g a hemocytometer, and r a d i o a c t i v i t y i n c o r p o r a t e d i n t o the c e l l n u c l e i was determined on 1.0 ml a l i q u o t s of the f r a c t i o n s by c o l l e c t i n g the c e l l s on g l a s s f i b e r f i l t e r s and washing w i t h 5% TCA-0.025% sodium t u n g s t a t e (see " M a t e r i a l s and Methods"). The numbers over the arrows r e f e r t o the sedimentation v e l o c i t i e s o f those f r a c t i o n s i n mm/hr. Regions of the g r a d i e n t which were pool e d and analyzed f o r 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 h i s t o n e s (see F i g . 34) are i n d i c a t e d by l e t t e r s . - 1 1 5 -160 120 8 0 -5 4 0 Q. O m 2 gl20 4 J] (-) l / l J i i_ a J u C 5 Jl * • 8 0 4 0 fi l,llb.,(I^Ub2jV T PROTAMINES P-lib, J UN FRACTIONATED CELLS j i u In 7.0 9.0 11.0 13.0 7.0 9.0 11.0 14.0 DISTANCE FROM ORIGIN, CM. 18.0 FIG. 34. Acetylation of histones i n d i f f e r e n t t e s t i s c e l l types. Fractions from the gradient i l l u s t r a t e d i n Fig.33 were pooled as indicated, and the c e l l s c o l l e c t e d on glass f i b e r f i l t e r s . The histones were then extracted from the c e l l s on the f i l t e r s with 0.4 N HCl, fractionated on a urea-aluminum lactate starch g e l , and analyzed for r a d i o a c t i v i t y by s l i c i n g and counting the gel as described i n "Materials and Methods". The i d e n t i t y of the various histone bands i s indicated i n the p r o f i l e from the un-fractionated c e l l s . P-IIbi r e f e r s to the phosphorylated form of histone IXb|. -116-the h i s t o n e s from the areas of the g r a d i e n t o f F i g u r e 33, and from the whole c e l l suspension a p p l i e d t o the g r a d i e n t . I t i s seen t h a t i n a l l c a s e s , the h i s t o n e s are l a b e l e d w i t h [ l * C ] - a c e t a t e . The p a t t e r n of l a b e l i n g seems t o be g e n e r a l l y s i m i l a r i n each of the major c e l l types examined, although i n F i g . 34C, which r e p r e s e n t s the h i s t o n e s from the spermatid peak of F i g . 33, ther e appears t o be an i n c r e a s e o f a c e t a t e l a b e l i n the r e g i o n o f a c e t y l - h i s t o n e IV. I t cannot be r u l e d out, t h e r e f o r e , t h a t d i f f e r e n c e s i n the l a b e l i n g o f s p e c i f i c h i s t o n e s may occur i n d i f f e r e n t c e l l t ypes, s i n c e the e l e c t r o p h o r e s i s o f t o t a l h i s t o n e s on s t a r c h g e l s does not r e s u l t i n the complete s e p a r a t i o n of a l l h i s t o n e f r a c t i o n s from each o t h e r . The a c e t y l a t i o n of h i s t o n e s i n spermatids was examined f u r t h e r at a l a t e r stage of spermatogenesis, 71 days a f t e r the s t a r t of i n j e c t i o n s w i t h p i t u i t a r y e x t r a c t , and the c e l l p r o f i l e i s shown i n F i g u r e 35. In t h i s case, s e d i m e n t a t i o n was allowed t o occur f o r 10 hours, which permits a p a r t i a l s e p a r a t i o n o f e a r l y and l a t e spermatids (with sedimentation v e l o c i t i e s of 1.5 and 1.0 mm/hr, r e s p e c t i v e l y ) . Peaks of [x l*C]-acetate l a b e l can be seen c o i n c i d e n t w i t h both c e l l types on the g r a d i e n t , and f r a c t i o n s were pooled as shown, f o r r a d i o a c t i v i t y a n a l y s i s o f the h i s t o n e s . I t i s c l e a r from F i g u r e 36 t h a t [ l l f C ] - a c e t a t e l a b e l i s i n c o r p o r a t e d i n t o the h i s t o n e s o f both the 1.5 and 1.0 mm/hr c e l l s . The proc e s s of h i s t o n e replacement by protamine, which begins i n the 1.5 mm/hr c e l l s (107,184), i s w e l l underway i n these samples, as shown by the l a r g e amounts of protamine p r e s e n t (compare w i t h F i g . 34). Thus -117-FIG. 35. I n c o r p o r a t i o n of t 1 ^ C ] - a c e t a t e i n t o t r o u t t e s t i s spermatids. T e s t i s c e l l s o b t a i n e d from a f i s h 71 days a f t e r the s t a r t of twice-weekly i n j e c t i o n s of salmon p i t u i t a r y ex-t r a c t were in c u b a t e d w i t h sodium t 1''C]-acetate (330 uCi/ml, 90 min.) and f r a c t i o n a t e d by v e l o c i t y s e d i m e n t a t i o n f o r 10 hrs i n a 1-3% bovine serum albumin g r a d i e n t (173). C e l l s were counted and analyzed f o r r a d i o a c t i v i t y as d e s c r i b e d i n " M a t e r i a l s and Methods". L e t t e r s r e f e r t o the r e g i o n s o f the g r a d i e n t which were pool e d f o r the a n a l y s i s of t 1 ^ 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 (see F i g . 36). The numbers over the arrows r e f e r t o the sedimentation v e l o c i t i e s o f the i n d i c a t e d c e l l s , i n mm/hr. -118-9.0 11.0 13.0 16.0 18.0 20.0 22.0 DISTANCE FROM ORIGIN, CM. FIG. 36. A c e t y l a t i o n of h i s t o n e s i n t r o u t t e s t i s spermatids. F r a c t i o n s from the g r a d i e n t i l l u s t r a t e d i n F i g . 35 were p o o l e d as shown, and the c e l l s c o l l e c t e d on g l a s s f i b e r f i l t e r s . The amount o f ^C ] - a c e t a t e i n c o r p o r a t e d i n t o the h i s t o n e s of these c e l l s was then determined 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 and r a d i o a c t i v i t y a n a l y s i s as d e s c r i b e d i n " M a t e r i a l s and Methods". P - I I b i r e f e r s t o the phosphorylated form of h i s t o n e I l b i . A i -IV and A 2 - I V r e f e r t o mono-acetyl IV and d i - a c e t y l IV, r e s p e c -t i v e l y . -119-h i s t o n e a c e t y l a t i o n seems t o occur i n a l l c e l l types of t r o u t t e s t i s which possess these p r o t e i n s . In r e c e n t experiments (179), the p a t t e r n of l a b e l i n g o f h i s t o n e IV w i t h r a d i o a c t i v e amino a c i d s was s t u d i e d , and i t was found t h a t the l a b e l f i r s t appeared i n the a c e t y l a t e d components of the p r o t e i n . T h i s suggested t h a t h i s t o n e IV was being a c e t y l a t e d soon a f t e r s y n t h e s i s . In or d e r t o see i f h i s t o n e a c e t y l a t i o n and s y n t h e s i s o c c u r r e d t o g e t h e r i n d i f f e r e n t c e l l t y p e s , we examined, by v e l o c i t y s e d i m e n t a t i o n , c e l l suspensions p r e v i o u s l y l a b e l e d w i t h I1 * C ] - a c e t a t e and [ 3 H ] - l y s i n e . The r e s u l t s of such an experiment are shown i n Fi g u r e " 37. There i s I1 ''C]-acetate l a b e l i n a l l c e l l s , as d i s c u s s e d aboye; t h e r e i s a l s o [ 3 H ] - l y s i n e i n both the 2.8-5.0 mm/hr r e g i o n and i n the spermatids (1.5 mm/hr). However, the p r o p o r t i o n o f [ 3 H ] - l y s i n e with, r e s p e c t t o [x **C]-acetate i s lower i n the l a t t e r c e l l s . When o n l y the l a b e l p r e s e n t s p e c i f i c a l l y i n h i s t o n e s i s examined, the d i f f e r e n c e i s more s t r i k i n g . F i g u r e 38 shows the s t a r c h g e l p r o f i l e s o f h i s t o n e s from c e l l s pooled over the i n d i c a t e d r e g i o n s of the g r a d i e n t shown i n F i g u r e 37. In order t o o b t a i n the r a t i o of i1 kC]-acetate/ [ 3 H ] - l y s i n e i n c o r p o r a t e d i n each sample, the counts were i n t e g r a t e d over the h i s t o n e r e g i o n s , and the r e s u l t s are shown i n Tabl e 9. The r a t i o o f I1 * * C ] - a c e t a t e / [ 3 H ] - l y s i n e i n the h i s t o n e s i s the same i n the c e l l s from d i f f e r e n t p a r t s of the 2.8-5.5 mm/hr r e g i o n , having a v a l u e o f 0.04. By c o n t r a s t , the r a t i o i n spermatids i s 1.1, a f a c t o r o f 27 times the r a t i o i n the e a r l i e r c e l l s . T h i s suggests t h a t a c e t y l a t i o n i n the spermatids -120-FRACTION NO.