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UBC Theses and Dissertations

Initial characterization of a new histone and role of methionine in protamine biosynthesis in trout testis Wigle, Donald Theodore 1970

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INITIAL CHARACTERIZATION OF A NEW HI ST ONE AND A ROLE FOR METHIONINE IN. PROTAMINE BIOSYNTHESIS IN TROUT TESTIS  by  DONALD THEODORE WIGLE M.D., U n i v e r s i t y o f Western. O n t a r i o , 1966  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR; THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the Department o f B i o c h e m i s t r y F a c u l t y o f Medicine  We accept t h i s  t h e s i s as conforming  to the r e q u i r e d s t a n d a r d  October, 1970 U n i v e r s i t y o f B r i t i s h Columbia  In p r e s e n t i n g  this  thesis  an a d v a n c e d d e g r e e a t the L i b r a r y I  further  for  agree  scholarly  by h i s of  shall  this  written  the U n i v e r s i t y  make  it  freely  that permission  for  It  of  Columbia,  British for  gain  of Columbia  the  requirements  reference copying o f  I agree and this  shall  that  not  copying or  for  that  study. thesis  by t h e Head o f my D e p a r t m e n t  is understood  financial  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  of  for extensive  permission.  Department  fulfilment  available  p u r p o s e s may be g r a n t e d  representatives. thesis  in p a r t i a l  or  publication  be a l l o w e d w i t h o u t my  -i-  ABSTRACT Histones are the b a s i c p r o t e i n s the chromosomes of e u k a r y o t i c described  histone  complexed w i t h MA  in  organisms. A p r e v i o u s l y  un-  ( h i s t o n e T.) was  discovered  i n chromatin  prepared from rainbow t r o u t (Salmo g a i r d n e r i i ) t e s t e s . Histone T was  p u r i f i e d by s e l e c t i v e e x t r a c t i o n and  exchange chromatography on C M - c e l l u l o s e . of t h i s p r o t e i n was  Histone T was  disc gel electrophoresis, urea starch and g e l f i l t r a t i o n chromatography.  homogeneous as  ent c r i t e r i a . The  judged by the above  m o l e c u l a r weight was  by the m o b i l i t y of formylated SDS  homogeneity  examined by p o l y a c r y l a m i d e d i s c g e l  e l e c t r o p h o r e s i s , SDS gel electrophoresis  The  ion-  g e l e l e c t r o p h o r e s i s . The  differ14,500  found to be  or a c e t y l a t e d h i s t o n e  Ton  amino a c i d composition and  the  N-terminal amino acid' were determined. P e p t i d e maps of t r y p t i c peptides  of histone  cated  that histone  major  histones.  T i s not  T and !  the major h i s t o n e s  the  indi-  a d e g r a d a t i o n product of  the  D e t a i l s of the mechanism o f i n i t i a t i o n of p r o t e i n s y n t h e s i s i n eukaryotes have only r e c e n t l y been  discovered.  Protamines are the s m a l l , h i g h l y b a s i c p r o t e i n s w i t h DNA The  i n the mature sperm c e l l s  synthesis  complexed  of most h i g h e r  of protamine i n t r o u t t e s t e s was  studied.  C e l l suspensions prepared from t r o u t t e s t e s at the mine stage of d i f f e r e n t i a t i o n were incubated  animals.  prota-  in vitro  and  -ii-  were found t o i n c o r p o r a t e i n t a c t , i s o t o p i c a l l y l a b e l l e d methionine i n t o the N - t e r m i n a l sequence,  Met-Pro-Arg...,  of protamine. Pulse-chase experiments r e v e a l e d t h a t the methionine r e s i d u e i s removed from protamine a f t e r c h a i n completion. Enzymatic a c t i v i t y capable of c l e a v i n g the d i p e p t i d e , Met-Pro, was  found i n t e s t i s c e l l  fractions.  The amino group of methionine i n c o r p o r a t e d i n t o bound nascent protamine was c o r p o r a t i o n was  ribosome-  not b l o c k e d . Methionine i n -  extremely s e n s i t i v e to i n h i b i t i o n by c y c l o -  heximide. The evidence obtained i n d i c a t e s a r o l e f o r methionine i n the i n i t i a t i o n of protamine b i o s y n t h e s i s i n the t r o u t , a eukaryote.  -iii-  ACKNOWLEDGMENT  The author wishes t o express a p p r e c i a t i o n t o P r o f . G.H. Dixon f o r h i s e x c e l l e n t guidance and encouragement during the course of t h i s work. A p p r e c i a t i o n i s a l s o expressed t o Drs. C-L. Hew, J e r g i l , B» Malchy, K. Marushige, M. Sung and Mr. A. 5  Louie, P. Candido and.J. Durgo f o r exchange o f ideas and technical assistance. The M e d i c a l Research C o u n c i l of 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 f o r the p e r i o d 1967-70 t o the author. The taxpayers o f Canada and B r i t i s h Columbia are thanked f o r t h e i r f i n a n c i a l support o f the U n i v e r s i t y .  -iv-  DEDICATION  ELIZABETH, JACQUELINE AND  JEFFREY  -V-  TABLE OE CONTENTS ABSTRACT .,  i  ACKNOWLEDGMENT  i i i  DEDICATION:  iv  LIST OF TABLES  x  LIST OF' FIGURES  x i i  PART ONE INTRODUCTION  1  E a r l y S t u d i e s on Chromosomes  1  E x t r a c t i o n and P u r i f i c a t i o n o f Histones  . . . .  3  .  4  C h a r a c t e r i s t i c s o f Histones S t u d i e s on Chromatin  6  Repressors  . .  Histones as Repressors "Masking" Hypothesis Chromosomal RNA  11 . . . . . . . . . . . .  S t r u c t u r e of Histones Proposed  DNA-binding S i t e i n Histone TV  MATERIALS AND METHODS Chemicals  . . . . .  Abbreviations Source  . . . . .  o f Trout Testes  9  11 12 13  . . . .  15 21 21 21 22  P r e p a r a t i o n - o f Chromatin  22  M a l e y l a t i o n of Chromatin  23  D i s s o c i a t i o n o f Histone T by S a l t  24  -vi-  A c i d E x t r a c t i o n of Chromatin Gel  24  E x c l u s i o n Chromatography  . .  Ion-exchange Chromatography  25 25  Polyacrylamide D i s c G e l E l e c t r o p h o r e s i s  . . .  26  M o l e c u l a r Weight Determination by SDS Gel  Electrophoresis  26  Amino A c i d A n a l y s i s  27  Tryptic Fingerprints  28  Ni-terminus Determination  30  RESULTS AND. DISCUSSION,  32  M a l e y l a t i o n o f Chromatin  32  D i s s o c i a t i o n o f Histone T: by S a l t E x t r a c t i o n o f Chromatin  37  w i t h \\4> TCA  37  P u r i f i c a t i o n of Histone T  37  M o l e c u l a r Weight D e t e r m i n a t i o n by SDS Gel  Electrophoresis  37  Amino A c i d Composition  47  Comparative  47  Peptide Maps  N-terminus Determination  .  52  . .  54  . . . .  57  R e l a t i v e Amount of Histone T i n T e s t i s Chromatin  a t Various Stages  of Maturation  Histone T i n T i s s u e s Other Than T e s t i s Conclusion  BIBLIOGRAPHY FOR PART ONE  57  60  -vii-  PART TWO INTRODUCTION  66  H i s t o r i c a l Development of Knowledge o f P r o t e i n Synthesis  . . . .  66  Information f o r P r o t e i n Synthesis  70  Ribosomes  75  '  I n i t i a t i o n of P r o t e i n Synthesis i n Prokaryotes  80  F-Met-tRNAf  90  Elongation  93  Termination Eukaryotic I n i t i a t i o n  100 .  102  Protamine B i o s y n t h e s i s  107  MATERIALS AND METHODS Chemicals  110  Abbreviations  110  Source  111  o f Trout Testes .  I n c u b a t i o n o f C e l l Suspensions  112  E x t r a c t i o n o f Protamine  113  Starch Gel Electrophoresis  113  Ion-exchange Chromatography  0  110  .  115  Edman Degradation  115  T h i n - l a y e r Chromatography of PTH Amino Acids .  115  High Voltage Paper E l e c t r o p h o r e s i s  116  Autoradiography  116  Counting Paper Electrophoretograms  117  -viii-  117  Formylation Deformylation .  119  S y n t h e s i s of L a b e l l e d Marker Peptides . . . .  119  Carboxypeptidase B D i g e s t s  123  Pulse-Chase Experiments  123  I n h i b i t o r Studies  127  Nascent Peptides  128  Enzyme Assays  132  RESULTS AND DISCUSSION  136  I n c o r p o r a t i o n of I n t a c t 14c-methylmethionine i n t o Protamine  . . .  136  Methionine i s N-terminal  HO  S t r u c t u r e of Methionine Peptide o f Protamine  143  G e n e r a l i t y o f Methionine I n c o r p o r a t i o n  . . .  147  T r a n s i e n t Nature of Methionine 151  Incorporation Nascent  Protamine  a) Puromycin-released Nascent Peptides b) Ribosome-bound Nascent Peptides  . .  . . . .  159 162  S e n s i t i v i t y of Methionine I n c o r p o r a t i o n to I n h i b i t i o n by A n t i b i o t i c s  168  He.-formate I n c o r p o r a t i o n i n t o Protamine  . .  171  Enzyme Assays a) Deacylase A c t i v i t y b) Methionine Aminopeptidase  174 Activity  . . .  179  -ix-  Oonclusion BIBLIOGRAPHY FOR. PART TWO  181 185  - X -  LIST OF TABLES PART..ONE  Page  1. Nomenclature  and C h a r a c t e r i s t i c s of the 5  Major Histones 2.  Chemical Composition of V a r i e d Chromatins  3. M o b i l i t i e s of Coloured Markers  .  on High  Voltage Paper E l e c t r o p h o r e s i s 4.  7  29  D i s s o c i a t i o n of Chromosomal P r o t e i n s by 36  Maleylation 5 . M o l e c u l a r Weights o f Trout T e s t i s Histones T and TTbo  as Estimated by M o b i l i t y During  E l e c t r o p h o r e s i s on P o l y a c r y l a m i d e Gels i n the  6.  Presence o f 0.1$ SDS  45  Amino A c i d Analyses o f H i s t o n e T . . . . . .  48  PART TWO 1 . E. c o l i Ribosomes  76  2.  86  P r o p e r t i e s of E. c o l i I n i t i a t i o n F a c t o r s . .  3 . Nomenclature  and C h a r a c t e r i s t i c s of 94  Elongation Factors 4.  Edman Degradation of  -methionine  L a b e l l e d Protamine  141  5.  Pulse-Chase Experiment B  157  6.  Edman Degradation of Ribosome-bound methionine L a b e l l e d Nascent Peptides . . . .  7.  E f f e c t of Aminopterin on 35s-methionine  169  -xi-  I n c o r p o r a t i o n i n t o Protamine  .  172  8. Deacylase A c t i v i t y 9.  Properties  of E. c o l i  176 K-acetyl-  ornithinase  177  10. Methionine-removing A c t i v i t y 11. P r o p e r t i e s  o f an Aminopeptidase  Ribosomes of E. c o l i  180 on 182  -xii-  LIST OP FIGURES PARI ONE  Page  1. H e l i c a l Wheel Arrangement of the N-terminal 18 Residues  of Histone TV/  1?  2. P o l y a c r y l a m i d e D i s c G e l E l e c t r o p h o r e s i s o f Chromosomal P r o t e i n s Released by  Chemical  M o d i f i c a t i o n by M a l e i c Anhydride  34  3 ( a ) . G e l F i l t r a t i o n of Histones on. a Column, of B i o - G e l P-10 (5.5X140 cm) Eluted" w i t h 0.0.1 IT HOI  38  3(b). Polyacrylamide Disc G e l E l e c t r o p h o r e s i s  . .  38  4 ( a ) . Ion-exchange Chromatography of 5$ TCA E x t r a c t a b l e Chromosomal P r o t e i n s on C M - c e l l u l o s e . 4(b). Polyacrylamide Disc Gel E l e c t r o p h o r e s i s  . .  39 39  5. Rechromatography of Histone T on a Column o f CM-52 (1 .8X50 cm)  .40  6. E l e c t r o p h o r e s i s on P o l y a c r y l a m i d e Gels i n the Presence  o f 0.1$ SDS  42  7. E l e c t r o p h o r e s i s on P o l y a c r y l a m i d e Gels in. the Presence  o f 0.1$ SDS  43  8. M o b i l i t y - o f Standard P r o t e i n s Versus the Logarithm  of t h e i r M o l e c u l a r Weights . . . .  9. Spectrophotometric  Scan of Histone T- . . . .  44 49  10. T r y p t i c F i n g e r p r i n t s S t a i n e d w i t h Cadmium Ninhydrin 11. T r y p t i c F i n g e r p r i n t s Showing A r g i n i n e -  .  50  -xiii-  c o n t a i n i n g Peptides  .  12. T h i n - l a y e r Chromatography o f Dansylated dues of Histone  T, and Standard M S  51  Resi-  Amino  Acids  53  13. D i s c Gel E l e c t r o p h o r e s i s of A c i d E x t r a c t s o f Trout T e s t i s Chromatin at Various ing  Stages Dur-  N a t u r a l Maturation  55  14. A P l o t of the R e l a t i v e Amount of Histone per mg; of DNA  T  i n Chromatin Prepared from Trout  T e s t i s at Various Stages of N a t u r a l Maturation  56  15. D i s c Gel E l e c t r o p h o r e s i s of A c i d E x t r a c t s of Chromatin from D i f f e r e n t Trout T i s s u e s Two  PART  and!  Guinea P i g Tissues  .  58  TWO  1 . Formation of I n i t i a t i o n Complexes i n E ^  -  •„  coli  85  2. Mechanism of Elongation, i n Rat L i v e r . . . .  95  3. Mechanism of E l o n g a t i o n i n E. c o l i  96  4. Sequences of Protamine from Salmo gairdnerii  . . . . . . . .  1  5. Counting Electrophoretograms  .  6. Autoradiogram of Electrophoretogram t i o n Products  i n the S y n t h e s i s  08 118  of Reac-  of M e t - C 1 4  Pro 7. H O m e t h y l - m e t h i o n i n e I n c o r p o r a t i o n i n t o  121  -xiv-  Trout T e s t i s N u c l e a r B a s i c P r o t e i n s  . . . .  137  8. E l e c t r o p h o r e t o g r a m o f H Q - m e t h y l - m e t h i o n i n e L a b e l l e d Protamine on a Starch-urea-aluminum 139  lactate Gel 9.  T h i n - l a y e r Chromatography  o f the Reaction.  Products o f Edman Degradation of 35S-methionine 142  Labelled. Protamine 10.  E l e c t r o p h o r e s i s a t pH 4 . 3 8 of the Products o f D a n s y l a t i o n of 35s-methionine L a b e l l e d .  Protamine  144  1 1 . Autoradiogram of E l e c t r o p h o r e t o g r a m (pH 3 . 6 ) of Enzyme D i g e s t s of 35s-methionine  Labelled 145  Protamine 1 2 . Autoradiogram o f E l e c t r o p h o r e t o g r a m (pH 3 . 