(7.0 ML) FIG. 37 I n c o r p o r a t i o n o f [l kC]-acetate and t 3 H ] - l y s i n e i n t o d i f f e r e n t c e l l types from t r o u t t e s t i s . A suspension of t e s t i s c e l l s o b t a i n e d from a f i s h 43 days a f t e r the s t a r t of i n j e c t i o n s w i t h salmon p i t u i t a r y e x t r a c t was i n c u b a t e d w i t h L- [ 3 H ] - l y s i n e (133 uCi/ml) and sodium 1- [ l *C]-acetate (330 yCi/ml) f o r 80 min a t 16°, and f r a c t i o n a t e d by v e l o c i t y s e d i m e n t a t i o n on a g r a d i e n t (1-3%) of bovine serum albumin, a t u n i t g r a v i t y (173). C e l l s were counted on a hemocytometer, and r a d i o a c t i v i t y was determined on an a l i q u o t o f each f r a c t i o n c o l l e c t e d on a m i l l i p o r e f i l t e r (see " M a t e r i a l s and Methods"). Numbers over the arrows r e f e r t o s e d i m e n t a t i o n v e l o c i t y , i n mm/hr. -121-- 120 5 O o - 60 "o1 7.0 10.0 DISTANCE 7.0 10.0 FROM ORIGIN, CM. FIG. 38. S y n t h e s i s and a c e t y l a t i o n of h i s t o n e s i n d i f f e r e n t c e l l types from t r o u t t e s t i s . C e l l s from the r e g i o n s of the g r a d i e n t i n d i c a t e d i n F i g . 37 were pooled, and the amount of i n c o r p o r a t i o n of [x l*C]-acetate and [ 3 H ] - l y s i n e i n t o the h i s t o n e s was determined 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 as d e s c r i b e d i n " M a t e r i a l s and Methods". The i d e n t i t y of the v a r i o u s h i s t o n e bands i s i n d i c a t e d i n F i g u r e 2a. P - I I b i r e f e r s t o the phos-p h o r y l a t e d form of h i s t o n e I l b i -122-T a b l e 9 R e l a t i v e i n c o r p o r a t i o n of [l ^ C l - a c e t a t e and [ 3 H ] - l y s i n e i n t o h i s t o n e s of d i f f e r e n t c e l l types from t r o u t t e s t i s . The t o t a l number of t 1''C]-acetate and [ 3 H ] - l y s i n e counts i n c o r p o r a t e d i n t o the h i s t o n e r e g i o n s of the g e l i l l u s t r a t e d i n F i g . 38 were i n t e g r a t e d , and the r a t i o of [l **C]- a c e t a t e / [3H] -l y s i n e i n c o r p o r a t i o n was c a l c u l a t e d f o r each sample, correspond-i n g t o the c e l l s pooled from the g r a d i e n t shown i n F i g . 37. F r a c t i o n [ x ''C]-acetate, of g r a d i e n t t o t a l net cpm i n h i s t o n e s [ 3 H ] - l y s i n e , t o t a l net cpm i n h i s t o n e s R a t i o I1 ^ C ] - a c e t a t e / [ 3 H ] - l y s i n e i n h i s t o n e s 40-52 (a) 56-63 (b) 64-73 (c) 78-84 (d) 498 638 912 404 11,929 12,007 23,429 378 0.043 0.041 0.039 1.1 -123-i s not n e c e s s a r i l y coupled to h i s t o n e s y n t h e s i s , as i t may be i n the e a r l i e r c e l l s . However, between the end of the i n c u b a t i o n w i t h i s o t o p e s , and the f i n a l p r o c e s s i n g of the c e l l s from.the g r a d i e n t , as much as 10 hours may e l a p s e , and the p o s s i b i l i t y t h e r e f o r e e x i s t e d t h a t the d i f f e r e n c e i n the t 1 **C]- a c e t a t e / [ 3H]-l y s i n e r a t i o s c o u l d be due t o d i f f e r e n t i a l t u r n o v e r of [ X I*C]-a c e t a t e between the e a r l y c e l l s and the spermatids; f o r i n s t a n c e , i f [ ^ C ] - a c e t a t e turned over r a p i d l y i n the former c e l l t y p e s , and s l o w l y or not a t a l l i n the l a t t e r , then the i n i t i a l r a t i o of I1 ^C]- a c e t a t e / [ 3 H ] - l y s i n e i n the e a r l i e r c e l l s would have been much h i g h e r . We t h e r e f o r e examined the r a t e of turnover of [x **C]-acetate i n r the p r e - s p e r m a t i d c e l l s , and the r e s u l t s are shown i n F i g . 39. The h a l f - l i f e of I1kC]-acetate, c a l c u l a t e d from these s t u d i e s , i s approximately 23 hours a t 16°, i n a c e l l suspension c o n s i s t i n g of more than 70% p r e - s p e r m a t i d c e l l s . S ince the c e l l sedimenta-t i o n s are c a r r i e d out a t 4 ° , and c e l l s are s t o r e d a t t h i s temp-e r a t u r e u n t i l counted and p r o c e s s e d f o r r a d i o a c t i v i t y a n a l y s i s , a c e t a t e t u r n o v e r under these c o n d i t i o n s would occur a t an even lower r a t e . The v i a b i l i t y o f the c e l l s d u r i n g pulse-chase e x p e r i -ments was shown by t h e i r l i n e a r uptake of [ 3 H ] - a r g i n i n e i n t o 5% TCA-tungstate i n s o l u b l e m a t e r i a l i n a p a r a l l e l i n c u b a t i o n mixture ( F i g . 40). T h i s experiment shows t h a t the d i f f e r e n c e i n the r a t i o o f [x kC]- a c e t a t e / [ 3 H ] - l y s i n e i n c o r p o r a t e d between e a r l y t e s t i s c e l l s and spermatids cannot be due to very r a p i d t u rnover of i 1 hC]-acetate i n the former c e l l t y p es. -124-d & 2.0 ui 5 CO ^ UJ 5 1.2 oc S o C 0.4 o o 0 4 8 12 16 20 24 T I M E , H R . FIG. 39. Turnover o f I1 **C]- a c e t y l groups i n t r o u t t e s t i s h i s t o n e s . T r o u t t e s t i s c e l l s (1 x 10 8 c e l l s ) were i n c u b a t e d f o r 90 min a t 16° w i t h sodium 1- [l *C]-acetate (286 uCi/ml) and QL- [ 3 H ] - a r g i n i n e (143 uCi/ml) i n 0.7 ml of TMKS-0.1% gl u c o s e c o n t a i n i n g 20% Waymouth's-Tris medium as d e s c r i b e d i n " M a t e r i a l s and Methods" (see "Pulse-chase s t u d i e s " ) . The c e l l s were then c o l l e c t e d by c e n t r i f u g a t i o n (1900 x g, 10 min) r e -suspended i n 20 ml of f r e s h , i s o t o p e - f r e e medium, and a l i q u o t s c o n t a i n i n g 5 x 10 6 c e l l s were taken at i n t e r v a l s and c o l l e c t e d on M i l l i p o r e g l a s s f i b e r f i l t e r s f o r the a n a l y s i s o f r a d i o -a c t i v i t y i n the h i s t o n 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 . The counts o f [ ^ C ] - a c e t a t e and [ 3 H ] - a r g i n i n e were i n t e g r a t e d over the h i s t o n e r e g i o n o f the g e l f o r each time sample, and the r a t i o o f t 1 " C ] - a c e t a t e / [ 3 H ] - a r g i n i n e was then c a l c u l a t e d f o r each sample. The h o r i z o n t a l l i n e r e p r e s e n t s the [*kC]- a c e t a t e / [ 3 H ] - a r g i n i n e r a t i o when h a l f o f the o r i g i n a l a c e t y l groups have been removed; the v a l u e o f t a t the i n t e r c e p t w i t h the graph i s th, the h a l f - l i f e o f [ l **C]-acetate. -125-I I I I I I TIME, HR. FIG. 40. I n c o r p o r a t i o n of [ 3 H ] - a r g i n i n e i n t o t r o u t t e s t i s n u c l e i w i t h time. A suspension o f t e s t i s c e l l s was i n c u b a t e d f o r 90 min a t 16° w i t h 286 uCi/ml of sodium 1- [l **C]-acetate and 143 uCi/ml o f D,L- [ 3 H ] - a r g i n i n e , as d e s c r i b e d i n the legend t o F i g . 39, p. 124. The c e l l 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 resuspended i n 20 ml of f r e s h , i s o t o p e - f r e e medium. H a l f of the sample was taken f o r measurement of [l **C]-acetate t u r n -over ( F i g . 39). To the o t h e r h a l f of the suspension was added 10 uCi/ml of D,L- [ 3 H ] - a r g i n i n e , and the uptake of i s o t o p e was f o l l o w e d f o r 12 hrs by a s s a y i n g samples on M i l l i p o r e f i l t e r s by TCA-tungstate p r e c i p i t a 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". The zero time p o i n t i n the f i g u r e t h e r e f o r e r e p r e s e n t s approximately 100 min a f t e r the s t a r t o f the i n i t i a l i n c u b a t i o n . -126-I t i s s t i l l p o s s i b l e t h a t d i f f e r e n c e s i n the a c e t a t e or amino a c i d p o o l s between spermatids and e a r l i e r c e l l s c o u l d be r e s p o n s i b l e f o r the d i f f e r e n c e s i n I1 **C]- a c e t a t e / [ 3 H 1 - l y s i n e r a t i o s observed, although the l a r g e changes (20-30 f o l d ) t h a t would be r e q u i r e d t o account f o r the r e s u l t s seem u n l i k e l y . The data p r e s e n t e d here suggest, t h e r e f o r e , t h a t a c e t y l a t i o n of h i s t o n e s i n t r o u t t e s t i s spermatids o c c u r s on molecules which have a l r e a d y been bound to chromatin f o r some time. In c o n t r a s t t o 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 of h i s t o n e s does not seem to occur t o any s i g n i f i c a n t e x t e n t i n t r o u t t e s t i s spermatids (107). ' T h i s suggests t h a t these two types of m o d i f i c a -t i o n may p l a y d i f f e r e n t r o l e s i n h i s t o n e f u n c t i o n , although o t h e r i n t e r p r e t a t i o n s are p o s s i b l e . These i d e a s w i l l be d i s c u s s e d i n d e t a i l below (see " D i s c u s s i o n " ) . -127-IV. Some P r o p e r t i e s of 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 from  Tr o u t T e s t i s . A d e t a i l e d understanding of the r o l e of a c e t y l a t i o n i n h i s t o n e f u n c t i o n w i l l r e q u i r e c o n s i d e r a b l e knowledge of the a c e t y l a t i n g enzymes themselves. We t h e r e f o r e undertook some p r e l i m i n a r y ex-periments designed to e l u c i d a t e some of the p r o p e r t i e s of these enzymes. I t was found t h a t a suspension of n u c l e i , prepared i n T r i s b u f f e r c o n t a i n i n g magnesium and potassium i o n s (TMK), would i n c o r -p o r a t e t 1 **C]-acetate i n t o a c i d - s o l u b l e p r o t e i n s d u r i n g an i n c u b a t i o n w i t h a c e t y l - 1 - t 1 "c]-coenzyme A ( F i g . 41). T h i s a c t i v i t y was a b o l i s h e d by h e a t i n g the n u c l e i at 100° f o r 2 minutes (Table 10). T h e r e f o r e t h i s suggested t h a t an enzyme a c t i v i t y , p r e s e n t i n n u c l e i , c o u l d t r a n s f e r a c e t a t e from a c e t y l coenzyme A ( a c e t y l CoA) t o endogenous h i s t o n e s . E x t e n s i v e washing of n u c l e i w i t h TMK by homogenization and c e n t r i f u g a t i o n d i d not remove the a c e t y l a t i n g a c t i v i t y , sug-g e s t i n g t h a t the enzyme(s) was t i g h t l y bound to the chromatin. When n u c l e o h i s t o n e was prepared i n 0.01M T r i s by a v a r i a t i o n of the technique of Marushige and Dixon (145) (see " M a t e r i a l s and Methods"), [ l **C]- a c e t a t e was i n c o r p o r a t e d i n t o a c i d - s o l u b l e p r o t e i n s from a c e t y l CoA when magnesium or potassium ions were added back t o the p r e p a r a t i o n ( F i g s . 42 and 43). Thus, i t seems l i k e l y t h a t the h i s t o n e a c e t y l a t i n g enzyme(s) i s bound to chromatin i n c l o s e ++ a s s o c i a t i o n w i t h i t s s u b s t r a t e , the h i s t o n e s , and r e q u i r e s Mg or K + i o n s f o r a c t i v i t y . When a suspension of n u c l e i , prepared i n TMK, was c e n t r i f u g e d -128-70 2 0 3 0 4 0 5 0 T I M E , M I N . FIG. 41. Uptake of [l H C ] - a c e t a t e i n t o a c i d - s O l u b l e p r o t e i n s of t r o u t t e s t i s n u c l e i . A suspension of n u c l e i was prepared i n TMK, 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 i q u o t s (0.200 ml) were then incubated w i t h 40,000 cpm of a c e t y l - 1 - [ l ^ C] coen-zyme A a t 19° and pH 8.0. Samples were taken a t i n t e r v a l s f o r the assay of [ l l ,C]-acetate i n c o r p o r a t e d i n t o a c i d s o l u b l e pro-t e i n s by the f i l t e r d i s c assay.as d e s c r i b e d i n " M a t e r i a l s and", • Methods".. . • ••^ •^';>:^'::i-'-.':. •'•>::'?,:../ A... -129-Table 10 Heat l a b i l i t y 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 a c t i v i t y i n t r o u t t e s t i s n u c l e i . A n u c l e a r suspension was prepared, u s i n g 14 g of f r o z e n t e s t i s ( s t o r e d at -80°) and 40 ml of TMK, pH 8.0, 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 i q u o t s (100 ul) of t h i s suspension were then i n c u b a t e d at room temperature f o r 30 min wit h 80,000 cpm of a c e t y l - 1 - [^C] coenzyme A, and assayed on f i l t e r d i s c s (see " M a t e r i a l s and Methods"). C o n t r o l Heated* I 1 "C]-acetate, 13,657 164 cpm i n a c i d s o l u b l e p r o t e i n * Suspension heated a t 100° f o r 2 min. -130-4 r 8 J 2 16 20 [ M g C I 2 ] , m M FIG. 42. Dependence 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 a c t i v i t y o f n u c l e o h i s t o n e on Mg i o n s . ' S o l u b l e ' n u c l e o h i s t o n e was prepared by s o n i c a t i o n of chromatin i n 10 mM T r i s , pH 7.4, 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 i q u o t s of the n u c l e o -h i s t o n e p r e p a r a t i o n (0.100 ml) were assayed i n the presence of 0.05 uCi of a c e t y l - 1 - [x kC]-coenzyme A a f t e r the a d d i t i o n o f v a r i a b l e amounts of 0.1 M MgCl 2, f o r 15 min a t room temperature, I n c u b a t i o n s were stopped w i t h 5 u l of cone. H Cl, the mixtures were p i p e t t e d onto f i l t e r d i s c s , and t r e 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". -131-CN I I | I o <10 i n U < 5 • i 1 — 1 25 50 75 [KCI] , m M FIG. 43. Dependence of 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 a c t i v i t y of n u c l e o h i s t o n e on K + i o n s . ' S o l u b l e ' n u c l e o h i s t o n e was pr e -pared by s o n i c a t i o n o f chromatin i n 10 mM T r i s , pH 7.4, and 0.100 ml a l i q u o t s were assayed i n the presence of 0.05 uCi o f a c e t y l - 1 - t 1''C]-coenzyme A and v a r i a b l e amounts o f 0.1M KCI to g i v e the d e s i r e d f i n a l s a l t c o n c e n t r a t i o n . The i n c u b a t i o n was performed a t room temperature f o r 15 min. -132-and the n u c l e i were resuspended i n the same volume of TMK con-t a i n i n g 0.4M NaCl, much of the a c e t y l a t i n g a c t i v i t y was l o s t (Table 11a). Although some of t h i s l o s s may be a t t r i b u t a b l e to i n a c t i v a t i o n of the enzyme, much of i t appears i n the 0.4M NaCl e x t r a c t , i n d i c a t i n g t h a t i t has been d i s s o c i a t e d from the chromatin (Table l i b ) . In order to measure t h i s s o l u b i l i z e d a c t i v i t y , p u r i f i e d h i s t o n e s i n s o l u t i o n are added to the enzyme p r e p a r a t i o n , whereas w i t h n u c l e i , the i n c o r p o r a t i o n i s i n t o chromatin-bound, endogenous h i s t o n e s ; t h e r e f o r e , s i n c e the c o n d i t i o n s of the assay i n the two cases are very d i f f e r e n t , the a c t i v i t i e s cannot be q u a n t i t a t i v e l y r e l a t e d to each o t h e r . P r e c i p i t a t i o n of the 0.4M NaCl e x t r a c t w i t h ammonium s u l f a t e (AS) r e v e a l e d t h a t most of the enzyme a c t i v i t y was p r e c i p i t a t e d between 30% and 60% s a t u r a t i o n (Table 12a), w i t h some a c t i v i t y i n the 0-30% f r a c t i o n . No a c t i v i t y was found w i t h h i g h e r concen-t r a t i o n s of ammonium s u l f a t e . For subsequent experiments, the 30-60% AS f r a c t i o n , or sometimes a 0-70% AS f r a c t i o n , was used. D i a l y s i s of the AS f r a c t i o n s a g a i n s t TMK alone r e s u l t e d i n a con-s i d e r a b l e l o s s of a c t i v i t y , which c o u l d be prevented by d i a l y s i s a g a i n s t TMK c o n t a i n i n g 0.4M NaCl (Table 12b). The i n c o r p o r a t i o n of I 1''C]-acetate i n t o h i s t o n e s from a c e t y l CoA was dependent upon the c o n c e n t r a t i o n o f enzyme, and the con-c e n t r a t i o n of added h i s t o n e ( F i g s . 44 and 45). A time course of the r e a c t i o n u s i n g the 0-70% AS f r a c t i o n r e s u l t e d i n a curve w i t h two d i s t i n c t s l o p e s , s u g g e s t i n g the presence of e i t h e r two d i f f e r e n t enzymes, or of a c e t y l a t i o n s i t e s w i t h d i f f e r e n t a f f i n i t i e s f o r the same enzyme ( F i g . 46). -133-Table 11 E x t r a c t i o n 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 a c t i v i t y from t r o u t t e s t i s n u c l e i by NaCl. (a) A suspension of n u c l e i i n TMK was prepared from f r o z e n t e s t i s , and assayed f o r a c e t y l t r a n s f e r a s e a c t i v i t y , as d e s c r i b e d i n " M a t e r i a l s and Methods". The n u c l e i were then c o l l e c t e d by cen-t r i f u g a t i o n (3000 x g, 10 min), resuspended i n an equal volume of TMK c o n t a i n i n g 0.4 M NaCl, and again assayed. F i n a l l y , the 0.4 M NaCl wash was removed by c e n t r i f u g a t i o n as b e f o r e , and the n u c l e i were assayed once aga i n i n TMK. A c i d S o l u b l e P r o t e i n , cpm* (i) N u c l e a r suspension i n TMK (100 y l ) 7,043 ( i i ) N u c l e a r suspension NaCl (100 y l ) i n TMK- 0.4 M 2,276 ( i i ) N u c l e a r suspension 0.4 M NaCl wash i n TMK a f t e r 2,789 (b) The 0.4 M NaCl e x t r a c t o f n u c l e i was assayed f o r a c e t y l -t r a n s f e r a s e a c t i v i t y i n the presence of added h i s t o n e . H i s t o n e , 5 mg/ml A c i d - s o l u b l e Pro-i n 0.4 M NaCl t e i n cpm* TMK-0.4 M NaCl e x t r a c t of n u c l e i (50 y l ) 50 y i 2,869 TMK-0.4 M NaCl e x t r a c t 50 y l 553 (50 y l ) , heated at 100° f