6 ) of Enzyme D i g e s t s o f C h e m i c a l l y Formylated 35s-methionine L a b e l l e d Protamine  . . . . .  1 3 . R e - e l e c t r o p h o r e s i s a t pH 1 . 9 of P e p t i d e A and the Marker P e p t i d e ,  148  f  P- 4-C-Met-Pro-Arg, 1  from P i g . 1 2  149  1 4 ( a ) . Autoradiogram of E l e c t r o p h o r e t o g r a m (pH 6 . 5 ) of P e p t i d e B .from P i g . 1 2  150  f  1 4 ( b ) . Autoradiogram of Descending Chromatogram o f Peptide B 15.  f  from P i g . 1 4 ( a )  Ion-exchange  Chromatography  150 of 35s-methionine 152  L a b e l l e d Protamine on C M - c e l l u l o s e 16.  Ion-exchange  Chromatography  of A l k a l i n e  Phos-  -XV-  phatase T r e a t e d 35s-methionine  Labelled Pro-  tamine on C M - c e l l u l o s e  153  17. Pulse-Chase Experiment A  155  18. Pulse-Chase Experiment B  156  19. Autoradiogram of E l e c t r o p h o r e t o g r a m (pE of 35s-methionine  3.6)  L a b e l l e d Products i n the  Cytoplasm o f T e s t i s C e l l s Incubated w i t h Puromycin  160  20. R e - e l e c t r o p h o r e s i s a t pHC6.5 o f 'Peptides' from F i g s . 19 & 21  161  21. Autoradiogram of E l e c t r o p h o r e t o g r a m (jpJi 3.6) o f Hc-formate L a b e l l e d Products i n the C y t o plasm o f T e s t i s C e l l s Incubated w i t h Puromycin  163  22. 35s_ -thion±ne L a b e l l e d Ribosomes F r a c t i o n me  ated on. Sucrose D e n s i t y G r a d i e n t s 23(a). E l e c t r o p h o r e s i s a t pH 3.6 D i g e s t s of 35s-methionine  164;  of Trypsin-C.pB; Labelled. Nascent  Peptides from Disome Region 23(b). R e - e l e c t r o p h o r e s i s (pH 1.9)  167 of the Peptides  from F i g . 23(a) 24. E f f e c t of I n h i b i t o r s on 35s-methionine c o r p o r a t i o n i n t o Protamine  167 In170  25. Assay f o r T e s t i s Deacylase ) E_35s-Met as S u b s t r a t e  175  b) Ac-35s-Met as S u b s t r a t e  175  a  PART ONE  INITIAL CHARACTERIZATION" OE A NEW (HISTONE T) FROM TROUT: TESTIS  HISTONE  -1 -  UTRODUQTION During the decade 1880-1890, t h e chromosomes o f c e l l n u c l e i were i d e n t i f i e d and? i n v e s t i g a t e d by h i s t o l o g i s t s . By 1900 t h e behaviour o f chromosomes i n c e l l d i v i s i o n and f e r t i l i z a t i o n was w e l l a p p r e c i a t e d  and in. 1903» W.S. Sutton gave  the f i r s t modern i n t e r p r e t a t i o n o f the r e l a t i o n s h i p between genes and chromosomes w i t h a c y t o l o g i c a l e x p l a n a t i o n gregation  of s e -  and independent assortment. Thus, a concrete  basis  was e s t a b l i s h e d f o r the phenomena o f i n h e r i t a n c e as d e s c r i b e d by the A u s t r i a n monk, Gregor Johann Mendel, i n a paper i n 1866.  The h i s t o l o g i c a l d e s c r i p t i o n o f t h e r e g u l a r and p r e c i s e  d u p l i c a t i o n and d i s t r i b u t i o n o f chromosomes d u r i n g c e l l d i v i s i o n plus t h e s e p a r a t i o n  ordinary  o f p a i r e d chromosomes at  meiosis a l l c o r r e l a t e d w e l l w i t h Mendel's observations and s t r o n g l y suggested t h a t chromosomes a r e t h e c a r r i e r s o f genes. The  f i n e s t r u c t u r e o f chromosomes c o u l d not be s t u d i e d  u n t i l t h e advent o f the e l e c t r o n microscope (EM). I n t h e l i g h t microscope, t h e fundamental element o f chromosomes i s the t h r e a d - l i k e chromonema; EM r e v e a l s t h a t chromonemata a r e a c t u a l l y bundles o f very fine f i b r e s o r single  f i b r e s which  are c o i l e d and s u p e r c o i l e d . These f i b r e s a r e 250 & i n d i a meter but d i s s o c i a t e i n t o two 100 $. f i b r i l s i n EDTA; digest i o n o f t h e f i b r i l B w i t h pronase r e l e a s e s one DHA duplex from each. The 100 £ f i b r i l B a r e c o i l e d and s u p e r c o i l e d ; removal o f h i s t o n e s  causes l o s s o f s u p e r c o i l i n g and r e p l a c e -  ment o f h i s t o n e s r e s t o r e s s u p e r c o i l i n g ( 1 ) .  Chemical s t u d i e s of the c e l l nucleus i n 1871  by F r i e d r i c h Miescher,  began w i t h a paper  a student o f Hoppe-Seyler. He  e x t r a c t e d n u c l e i of pus c e l l s w i t h a l k a l i and f r a c t i o n a t e d the e x t r a c t by a simple a c i d p r e c i p i t a t i o n s t e p . The p h a t e - r i c h p r e c i p i t a t e , now  known to be DNA,  phos-  seemed t o be  c h a r a c t e r i s t i c o f a l l n u c l e i t h a t he examined and thus  the  name " n u c l e i n " was  dis-  assigned to i t . Miescher  went on t o  cover protamine i n salmon spermatozoa but concluded was  a simple n i t r o g e n - r i c h base. His work was  A l b r e c h t K o s s e l who  that i t  continued  by  d i s c o v e r e d the h i s t o n e s , r e d i s c o v e r e d  1  protamine and d i d s t r u c t u r a l s t u d i e s on the n u c l e i c a c i d s ( 2 ) . The n u c l e i c a c i d bases and the b a s i c amino a c i d s o f h i s tones and  protamine were h i s p r i n c i p a l i n t e r e s t s . He f e l t  that  h i s t o n e s and protamine both arose from amino a c i d s r e l e a s e d from muscle p r o t e i n as i t wasted away d u r i n g the spawning m i g r a t i o n of salmonids.  The h i s t o n e s o f the u n r i p e  testis  c e l l n u c l e i were b e l i e v e d to be s i m p l i f i e d d u r i n g the r i p e n i n g process and sperm c e l l s . The Lilienfeld  d i r e c t l y converted t o the protamine o f mature term " n u c l e o h i s t o n e " was  g i v e n i n 1892  (3) t o the m a t e r i a l e x t r a c t e d from l e u k o c y t e s  by. or  minced thymus w i t h d i s t i l l e d water and p r e c i p i t a b l e i n a c e t i c acid. A f t e r Kossel's book i n 1928  ( R e f . 4 ) , l i t t l e work was  done  on h i s t o n e s f o r the next 25 y e a r s . Renewed i n t e r e s t i n t h i s area came w i t h the s u g g e s t i o n by Stedman and Stedman i n (Ref.5) t h a t gene a c t i v i t y i s suppressed  during  1950  differentia-  -3-  t i o n audi t h a t t h i s might be done by h i s t o n e s . I n V i t r o s t u d i e s (6-8)  have shown t h a t D M  coinplexed w i t h h i s t o n e s has a r e -  duced c a p a c i t y t o serve as a template pared to pure DNA.  f o r RNA  polymerase com-  S i m i l a r l y , removal of h i s t o n e s from c h r o -  matin by exposure t o h i g h i o n i c s t r e n g t h or a c i d i c s o l u t i o n s i n c r e a s e s the template  (9).  a c t i v i t y of the r e s i d u a l chromatin  These r e l a t i v e l y crude experiments suggest indeed r e p r e s s t r a n s c r i p t i o n o f DNA  that histones  can  but the exact mechanism  by which t h i s comes about i n v i v o remains a g r e a t gap i n c u r r e n t knowledge. Biochemical  s t u d i e s were l i m i t e d u n t i l r e c e n t l y by  dif-  f i c u l t y i n 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 s ; p r o t e o l y s i s during e x t r a c t i o n and c r o s s - l i n k i n g o f h i s t o n e s by d i s u l p h i d e bonds between c y s t e i n e r e s i d u e s both gave r i s e t o a l a r g e number o f components upon e l e c t r o p h o r e s i s and thus to the i d e a t h a t h i s t o n e s might be v e r y heterogeneous. L a r g e - s c a l e f r a c t i o n a t i o n by s e l e c t i v e e x t r a c t i o n and p r e c i p i t a t i o n developed  was  by Johns and co-workers (10-12) but t h i s method does  not y i e l d pure f r a c t i o n s . Ion-exchange chromatography on umns o f Amberlite and Luck ( 1 3 ) ;  IRC-50 was  col-  f i r s t used by Rasmussen, Murray  t h i s method has been used e x t e n s i v e l y by  Cole  and co-workers (14) f o r s t u d i e s o f h i s t o n e T ( f o r nomenclat u r e , see Table 1 ) w i t h good r e s u l t s but the other h i s t o n e f r a c t i o n s are  cross-contaminated.  Sung and Dixon. (15)  have been a b l e to p u r i f y each o f the  major h i s t o n e s by g e l f i l t r a t i o n on l o n g columns o f B i o - G e l  -4-  P-10  e l u t e d w i t h 0.01  N! HOI.  T h i s method can. he combined  w i t h another s u c h as ion-exchange chromatography to o b t a i n r e l a t i v e l y l a r g e amounts o f i n d i v i d u a l pure h i s t o n e s . B a i l e y and Dixon (unpublished r e s u l t s ) have d e v i s e d an  efficient  method f o r l a r g e - s c a l e p u r i f i c a t i o n o f h i s t o n e s  T  and  TTb-|.  The method i n v o l v e s a c i d e x t r a c t i o n of t o t a l h i s t o n e from chromatin,  a d s o r b t i o n and washing at low s a l t  concentration  on carboxymethyl c e l l u l o s e ( C M - c e l l u l o s e ) columns to remove n u c l e i c a c i d s , non-histone  p r o t e i n s and h i s t o n e T,  w i t h cyanogen bromide (w/w  i n 75$  20 h) and, HOI.  treatment  formic a c i d at R.T.  f i n a l l y , g e l f i l t r a t i o n on B i o - G e l P-10  for  i n 0.01  Cyanogen bromide cleaves the m e t h i o n i n e - c o n t a i n i n g  tones  (Tfbp.  111  and  T7)  Hi  his-  i n t o fragments s m a l l enough to be  r e t a r d e d d u r i n g g e l f i l t r a t i o n and thereby s e p a r a t e d the other h i s t o n e s (T and! TTb-|  from  ).  Table 1 summarizes t h r e e of the common nomenclature s y s tems f o r h i s t o n e s and g i v e s some of t h e i r s t u c t u r a l  charac-  t e r i s t i c s ; the nomenclature of Rasmussen, Murray and Luck w i l l be f o l l o w e d i n t h i s t h e s i s . One  may  (13)  note t h a t a l l the  h i s t o n e s are r e l a t i v e l y s m a l l b a s i c p r o t e i n s which would e x i s t as p o l y c a t i o n s at p h y s i o l o g i c a l pH.  In most t i s s u e s , the  his-  tone content of b a s i c r e s i d u e s (25-30 molest l y s i n e + a r g i n i n e ) i s s u f f i c i e n t to n e u t r a l i z e a l l of the n e g a t i v e l y c h a r g ed phosphates of DNA  (see Table 2 ) . The t o t a l number of h i s -  tone f r a c t i o n s i s q u i t e l i m i t e d i n a l l organisms s t u d i e d furthermore,  the s t u c t u r e of i n d i v i d u a l h i s t o n e  fractions  and,  -5-  TABLE 1 Nomenclature and C h a r a c t e r i s t i c s of the Major  Class  Rasmussen, Murray and Luck (13)  Lysinerich  la  Slightly lysinerich  Johns and B u t l e r (10)  M.W.  Moles$ Lys+Arg  T e r m i n i (60.61 )  f1  21,000 (Ref.62)  28 (Ref.64)  Frblocked C:lysine  TTb-,  f2a2  14,000**  23 (Ref.59)  Frblocked C:lysine  li 2  f2b  13,774 (Ref.63)  28 (Ref.63)  K: p r o l i n e C:lysine  TTT  f3  14,000**  21 (Ref.59)  BT:alanine C:alanine  1V  f2a1  11 ,282 (Ref.39)  25 (Ref.39)  N: Ac-Ser C:glycine  Tb  b  Argininerich  Histones*  * C a l f thymus ** Estimated from behaviour on e l e c t r o phoresis  and g e l f i l t r a t i o n  -6-  seems t o be s i m i l a r r e g a r d l e s s o f the o r i g i n ? the l a t t e r w i l l be d i s c u s s e d i n more d e t a i l  fact  later.  The N-termini are l i m i t e d t o p r o l i n e , a l a n i n e or a r e s i due blocked by an acetylfrgr/oup; p r o l i n e and blocked r e s i d u e s i n t h i s p o s i t i o n have been p o s t u l a t e d t o serve as p r o t e c t i o n f o r the p r o t e i n a g a i n s t d e g r a d a t i o n by aminopeptidases This may  (16).  be p a r t i c u l a r l y important f o r h i s t o n e s s i n c e they  are b e l i e v e d t o l a c k t e r t i a r y s t r u c t u r e (17) and t h e i r  N-  t e r m i n i would not be p r o t e c t e d by f o l d i n g as i n g l o b u l a r p r o teins. The term "chromatin" r e f e r s t o the extended  form  chromosomes present i n the c e l l nucleus between c e l l  of divi-  s i o n s and t h i s i s the form o f the chromosomes i n which r e p l i c a t i o n and DNA-dependent RNA  s y n t h e s i s are b e l i e v e d t o  occur. S e v e r a l groups (18-21 ) have developed f o r i s o l a t i o n of chromatin  DNA  s i m i l a r methods  from c e l l n u c l e i i n order t o p e r -  mit chemical and p h y s i c a l s t u d i e s t o l e a r n something about i t s o r g a n i z a t i o n . Table 2 i l l u s t r a t e s some c h a r a c t e r i s t i c s of chromatin and was  taken from a paper by Bonner et a l . ( 2 2 ) .  The mass r a t i o s r e l a t i v e t o MA t e i n s and RNA  of h i s t o n e s , non-histone  pro-  are g i v e n f o r s e v e r a l chromatin p r e p a r a t i o n s ;  the a c t i v i t y o f the chromatin as a template  f o r RNA  polymer-  ase i s g i v e n f o r each case but does not c o r r e l a t e w i t h the content of any o f the t h r e e components mentioned above. Most chromatin p r e p a r a t i o n s have a h i s t o n e : M A mass r a t i o c l o s e to u n i t y ; the average  content of 25-30 molest o f b a s i c r e s i -  •7-  TABLE 2 Chemical Compositions o f V a r i e d Chromatins -(22)  Mass R a t i o s * * Source o f Chromatin  DNA  Histone  Non-histone Protein RNA  Template* Activity  Pea embryo axis  1.03  0.29  0.26  12$  Pea bud  1.30  0.10  0.11  6  Pea growing cotyledon  0.76  0.36  0.13  32  Rat  1.00  0.67  0.04  20  Rat a s c i t e s tumour  1.16  1.00  0.13  10  Human HeLa cells  1.02  0.71  0.09  10  Cow thymus  1.14  0.33  0.01  15  Sea u r c h i n biastula  1.04  0.48  0.04  10  Sea u r c h i n pluteus  0.86  1.04  0.08  20  vegetative  liver  * Relative to MA ** R e l a t i v e t o M A  (deproteinized) as 1  -8-  dues can then be c a l c u l a t e d as being s u f f i c i e n t t o n e u t r a l i z e a l l t h e DNA phosphates  as mentioned  earlier.  