o r 30 sec. * Each assay mixture c o n t a i n e d 80,000 cpm of a c e t y l - 1 - [^C] coenzyme A. -134-Table 12 Ammonium sulfate p r e c i p i t a t i o n of histone acetyltransferase a c t i v i t y , and e f f e c t of d i a l y s i s on enzyme a c t i v i t y . (a) Histone acetyltransferase a c t i v i t y was extracted from trout t e s t i s n u c l e i with TMK containing 0.4 M NaCl, and was fractionated by ammonium sulf a t e (AS) p r e c i p i t a t i o n . The AS p r e c i p i t a t e s were dialyzed against TMK, pH 8.0, and aliquots assayed for enzyme ac-t i v i t y i n the presence of added histone, i n a f i n a l volume of 100 y l . Histone added Acid-soluble Pro-t e i n cpm* 0-30% AS f r a c t i o n (50 yl) 0.100 mg 1,885 .3.0-6.0*. AS f r a c t i o n (50 yl) 0.100 mg 5,505 (b) A 70% AS f r a c t i o n was obtained from a 0.4 M NaCl extract of nu c l e i , and divided into two aliquots. One aliquot was dialyzed against TMK, and the other against-TMK containing 0.4 M NaCl. Aliquots (25 yl) of both solutions were then assayed i n TMK-0.4 M NaCl, pH 8.0, i n a f i n a l volume of 100 y l . Histone added Acid-soluble Pro-t e i n cpm* 70% AS f r a c t i o n (25 yl) , 0.250 mg 1,011 TMK d i a l y s i s 70% AS f r a c t i o n (25 yl) 0.250 2,363 TMK-0.4 M NaCl d i a l y s i s * Each assay mixture contained 0.05 yCi of acetyl-1- [*kC] coenzyme A, and incubations were done at room temperature for 15 min. -135-i i FIG. 44. Dependence o f h i s t o n e a c e t y l t r a n s f e r a s e a c t i v i t y on enzyme c o n c e n t r a t i o n . The enzyme s o l u t i o n was o b t a i n e d by p r e c i p i t a t i o n o f a 0.4 M NaCl e x t r a c t o f chromatin w i t h ammonium s u l f a t e a t 30-60% s a t u r a t i o n . Assays were done i n a t o t a l volume of 100 u l of TMK, pH 8.0, c o n t a i n i n g 0.135 mg of whole h i s t o n e , 0.05 u C i o f a c e t y l - 1 - [ l ''C] coenzyme A, and v a r i a b l e amounts of enzyme s o l u t i o n . - The i n c u b a t i o n was c a r r i e d out a t room temp-e r a t u r e f o r 15 min. -136-16| 1 r 1 r C M I HISTONE CONC, mg/ml i FIG. 45. Dependence 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 a c t i v i t y on h i s t o n e c o n c e n t r a t i o n . The enzyme s o l u t i o n was prepared by p r e c i p i t a t i o n o f a 0.4 M NaCl e x t r a c t of chromatin w i t h ammonium s u l f a t e a t 30-60% s a t u r a t i o n (see " M a t e r i a l s and Methods"). Each i n c u b a t i o n mixture ( t o t a l volume, 100 y l ) c o n t a i n e d 50 y l o f enzyme s o l u t i o n , 4*1 x 10** cpm a c e t y l - 1 -t 1''C]-coenzyme A, v a r i a b l e amounts of h i s t o n e , 50 mM T r i s - H C l , pH 7.4, 1 mM MgCl2, and 25 mM KCI. The i n c u b a t i o n time was 15 min a t room temperature (ca. 2 5 ° ) . The r e a c t i o n was stopped by the a d d i t i o n o f 5 y l of cone. HCl, and 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 p r o t e i n s was determined by the f i l t e r d i s c t e chnique, as d e s c r i b e d i n " M a t e r i a l s and Methods". -137-2 0 4 0 6 0 TIME, MIN. FIG. 46. Time course o f h i s t o n e a c e t y l t r a n s f e r a s e a c t i v i t y . The enzyme was prepared by p r e c i p i t a t i o n of a 0.4 M NaCl e x t r a c t o f chromatin w i t h 70% s a t u r a t e d ammonium s u l f a t e , and d i a l y s i s a g a i n s t TMK-0.4 M NaCl. Each assay mixture c o n t a i n e d , i n 100 u l of TMK-0.4 M NaCl pH 8.0, 50 y l of enzyme s o l u t i o n , 0.250 mg o f h i s t o n e , and 0.05 uCi of a c e t y l - 1 - [ x ^ C ] coenzyme A. The i n c u -b a t i o n was c a r r i e d out a t 15° i n a g y r a t o r y water bath. -138-A study of the enzyme a c t i v i t y at d i f f e r e n t pH values (Fig. 47) revealed a pH optimum of approximately 8.5, a r e s u l t similar to that observed for histone acetyltransferases from rat l i v e r n u c l ei (59). I t i s important to note that at a l k a l i n e pH values above pH 8.0, non-enzymatic transfer of acetyl groups from acetyl CoA to histones can occur (185), and hence i n the study of the pH dependence of the acetyltransferase a c t i v i t y , a blank incubation ( i . e . without enzyme) must be c a r r i e d out at each pH value, and the r e s u l t subtracted from the experimental value to obtain the net enzymatic a c t i v i t y . For the same reason, although the pH optimum of the enzyme i s 8.5, assays were usually c a r r i e d out at pH 7.4 or pH 8.0. Attempts were made to p u r i f y the enzyme a c t i v i t y by column chromatography. DEAE c e l l u l o s e chromatography of the ammonium sul f a t e fractions usually resulted i n complete loss of the a c t i v i t y of the preparation. In cases where a small amount of a c t i v i t y was recovered, i t emerged with the bulk of the protein, and ho pur-i f i c a t i o n was achieved. Gel f i l t r a t i o n chromatography of a 70% AS f r a c t i o n on Sephadex G-200 equ i l i b r a t e d with TMK and 0.4M NaCl resulted i n the emergence of the enzyme a c t i v i t y i n the void volume of the column, with most of the other protein (Fig. 48). The fractions containing the a c t i v i t y were opalescent i n appear-ance, suggesting that t h i s material was probably not i n true solution, and might consist of very large aggregates of protein. Thus, although approximately 60% of the histone acetyl trans-ferase a c t i v i t y can be extracted with 0.4M NaCl and p r e c i p i t a t e d -139-7.0 7.5 8X) 8.5 9.0 9.5 PH FIG. 47. pH dependence of the histone acetyltransferase ac-t i v i t y from trout t e s t i s chromatin. Histone acetyltransferase a c t i v i t y was extracted from chromatin with 0.4 M NaCl, pre-c i p i t a t e d with ammonium sulf a t e (70% saturation), and dialyzed against TMK-0.4 M NaCl, pH 8.0. Aliquots (0.01 ml) of t h i s preparation were incubated with 0.02 ml of histone solution (5 mg/ml, i n 0.4 M NaCl), 0.05 uCi of acetyl-1- t1 **C]-coenzyme A, and 0.08 ml of either 50 mM Tris-HCl or 50 mM Glycine buffer, containing 1 mM MgCl 2 and 25 mM KCI. The incubation was per-formed at 19° for 60 min. A blank incubation (without enzyme) was done f o r each pH value, and the r e s u l t i n g incorporation was subtracted from the experimental values to y i e l d the net enzy-matic incorporation. -140-10 20 FRACTION NO.,5ml FIG. 48. Sephadex G-200 chromatography 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 a c t i v i t y . The enzyme was prepared by p r e c i p i t a -t i o n of a 0.4 M NaCl e x t r a c t o f chromatin w i t h ammonium s u l -f a t e at 70% s a t u r a t i o n , and d i a l y s i s o f the p r e c i p i t a t e a g a i n s t TMK-0.4 M NaCl. An a l i q u o t (7.0 ml) of t h i s p r e p a r a t i o n was a p p l i e d t o a column (2.5 x 22 cm) of Sephadex G-100 e q u i l i b r a t e d and e l u t e d w i t h TMK-0.4 M NaCl, pH 8.0. Enzyme a c t i v i t y was determined on 0.05 ml a l i q u o t s o f each column f r a c t i o n , i n the presence o f 0.05 u C i of acety 1-1- I 1 **C)-coenzyme A and 0.25 mg of h i s t o n e , i n a f i n a l volume o f 0.100 ml of TMK-0.4 M NaCl. The i n c u b a t i o n was c a r r i e d out a t 19° f o r 90 min. -141-w i t h ammonium s u l f a t e , the r e s u l t i n g p r o t e i n p r e p a r a t i o n i s h i g h l y aggregated. T h i s problem might be avoided by i s o l a t i n g the enzyme i n the presence of low c o n c e n t r a t i o n s of n o n - i o n i c d e t e r g e n t s such as T r i t o n X-100 or Nonidet P-40, although the e f f e c t of these d e t e r g e n t s on the a c t i v i t y of the enzyme has not y e t been e x p l o r e d . G a l l w i t z (59) has e x t r a c t e d 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 from r a t l i v e r n u c l e i i n a b u f f e r c o n t a i n i n g 1M ammonium s u l f a t e and 20% g l y c e r o l . The chromatin, which became h i g h l y v i s c o u s i n t h i s medium, was then s o n i c a t e d , and the p r o t e i n s were p r e c i p i t a t e d w i t h 3.5M (^70% s a t u r a t i o n ) ammonium s u l f a t e . A v a r i a t i o n of t h i s method was t r i e d w i t h t r o u t t e s t i s n u c l e i i n which 0.4M NaCl was used i n s t e a d o f the i n i t i a l 1M ammonium s u l f a t e , and 10% r a t h e r than 20% g l y c e r o l was p r e s e n t . D i f f i c u l t y was experie n c e d i n c e n t r i f u g a t i o n of the 70% ammonium s u l f a t e p r e c i p i t a t e through the v i s c o u s medium, and l i t t l e a c t i v i t y was recovered. However, f u r t h e r study o f t h i s method might prove f r u i t f u l i n i s o l a t i n g the h i s t o n e a c e t y l a t i n g enzymes from t r o u t t e s t i s n u c l e i . -142-D i s c u s s i o n We have shown t h a t t 1 k C ] - a c e t a t e i s i n c o r p o r a t e d as e-N-acetyl groups i n t o t r o u t t e s t i s h i s t o n e s I l b i , IIb2i H I and IV. A l l of the a c e t y l a t e d l y s y l r e s i d u e s which have been i d e n t i f i e d are i n the very b a s i c amino-terminal r e g i o n s of these h i s t o n e s . The extent of a c e t y l a t i o n of i n d i v i d u a l h i s t o n e s i s r e f l e c t e d i n the degree of h e t e r o g e n e i t y e v i d e n t i n the s t a r c h g e l p a t t e r n s o f the p u r i -f i e d p r o t e i n s , and the number of major a c e t y l a t e d s p e c i e s can be de t e c t e d by autoradiography of the d r i e d g e l s ( F i g s . 13 and 14). Thus, the presence of up to fo u r a c e t y l a t e d l y s y l r e s i d u e s i n h i s t o n e IV ( F i g . 17) r e s u l t s i n the appearance of fo u r [* **C]-acetate l a b e l e d bands i n the g e l p a t t e r n of t h i s h i s t o n e . The s i n g l e major a c e t y l a t e d l y s y l r e s i d u e o f h i s t o n e I l b i ( F i g . 23) i s a l s o a s s o c i a t e d w i t h a r a d i o a c t i v e band on s t a r c h g e l s , and another, f a i n t l y v i s i b l e band c o r r e l a t e s w i t h the suspected presence of another a c e t y l a t i o n s i t e i n t h i s h i s t o n e ( F i g s . 14 and 24). In h i s t o n e I I I , o n l y two of the fo u r a c e t y l a t e d l y s y l r e s i d u e s are m o d i f i e d e x t e n s i v e l y enough to be r e f l e c t e d i n the s t a r c h g e l p a t t e r n (although f a i n t l a b e l i n g over a t h i r d band, i . e . behind "A 2" i n F i g . 14, i s sometimes seen). The same s i t u a t i o n holds f o r h i s t o n e I I b 2 . The f a c t t h a t some l y s y l r e s i d u e s are found t o be more e x t e n s i v e l y m o d i f i e d than o t h e r s probably r e f l e c t s the e x i s t -ence of v a r i a t i o n s between the r a t e s o f 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 a t d i f f e r e n t s i t e s . Thus, a l y s y l r e s i d u e which i s r a p i d l y a c e t y l a t e d , but s l o w l y d e a c e t y l a t e d , w i l l show a h i g h e r steady -143-s t a t e of m o d i f i c a t i o n than one which i s more s l o w l y a c e t y l a t e d , but d e a c e t y l a t e d a t the same r a t e as the former s i t e . Although h e t e r o g e n e i t y of h i s t o n e IV from c a l f thymus had been observed on long acrylamide d i s c g e l s (186), and one of the two bands seen was b e l i e v e d to be due to a c e t y l a t i o n , t h i s has not been shown by a u t o r a d i o g r a p h i c s t u d i e s . In c a l f thymus h i s t o n e IV, DeLange e t a l . (26) found a s i n g l e a c e t y l a t e d l y s y l r e s i d u e a t p o s i t i o n 16. In pea c o t y l e d o n h i s t o n e IV, the same workers found, i n a d d i t i o n , a c e t y l a t i o n a t one of p o s i t i o n s 5, 8 or 12, the exact l o c a t i o n b e i ng u n c e r t a i n . Two a c e t y l a t e d l y s y l r e s i d u e s were a l s o found i n c a l f thymus h i s t o n e I I I by DeLange e t a l . a t positions 14 and 23. No s i t e s of a c e t y l a t i o n have y e t been r e p o r t e d f o r h i s t o n e s IIb2 and I l b i from o t h e r sources. The p a t t e r n of t r y p t i c a c e t y l a t e d p e p t i d e s from h i s t o n e IV ( F i g . 16) suggests t h a t a g i v e n sample of t h i s p r o t e i n c o n s i s t s o f a very heterogeneous p o p u l a t i o n of molecules, r e p r e s e n t i n g a c e t y l a t i o n a t f o u r l y s y l r e s i d u e s (5,8,12 and 16) i n a l l p o s s i b l e combinations. I f t h i s i s so, then band " A i " ( F i g s . 13 arid 14) i s a c t u a l l y made up of f o u r d i f f e r e n t types of molecule, of i d e n t i c a l sequence and charge, d i f f e r i n g o n l y i n the l o c a t i o n o f the s i n g l e a c e t y l group a t one of the f o u r l y s y l r e s i d u e s . S i m i l a r l y , i t i s r e a d i l y seen t h a t band "A 2" would c o n t a i n s i x , band " A 3 " f o u r , and band " A i / ' one type of h i s t o n e IV molecule. Such p o p u l a t i o n s of molecules c o u l d r e s u l t from random (or p a r t i a l l y random) a c e t y l -a t i o n or d e a c e t y l a t i o n o f the h i s t o n e a t each o f the f o u r s i t e s . The p h o s p h o r y l a t e d components of h i s t o n e s I l b i and IV migrate -144-slower than a l l of 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 s p e c i e s on s t a r c h g e l s ( F i g . 14). These g e l s are run at pH 3.1-3.5, and under these c o n d i t i o n s , the phosphate moiety a t t a c h e d t o a s e r i n e h y d r o x y l group would be expected t o c a r r y a charge of -1; however, phos-p h o r y l a t e d h i s t o n e IV migrates behind t e t r a - a c e t y l IV ( F i g . 14) , which d i f f e r s i n charge from unmodified h i s t o n e IV by -4. Since t h e r e i s onl y a s i n g l e s i t e of p h o s p h o r y l a t i o n i n h i s t o n e IV (81, 91), the l a r g e m o b i l i t y d i f f e r e n c e a s s o c i a t e d w i t h p h o s p h o r y l a t i o n must be due to some other change i n the molecule, such as an a l t e r -a t i o n i n conformation. S i m i l a r l y , phospho-IIbi has a much lower m o b i l i t y than expected from i t s charge on s t a r c h g e l s . Dr. M. Sung has o b t a i n e d evidence t h a t a c o n f o r m a t i o n a l change may be i n v o l v e d i n t h i s case (154). A comparison o f the N H 2 - t e r m i n a l r e g i o n of t r o u t t e s t i s and c a l f thymus h i s t o n e I l b i w i t h t h a t o f h i s t o n e IV r e v e a l s exten-s i v e areas of homology between the two sequences ( F i g . 23, p. 93). Residues 1-5 of the two molecules are i d e n t i c a l , i n c l u d i n g the phos-p h o r y l a t i o n s i t e at s e r y l 1, and the a c e t y l a t i o n s i t e at l y s y l 5. I f a d e l e t i o n i s i n t r o d u c e d a f t e r l y s y l 5 i n h i s t o n e IV, the two sequences show e x t e n s i v e homology up to a l a n y l r e s i d u e 10 of I l b i . Such s i m i l a r i t i e s suggest common e v o l u t i o n a r y o r i g i n s f o r these sequences, and a l s o imply a s i m i l a r i t y i n t h e i r f u n c t i o n s . I t i s of i n t e r e s t t o note t h a t the sequence of c a l f thymus I l b i (35 ) con-t a i n s a g l u t a m i n y l r e s i d u e at p o s i t i o n 6 i n p l a c e of the t h r e o n y l r e s i d u e observed i n t r o u t t e s t i s I l b i . Thus the sequence around the a c e t y l a t i o n s i t e of t r o u t t e s t i s I l b i i s G l y - L y s ( A c ) - T h r - G l y , -145-whereas i n c a l f thymus i t i s G l y - L y s - G l n - G l y . I t would be of great i n t e r e s t t o see whether the a c e t y l a t i n g enzyme(s) from t r o u t t e s t i s c o u l d modify l y s y l r e s i d u e 5 i n c a l f thymus I l b i , and v i c e v e r s a . Such an experiment might i n d i c a t e whether these enzymes r e c o g n i z e s p e c i f i c sequences i n h i s t o n e s , or whether other f a c t o r s , such as the conformation of the h i s t o n e s i n chromatin, p l a y a r o l e . The b a s i s f o r the s p e c i f i c i t y of the a c e t y l a t i n g enzymes w i l l be d i s -cussed f u r t h e r below. The g e n e t i c code shows t h a t a change from t h r e o n i n e (ACX) to glutamine (CA —) cannot r e s u l t from a simple s i n g l e p o i n t mutation, A A but c o u l d be a r e s u l t e i t h e r of an i n v e r s i o n , AC ^ t o CA ^, or a double mutation, AC ^ + (AA ~) •*• CA or AC ~ •*• (CC h -> AC ~. Ka \3 o V J vjr Qa In the l a t t e r case, the i n t e r m e d i a t e amino a c i d at p o s i t i o n 6 would A A be e i t h e r l y s i n e (AA ^) o r p r o l i n e (CC -x) , and i t would be i n t e r -im G e s t i n g to see i f another s p e c i e s (perhaps an amphibian or b i r d ) might show one of these r e s i d u e s at t h i s p o s i t i o n . From the data p r e s e n t e d here, i t appears t h a t h i s t o n e I l b i i s the l e a s t m o d i f i e d by a c e t y l a t i o n of a l l the t r o u t t e s t i s h i s t o n e s . However, the d e t e r m i n a t i o n of the s i t e of a c e t y l a t i o n i n t h i s h i s t o n e i n v o l v e d the i s o l a t i o n and manual sequencing of t r y p t i c p e p t i d e s , and p e p t i d e s r e s u l t i n g from low degrees of a c e t y l a t i o n a t o t h e r s i t e s i n the molecule may e a s i l y have gone undetected. For i n s t a n c e , the low e x t e n t of m o d i f i c a t i o n of l y s y l r e s i d u e s 9 and 18 i n h i s t o n e I I I , and o f number 5 i n I I b 2 , would almost c e r t a i n l y have gone undetected i f c o n v e n t i o n a l t e c h -niques had been used. In view of t h i s c o n s i d e r a t i o n , i t i s pos--146-s i b l e t h a t a c e t y l a t i o n o f I l b i occurs at oth e r s i t e s i n the amino-t e r m i n a l r e g i o n , analogous t o the s i t u a t i o n i n h i s t o n e s I I b 2 , I I I , and IV. L y s y l r e s i d u e s 9, 13 and 15 would seem t o be l i k e l y c a ndidates f o r a c e t y l a t i o n i n h i s t o n e I l b j . , i n a d d i t i o n t o l y s y l r e s i d u e 5. For convenience, the amino-terminal sequences o f t r o u t t e s t i s h i s t o n e s I l b i , I I b 2 , I I I and IV are i l l u s t r a t e d i n F i g . 49 and a l l known s i t e s of a c e t y l a t i o n and p h o s p h o r y l a t i o n o f these p r o t e i n s are i n d i c a t e d . Reference w i l l be made to t h i s f i g u r e i n the d i s -c u s s i o n t o f o l l o w . When the sequences around a l l the known a c e t y l a t e d l y s y l r e s -idues i n h i s t o n e s are compared (Table 13), some s t r i k i n g p a t t e r n s emerge. The 13 sequences f a l l i n t o two major c a t e g o r i e s : (a) an a c e t y l a t e d l y s y l r e s i d u e bordered on e i t h e r s i d e by an amino a c i d w i t h a very s m a l l , n e u t r a l s i d e c h a i n ; t h i s i s most o f t e n g l y c i n e , but i n some cases i t i s t h r e o n i n e , a l a n i n e , or s e r i n e ; (b) an a c e t y l a t e d l y s y l r e s i d u e p r e s e n t as a member of a Lys-Arg, Arg-Lys or Lys-Lys p a i r , w i t h the a c e t y l a t e d l y s y l r e s i d u e bordered on one s i d e by s e r i n e or a l a n i n e , except t h a t f o r l y s y l r e s i d u e 18 of h i s t o n e I I I , the adj a c e n t r e s i d u e i s glutamine (33). The presence of amino a c i d s w i t h s m a l l s i d e c h a i n s next to a l y s y l r e s i d u e may enable t h a t l y s y l r e s i d u e to b i n d r e a d i l y i n the major groove of DNA (81), without the s t e r i c hindrance which might r e s u l t i f o t h e r amino a c i d s were p r e s e n t . The codons of the amino a c i d s u s u a l l y found ad j a c e n t t o a c e t y l a t e d l y s y l r e s i d u e s can be i n t e r -c o nverted by s i n g l e base changes, as f o l l o w s : -147-Hlstone IV Histone lib. Comparison _of_N-Terminal Regions of Histones of Trout Testis Ac Ac-Ser-OlyArg-Gly-Lysl 1 S T Ac-Ser-Gly-Arg-Cly-Lys Ac C l y - C l y - L y j -I Gly-LrHl-Gly-\ 10 -Thr-Gly-Gly-Lya C / n -Ac i i ! Lys-GlyCly-j •Ala-Anj-AlajLys^ — to Ac ' Ma I: ! Ala-Lys-Arg-Hls-Arg-Lya" IS | 20 I I Ala-Lys-Thr-Anj-(Anj)-Sar~ I (8 Histone III Ac Ac Ac Ac I I I ' 10 15 20 28 Ac I Ac I Ac I Ac .1 Histone I Ib^ Pro^lx-Pro-Ala<ls-Sar-Ala^ro-Lys-Lyi^ly{SaryLys-Lr9-Ala-Val-Thr-Lya.?.A 1 8 10 18 20 FIG. 49. Summary of N - t e r m i n a l sequences and s i t e s o f mod-i f i c a t i o n i n t r o u t t e s t i s h i s t o n e s IV, l i b ! , I l l and I I b 2 . The sequence o f h i s t o n e IV and the s i t e o f m e t h y l a t i o n a t l y s y l r e s i d u e 20 are from c a l f thymus, a c c o r d i n g t o DeLange e t a l . (26); the s i t e s o f p h o s p h o r y l a t i o n i n IV and I l b i are from Sung and Dixon (81), and the sequence o f I l b i from B a i l e y and Dixon (36). -148-Table 13 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 o f t r o u t t e s t i s h i s t o n e s A B IV G l y - L y s ( A c ) - G l y 5 A l a - L y s ( A c ) - A r g 16 G l y - L y s ( A c ) - G l y 8 G l y - L y s ( A c ) - G l y 12 I l b i G l y - L y s ( A c ) - T h r 5 I I I G l y - L y s ( A c ) - A l a 14 Arg - L y s ( A c ) - ( S e r ) 9 T h r - L y s ( A c ) - A l a 23 A r g - L y s ( A c ) - G l x 18 I I b 2 A l a - L y s ( A c ) - S e r 5 T h r - L y s < A c ) - ^ > L y s - L y s ( A c ) - G l y 10 (Ser) -Lys (Ac) -Lys 13 -149-GGX (Gly) -*• GCX (Ala) -> ACX (Thr) •* UCX (Ser) , and GCX (Ala) -> UCX ( S e r ) . In view o f the s i m i l a r i t i e s i n a c e t y l a t i o n s i t e s , and the sequence homologies noted i n h i s t o n e s IV, I l b i , and I I I by DeLange e t a l . (33), i t seems p o s s i b l e t h a t the d i f f e r e n t h i s t o n e sequences may have common o r i g i n s . In p a r t i c u l a r , the r e c u r r e n c e of c e r t a i n s h o r t sequences w i t h i n the same h i s t o n e molecule (26), and a l s o i n sequences of d i f f e r e n t h i s t o n e s (33), suggests t h a t these p r o t e i n s may have e v o l v e d by a s e r i e s of p a r t i a l gene d u p l i c a t i o n s and mutations of some s h o r t e r , a n c e s t r a l sequences (187). I t i s p o s s i b l e t h a t the sequence s i m i l a r i t i e s i n the s i t e s of a c e t y l a t i o n noted i n Tab l e 13 pro v i d e some b a s i s f o r the s p e c i f i c i t y o f the h i s t o n e a c e t y l a t i n g enzymes. However, these sequences are u n l i k e l y t o p r o v i d e the onl y r e c o g n i t i o n s i g n a l s f o r the enzymes, s i n c e some i d e n t i c a l sequences i n other r e g i o n s of h i s t o n e s are not a c e t y l a t e d ; f o r i n s t a n c e , the sequence Thr-L y s - A l a ( r e s i d u e s 46-48, F i g . 3) appears i n both r a b b i t thymus and c a l f thymus h i s t o n e I s u b f r a c t i o n s (37), but no a c e t y l a t i o n of t h i s h i s t o n e has been observed (30). A l s o , the sequence G l y - G l y - L y s -A l a appears i n both c a l f thymus (35) and t r o u t t e s t i s h i s t o n e I l b i a t p o s i t i o n s 7-10, and i n h i s t o n e I I I from both sources a t p o s i t i o n s 12-15; i n the l a t t e r , the l y s y l r e s i d u e i s e x t e n s i v e l y a c e t y l a t e d , whereas i n the former i t i s e i t h e r not m o d i f i e d at a l l , or o n l y m o d i f i e d t o a s m a l l e x t e n t , i n t r o u t t e s t i s . Thus, whether o r not a g i v e n l y s y l r e s i d u e i n a h i s t o n e molecule i s a c e t y l a t e d p r o b a b l y depends both upon the r e c o g n i t i o n of s p e c i f i c sequences -150-by the a c e t y l a t i n g enzymes, and upon c e r t a i n as y e t unknown aspects of h i s t o n e secondary s t r u c t u r e . T h i s s t r u c t u r e p r o b a b l y i n v o l v e s the i n t e r a c t i o n of the h i s t o n e s w i t h DNA. The automatic p r o t e i n sequencer has been used to determine the l o c a t i o n of H c y s t i n e r e s i d u e s i n neurophysin f o l l o w i n g t h e i r chem-i c a l m o d i f i c a t i o n w i t h [ x''C]-iodoacetamide by Capra e t a l . (188), and i n the p r e s e n t work a s i m i l a r approach has been used to l o c a t e the s i t e s of i n v i v o enzymatic a c e t y l a t i o n of h i s t o n e s I I b 2 and I I I . S i m i l a r techniques might a l s o be used i n the study of the s i t e s of other types of m o d i f i c a t i o n , such as m e t h y l a t i o n of l y s y l , a r g i n y l , or h ' i s t i d y l r e s i d u e s which have been r e p o r t e d i n h i s t o n e s (30). A l s o , c e r t a i n p r o s t h e t i c groups which occur i n p r o t e i n s , such as the b i o t i n p r o s t h e t i c group, which i s l i n k e d t o an e-NH2 group of i t s c a r r i e r p r o t e i n by an amide l i n k a g e (189), might be l o c a t e d by these methods. The requirements which must be met i n order f o r any such study t o be f e a s i b l e are as f o l l o w s : (1) the m o d i f i c a t i o n being s t u d i e d