I s o l a t e d chromatin c o n t a i n s bound RNA polymerase  and c a t a -  l y z e s the s y n t h e s i s o f RNA from the f o u r r i b o n u c l e o t i d e  tri-  phosphates. The enzyme has been p u r i f i e d from t h e chromatin o f pea p l a n t s ( 7 ) and v a r i o u s mammalian t i s s u e s ( 2 3 ) . The a c t i v i t y o f t h e endogenous enzyme i s r e l a t i v e l y low but c h r o matin can serve as a template f o r s y n t h e s i s o f RNA by added enzyme; r e a c t i o n mixtures w i t h excess E. c o l i RNA  polymerase  can s y n t h e s i z e RNA i n amounts up t o f i f t y times t h a t o f the template DNA s u p p l i e d . RNA s y n t h e s i z e d i n t h i s manner can serve as a template f o r p r o t e i n s y n t h e s i s w i t h products r e p r e s e n t a t i v e o f t h e t i s s u e used t o prepare t h e chromatin ( 2 4 ) . A l l chromatins show r e s t r i c t i o n o f template a c t i v i t y f o r RNA s y n t h e s i s compared t o d e p r o t e i n i z e d DNA from the same source; f o r example, Table 2 shows t h a t l i v e r chromatin has a template a c t i v i t y 20% that o f l i v e r DNA. The template a c t i v i t y o f a g i v e n chromatin p r e p a r a t i o n has been found t o be p r o p o r t i o n a l t o t h e a c t i v i t y o f t h e t i s s u e o f o r i g i n i n RNA s y n t h e s i s ; t h i s suggests t h a t the i n v i v o p r o p e r t i e s o f chromatin a r e preserved i n v i t r o . The RNA t r a n s c r i b e d from chromatin i n v i t r o has a base composition d i f f e r e n t t r a n s c r i b e d on d e p r o t e i n i z e d DNA ( 9 ) , i n d i c a t i n g same DNA sequences  from t h a t  t h a t the  a r e not t r a n s c r i b e d i n both eases. Paul  and G-ilmour (25) used the technique o f h y b r i d i z a t i o n t o show t h a t a r e s t r i c t e d p o r t i o n o f DNA i n chromatin i s t r a n s c r i b e d by RNA polymerase;  i n t h e case o f thymus chromatin they  -9-  found template a c t i v i t y 7$ t h a t u s i n g d e p r o t e i n i z e d  DNA.  In f u r t h e r experiments (26), i n v o l v i n g c o m p e t i t i o n h y b r i d i z a t i o n , these authors showed t h a t the p o r t i o n of M A  availa-  b l e f o r t r a n s c r i p t i o n i n chromatin from v a r i o u s t i s s u e s i s not the same. They a l s o e s t a b l i s h e d t h a t the RNA  transcrib-  ed i n v i t r o on chromatin i s i d e n t i c a l to t h a t t r a n s c r i b e d i n v i v o i n the same t i s s u e . T h i s i s good a d d i t i o n a l e v i dence t h a t chromatin maintains i t s o r i g i n a l p r o p e r t i e s a f t e r I s o l a t i o n and  assay i n v i t r o .  Development has been d e f i n e d as the s y n t h e s i s of a s p e c i f i c p r o t e i n a t a s p e c i f i c time (27). A s i m i l a r d e f i n i t i o n c o u l d apply to d i f f e r e n t i a t i o n which i s r e a l l y p a r t of development. This means t h a t the c e l l must be able t o s p e c i f i c a l l y a c t i v a t e those genes r e q u i r e d at a c e r t a i n time and i n a c t i v a t e or keep i n a c t i v a t e d those genes which are  not  r e q u i r e d ; thus, a r e t i c u l o c y t e employs i t s hemoglobin genes but not i t s myosin gene(s).  T h i s a b i l i t y of c e l l s has  long  i n t r i g u e d developmental b i o l o g i s t s and people i n r e l a t e d d i s c i p l i n e s and  some d e t a i l s of the mechanisms probably i n -  volved have been d i s c o v e r e d . Examples are the r o l e of i n ducers d u r i n g development and  the a b i l i t y of hormones t o  s t i m u l a t e s y n t h e s i s of s p e c i f i c p r o t e i n s i n c l u d i n g enzymes and s t r u c t u r a l p r o t e i n s . The  complete s e r i e s of steps i n -  volved i n the a c t i o n of inducers  or hormones are not known  but i n the case of the l a t t e r , i n c r e a s e s i n both the and  amounts of RNA  synthesized  i s one  kinds  of the e a r l i e s t e f f e c t s  observed i n many systems. "Repressors'*  w i l l be d e f i n e d as the agents r e s p o n s i b l e  f o r the s p e c i f i c and d i r e c t i n a c t i v a t i o n o f a gene or f a m i l y of genes i n a d i f f e r e n t i a t e d c e l l . Such agents have not been demonstrated y e t i n eukaryotes but a d i s c u s s i o n o f some o f t h e i r expected f e a t u r e s might be u s e f u l . I f one accepts  that  e u k a r y o t i c c e l l s have about 5X10-* genes (28) and t h a t each r e p r e s s o r c o n t r o l s as many as 10 genes ( e . g . f o r the enzymes i n a metabolic  pathway), then t h e r e would be the order of 5  5X10^ i n d i v i d u a l r e p r e s s o r s . E u k a r y o t i c c e l l s c o n t a i n about 5X10  12  daltons o f DNA and thus about 1 0  1 0  base p a i r s ; t o be  unique, a sequence would have to be 16 base p a i r s l o n g . A r e p r e s s o r must then r e c o g n i z e a DNA sequence a t l e a s t t h i s  long  and the r e c o g n i t i o n should be h i g h l y s p e c i f i c . T h i s could be :  accomplished by a p r o t e i n or a n u c l e i c a c i d but there are obvious problems w i t h e i t h e r s u g g e s t i o n . RNA has the d e s i r e d a b i l i t y by v i r t u e o f base p a i r i n g but t h i s r e q u i r e s e i t h e r s e p a r a t i o n of DNA  strands  or f o r m a t i o n  of a t r i p l e x i n this  r e g i o n . There might be problems w i t h removal o f an RNA r e p re ss or s i n c e the minimum of 16 base p a i r s would g i v e a s t a b l e complex. On the other hand, a p r o t e i n r e p r e s s o r might be removed a f t e r undergoing a hormone or other "inducer" duced conformational  change. One wonders i f there c o u l d have  evolved 50,000 i n d i v i d u a l p r o t e i n s each capable i n g a d i f f e r e n t DNA  pro-  of recogniz-  sequence; r e c o g n i t i o n o f a s i n g l e base  p a i r change i n a sequence of 16 or more base p a i r s i s a s k i n g  -11-  a l o t from a p r o t e i n . Of course, the r e p r e s s i o n mechanism may  not i n v o l v e these proposed r e p r e s s o r s . Histones appear t o l a c k the requirements  s o l e r e p r e s s o r s i n eukaryotes. fewer than 15  o f being  There are almost  the  certainly  i n d i v i d u a l histones i n a given tissue  (29)  and there i s l i t t l e t i s s u e or s p e c i e s v a r i a t i o n i n numbers or types of h i s t o n e s . Histones  w i t h h i s t o n e T p r e f e r r i n g r e g i o n s r i c h i n A-T  t i o n of D M  base p a i r s and h i s t o n e s 111 (30).  show o n l y l i m i t e d r e c o g n i -  and TY p r e f e r r i n g G-C  At h i g h i o n i c s t r e n g t h , p o l y l y s i n e binds  l y to A-T  p a i r s and p o l y a r g i n i n e to G-0  regions  preferential-  pairs (31).  The  sig-  n i f i c a n c e of these i n t e r a c t i o n s seems l i m i t e d i n view of the f a c t t h a t each h i s t o n e f r a c t i o n can completely DNA  in vitro  repress  (32).  Bonner and co-workers (22,24) found by s e v e r a l methods that s e l e c t i v e removal o f h i s t o n e s from chromatin i t s template  increases  a c t i v i t y to almost t h a t of d e p r o t e i n i z e d  DNA.  The s i m p l e s t i n t e r p r e t a t i o n o f the above f a c t i s t h a t h i s tones  are the s o l e r e p r e s s o r s of DNA  t r a n s c r i p t i o n i n chro-  matin; however, i t i s not p o s s i b l e to separate the e f f e c t s  of  the methods used t o remove the h i s t o n e s from t h e i r removal per s e , s i n c e both operations probably cause d r a s t i c changes i n the o r g a n i z a t i o n of the chromatin.  I f the r e s u l t s above  are v a l i d , then the non-histone  p r o t e i n s remaining  chromatin  transcription.  The  cannot alone r e s t r i c t  "masking" hypothesis  i n the  (33) r e q u i r e s the p r o t e c t i o n  -12-  o f c e r t a i n genes, perhaps by non-histone p r o t e i n s , while the remaining genes are r e p r e s s e d or masked by the h i s t o n e s . Thus h i s t o n e s would a c t i n a r e l a t i v e l y n o n s p e c i f i c manner but s t i l l an e s s e n t i a l one. Involvement  o f the non-histone p r o -  t e i n s i n the s p e c i f i c i t y o f r e p r e s s i o n i s i m p l i e d by the work of P a u l and Gilmour ( 3 4 ) . These authors r e c o n s t i t u t e d chromatin from i t s components and then assayed by t i o n h y b r i d i z a t i o n the RNA  product produced  p l a t e s under the a c t i o n of RNA  competi-  on these tem-  polymerase. They found t h a t  the non-histone p r o t e i n s are e s s e n t i a l i n the r e c o n s t i t u t i o n of a chromatin w i t h the same.properties as the ginal  ori-  chromatin.  There i s some evidence f o r ft. RNA which may  component i n chromatin  be c o v a l e n t l y bound t o a chromosomal p r o t e i n , ( 3 5 )  and seems to be r e q u i r e d f o r s p e c i f i c r e c o n s t i t u t i o n 37). T h i s chromosomal RNA  (36,  (cRNA) i s 40-60 n u c l e o t i d e s l o n g  by endgroup a n a l y s i s and c o n t a i n s 8-10  moles$ of d i h y d r o -  u r i d y l a t e or dihydrpthymidylate depending  on the s p e c i e s .  cRNA can be prepared by d i s s o l v i n g p u r i f i e d chromatin i n 4 M C s C l and c e n t r i f u g i n g a t highspeed; a p e l l i c l e forms a t the top of the tube and c o n t a i n s RNA  and p r o t e i n . The cRNA  i s r e c o v e r e d from the p e l l i c l e by phenol e x t r a c t i o n a f t e r pronase  digestion.  Huang (38) found t h a t p r i o r t o pronase d i g e s t i o n , cRNA i s a s s o c i a t e d w i t h a p r o t e i n i n a complex s t a b l e t o h i g h s a l t and agents capable of d i s r u p t i n g H-bonds; t h i s  implies  -13-  but does not prove the presence of a c o v a l e n t cRNA and  p r o t e i n . Pronase d i g e s t i o n and  bond between  alkaline hydrolysis  o f the cRNA:protein complex r e l e a s e d a small peptide  bound  to d i h y d r o u r i d y l a t e ; a c i d h y d r o l y s i s of t h i s " l i n k e r " p r o duced b e t a - a l a n i n e  and s e r i n e . Huang t h e r e f o r e  that cRNA i s l i n k e d t o a p r o t e i n by a peptide a s e r i n e r e s i d u e and u r e i d o p r o p i o n i c ring-opening  of  concluded bond between  a c i d , the product  of  dihydrouridylate.  Bekhor, Kung and Bonner (36) i z e s t o n a t i v e DNA  showed t h a t cRNA h y b r i d -  from the same s p e c i e s i n the presence of  5 M u r e a a t 1 °0 and  t h a t a mRNA p r e p a r a t i o n  source does not h y b r i d i z e to DNA  from the same  under these c o n d i t i o n s . I f  the work above can be confirmed w i t h the cRNA-protein complex c h a r a c t e r i z e d more completely, one would a n t i c i p a t e a v e r y important r o l e f o r the complex i n the c o n t r o l of gene  expression. Histones from widely d i f f e r e n t sources are remarkably  s i m i l a r i n behaviour on ion-exchange chromatography and amino a c i d composition, t e r m i n a l r e s i d u e s  and  t i c m o b i l i t y . Histone TV  from pea s e e d l i n g s  have been sequenced (39)  and  p o s i t i o n s and  and  electrophorec a l f thymus  d i f f e r i n sequence at only  even these s u b s t i t u t i o n s are c o n s e r v a t i v e ;  only other d i f f e r e n c e s are the number and t i o n and m e t h y l a t i o n  in  two the  s i t e s of a c e t y l a -  of e p s i l o n amino groups o f l y s i n e r e s i -  dues . Histone TV i s , t h e r e f o r e , the p r o t e i n whose s t u c t u r e i s most r i g i d l y conserved d u r i n g e v o l u t i o n o f any  yet  studied;  -14-  only w i t h cytochrome c has the sequence o f another  protein  from both a p l a n t (40) and an animal ( 4 1 ) been e s t a b l i s h e d and i n t h a t case more than o n e - t h i r d o f the r e s i d u e s d i f f e r * The r i g i d s e l e c t i o n r e q u i r e d t o preserve the 102 r e s i d u e s e quence of h i s t o n e T 7 suggests t h a t the e n t i r e molecule i s important i n i t s f u n c t i o n and any changes must be ous t o the organism.  