must be s t a b l e under the temperature and pH c o n d i t i o n s used i n automated sequencing procedures; (2) one must have a s e n s i t i v e means of d e t e c t i n g the m o d i f i e d r e s i d u e , e.g., a r a d i o a c t i v e l a b e l ; (3) the m o d i f i c a t i o n must be p r e s e n t near the f r e e amino terminus of the p r o t e i n or p e p t i d e , i . e . , w i t h i n approximately the f i r s t 25-50 r e s i d u e s , depending on the r e p e t i t i v e y i e l d s o b t a i n a b l e w i t h the p a r t i c u l a r sample, the con-d i t i o n s b e i ng used, and the s p e c i f i c a c t i v i t y of the r a d i o a c t i v e l a b e l . The sequence s t u d i e s r e p o r t e d here support the i d e a t h a t the -151-a r g i n i n e - r i c h h i s t o n e s I I I and IV have remained much more con-s e r v a t i v e d u r i n g the course of e v o l u t i o n than the o t h e r h i s t o n e s . Histone IV from pea s e e d l i n g s , f o r i n s t a n c e , d i f f e r s i n only two c o n s e r v a t i v e amino a c i d replacements from c a l f thymus h i s t o n e IV out of a t o t a l of 102 r e s i d u e s , making i t the most c o n s e r v a t i v e p r o t e i n sequence known to date (131). The s t u d i e s r e p o r t e d here on the a c e t y l a t e d p e p t i d e s of t r o u t t e s t i s h i s t o n e IV suggest t h a t the f i r s t 17 r e s i d u e s of t h i s h i s t o n e are i d e n t i c a l to those of the c o r r e s p o n d i n g c a l f thymus and pea h i s t o n e s . The sequence of the f i r s t 25 r e s i d u e s of h i s t o n e I I I , as w e l l as two of the s i t e s o f a c e t y l a t i o n ( l y s y l r e s i d u e s 14 and 23) are i d e n t i c a l i n both the c a l f thymus and t r o u t t e s t i s p r o t e i n s . On the other hand, the ' s l i g h t l y l y s i n e - r i c h ' h i s t o n e s I l b i and I I b 2 c o n t a i n s e v e r a l s p e c i e s - s p e c i f i c v a r i a t i o n s i n t h e i r amino-terminal sequences, as o u t l i n e d above. The l y s i n e r i c h h i s t o n e I , which i s not m o d i f i e d by a c e t y l a t i o n , shows sequence h e t e r o g e n e i t y i n d i f f e r e n t t i s s u e s as w e l l as d i f f e r e n t s p e c i e s (37). These d i f f e r e n c e s i n the e v o l u t i o n a r y c o n s e r v a t i s m of h i s t o n e sequences are s u g g e s t i v e of the e x i s t e n c e o f d i f f e r e n t r o l e s f o r d i f f e r e n t c l a s s e s of h i s t o n e s i n the maintenance of chromosome f u n c t i o n . Some of these r o l e s would seem to r e q u i r e very s t r i n g e n t s t r u c t u r a l s p e c i f i c a t i o n s f o r h i s t o n e s , whereas o t h e r s r e q u i r e r e l a t i v e l y l e s s constancy of s t r u c t u r e . F i g u r e 49 shows t h a t a l l known s i t e s of m o d i f i c a t i o n by a c e t y l a t i o n and p h o s p h o r y l a t i o n of t r o u t t e s t i s h i s t o n e s occur i n the b a s i c amino-terminal r e g i o n s of these p r o t e i n s . These -152-r e g i o n s , being the most b a s i c p a r t s of the molecules, are l i k e l y s i t e s of i n t e r a c t i o n w i t h the sugar-phosphate backbone of DNA (25,26,30). Sung and Dixon (81), and S h i h and Bonner (122) have shown t h a t by a r r a n g i n g the amino-terminal r e g i o n of h i s t o n e IV i n the a l p h a - h e l i c a l conformation, t h i s p a r t of the molecule can be accomodated i n the major groove of DNA, w i t h the b a s i c l y s y l and a r g i n y l r e s i d u e s i n t e r a c t i n g with phosphate groups of the DNA backbone. When the amino-terminal r e g i o n of h i s t o n e IV i s r e p -r e s e n t e d i n the h e l i c a l wheel arrangement of S c h i f f e r and Edmundson (119) ( F i g . 50) the numerous g l y c y l r e s i d u e s are s i t u a t e d i n p o s i t i o n s which permit c l o s e f i t t i n g of the p r o t e i n i n the major groove of DNA, where o t h e r , b u l k i e r r e s i d u e s might cause s t e r i c problems. I t was proposed t h a t a c e t y l a t i o n of the l y s y l r e s i d u e s , and p h o s p h o r y l a t i o n of the N H 2 - t e r m i n a l s e r y l r e s i d u e c o u l d cause t h i s r e g i o n of h i s t o n e IV to detach i t s e l f from DNA, s i n c e the charge would be changed from +6 to almost zero ( f o r r e s i d u e s 1-18). The g r e a t s i m i l a r i t y of the amino-terminal r e g i o n of h i s t o n e I l b i t o t h a t of h i s t o n e IV suggests t h a t i t c o u l d b i n d t o DNA i n a s i m i l a r manner, and model b u i l d i n g experiments conducted by Mr. A. L o u i e have shown t h a t i n the a l p h a - h e l i c a l conformation, the amino-terminal r e g i o n of t h i s h i s t o n e can a l s o be accomodated i n the major groove of DNA (105). In h i s t o n e I I I , however, t h e r e i s a p r o l y l r e s i d u e between two o f the a c e t y l a t e d l y s y l r e s i d u e s (14 and 18), and i n h i s t o n e I I b 2 from t r o u t t e s t i s t h e r e are t h r e e p r o l y l r e s i d u e s i n the amino-terminal r e g i o n . S i n c e p r o l y l r e s i d u e s are known to d i s r u p t - 1 5 3 -P04 Ac I I FIG. 50. R e p r e s e n t a t i o n of the f i r s t 18 r e s i d u e s o f h i s t o n e IV i n the °<-helical conformation a c c o r d i n g t o S c h i f f e r and Edmundson (119). The s i t e of p h o s p h o r y l a t i o n (91) and the s i t e s of 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 h i s t o n e IV are i n d i c a t e d . Note the sequence of g l y c y l r e s i d u e s i n the lower l e f t - h a n d quadrant, and the presence of a l l the m o d i f i e d r e s i d u e s i n the upper l e f t - h a n d quadrant. -154-a l p h a - h e l i c a l s t r u c t u r e i n t h e i r immediate v i c i n i t y (120), i t i s u n l i k e l y t h a t the amino-terminal r e g i o n s of these h i s t o n e s can con-t a i n e x t e n s i v e a l p h a - h e l i c a l s t r u c t u r e i n the chromatin, e s p e c i a l l y i n the case of h i s t o n e I I b 2 . The s i n g l e p r o l y l r e s i d u e i n the h i s t o n e I I I amino-terminal r e g i o n , however, might serve to separate two s h o r t segments of alpha h e l i x , and thus a l l o w the molecule to bend around a t u r n of the DNA double h e l i x , s i n c e the alpha h e l i c a l s t r u c t u r e of a p o l y p e p t i d e c h a i n i s very r i g i d . The s t u d i e s r e p o r t e d here on the s i t e s of a c e t y l a t i o n i n t r o u t t e s t i s h i s t o n e s I l b i , I I b 2 , I I I and IV suggest t h a t a c e t y l -a t i o n i s . a means of r e g u l a t i n g the charge d e n s i t y of the most b a s i c r e g i o n s of these molecules, and hence the t i g h t n e s s of t h e i r b i n d i n g t o DNA. The r o l e of t h i s r e g u l a t i o n mechanism i n the f u n c t i o n i n g of the c e l l nucleus w i l l be d i s c u s s e d below. I t i s worth n o t i n g t h a t h i s t o n e I , which has the h i g h e s t number of l y s y l r e s i d u e s , i s not a c e t y l a t e d i n t r o u t t e s t i s . The most b a s i c p a r t of the h i s t o n e I molecule i s i n the c a r b o x y - t e r m i n a l r e g i o n (40), and t h i s h i s t o n e i s e x t e n s i v e l y p h o s p h o r y l a t e d i n regen-e r a t i n g r a t l i v e r (84-88), and i n t r o u t t e s t i s (81). These obser-v a t i o n s are i n t r i g u i n g , i n view of the f a c t t h a t h i s t o n e I i s b e l i e v e d by some workers t o l i e i n a more exposed l o c a t i o n on the chromatin (190,191), and t h e r e f o r e may not be i n d i r e c t c o n t a c t w i t h DNA over most of i t s l e n g t h (192). We may now ask the q u e s t i o n , "What aspects of n u c l e a r met-aboli s m c o u l d r e q u i r e the r e g u l a t i o n of h i s t o n e b i n d i n g t o DNA?" - 1 5 5 -Recent experiments i n t h i s l a b o r a t o r y (performed by Mr. A. L o u i e ) , i n which the p a t t e r n of l a b e l i n g of h i s t o n e IV w i t h r a d i o a c t i v e amino a c i d s was s t u d i e d , showed t h a t the amino a c i d l a b e l appeared f i r s t i n the a c e t y l a t e d bands of h i s t o n e IV, and o n l y l a t e r d i