deleteri-  The authors s t a t e t h a t s i n c e the o n l y  obvious constant f e a t u r e s of DNA  from pea s e e d l i n g s and  thymus are the sugar-phosphate backbone and the double c a l n a t u r e , h i s t o n e T 7 may  calf heli-  be u n i q u e l y s u i t e d t o i n t e r a c t  w i t h the h e l i c a l backbone of DNA;  both DNAs c o n t a i n the  same 4 bases, o f course, but these must be present i n the same sequences v e r y seldomly. T h i s h i s t o n e i s a l s o i n g because of the d i s t r i b u t i o n of b a s i c and  interest-  hydrophobic  r e s i d u e s ; 17 of the 27 b a s i c amino a c i d s are i n the N - t e r minal h a l f o f the molecule  and 5 of the 6 aromatic amino  a c i d s are i n the G-terminal h a l f . T h i s asymmetric " d i s t r i b u t i o n has been taken t o i n d i c a t e a DNA-binding s i t e i n the N-terminal h a l f and a hydrophobic  b i n d i n g s i t e i n the  C-  t e r m i n a l h a l f p o s s i b l y f o r i n t e r a c t i o n w i t h other p r o t e i n s (42). B u s t i n and Cole (43) c l e a v e d r a b b i t thymus h i s t o n e T a t i t s s i n g l e t y r o s i n e r e s i d u e w i t h N-bromosuccinimide; amino a c i d analyses and endgroup determinations o f the whole h i s t o n e and the two  fragments were done t o e s t a b l i s h the  o r i g i n of the fragments.  The N-terminal fragment  (M.W.  - 1 5 -  about 6000 from amino a c i d a n a l y s i s ) had 2 3 moles$ b a s i c r e s i dues and the C-terminal fragment (M.W. amino a c i d a n a l y s i s ) had  3 1 molest b a s i c r e s i d u e s . On  b a s i s of t h i s r a t h e r unimpressive suggested  about 15,000 from  asymmetry, the  the  authors  a DNA-binding s i t e i n the C - t e r m i n a l fragment;  s i n c e t h i s r e g i o n i s 2 . 5 times l a r g e r than the N-terminal p i e c e , they may  w e l l be c o r r e c t .  Cole and co-workers ( 4 4 - 4 7 ) have f r a c t i o n a t e d h i s t o n e T from v a r i o u s sources i n t o as many as 5 components by i o n exchange chromatography on Amberlite IRC-50; these components are a l l s i m i l a r i n s i z e and amino a c i d  composition.  One  of the p u r i f i e d components of h i s t o n e T from r a b b i t t h y -  mus  was  compared w i t h whole h i s t o n e T from the same source  by performing the N-bromosuccinimide cleavage and mapping of the fragments.  peptide  The N-terminal fragments showed  s e v e r a l d i f f e r e n c e s w h i l s t the C-terminal fragments were n e a r l y i d e n t i c a l . T h i s suggests t h a t h i s t o n e T has a v a r i able N-terminal r e g i o n and a constant C-terminal r e g i o n ; t h i s would be analogous  t o the i n d i v i d u a l chains of immunoglobu-  l i n s . Sequence a n a l y s i s of h i s t o n e T components should g i v e the f i n a l answer t o the nature of the h e t e r o g e n e i t y of h i s tone T and, h o p e f u l l y , some i n s i g h t i n t o i t s f u n c t i o n . Supporting evidence f o r the presence  o f DNA-binding s i t e s  i n h i s t o n e s comes from the work of Dixon and co-workers. At the stage of spermatogenesis are r e p l a c e d by protamine,  i n t r o u t t e s t i s when h i s t o n e s t h e r e i s a marked p h o s p h o r y l a t i o n  -16-  of h i s t o n e s which occurs independently sis  of p r o t e i n  (48,49). The s i t e s o f p h o s p h o r y l a t i o n  synthe-  i n the i n d i v i d u a l  h i s t o n e s were i n v e s t i g a t e d and r e c e n t l y these s t u d i e s were extended by Sung and Dixon (15,50). With t h e p o s s i b l e except i o n o f h i s t o n e TTbp, there appears t o be a s i n g l e s i t e o f phosphorylation  i n each of t h e h i s t o n e s . The s i t e i n h i s t o n e  T i s i n the C-terminal cleavage  fragment a f t e r N-bromosuccinimide  and i s i n the sequence Lys-Ser(P04 )-Pro-kLys; the  C-terminal  fragment was the r e g i o n suggested by B u s t i n and  Cole as a DNA-binding s i t e  (43). I n h i s t o n e s TTb-j and TV  the s i t e i s present i n the i d e n t i c a l N-iterminal sequence, N-Ac-Ser(P04)-Gly-Arg-Gly-Lys-Gly; t h i s evidence i n d i c a t e s a r e g i o n o f homology between these h i s t o n e s and. a l s o l o c a l i z e s the s i t e o f p h o s p h o r y l a t i o n  i n h i s t o n e TV i n the h a l f  o f the molecule r i c h i n b a s i c r e s i d u e s ( 4 2 ) . Sung and Dixon (15,50) have proposed a model f o r a DNAb i n d i n g s i t e i n t h e N-terminal  18 r e s i d u e s o f h i s t o n e T7 .  They assumed t h a t t h i s r e g i o n i s i n the a l p h a - h e l i c a l conformation when a s s o c i a t e d with DNA and arranged  t h e 18 r e s i -  dues i n t h e form o f a h e l i c a l wheel as proposed by S c h i f f e r and Edmundson (51) and i l l u s t r a t e d i n F i g . 1. The circumference of t h e wheel represents a right-handed terminus;  the p o l y p e p t i d e backbone of  a l p h a - h e l i c a l p r o t e i n as viewed from t h e C-  the p r o j e c t i o n s are t h e amino a c i d s i d e c h a i n s o f  consecutive r e s i d u e s spaced a t 100° i n t e r v a l s s i n c e there are 3.6 r e s i d u e s per t u r n i n an a l p h a - h e l i x . The s e r i n e r e s i d u e  Fig. 1 H e l i c a l Wheel Arrangement o f the N-terminal Residues of Histone  HELICAL  WHEEL OF RESIDUES  18  T7  1-18  OF  HISTONE  IV  Note: T h i s i l l u s t r a t i o n was taken from the work o f Sung and Dixon ( 5 0 ) . Consecutive  r e s i d u e s are numbered s t a r t i n g  from the N-terminus as #1. The l e f t  s i d e o f the f i g u r e  shows the h i s t o n e m o d i f i e d only by a c e t y l a t i o n o f the N-terminal  s e r i n e ; the r i g h t s i d e shows complete  mo-  d i f i c a t i o n a f t e r p h o s p h o r y l a t i o n and a c e t y l a t i o n . F u r t h e r e x p l a n a t i o n i s g i v e n i n the t e x t .  -18-  which i s phosphorylated  i n v i v o (48) i s s i t u a t e d on t h e  same s i d e of the h e l i x as a c l u s t e r o f f o u r l y s i n e s ; the s i d e s adjacent t o the c l u s t e r above a r e occupied by an a r c of 4 g l y c i n e s and an a r c i n which 4 out o f 6 r e s i d u e s are g l y c i n e . A molecular as a right-handed  model o f t h e sequence was c o n s t r u c t e d  a l p h a - h e l i x and was found t o f i t n e a t l y  i n t o the major groove o f a model o f double h e l i c a l M A i n the B form. The 4 amino groups o f t h e s i d e c h a i n s o f l y s i n e s 5, 8, 12 and 16 and the guanidino  group o f a r g i n i n e 17 a l l  make c l o s e contact w i t h 5 consecutive  phosphate groups on  the same DNA s t r a n d ; a r g i n i n e 3 i n t e r a c t s w i t h a phosphate group on the opposite M A  s t r a n d . The predominance o f g l y -  c i n e r e s i d u e s i n the arcs adjacent  t o the l y s i n e - s e r i n e a r c  allows the h i s t o n e sequence t o f i t completely  w i t h i n the ma-  j o r groove. At the time o f h i s t o n e replacement i n t r o u t t e s t i s ,  there  i s a l s o e x t e n s i v e a c e t y l a t i o n o f e p s i l o n amino groups o f l y s i n e r e s i d u e s i n the N-terminal  region of histone TV  (Oandido and Dixon, i n p r e p a r a t i o n ) . I f t h e l y s i n e - s e r i n e a r c i s completely  m o d i f i e d by a c e t y l a t i o n and  phosphoryla-  t i o n , the n e t charge o f the h i s t o n e up t o p o s i t i o n 18 w i l l change from +6 t o +0.3, assuming a charge o f -1.7 on the s e r i n e phosphate a t p h y s i o l o g i c a l pH. The model of Sung and Dixon i s supported  by t h e r e c e n t  work o f Wagner (80). One o f the assumptions o f the model i s that the N-terminal  region i s a c t u a l l y i n the a l p h a - h e l i c a l  -19-  conformation  i n a s s o c i a t i o n w i t h DNA;  charge on the l y s i n e s by the DNA  the n e u t r a l i z a t i o n of  phosphates would be r e q u i r e d  s i n c e charge r e p u l s i o n would d e s t a b i l i z e a h e l i x . Wagner found t h a t c a l f thymus h i s t o n e TV undergoes a marked c o n f o r mational or DNA  change i n the presence of p o l y v i n y l p h o s p h a t e  as i n d i c a t e d by changes i n the c i r c u l a r  (PVP)  dichroism  spectrum. The r e s u l t s i n d i c a t e d an i n c r e a s e i n the r a t i o of a l p h a - h e l i x to random c o i l i n the h i s t o n e upon i n t e r a c t i o n w i t h e i t h e r polymer. The a c t u a l i n c r e a s e i n h e l i c a l was  content  estimated t o be 10-23%; t h i s would i n v o l v e 10-23  102 r e s i d u e s i n the p r o t e i n and  o f the  f i t s n i c e l y w i t h the 18 r e -  sidue b i n d i n g s i t e o f the model. E v i d e n t l y , h i s t o n e T does not undergo a conformational DNA  change when a s s o c i a t e d w i t h  (81); one would then expect  a d i f f e r e n t binding s i t e f o r  t h i s h i s t o n e and the sequence data suggest (15). T h i s s i t e must be i n the proper a s s o c i a t i n g w i t h DNA  t h a t t h i s i s so  conformation  before  or e l s e the change i s too s m a l l to be  detected by the methods used. I f the homology between h i s t o n e s TTb-j and TV extends as f a r as 18 r e s i d u e s without  s i g n i f i c a n t change, then the  mo-^  d e l would apply to h i s t o n e TTb-j as w e l l . Thus, at l e a s t  two  of the h i s t o n e s have a s i t e s u i t e d f o r b i n d i n g t o DNA m o d i f i c a t i o n of t h i s r e g i o n by a c e t y l a t i o n and l a t i o n may  and  phosphory-  allow r e v e r s a l of b i n d i n g . S t r u c t u r a l s t u d i e s on  h i s t o n e s have t h e r e f o r e g i v e n c l u e s to the b a s i s o f t h e i r haviour i n chromatin a i d i n determining  and f u r t h e r s t u d i e s w i l l undoubtedly  their function.  be-  -20-  Part One of t h i s t h e s i s w i l l d e s c r i b e the work done on a h i s t o n e , t e n t a t i v e l y named h i s t o n e I , d i s c o v e r e d i n . t r o u t t e s t i s chromatin; t h i s h i s t o n e has not been d e s c r i b e d  pre-  viously-. Histone T was found t o be s i m i l a r t o h i s t o n e  TTbg  i n s i z e , N-terminal  r e s i d u e and amino a c i d composition.  How-  ever, s t r u c t u r a l s t u d i e s of the new h i s t o n e r e v e a l e d t h a t it  can not be a degradation  tones .  product  of any of the major h i s -  -21-  MATERIALS AMD METHODS Chemicals and A b b r e v i a t i o n s a) Chemicals Commonly used chemicals were o b t a i n e d commercially and were o f reagent grade. B i o - G e l P-10 and C M - c e l l u l o s e were obtained from Bio-Rad L a b o r a t o r i e s ; CM-52 and chromatography paper (3 MM) from Whatman; and p o r c i n e t r y p s i n from Novo I n d u s t r i . b) A b b r e v i a t i o n s Most a b b r e v i a t i o n s a r e those recommended by the J o u r n a l of B i o l o g i c a l Chemistry (J.Biol.Chem., 24-5, 1 , 1970). £ : Angstrom ( 1 0 ~  1 0  m)  h: hours R.T. : room temperature M.W. : molecular weight w/w: weight p e r weight v/v: volume p e r volume Ac:  acetyl  A-T: adenine-thymine base G-C:  guanine-cytosine base  pair pair  EDTA: e t h y l e n e d i a m i n e t a t r a a c e t a t e TRIS: tris(hydroxymethyl)aminomethane TCA: t r i c h l o r a c e t i c a c i d N-terminus: amino terminus C-terminus: c a r b o x y l terminus  -22-  DNSC1: 1-dimethylaminonaphthalene-5-sulphonyl DNS:  chloride  dansyl  Trout T e s t i s N a t u r a l l y maturing rainbow?trout t e s t i s  (Salmo  gairdnerii)  was k i n d l y donated by Mr. and Mrs. Hans Lehmann of the Sun V a l l e y Trout Farm a t M i s s i o n , B.C.  Testes were t r a n s p o r t e d (1  on i c e , washed and f r o z e n at -80 °C u n t i l the time of use. Experimental Methods 1. P r e p a r a t i o n o f Chromatin Chromatin was  i s o l a t e d from t r o u t t e s t i s by the method of  Marushige and Bonner (9) w i t h a l l steps performed a t 4 °C. Frozen t e s t e s were p a r t i a l l y thawed and homogenized i n a Waring blendor w i t h 3 volumes of saline-EDTA (0.075 M NaCl, 0.024 M EDTA, pH 8.0) a t highspeed f o r 2 minutes,  filtered  through s e v e r a l l a y e r s o f washed c h e e s e c l o t h and c e n t r i f u g e d at 1000 g f o r 10 minutes. The n u c l e a r p e l l e t was washed once by resuspending i n saline-EDTA and c e n t r i f u g a t i o n and then l y s e d i n 0.01 sedimented  M THIS b u f f e r (pH 8.0). Chromatin  was  at 10,000 g f o r 10 minutes and washed once w i t h  the same b u f f e r . Sheared chromatin was  prepared by homo-  g e n i z i n g chromatin i n 0.01  b u f f e r (pH 8.0)  i n 0.01  M phosphate  or  M TRIS b u f f e r (pH 8.0) a t highspeed i n the Waring  blendor f o r 2 minutes and then c e n t r i f u g i n g a t 10,000 g f o r 15 minutes  a f t e r which the sheared chromatin remains i n the  supernatant. In some experiments, chromatin was  further  h)  -23-  p u r i f i e d by l a y e r i n g 6 ml o f sheared chromatin over 25 ml o f 1.7 M sucrose underlayed w i t h 4 ml o f 2 M s u c r o s e . The upper o n e - t h i r d o f the tube was g e n t l y mixed and then c e n t r i f u g e d at 22,000 rpm f o r 2 h i n the SW-27 r o t o r o f a Spinco L2-65 p r e p a r a t i v e u l t r a c e n t r i f u g e . The chromatin p e l l e t was r e c o v ered by c a r e f u l l y a s p i r a t i n g t h e supernatant. 2. M a l e y l a t i o n Sheared chromatin was prepared i n 0.01 M phosphate b u f f e r (pH 8.0) and d i l u t e d t o 1 mg/ml as estimated by absorpt i o n a t 260 nm (1 mg chromatin DNA/ml has an A26O = 2 0 ) . The chromatin s o l u t i o n was s t i r r e d m a g n e t i c a l l y a t 4  and  f i n e l y powdered m a l e i c anhydride was s l o w l y added and t h e pH was maintained a t 8.0 by a d d i t i o n o f 0.1 N NaOH w i t h a m i c r o p i p e t t e . The end o f t h e r e a c t i o n was i n d i c a t e d by a constant pH a f t e r adding a l l t h e maleic anhydride and s t i r r i n g was continued f o r 30 minutes a f t e r t h i s time. The r e a c t i o n mix was then c e n t r i f u g e d a t 22,00.0 rpm f o r 2 h i n t h e SW-27 r o t o r ; the supernatant then c o n t a i n e d any p r o t e i n s r e l e a s e d by m a l e y l a t i o n . Chromatin c o u l d be removed q u i c k l y at the end o f t h e r e a c t i o n by adding a c e t i c a c i d t o lower the pH t o 4.5 which causes t h e chromatin t o aggregate, and i t can then be sedimented  by c e n t r i f u g a t i o n a t 10,000 g - f o r 1Q  minutes. A f t e r e i t h e r method, t h e supernatant was then 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 a t 37 °C f o r f o u r days o r 50 °C f o r two days i n order t o h y d r o l y z e t h e a c i d - l a b i l e maleyl groups. C o n t r o l h i s t o n e samples showed no d e t e c t a b l e  -24-  d e g r a d a t i o n a f t e r t h i s treatment when e l e c t r o p h o r e s e d on polyacrylamide d i s c g e l s . 3. S a l t D i s s o c i a t i o n 1.5 M NaCl i n 0.01 M TRIS (pH 8.0) was added s l o w l y w i t h vigorous s t i r r i n g t o sheared chromatin u n t i l the s a l t  con-  c e n t r a t i o n was 0.3 M. The s o l u t i o n was then a d j u s t e d t o pH 4.5 w i t h a d d i t i o n of a c e t i c a c i d and aggregated sedimented  chromatin  a t 10,000 g f o r 10 minutes. The supernatant was  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 a t 4 °C and l y o p h i l i z e d . 4.  Acid  Extraction  a) 5$ TOA Chromatin was homogenized a t lowspeed  i n a Waring  blendor while 10 or 20$ TCA was s l o w l y added u n t i l a f i n a l c o n c e n t r a t i o n of 5$ TCA was reached ( v / v ) ; s t i r r i n g was continued  f o r another minute. The e x t r a c t was then  centrifuged  twice at 20,000 g f o r 20 minutes. The supernatant was then adjusted t o 20$ i n TCA by a d d i t i o n o f s o l i d TCA w i t h r i n g and a heavy white p r e c i p i t a t e formed  stir-  immediately.  After  s t a n d i n g a t 4 °C f o r 1 h, t h e p r e c i p i t a t e d p r o t e i n was r e covered by c e n t r i f u g i n g a t 10,000 g f o r 20 minutes. The p e l l e t was washed once or twice w i t h ether and d r i e d i n vacuo. b) 0.2 M H S 0 2  4  Chromatin was e x t r a c t e d by homogenizing H S0 2  4  i n the Waring blendor f o r 2 minutes  w i t h 0.2 M  a t highspeed  fol-  lowed by c e n t r i f u g a t i o n a t 10,000 g f o r 20 minutes.twice.  -25-  The supernatant was  saved, 3-4  volumes o f c o l d 95% ethanol  was  added and p r e c i p i t a t i o n allowed to occur overnight at  -20  °C. The p r e c i p i t a t e was  r e c o v e r e d by c e n t r i f u g a t i o n ,  washed once w i t h ethanol and d r i e d i n vacuo. 5. Gel E x c l u s i o n Chromatography Histones were f r a c t i o n a t e d by chromatography on B i o - G e l P-10  or Sephadex G-100  i n g the A230  e l u t i n g w i t h 0.01  N HC1  and  monitor^  the e l u a t e .  o f  6. Ion-exchange Chromatography C M k i e l l u l o s e r e s i n was 0.5  N NaOH f o r 30 minutes, washed w i t h d i s t i l l e d water u n t i l  n e u t r a l , s t i r r e d i n 0.5 n e u t r a l . The HC1 s t i r r e d i n 1-2 10-15  t r e a t e d as f o l l o w s : s t i r r e d i n  N HC1  s t e p was  f o r 30 minutes and washed u n t i l  repeated and then the r e s i n  was  M L i C l f o r 10 minutes, allowed to s e t t l e f o r  minutes, decanted,  the L i C l step repeated 3-4  and the column poured. The column was 0.15 M L i C l i n 0.01  times  washed o v e r n i g h t w i t h  M l i t h i u m a c e t a t e pH  5.0.  The 5% TCA e x t r a c t a b l e p r o t e i n s were d i s s o l v e d i n 0.01 l i t h i u m a c e t a t e pH 5 . 0 umn  and a p p l i e d to the C M - c e l l u l o s e  (3X30 cm). The column was  col-  washed w i t h 50-100 ml of  s t a r t i n g b u f f e r and then a l i n e a r g r a d i e n t of L i C l was ed. The g r a d i e n t was  M  start-  generated from 1000 ml each of 0.15  and 0.75  M L i C l i n 0.01  M l i t h i u m a c e t a t e pH 5 . 0 .  r a t e was  about 100 ml/h and the A ^ Q of the e l u a t e was  The  2  t o r e d . S a l t c o n c e n t r a t i o n i n every f i f t h f r a c t i o n was  M  flow monimeasured  -26-  w i t h a c o n d u c t i v i t y meter making r e f e r e n c e t o standard s o l u t i o n s of l i t h i u m  chloride.  OM-52 r e s i n was obtained i n a p r e c y c l e d and p r e s w o l l e n form but f i n e s were removed by suspending  the r e s i n i n 1-2 M  L i O l and a l l o w i n g the l a r g e r p a r t i c l e s t o s e t t l e f o r about 30 minutes. A column was poured  and subsequent steps were  as above except f o r s a l t c o n c e n t r a t i o n and the f l o w r a t e was much slower. 7. P o l y a c r y l a m i d e D i s c G e l E l e c t r o p h o r e s i s The method o f Bonner et a l . ( 1 8 ) was f o l l o w e d f o r f r a c t i o n a t i n g h i s t o n e s on polyacrylamide d i s c g e l s . M o d i f i c a t i o n s i n c l u d e d t h e use o f l o n g e r g e l s (7 cm) and use o f methyl green t o judge t h e l e n g t h of e l e c t r o p h o r e s i s ; the l a t t e r dye was found t o migrate s l i g h t l y f a s t e r than p r o t a mine. The power s u p p l y was a d j u s t e d t o 4-5 m i l l i a m p e r e s per g e l and the p o t e n t i a l was u s u a l l y 25 v o l t s / c m . Gels were s t a i n e d i n 1$ Amido B l a c k 1GB  i n destaining solution  (7$ a c e t i c a c i d i n 40$ e t h a n o l ) f o r a t l e a s t 1 h and des t a i n e d w i t h s e v e r a l changes o f t h e d e s t a i n i n g s o l u t i o n . P r o t e i n samples were d i s s o l v e d a t 1 mg/ml i n 6 M u r e a and 0 . 0 1 - 0 . 0 3 ml was a p p l i e d t o each g e l a f t e r the apparatus was  completely arranged  and f i l l e d w i t h b u f f e r .  8. M o l e c u l a r Weight E s t i m a t i o n by SDS G e l E l e c t r o p h o r e s i s The method o f Weber and Osborn (52) was f o l l o w e d . Samples and standards were d i s s o l v e d i n 6 M u r e a i n d i a l y s i s  -27-  b u f f e r (0.01  M phosphate b u f f e r pH 7.0,  2-mereaptoethanol) at 0.5 1 h. 10$  mg/ml and  0.005 ml concentrated bromphenol blue and  SDS  incubated  or 15$ g e l s were prepared and  tus w i t h b u f f e r ; 0.01-0.05 ml  0.1$  and  at 37 °C f o r  p l a c e d i n the  of each sample was  2-mercaptoethanol and  appara-  mixed w i t h  0.005 ml  a p p l i e d to the a p p r o p r i a t e  l a y i n g the b u f f e r . E l e c t r o p h o r e s i s was  0.1$  0.05$  g e l by under-  a t about 5  volts/cm  f o r about 5 h or u n t i l the bromphenol blue marker had a l most reached the end  of the g e l s . Gels were then s t a i n e d i n  0.25$ Ooomassie B r i l l i a n t Blue i n 10$ methanol overnight 7.5$  and  destained  d i s t a n c e migrated  the g e l l e n g t h were measured  s t a i n i n g ; a f t e r s t a i n i n g , the g e l l e n g t h and  distance  grated by the p r o t e i n sample were measured. The the sample was  =  This formula allows  and  before mi-  m o b i l i t y of  l e n g t h of g e l before staining X  d i s t a n c e of dye migration  across  by  c a l c u l a t e d by the f o l l o w i n g f o r m u l a : distance of p r o t e i n migration  Mobility  50$  w i t h s e v e r a l changes o f  a c e t i c a c i d i n 5$ methanol. The  the bromphenol blue and  acetic acid i n  g e l s and  :  '—  l e n g t h of g e l a f t e r destaining  f o r s l i g h t d i f f e r e n c e s i n the p o t e n t i a l  f o r s w e l l i n g or s h r i n k i n g d u r i n g s t a i n i n g  destaining.  8. Amino A c i d  Analysis  Samples were hydrolyzed  i n 0.5  ml of 6.If HC1  i n evacuat-  -28-  ed s e a l e d g l a s s tubes a t 110  0 f o r various, times and then  d r i e d i n vacuo over NaOH. A f t e r r e d i s s o l v i n g i n 0.2 N s o d i um c i t r a t e b u f f e r (pH 2.2), a l i q u o t s were a p p l i e d t o the l o n g or s h o r t coluntns o f a Beckman Model 1200 amino a c i d a n a l y z e r u s i n g the 2 h procedure  (53). A few samples were analyzed  by the s i n g l e column method o f Devenyi  (54).  9. T r y p t i c F i n g e r p r i n t s Samples were d i g e s t e d w i t h p o r c i n e t r y p s i n (Novo t r i ) w i t h an enzyme:substrate at 37 °0 i n 0.2 M NH HC0 4  3  Indus-  r a t i o o f 1:25 (w/w) f o r 4 h  (pH 8.0). A f t e r l y o p h i l i z i n g and  r e l y o p h i l i z i h g from d i s t i l l e d water, the sample was r e d i s s o l v e d i n pH 6.5 b u f f e r ( p y r i d i n e / a c e t i c acid/water, 100: 4:900, v / v ) and a l i q u o t s equal t o 0.5 A-230  -^  vaii  a  o  f  Protein  were a p p l i e d t o Whatman 3 MM paper. E l e c t r o p h o r e s i s was at about 40 v o l t s / c m f o r about 1 h, the exact time being determined by the m i g r a t i o n of c o l o u r e d r e f e r e n c e dyes whose c h a r a c t e r i s t i c s are o u t l i n e d i n Table 3. The paper was d r i e d and the s t r i p w i t h the sample peptides was c u t out and sewn i n t o a new sheet o f Whatman 3 MM paper. Descending tography was then performed  chroma-  f o r 20 h w i t h t h e s o l v e n t s y s -  tem b u t a n o l / w a t e r / a c e t i c a c i d / f o r m i c a c i d , 45 15:15:1* v/v. :  The sheets were d r i e d and s t a i n e d w i t h cadmium n i n h y d r i n (55)  or phenanthrene quinone ( 5 6 ) . The l a t t e r  procedure  i n v o l v e s a f r e s h l y prepared s p r a y c o n s i s t i n g o f equal  vol-  umes o f 0.02$ phenanthrene quinone i n ethanol and 10$ NaOH  -29-  I  TABLE 3 Mobilities  o f Coloured Markers Paper  Electrophoresis  Dye  pH 2.1  DHP l y s i n e  Ser  DNP  Agmatine  Xylene Cyanol PF  Crystal Violet  pH 3.6  pH  6.5  u= 0.56  u= 0  u=  Ser  Lys  Asp  u= 1.1  u= 0.67  u=  Ser  Lys  Asp  u= -0.3  u= -0.43  u=  Lys  Asp  u= -0.86  u=  Ser  Lys  Asp  u= 2.0  u= 1.05  u=  Ser  Lys  Asp  u= 0.82  u= 0.46  u=  Orange G  Methyl Green  on H i g h V o l t a g e  0  -0.57  0.35  0.92  -0.91  -0.37  -30-  i n 60% e t h a n o l ; the chromatogram i s sprayed, d r i e d and examined under UV i l l u m i n a t i o n . A r g i n i n e - c o n t a i n i n g peptides f l u o r e s c e s t r o n g l y and may be c i r c l e d and photographed w i t h a P o l a r o i d camera w i t h a UV f i l t e r as d e s c r i b e d under "N-terminus d e t e r m i n a t i o n " below. The same chromatogram may be s t a i n e d w i t h cadmium n i n h y d r i n a f t e r washing w i t h ethanol 3 times and once w i t h 5% a c e t i c a c i d i n acetone. 10.  N-terminus The  Determination  d a n s y l a t i o n method o f Gray ( 5 7 ) was used t o i d e n t i -  f y the N-terminus o f h i s t o n e T. One mg o f the p r o t e i n was d i s s o l v e d i n 0 . 0 5 ml o f 0.1 M NaHCO^, l y o p h i l i z e d t o remove NH3, r e d i s s o l v e d i n d i s t i l l e d water and t h e pH checked  with  l i t m u s paper t o assure a pH c l o s e t o 8 . 0 . DNS-01 was d i s solved a t 50 mg/ml i n acetone was  j u s t b e f o r e use and 0.05 ml  added w i t h mixing t o t h e sample; the tube was s e a l e d  and incubated a t 37 °C f o r 3 h. A p r e c i p i t a t e formed d u r i n g the r e a c t i o n and was saved; t h e supernatant was d e s a l t e d by gel  f i l t r a t i o n on a column o f Sephadex G-10 (1X15 cm) e l u t e d  w i t h 0.01 N HC1 and then l y o p h i l i z e d . The p r e c i p i t a t e and supernatant f r a c t i o n s were both h y d r o l y z e d i n 6 N H01 a t 110 °0 f o r 8 h i n s e a l e d , evacuated g l a s s tubes. A f t e r d r y ing  i n vacuo over NaOH, the samples were r e d i s s o l v e d i n 0.025  ml o f 1 M NH4OH and 0.005 ml a p p l i e d t o each pf two t h i n l a y e r p l a t e s . These p l a t e s had been prepared by spreading a s l u r r y o f s i l i c a g e l G (25 g i n 50 ml water) i n a l a y e r  0.25  mm  t h i c k on g l a s s p l a t e s (20X20 cm)  Desaga spreader. The 100 C G  w i t h the a i d o f a  p l a t e s were a i r d r i e d and heated t o  f o r 15 minutes j u s t b e f o r e use. The  employed were those d e s c r i b e d p o l a r DNS  by Black and Dixon ( 5 8 ) ;  d e r i v a t i v e s are separated  (chloroform/methanol/acetic  s o l v e n t systems non-  i n s o l v e n t system 1  a c i d , 95:10:1, v/v) and  polar  d e r i v a t i v e s are r e s o l v e d i n s o l v e n t system 2 ( n - p r o p a n o l / concentrated  NH4OR", 80:20, v / v ) . The  about 1£ h and  the second about 2-2%  f i r s t system r e q u i r e s h t o g i v e adequate  s e p a r a t i o n . A f t e r d r y i n g , the p l a t e s were examined under i l l u m i n a t i o n and Model 160 oid  UV  photographed immediately w i t h a P o l a r o i d  camera w i t h a copy l e n s and UV  Land P i c t u r e R o l l Type 47  f i l t e r and  (3000 s p e e d ) .  