d l a b e l appear over the unmodified h i s t o n e IV band (Ao). In the same way, i t was shown t h a t the p h o s p h o r y l a t e d band of h i s t o n e I l b i c o n t a i n e d 2 0 % of the [ 3 H ] - a r g i n i n e l a b e l a f t e r 1 hour of i n -c u b a t i o n of a c e l l suspension. These r e s u l t s i n d i c a t e d t h a t at l e a s t some newl y - s y n t h e s i z e d h i s t o n e s were m o d i f i e d soon a f t e r s y n t h e s i s , and i t was p o s t u l a t e d t h a t m o d i f i c a t i o n of h i s t o n e s I l b i and I V by a c e t y l a t i o n and p h o s p h o r y l a t i o n was necessary to ensure the c o r r e c t b i n d i n g of the n e w l y - s y n t h e s i z e d p r o t e i n s to DNA ( 1 0 5 ) . The p r o b a b i l i t y t h a t a p o l y - c a t i o n i c r e g i o n such as the N H 2 - t e r m i n a l of h i s t o n e IV or I l b i would immediately b i n d c o r r e c t l y t o a s e r i e s of phosphate groups on DNA would seem to be r low. A f t e r n e u t r a l i z a t i o n of some of the p o s i t i v e charges by a c e t y l a t i o n and p h o s p h o r y l a t i o n , the h i s t o n e would be l e s s s t r o n g l y bound and might be a b l e t o assume the c o r r e c t b i n d i n g mode; f i n a l l y , d e a c e t y l a t i o n and d e p h o s p h o r y l a t i o n would l o c k the now c o r r e c t l y -bound h i s t o n e i n p l a c e . Hence, the m o d i f i e d forms may be i n t e r m e d i a t e s i n the path-way of b i n d i n g of the newly s y n t h e s i z e d h i s t o n e s t o DNA. I f t h i s i s the case, one would expect the observed amount of a c e t y l a t i o n of the h i s t o n e s from a g i v e n t i s s u e t o be p r o p o r t i o n a l to the r a t e of c e l l d i v i s i o n i n t h a t t i s s u e , s i n c e h i s t o n e s y n t h e s i s accompanies -156-DNA synthesis (63). Thus, i n a tissue which i s undergoing extensive and r e l a t i v e l y synchronous c e l l d i v i s i o n , such as developing trout t e s t i s , one would detect acetylation of histones more r e a d i l y than i n a tissue such as l i v e r or thymus, i n which only a very small proportion of the t o t a l c e l l population i s undergoing d i v i s i o n at any given time. The f a i l u r e to observe acetylation of histones I l b i and I I b 2 , and extensive acetylation of histone IV i n the l a t t e r tissues could then be due to a lack of c e l l s i n a suitable metabolic state rather than to inherent differences i n enzymatic s p e c i f i c i t i e s . Application to other systems of the techniques used i n elucidating the acetylation s i t e s of trout t e s t i s histones could be useful i n c l a r i f y i n g these questions. Another process which may require the modulation of histone binding to DNA i s the replacement of histones by protamines, which occurs at the late spermatid stage of trout t e s t i s development (145, 146). The binding of the polybasic regions of histones to DNA i s very t i g h t , as shown by the high s a l t concentrations required to detach them (1.2-2.0M). In f a c t , histones III and IV are more d i f f i c u l t to dissociate from chromatin than the very basic protamine i t s e l f (193). Therefore, some mechanism must be available for loosening the t i g h t l y bound histones from the DNA; acetylation of the e-NH2 groups of l y s y l residues i n the most basic region of the molecule could f u l f i l l t h i s function. The p a r t i a l loosening of the histone-DNA complex i n t h i s manner might then allow degradation of these proteins to take place, by the action of a s p e c i f i c nuclear protease (194-196). -157-We have shown t h a t middle and l a t e (1.5 and 1.0 mm/hr) sperm-a t i d s a c t i v e l y a c e t y l a t e h i s t o n e s . Furthermore, the a c e t y l a t i o n of h i s t o n e s i n these c e l l types does not seem to c o r r e l a t e w i t h h i s t o n e s y n t h e s i s . In Tabl e 14, the r e l a t i v e i n c o r p o r a t i o n o f t 1 1*C]-acetate i n t o h i s t o n e s per c e l l i s c a l c u l a t e d f o r each of the re g i o n s of the g r a d i e n t s from the experiments i l l u s t r a t e d i n F i g s . 34 and 38 (pp. 115 and 121). I t i s seen t h a t f o r spermatids, the r e l a t i v e [x l f C ] - a c e t a t e i n c o r p o r a t i o n per c e l l i s 0.70-0.81 times the r a t i o f o r the l a r g e r (3.5 and 2.8 mm/hr) d i p l o i d c e l l s . I f a l l o w -ance i s made f o r the f a c t t h a t the spermatids are h a p l o i d , and hence have h a l f the DNA and h i s t o n e content of the d i p l o i d c e l l s , then the r e l a t i v e I 1''CJ-acetate i n c o r p o r a t i o n per spermatid ( i . e . 1.5 mm/hr c e l l s ) i s 1.4 to 1.6 times t h a t of the d i p l o i d c e l l s . T h i s i s i n c o n t r a s t t o the r e s u l t s o b t a i n e d from measurements of h i s t o n e p h o s p h o r y l a t i o n , where the 3 2 P 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 per spermatid was found t o be on l y 0.44 times t h a t of the 2.8 mm/hr c e l l s , and only 0.15 times t h a t of the val u e f o r the 3.5 and 5.0 mm/hr c e l l s (107), a l l o w i n g f o r the h a p l o i d h i s t o n e content of the spermatids. Furthermore, p h o s p h o r y l a t i o n o f h i s t o n e s c o r r e l a t e d w e l l w i t h h i s t o n e s y n t h e s i s i n a l l c e l l types (107) . Histon e a c e t y l a t i o n , then, must be c o n s i d e r e d as a p o s s i b l e o p e r a t i v e mechanism i n the t r a n s f o r m a t i o n p r o c e s s o c c u r r i n g on the chromatin o f t r o u t t e s t i s spermatids. Thus, a c e t y l a t i o n o f h i s t o n e s may be i n v o l v e d i n the detachment o f h i s t o n e s from DNA, as w e l l as i n the c o r r e c t placement o f newly s y n t h e s i z e d h i s t o n e s T a b l e 14 [ l ^ C ]-acetate i n c o r p o r a t i o n per c e l l f o r d i f f e r e n t t r o u t t e s t i s c e l l t y p e s ( * ) C e l l Type (Sedimentation V e l o c i t y , i n mm/hr) 3.5 2.8 1.5 I 1 **C]-acetate, cpm/ 10 7 c e l l s (a) Gradient o f F i g . 33 (p. 114) 3,060 (1.0)t 3,000 (0.98) 2,220 (0.70) 5.5-5.0 4.0-3.5 2.8 1.5 t 1 ""C]-acetate, cpm/ 10 7 c e l l s (b) G r a d i e n t of F i g . 37 (p. 120) 4,300 (1.6) 2,600 (1.0) 2,100 (0.81) 2,100 (0.81) 1'*C counts from the h i s t o n e r e g i o n o f F i g s . 34 and 38 were summed and d i v i d e d by the number of c e l l s on each f i l t e r . Numbers i n parentheses are the r e l a t i v e r a t i o s . -159-on the DNA. The t o t a l l a c k of s e n s i t i v i t y of h i s t o n e a c e t y l a t i o n t o c y c l o h e x i m i d e suggests t h a t i n c e l l s where p r o t e i n s y n t h e s i s has been i n h i b i t e d , t h e r e i s a p o o l of r e c e n t l y s y n t h e s i z e d h i s t o n e which has not y e t been p r o p e r l y bound t o DNA and which i s respon-s i b l e f o r the a c e t a t e i n c o r p o r a t i o n observed under these c o n d i t i o n s . In view of the p o s s i b l e e x i s t e n c e of two d i f f e r e n t r o l e s f o r h i s t o n e a c e t y l a t i o n i n d i v i d i n g c e l l s and spermatids from t r o u t t e s t i s , i t would be i n t e r e s t i n g t o see whether th e r e are any d i f -f e r e n c e s between 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 o f pre-spermatid and spermatid c e l l s . Another phenomenon which may i n v o l v e changes i n the s t a t e of the h i s t o n e s i n chromatin i s the pronounced i n c r e a s e i n n u c l e a r volume which has been observed to precede the ' a c t i v a t i o n ' of c e r t a i n n u c l e i . Gurdon (197,198) has observed t h a t b r a i n n u c l e i t r a n s -p l a n t e d i n t o e n u c l e a t e d oocytes undergo a l a r g e i n c r e a s e i n volume (up t o 2 0 - f o l d ) , and t h a t t h i s precedes the m i g r a t i o n of c y t o p l a s m i c p r o t e i n s i n t o the n u c l e i , and the onset of DNA and RNA s y n t h e s i s . H a r r i s (199) has observed a s i m i l a r phenomenon when hen e r y t h r o c y t e n u c l e i are a c t i v a t e d by being p l a c e d i n HeLa c e l l cytoplasm i n c e l l f u s i o n experiments. Pogo e t a l . 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