Polar-  -32-  RESULTS AND  DISCUSSION  The m a l e y l a t i o n of chromatin was  s t u d i e d t o determine  whether chemical m o d i f i c a t i o n of h i s t o n e s i s alone c i e n t to promote t h e i r d i s s o c i a t i o n from DNA  suffi-  i n analogy  to the p o s t u l a t e d r o l e of the i n v i v o m o d i f i c a t i o n s o f h i s tones by s p e c i f i c enzymes. These i n v i v o m o d i f i c a t i o n s i n clude p h o s p h o r y l a t i o n  of s e r y l (48,66) and t h r e o n y l  groups, a c e t y l a t i o n of e p s i l o n - (67) and groups (68,69) and m e t h y l a t i o n (70,71 ) or guanidino  (66)  alpha-amino  o f epsilon-amino  groups  groups ( 7 2 ) . These m o d i f i c a t i o n s  b e l i e v e d t o be important  are  d u r i n g periods o f gene a c t i v a t i o n  (73,74) and at the time of h i s t o n e replacement by protamine as d i s c u s s e d i n the " I n t r o d u c t i o n " . P h o s p h o r y l a t i o n duces about 1.7  negative  charges per s e r i n e or  intro-  threonine  m o d i f i e d w h i l s t a c e t y l a t i o n removes the s i n g l e p o s i t i v e charge of amino groups. M e t h y l a t i o n may three methyl groups on the epsilon-amino  i n t r o d u c e up  to  group o f l y s i n e  but does not change the charge; m e t h y l a t i o n  i n c r e a s e s the  bulk of the s i d e c h a i n and the b a s i c i t y of the n i t r o g e n .  The  l a t t e r would not be s i g n i f i c a n t  the  former may  be very important  i n i t s i n f l u e n c e on the  a c t i o n o f h i s t o n e s w i t h other M a l e i c anhydride was s i n c e t h i s reagent  at p h y s i o l o g i c a l pH but  inter-  molecules.  chosen f o r chemical m o d i f i c a t i o n  r e a c t s r a p i d l y w i t h uncharged amino  groups; m o d i f i c a t i o n of a l y s i n e s i d e c h a i n by t h i s means w i l l change i t s charge from +1  to -1. Since h i s t o n e s  con-  t a i n 10-25 anhydride  moles% of l y s i n e , complete m o d i f i c a t i o n by  maleic  would reduce the net charge by 20-50 u n i t s per  molecule of 100  r e s i d u e s . By use  of r e l a t i v e l y m i l d c o n d i -  t i o n s and perhaps i s o t o p i c a l l y l a b e l l e d m a l e i c anhydride i t might prove p o s s i b l e to determine the s i t e s and numbers of l y s i n e s which must be m o d i f i e d t o allow a h i s t o n e to d i s s o c i ate from  DNA.  Chromatin from September stage n a t u r a l l y maturing t r o u t t e s t i s was  prepared  and m a l e y l a t e d  mental Methods". M a l e i c anhydride  as d e s c r i b e d i n " E x p e r i was  used at 10 or 20 times  molar excess c a l c u l a t e d on the b a s i s of 1 mg mg  of chromatin DNA  of h i s t o n e  t i c a l l y but e i t h e r without m a l e i c anhydride  iden-  or w i t h sodium  maleate i n s t e a d . P r o t e i n s r e l e a s e d from chromatin by treatment  above were demaleylated  polyacrylamide  15  and an average h i s t o n e content o f  moles% of l y s i n e . The c o n t r o l samples were processed  and  per  the  electrophoresed  d i s c gels w i t h the r e s u l t shown i n P i g .  The major p r o t e i n r e l e a s e d by m a l e y l a t i o n ( g e l s 3 and  on 2. 4)  i s a r a p i d l y m i g r a t i n g band w i t h twice the m o b i l i t y of h i s tone T.  Lesser amounts of h i s t o n e s T,  r e l e a s e d . The  1Tb  and TTb  1  f a s t component had been noted  a minor f r a c t i o n o f t r o u t h i s t o n e s but was  2  are a l s o  p r e v i o u s l y as not f u r t h e r s t u -  d i e d at t h a t time. A h i s t o n e component m i g r a t i n g i n t h i s p o s i t i o n has not been d e s c r i b e d i n other organisms and i t w i l l be r e f e r r e d t o below as h i s t o n e T ( f o r t r o u t ) . The  complete absence of h i s t o n e s 111  a n d T 7 i n the ?  -34-  Fig.  2  Polyacrylamide d i s c g e l e l e c t r o p h o r e s i s o f chromosomal p r o t e i n s r e l e a s e d by chemical m o d i f i c a t i o n by maleic  anhydride.  I  -  1  M i g r a t i o n was  2  3  4  5  from top to bottom and  6  7  8  the i d e n t i t y  of  the bands i s i n d i c a t e d by the l i n e s and numbers at l e f t . The 1 and  samples are numbered at the bottom and  5, t o t a l t r o u t t e s t i s h i s t o n e s ; 2, no  3 and 4, ten and anhydride; 6-8,  include:  additions;  twenty times molar excess of ten, twenty and  the  maleic  f i f t y times molar excess  of sodium maleate. 111d  i s the dimer o f h i s t o n e  to d i s u l p h i d e formation  between c y s t e i n e r e s i d u e s  1 c y s t e i n e r e s i d u e per molecule of h i s t o n e  111  111  due (Note:  i n trout).  -35-  d i s s o c i a t e d m a t e r i a l suggests t h a t they are e i t h e r u n a v a i l able t o the maleic  anhydride or e l s e chemical  modification  of amino groups alone i s not s u f f i c i e n t t o d i s s o c i a t e them. In a d d i t i o n , the r e l a t i v e amount of l y s i n e i s l e s s i n the a r g i n i n e - r i c h histones  i 11  and TV  and  the e f f e c t of maley-  l a t i o n would be l e s s than i n the other h i s t o n e s . The d i s s o c i a t i o n of h i s t o n e T would thus imply  facile  that i t i s r i c h  i n l y s i n e and as w i l l be shown l a t e r , t h i s i s t r u e . Gels 2 and  6-8  i n F i g . 2 show t h a t h i s t o n e s  from chromatin under the experimental sence of m a l e i c  anhydride. The  are not d i s s o c i a t e d c o n d i t i o n s i n the  a c t u a l amounts of p r o t e i n  d i s s o c i a t e d from chromatin by the experimental was  measured and  i s g i v e n i n Table  that d i s s o c i a t i o n of h i s t o n e s not due  ab-  conditions  4. These f i g u r e s  i s due  to m a l e y l a t i o n  confirm and  to the s a l t produced upon h y d r o l y s i s of excess  ma-  l e i c anhydride and n e u t r a l i z a t i o n w i t h NaOH. Although i t i s i n t e r e s t i n g t o note t h a t m a l e y l a t i o n i s s u f f i c i e n t t o d i s s o c i a t e c e r t a i n h i s t o n e s , the mechanism probably  i n v o l v e s extensive  and  random m o d i f i c a t i o n of l y -  s i n e s ; a l s o , the m o d i f i c a t i o n causes a l a r g e charge (+1 -1 ) and s i z e change of the l y s i n e s i d e c h a i n s . As  to  discussed  i n the " I n t r o d u c t i o n " , i n v i v o h i s t o n e m o d i f i c a t i o n i s c h a r a c t e r i z e d by r e s t r i c t i o n t o c e r t a i n s i t e s i n these t e i n s . Because of t h e i r l i m i t a t i o n s , the chemical t i o n s t u d i e s were d i s c o n t i n u e d T) was  further characterized.  and  the new  histone  pro-  modifica(histone  -36-  TABLE 4 D i s s o c i a t i o n of Chromosomal P r o t e i n s by M a l e y l a t i o n  Treatment  Mg P r o t e i n R e l e a s e d *  No a d d i t i o n s  1.0  Sodium maleate 2:1  1.0  10:1  0.8  20:1  0.9  50:1  1.0  M a l e i c anhydride 2:1  0.9  10:1  2.3  20:1  2.5  50:1  8.7  * Mg o f p r o t e i n r e l e a s e d i n t o the supernatant and measured by the Lowry method ( 6 5 ) .  -37-  In a d d i t i o n t o i t s r e l e a s e upon l i m i t e d m a l e y l a t i o n , h i s t o n e T c o u l d be e x t r a c t e d from chromatin at pH 4.5 was  b u t " i t was  by 0,3 M NaCl  found t h a t e x t r a c t i o n by 5% TCA  s i m p l e r and o f f e r e d the advantage of a v o i d i n g  l y t i c degradation TCA was  found  (75)  proteo-  during the p r o c e s s . E x t r a c t i o n w i t h  5%  to remove h i s t o n e s T and T as w e l l as some  protamine; a l l of the p r o t e i n i n the e x t r a c t c o u l d be c i p i t a t e d by a d d i t i o n of s o l i d TCA 20% P-10  (w/v). F u r t h e r p u r i f i c a t i o n was i n 0.01  N HC1  pre-  to a c o n c e n t r a t i o n of done e i t h e r on B i o - G e l  as i n F i g . 3 or on OM-cellulose  eluted  w i t h a l i n e a r g r a d i e n t of L i C l as i n F i g . 4. H i g h l y p u r i f i e d h i s t o n e T e l u t e s as a s i n g l e peak a f t e r h i s t o n e T B i o - G e l P-10  on  or before h i s t o n e T on C M - c e l l u l o s e . In Octo-  ber stage n a t u r a l l y maturing t r o u t t e s t i s , h i s t o n e T found to form about 0.5%  was  of the t o t a l h i s t o n e . Rechromato-  graphy of h i s t o n e T on CM-52 ( m i c r o g r a n u l a r  CM-cellulose,,  Whatman) i s shown, i n F i g . 5 and r e v e a l s a s i n g l e peak a t 230 nm w i t h a shoulder on the ascending  l i m b ; no  ences c o u l d be found by amino a c i d a n a l y s i s or  differ-'  peptide  mapping i n a l i q u o t s taken from e i t h e r s i d e of the peak. The molecular weight of h i s t o n e T was  estimated  by  the method of Weber and Osborn (52) which i n v o l v e s measuri n g the m o b i l i t y of the sample together w i t h standard  pro-  t e i n s of known molecular weight during e l e c t r o p h o r e s i s on polyacrylamide g e l s i n the presence of the detergent, ium dodecyl s u l p h a t e  sod-  (SDS). S i n c e h i s t o n e s are q u i t e b a s i c  -38-  Fig.  3  a) G e l f i l t r a t i o n of h i s t o n e s on a column o f B i o - G e l (5.5XHO cm) e l u t e d w i t h 0.01  BIO-GEL  IO  20  30  P-IO  I f HC1.  (5-5XI40cmJ  40  FRACTION  50  NO  60  P-10  70  ELUTE  WITH  6 Ol N  HCL  80  90  I00  110  I20  V0L«l4ml.  b) Polyacrylamide d i s c g e l e l e c t r o p h o r e s i s 1  2  3  Samples a r e : 1, t o t a l trout testis  histones;  2, peak A from P i g . 3a; and 3» peak B from P i g . 3a.  -39fig.  4  a) Ion-exchange chromatography o f 5$ TCA chromosomal p r o t e i n s  on  CM-cellulose.  FRACTION The column (3X30 cm) was of  extractable  NO-  eluted with a l i n e a r  gradient  LiCl.  b) P o l y a c r y l a m i d e d i s c g e l e l e c t r o p h o r e s i s 1  2  3 Samples a r e : 1, t o t a l trout testis  his-  tones ; 2, peak A from P i g . 4a; and 3, peak B from P i g . 4a.  Pig.  5  Rechromatography o f h i s t o n e T on a column of CM-52 (1.8X50  cm)  I-5-  l  o Li  IO-  >-  o  IO CM  <  -0-5  0-5-  ..•-B-B-  -0-3  .Q--D-  0  -Ol  O  20  40  60  80  I00  120  F R A C T I O N NO-  The column was  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 L i C l  generated from 300 ml each o f 0.2 M and 0.45 M L i C l i n 0.01  M l i t h i u m a c e t a t e pH  5.0.  cr <  -4(1  and might behave anomalously, a l i q u o t s of h i s t o n e T were e i t h e r f o r m y l a t e d by the mixed anhydride  method of Sheehan  and Yang (76) or a c e t y l a t e d as d e s c r i b e d i n  "Experimental  Methods" i n P a r t 2 of t h i s t h e s i s . E i t h e r of the above m o d i f i c a t i o n s would block a l l o f the f r e e amino groups of a h i s t o n e and markedly reduce i t s net charge at pH 7, pHi a t which the SDS  g e l s are e l e c t r o p h o r e s e d . I t was  served t h a t unmodified  h i s t o n e s are i n s o l u b l e i n the  the obSDS  b u f f e r u n l e s s 6 M u r e a i s p r e s e n t ; however, the m o d i f i e d h i s t o n e s are s o l u b l e even i n the absence o f u r e a . T y p i c a l g e l s are shown i n P i g . 6 and P i g . 7. A graph of m o b i l i t y versus the l o g a r i t h m of the molecular weight i s shown i n P i g . 8 and Table 5. Untreated  a summary of the r e s u l t s i s g i v e n i n h i s t o n e T gave a m o l e c u l a r  weight of  16,400 whereas formylated h i s t o n e T gave 14,500 and  acety-  l a t e d p r e p a r a t i o n s gave H , 1 ° Q . A c e t y l a t e d h i s t o n e TTbo gave a value of 14,400 which i s o n l y 4$ h i g h e r than  the  value assigned from the sequence data ( 6 3 ) .  method  The SDS  t h e r e f o r e seems t o be s u i t a b l e f o r e s t i m a t i o n of the molec u l a r weights of h i s t o n e s i f they are m o d i f i e d t o reduce t h e i r net  charge.  Weber and Osborn (52) d i d a c a r e f u l study on f o r t y t e i n s of known molecular weight u s i n g the SDS  pro-  method. They  found t h a t the m o b i l i t i e s were independent of i s o e l e c t r i c p o i n t or amino a c i d composition  and  depended s o l e l y on the  molecular weight. The r e s u l t s were e x c e l l e n t not o n l y f o r  -42-  *±g. 6 E l e c t r o p h o r e s i s on polyacrylamide  g e l s i n the presence o f  0.1$ SDS a c c o r d i n g t o Weber and Osborn ( 5 2 ) .  1  2  3  The samples a r e : 1,  4  5  35,000); 5, r i b o n u c l e a s e (M.W.  17,200); 4 , pepsin  8  (M.W.  13,700); 6, lysozyme  14,300); 7, t r y p s i n (M.W. 2 3 , 3 0 0 ) ;  trypsinogen  7  u n t r e a t e d h i s t o n e T; 2, formylated  h i s t o n e T; 3, myoglobin (M.W.  (M.W.  6  and 8, chymo-  (M.W. 25,700). There are some i m p u r i t i e s  i n the myoglobin sample and the r i b o n u c l e a s e sample. The chymotrypsinogen has p a r t i a l l y a u t o l y z e d . These are 15$ g e l s .  -43*ig. 7 E l e c t r o p h o r e s i s on polyacrylamide  g e l s i n the presence o f  0.1% SDS a c c o r d i n g t o Weber and Osborn ( 5 2 ) .  II »;  I  —  2  Samples a r e : 1, pepsin and lysozyme; 2, r i b o n u c l e a s e and chymotrypsinogen; 3 , myoglobin; 4 , t r y p s i n ; 5, a c e t y l a t e d h i s t o n e T; 6, formylated h i s t o n e T; 7, acetylated histone  Tib?.  There i s a slow contaminant  i n p e p s i n and the t r y p s i n i s p a r t i a l l y There i s a t r a c e o f h i s t o n e p l e . These are 15% g e l s .  T  autolyzed.  i n the h i s t o n e  TTbo  sam-  -44-  Fig. 8 M o b i l i t y of standard p r o t e i n s versus molecular  i  0-2  1  the l o g a r i t h m o f t h e i r  weights  1  0-4  1  i  1  0-6  i  0-8  r  MOBILITY  The m o b i l i t y o f thei standard  p r o t e i n s was  measured  and p l o t t e d a g a i n s t t h e i r e s t a b l i s h e d molecular as r e p o r t e d by Weber and Osborn ( 5 2 ) . The  p o i n t s above  r e p r e s e n t : 1, p e p s i n ; 2, chymotrypsinogen; 3, 4, myoglobin; 5, r i b o n u c l e a s e ; and 6,  weights  trypsin;  lysozyme.  -45-  TAB1E 5 M o l e c u l a r weights o f t r o u t t e s t i s h i s t o n e s T and TTb2 as estimated by m o b i l i t y d u r i n g e l e c t r o p h o r e s i s on p o l y a c r y l a mide g e l s i n the presence o f 0.1% SDS.  Apparent M.W.  Histone  M.W. c o r r e c t e d f o r mass o f modif y i n g groups  I. 1 Unmodified ii iii  Hb  16,400  16,400  Pormylated  15,300  14,500  Acetylated  15,300  14,100  2  15,300  14,400*  * This value f o r h i s t o n e 11 bo i s o n l y 4% h i g h e r than t h a t from the sequence data ( 6 3 ) .  -46-  g l o b u l a r p r o t e i n s but a l s o f o r h i g h l y h e l i c a l , rodrshaped molecules such as myosin and  tropomyosin. For 37  proteins  over the molecular weight range 10,000 t o 70,000 the maximal  d e v i a t i o n was  l e s s than 10$  and u s u a l l y l e s s than t h i s .  No s e r i o u s d e v i a t i o n s Were found. Dunker and Rueckert s t u d i e d 24 p r o t e i n s on SDS  (77)  g e l s and the e f f e c t of chemical  m o d i f i c a t i o n upon m o b i l i t y . They found t h a t 10$ g e l s gave good r e s u l t s over the molecular weight range 10,000 to 100,000 daltons w i t h an accuracy w i t h i n 5-6$ values  pf the accepted  f o r most p r o t e i n s . Ribonuclease, a b a s i c p r o t e i n ,  gave the o n l y s e r i o u s d e v i a t i o n by behaving 21$  too  largei  however, cytochrome c i s a l s o a b a s i c p r o t e i n and  d i d not  g i v e a s e r i o u s d e v i a t i o n . Lysozyme was  modified  chemically  t o c a r r y 8 e x t r a p o s i t i v e charges, 8 e x t r a n e u t r a l groups or 8 e x t r a negative  charges; the change i n m o b i l i t y w i t h  a charge change of 16 u n i t s was r i b o n u c l e a s e was  only 12$.  The  d e v i a t i o n of  found to be probably r e l a t e d to f o l d i n g  of the p r o t e i n s i n c e a l k y l a t i o n or d i s u l p h i d e reduced the d e v i a t i o n to l e s s than 10$; t e i n p r o b a b l y can b i n d more SDS  and  interchange  the unfolded  pro-  t h e r e f o r e migrate f a s -  t e r . I t seems s a f e to assume no problems i n f o l d i n g w i t h h i s t o n e T which has  no d i s u l p h i d e bonds so t h a t the mole-  c u l a r weight must be c l o s e t o 14,500. This f i g u r e i s c o n s i s t e n t w i t h the behaviour of h i s t o n e T d u r i n g g e l t i o n on Sephadex G-100  or on B i o - G e l P-10  or P-60  filtrasince i t  e l u t e s w i t h h i s t o n e s TT which have m o l e c u l a r weights c l o s e  •  -47-  to 14,000. Amino a c i d analyses  were performed on samples of p u r i -  f i e d M s tone T hydrolyzed  f o r v a r i o u s times w i t h the r e s u l t s  summarized i n Table 6. The number o f r e s i d u e s was c a l c u l a t e d assuming a molecular  weight c l o s e t o 14,500 and an i n t e g r a l  number o f a s p a r t i c a c i d r e s i d u e s . The main f e a t u r e s of the analyses  are the l a r g e amounts o f l y s i n e and a l a n i n e which  account f o r 50% o f the r e s i d u e s complete absence o f aromatic a c i d s . A spectrophotometric  i n t h e p r o t e i n and a l s o the  or s u l p h u r - c o n t a i n i n g amino scan  ( P i g . 9) o f a s o l u t i o n o f  h i s t o n e T showed no absorbance i n the 260-300 nm range and thus confirmed  the absence o f aromatics  including trypto-  phan., Analyses  o f the b a s i c amino a c i d s performed on a l o n g  b a s i c column showed a complete absence of h i s t i d i n e and methylated l y s i n e . The net charge on h i s t o n e T could be as h i g h as +37 a t pH 7 or as low as +21 i f none o f the a c i d i c s e x i s t as amides; a r e l a t i v e l y h i g h charge seems l i k e l y i n view o f the r a p i d m i g r a t i o n o f h i s t o n e T on  polyacrylamide  g e l s a t pH 4. T r y p t i c f i n g e r p r i n t s of h i s t o n e T were compared w i t h those  of h i s t o n e s T, TTb  1  and TTW w i t h the r e s u l t s shown  i n P i g s . 10 and 11. A f t e r c a r e f u l comparison, 13 of the 27 peptides  o f h i s t o n e T were found t o be unique t o i t and  these are marked i n P i g . 10; the remaining not be d i s t i n g u i s h e d from some present  peptides  i n other  could  histones.  A r g i n i n e - c o n t a i n i n g peptides were l o c a t e d by t h e phenan-  -48-  TABLE 6 Amino A c i d Analyses  of Histone T  Residue  Moles Percent 21 h 42 h 70 h  Lysine  23.1  23.4  22.1  27.2  0  0  0  0  Arginine  7.2  7.9  7.4  9.1  Aspartic  6.7  6.8  6.5  8.1  Threonine  1.6  1.6  1.5  2.0  Serine  5.6  5.2  5.6  7.1  Glutamic  6.1  6.0  6.7  8.1  Proline  12.3  11.4  11.4  14.1  G  0  0  0  Glycine  7.4  6.8  7.3  9.1  Alanine  25.4  25.5  26.3  31.2  3.4  3.9  4.0  5.0  Methionine  0  0  0  0  Isoleucine  0  0  0  0  1.2  1.4  1.3  2.0  Tyrosine  0  0  0  0  Phenylalanine  G  0  0  0  Histidine  Cysteine  Valine  Leucine  jr residues f o r M.W. = 0 <  o  Total  f  122  -49-  *ig. 9 S p e c t r o p h o t o m e t r y Scan of H i s t o n e T  WAVELENGTH  Histone TI was  nm  d i s s o l v e d i n water at about 0.5 mg/ml  and scanned i n a Unicam spectrophotometer (Model S P 800 B ) .  -5010  Pig.  T r y p t i c f i n g e r p r i n t s s t a i n e d w i t h cadmium n i n h y d r i n .  HISTONE  HISTONE  HISTONES  E l e c t r o p h o r e s i s was  T  I  II  a t pH 6.5  o r i g i n being marked w i t h an  i n the h o r i z o n t a l plane, the 0.  rt  M  Chromatography was  perform-  ed v e r t i c a l l y . Peptides shaded i n b l a c k were p o s i t i v e f o r a r g i n i n e by the phenanthrene quinone t e s t (see P i g . Peptides unique dash.  11).  to h i s t o n e T are marked w i t h a h o r i z o n t a l  -51 -  F i g . 11 T r y p t i c f i n g e r p r i n t s showing a r g i n i n e - c o n t a i n i n g  peptides.  HISTONE T  HISTONE  HISTONES  I  H  Chromatograms were sprayed w i t h phenanthrene quinone (56), d r i e d and photographed under UV The f l u o r e s c e n t  illumination.  spots are the peptides c o n t a i n i n g  arg-  i n i n e . These f i n g e r p r i n t s were prepared i d e n t i c a l l y to those i n P i g . 10 and as o u t l i n e d i n s e c t i o n #9 o f "Methods".  -52-  threne  quinone t e s t (56) as shown i n P i g . 11 i n which the  p o s i t i v e peptides  are r e v e a l e d as f l u o r e s c e n t s p o t s .  presence of 9 a r g i n i n e peptides  The  i n histone T i s consistent  w i t h the number of a r g i n i n e r e s i d u e s determined by amino a c i d a n a l y s i s and  assuming a m o l e c u l a r  the l a t t e r method i n d i c a t e d 9.1  weight of 14,500;  arginine residues  Table 6 ) . These f i n g e r p r i n t s a l s o e l i m i n a t e the i t y that histone T i s a degradation 11b.j  or 11b . 2  product  (see  possibilT,  of h i s t o n e s  Histone TV i s too s m a l l to g i v e r i s e to h i s -  tone T by degradation  and both the former and h i s t o n e  111  lack s u f f i c i e n t lysine for t h i s . On the b a s i s o f 27 l y s i n e and 9 a r g i n i n e r e s i d u e s i n h i s t o n e T one would expect up t o 37 peptides f i n g e r p r i n t . The  on a t r y p t i c  presence of o n l y 27 peptides on  the  f i n g e r p r i n t s performed c o u l d be due t o s e v e r a l reasons i n c l u d i n g incomplete  f r a c t i o n a t i o n of peptides by the  g e r p r i n t method, r e s i s t a n t cleavages The  presence of 14 peptides  or repeated  sequences.  i n histone T t r y p t i c digests  t h a t are i n d i s t i n g u i s h a b l e from those examined may  fin-  i n the other  histones  i n d i c a t e p a r t i a l homology between h i s t o n e T  and  the o t h e r s . This i s not s u r p r i s i n g i n view o f the homology between p o r t i o n s o f h i s t o n e s TTb  1  the  and TV as d i s c u s s e d i n  "Introduction". The N-terminus of h i s t o n e T was  investigated using  the  d a n s y l a t i o n method o f Gray (57) w i t h the r e s u l t s shown i n P i g . 12. The  t h i n - l a y e r p l a t e s r e v e a l DNS-Pro a f t e r  the  P i g . 12. T h i n - l a y e r chromatography of d a n s y l a t e d r e s i d u e s of h i s t o n e T and standard  DNS  SYSTEM I  amino a c i d s . The  p l a t e s were photographed under UV i l l u m i n a t i o n . Samples i n c l u d e : 1, 4, 7 and 11, DNS-Val; 2, 6 and 9,  DNS-  Pro; 3 and 10, mixture o f mono- and di-DNS-Lys; 5, p r e c i p i t a t e from dans y l a t i o n of h i s t o n e T; 8, s o l u b l e por t i o n a f t e r d a n s y l a t i o n of h i s t o n e  T;  SYSTEM 2  and 12, DNS-Arg.  I 2345678  9IOIII2  -5:4-  f i r s t s o l v e n t system and a l a r g e amount o f mono-DNS-Lys a f t e r the second s o l v e n t system i n both the p r e c i p i t a t e and  the s o l u b l e p o r t i o n a f t e r d a n s y l a t i o n  o f h i s t o n e T.  The  N-terminus must t h e r e f o r e be p r o l i n e and the mono-DNS-  Lys  a r i s e s from the l a r g e number o f i n t e r n a l l y s i n e r e s i -  dues. E a r l i e r experiments, u s i n g h y d r o l y s i s times o f 2024 h, were negative ty  probably because of the known  labili-  o f DNS-Pro t o a c i d h y d r o l y s i s . P o s i t i v e r e s u l t s were  obtained  a f t e r h y d r o l y s i s times o f 6-8 h. Histone T thus  resembles h i s t o n e TTbg i n i t s N-terminus, s i z e and l y s i n e : arginine r a t i o  (3 1). :  Chromosomal b a s i c p r o t e i n s were e x t r a c t e d from t r o u t t e s t i s a t d i f f e r e n t months o f n a t u r a l m a t u r a t i o n and e l e c trophoresed on polyacrylamide i n P i g . 13. The major h i s t o n e s  g e l s w i t h the r e s u l t s shown and h i s t o n e T-are a l l p r e -  sent a t a l l stages examined s i n c e the t e s t e s always c o n t a i n some immature c e l l s . The r e l a t i v e amount o f h i s t o n e T per u n i t weight o f DNA a t the v a r i o u s stages was measured by scanning the g e l s i n P i g . 13 w i t h the a i d o f a Jbyce-Loebl Chromoscan w i t h an i n t e g r a t o r ; the amount o f DNA from which each sample was e x t r a c t e d was measured by the Burton m o d i f i c a t i o n o f the Dische method ( 7 9 ) . The r e s u l t s i n P i g . 14 i n d i c a t e t h a t the r e l a t i v e amount o f h i s t o n e T reaches a maximum i n n a t u r a l maturing t e s t i s a t 7 weeks ( e a r l y October) and d e c l i n e s as protamine replacement occurs. r e s u l t s a r e ' s i m i l a r t o the o b s e r v a t i o n  These  o f Marushige and  Fig.  13  Disc g e l e l e c t r o p h o r e s i s o f a c i d e x t r a c t s o f t r o u t  testis  chromatin at v a r i o u s stages d u r i n g n a t u r a l m a t u r a t i o n .  0-2 M H S 0 2  4  5% TCA  TOTAL HISTONE • I  i i  |  AUG SEPT OCT NOV DEC  Chromatin was f i r s t  e x t r a c t e d w i t h 5% TCA and then w i t h  0 . 2 M H2SO4 as d e s c r i b e d i n "Methods". The d i r e c t i o n o f e l e c t r o p h o r e s i s was from r i g h t to l e f t .  -56-  Fig.  H  A p l o t o f the r e l a t i v e amount o f h i s t o n e T per mg of  DM  i n chromatin prepared from t r o u t t e s t i s a t v a r i o u s stages of n a t u r a l maturation.  The g e l s shown i n P i g . 13 were scanned u s i n g a Joyce-Loebl Ohromoscan w i t h an. i n t e g r a t o r ; the amount o f DNA i n the chromatin samples was measured by the Burton m o d i f i c a t i o n o f t h e Dische method ( 7 9 ) .  Dixon (78) t h a t the mass r a t i o o f histone:DUA reaches a maximum of 1.4  b e f o r e protamine appears and d e c l i n e s l a t e r  w i t h 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 d e c r e a s i n g at d i f f e r e n t r a t e s . T h i s i n c r e a s e i n histone:DNA r a t i o d u r i n g e a r l y  ma-  t u r a t i o n i s s u r p r i s i n g since i t implies e i t h e r synthesis h i s t o n e s independently of DNA.  of DNA  s y n t h e s i s or e l s e  of  degradation  The former p o s s i b i l i t y seems u n l i k e l y because h i s -  tone s y n t h e s i s has  been shown to be t i g h t l y coupled to  DNA  s y n t h e s i s (82-85); the l a t t e r p o s s i b i l i t y cannot be e l i m i n ated a t  present.  P i g . 15 shows t h a t h i s t o n e T i s present  i n other t r o u t  t i s s u e s i n c l u d i n g l i v e r , h e a r t and s p l e e n and t h e r e f o r e i s not t i s s u e - s p e c i f i c . The the guinea p i g and T i s present  o n l y other s p e c i e s examined  was  a component w i t h the m o b i l i t y of h i s t o n e  i n the t e s t e s of t h i s animal. The  only  tissue-  s p e c i f i c and s p e c i e s - s p e c i f i c h i s t o n e known i s the  lysine-  r i c h , s e r i n e - r i c h h i s t o n e present  erythro-  i n the n u c l e a t e d  cytes of s e v e r a l b i r d s (86). Conclusion A p r e v i o u s l y undescribed  h i s t o n e f r a c t i o n has  been p u r i -  f i e d from t r o u t t e s t i s chromatin and p a r t i a l l y c h a r a c t e r i z e d . This p r o t e i n , designated of about 14,500 and has  h i s t o n e T, has  a molecular  an unusual amino a c i d  even f o r a h i s t o n e . F i f t y percent  composition  of the r e s i d u e s are e i t h e r  l y s i n e or a l a n i n e and s u l p h u r - c o n t a i n i n g and acids are t o t a l l y absent. Histone  weight  aromatic  amino  T i s a minor component of  -58-  Fig.  15  D i s c g e l e l e c t r o p h o r e s i s of a c i d e x t r a c t s of chromatin  from  d i f f e r e n t t r o u t t i s s u e s and two g u i n e a p i g t i s s u e s .  0-2 M H S 0 2  4  5 % TCA ti I  TOTAL HISTONE GP- LIVER GP- TESTIS TROUT LIVER TROUT HEART TROUT SPLEEN HISTONE T  Chromatin was f i r s t  e x t r a c t e d w i t h 5$ TCA and then  w i t h 0.2 M H2SO4. The d i r e c t i o n of e l e c t r o p h o r e s i s was from r i g h t to l e f t . G.P. M  M  r e f e r s to g u i n e a p i g .  about 0 . 5 % of the t o t a l a t  t r o u t t e s t i s h i s t o n e s , forming the October stage  of n a t u r a l maturation.  This histone i s  r e a d i l y d i s p l a c e d from i t s a s s o c i a t i o n w i t h DNA  i n chroma-  t i n e i t h e r by chemical m o d i f i c a t i o n w i t h maleic  anhydride  or the presence of r e l a t i v e l y low  s a l t concentrations.  The  presence of t h i s h i s t o n e i n h i g h l y p u r i f i e d chromatin e l i m i n a t e s the p o s s i b i l i t y t h a t i t o r i g i n a t e s elsewhere i n the c e l l . Experimental  evidence  has  been d e s c r i b e d t h a t r u l e s  out the p o s s i b i l i t y t h a t h i s t o n e T might be a product  degradation  of the major h i s t o n e s . H i s t o n e T i s present  in  maximal amount at a stage before r a p i d h i s t o n e removal o c c u r s ; a degradation  product  would be maximal d u r i n g  the  time of h i s t o n e removal. Peptide maps r e v e a l e d s e v e r a l pept i d e s unique t o h i s t o n e T; a d e g r a d a t i o n have one  product  could only  or two unique p e p t i d e s .  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N e e l i n , J.M., and B u t l e r , C.G., Can.J.Biochem.Physiol., 39, 485 (1961).  PART TWO  A ROLE FOR METHIONINE IN THE INITIATION OF PROTAMINE BIOSYNTHESIS IN TROUT TESTIS  -66-  INTRODUQTION PROTEIN BIOSYNTHESIS The  i n t e r e s t i n p r o t e i n "biosynthesis i n r e c e n t years i s  e a s i l y explained  by the importance o f these molecules i n the  f u n c t i o n i n g of a l l b i o l o g i c a l systems. The complement o f p r o t e i n s i n a c e l l provides  much o f the b a s i s f o r the appearance  and behaviour of the c e l l , t h a t i s , the phenotype or s t a t e o f d i f f e r e n t i a t i o n . T h i s i s l a r g e l y a r e f l e c t i o n o f the a b i l i t y of p r o t e i n s t o i n t e r a c t i n a h i g h l y s p e c i f i c manner w i t h molecules^including  other  other p r o t e i n s . Examples o f such i n t e r a c -  t i o n s are the many enzymes w i t h a great v a r i e t y o f s u b s t r a t e s , subunits  o f enzymes, subunits  o f s t r u c t u r e s such as m i c r o t u -  bules and v i r u s e s , and r e p r e s s o r p r o t e i n s w i t h s p e c i f i c DNA sequences. P r o t e i n s perform key s t r u c t u r a l and r e g u l a t o r y r o l e s i n the c e l l and t h e i r b i o s y n t h e s i s must be c a r e f u l l y c o n t r o l l e d and p r e c i s e l y performed. The  c r y s t a l l i z a t i o n of some enzymes i n the l a t e 1920's  and  e a r l y 1930's showed t h a t p r o t e i n s a r e w e l l - d e f i n e d  cal  e n t i t i e s and hence amenable t o chemical and p h y s i c a l ana-  lysis.  chemi-  Sanger's work on i n s u l i n (1) r e v e a l e d f o r the f i r s t  time the complete sequence o f amino a c i d s i n a p r o t e i n ; the complexity  o f the sequence c l e a r l y e l i m i n a t e d o l d e r ideas  that  p r o t e i n s might c o n s i s t o f simple r e p e a t i n g amino a c i d s e quences ( 2 ) . As r e c e n t l y as 1951  some thought that p r o t e i n  s y n t h e s i s might proceed by r e v e r s a l o f p r o t e o l y s i s ( 3 ) or by  ;  -67-  the a c t i o n o f t r a n s p e p t i d a s e s  on e i t h e r amino a c i d s or pep-  t i d e s ( 4 ) . Others, however, r a i s e d o b j e c t i o n s  to these p r o -  posals s i n c e t h e e q u i l i b r i u m p o s i t i o n w i t h proteases i s f a r t o t h e s i d e o f h y d r o l y s i s ( 5 , 6 ) and these enzymes a r e not abs o l u t e l y s p e c i f i c i n d i s t i n g u i s h i n g between the s i d e c h a i n s o f amino a c i d s which are s i m i l a r ( 7 ) . C l e a r l y missing  was an understanding o f the r e l a t i o n -  s h i p between p r o t e i n s t r u c t u r e and g e n e t i c  information.  Beadle  h i n t e d a t such a r e l a t i o n s h i p i n h i s "one gene, - one enzyme" hypothesis ( 8 ) but d i d not make c l e a r t h e n a t u r e of the r e l a t i o n s h i p ; t h i s was o b v i o u s l y  due t o t h e l a c k o f knowledge about  the chemical b a s i s f o r genes a t t h i s time. The "sequence" hypot h e s i s was probably f i r s t s t a t e d by Dounce ( 9 ) i n 1952; he postulated  t h a t amino acids were ordered by s p e c i f i c  inter-  a c t i o n w i t h a n u c l e i c a c i d template and then polymerized i n t o a p r o t e i n . With t h e p u b l i c a t i o n o f t h e s t r u c t u r e o f D M i n 1953  (Ref.10) much i n t e r e s t i n g e n e t i c  coding was aroused and  Gramow (11) proposed t h a t t h e amino a c i d s f i t i n t o pockets on DNA formed by t h e base p a i r s i n a s p e c i f i c manner determined by stereochemical  r u l e s . Thus,people were beginning t o t h i n k  of the g e n e t i c i n f o r m a t i o n  f o r protein biosynthesis  as being  s t o r e d i n the base sequence o f a n u c l e i c a c i d . A h i n t as t o the nature o f t h e template was g i v e n as e a r l y as 1941 i n t h e work o f Caspersson ( 1 2 , 1 3 ) and Brachet ( 1 4 ) who both found a good c o r r e l a t i o n between the amount o f r i b o n u c l e o p r o t e i n and the r a t e o f p r o t e i n s y n t h e s i s  i n various  cells.  -68-  The  d i s c o v e r y of H Q by Ruben and Kamen (15)  by the p r e p a r a t i o n  of amino a c i d s l a b e l l e d w i t h t h i s  (16) gave a g r e a t a i d to those s t u d y i n g The  followed  protein  isotope  biosynthesis.  systems used progressed from whole animals to i n v i t r o  s t u d i e s on organs, t i s s u e s l i c e s , c e l l suspensions and f r e e systems. The  cell-  e a r l y c e l l - f r e e systems were prepared from  t i s s u e s such as r a t l i v e r s i n c e there were problems i n removi n g any  i n t a c t c e l l s i n the b a c t e r i a l systems. S i e c k e v i t z  Zamecnik (17) found t h a t p r o t e i n s y n t h e s i s  and  in a cell-free  system from r a t l i v e r r e q u i r e d simultaneous o x i d a t i v e phosp h o r y l a t i o n by m i t o c h o n d r i a present  i n the homogenate and  the microsomes were most a c t i v e i n the s y n t h e s i s . T h i s ed e a r l i e r work (18) i n which d i n i t r o p h e n o l (DNP) i n h i b i t protein synthesis  was  i n mammalian c e l l s and DNP  ready known to d i s s o c i a t e o x i d a t i v e p h o s p h o r y l a t i o n p i r a t i o n (19). l a t e d i n 1941  Lipmann (20)  and K a l c k a r  (21) had  that  confirmfound t o  was  al-  from r e s -  both p o s t u -  t h a t phosphorylated d e r i v a t i v e s of amino a c i d s  were r e q u i r e d f o r p r o t e i n b i o s y n t h e s i s . Zamecnik (22)  found that ATP  or an ATP-generating system  c o u l d r e p l a c e the need f o r a c t i v e m i t o c h o n d r i a i n a c e l l - f r e e system; t h i s e l i m i n a t e d the p o s s i b i l i t y t h a t products other than ATP (23)  mitochondrial  were r e q u i r e d i n h i s system. Hoagland  s t u d i e d the mechanism of amino a c i d a c t i v a t i o n by  by l o o k i n g f o r ^ P-pyrophosphate exchange i n t o ATP 2  sence of amino a c i d s and pH'5" enzymes. Exchange H  and was  ATP  i n the occurred  shown to be dependent on the presence of an amino  pre-  -69-  a c i d . The  formation  the formation  of an aminoacyl adenylate was  by  of aminoacyl hydroxamates upon a d d i t i o n of  hydroxylamine and was AMP  implied  confirmed by the s y n t h e s i s  of 1 - l e u c y l -  (24); the l a t t e r compound, i n the presence of pyrophos-  phate and The  "pH  5" enzymes, l e d to the s y n t h e s i s  of  ATP.  f a t e o f the a c t i v a t e d aminoacyl-AMP d e r i v a t i v e s  was  unknown. H o l l e y (25) demonstrated an RNase s e n s i t i v e s t e p between a c t i v a t i o n of amino a c i d s and  their  incorporation  i n t o p r o t e i n . In the same year, Zamecnik et a l . (26) the d i s c o v e r y of ^ C - l e u c i n e  t i g h t l y coupled to an RNA  1  ponent present  reported com-  i n the highspeed supernatant (HSS); t h i s  f r a c t i o n contained  what i s now  known as t r a n s f e r RNA  RNA  (tRNA).  Hoagland e t a l . ( 2 7 ) showed the important r o l e of a m i n o a c y l tRNA i n p r o t e i n s y n t h e s i s ; Zachau, Acs  and  Lipmann (28)  found  that the c a r b o x y l group of the amino a c i d moiety i s l i n k e d by an e s t e r bond to the 2' or 3 moiety of adenosine a t the V  1  hydroxyl  group of the  ribose  terminus of the tRNA. The  p u r i f i c a t i o n o f an a c t i v a t i n g enzyme (29) l e d to the  first  discovery  that the same enzyme i s r e s p o n s i b l e both f o r the a c t i v a t i o n of an amino a c i d and  i t s c o u p l i n g t o the c o r r e c t tRNA.  C r i c k ' s adaptor hypothesis (30)  s t a t e d t h a t tRNA's were  adaptor molecules f o r c a r r y i n g amino a c i d s to the c o r r e c t posi t i o n on a h y p o t h e t i c a l RNA  template. D i s c o v e r y  of ayRNA f r a c -  t i o n w i t h the expected p r o p e r t i e s of a template f o r p r o t e i n s y n t h e s i s was i s now  made by s e v e r a l groups ( 3 1 - 3 3 ) ; t h i s f r a c t i o n  termed "messenger RNA"  or mRNA. The  assignment o f  co-  -70-  dons f o r each amino a c i d was  the climax of t h i s work  (34-  36). With the development of a c e l l - f r e e system from E. (37),  b a c t e r i a l p r o t e i n s y n t h e s i s has been s t u d i e d  l y and i s now  coli  extensive-  understood i n more d e t a i l than e u k a r y o t i c  pro-  t e i n s y n t h e s i s . Some of the known steps of p r o t e i n s y n t h e s i s i n b a c t e r i a l systems w i l l be d i s c u s s e d below and  eukaryotic  systems w i l l be r e f e r r e d to where they are known to d i f f e r from the former.  INFORMATION FOR  PROTEIN BIOSYNTHESIS  A l l b i o l o g i c a l systems, w i t h the e x c e p t i o n of RNA  phage  and RNA  v i r u s e s , c a r r y t h e i r g e n e t i c i n f o r m a t i o n i n the form  of DNA.  P r o t e i n s y n t h e s i s takes p l a c e on RNA  are u s u a l l y c o p i e d from DNA RNA  polymerase. The  by the enzyme(s), DNA-dependent  E. c o l i enzyme has been p u r i f i e d ( 3 8 )  c o n s i s t s o f 5 d i f f e r e n t subunits of about One  templates which  w i t h a t o t a l molecular  weight  330,000. o f the subunits  of the E. c o l i enzyme, r e f e r r e d to  as sigma f a c t o r , i s d i s s o c i a t e d from the enzyme a f t e r t i o n of RNA  s y n t h e s i s on a DNA  template ( 3 9 ) .  l a r f a c t o r s i n other b a c t e r i a ( 4 0 ) c o r r e c t i n i t i a t i o n of RNA a DNA  and  template.  initia-  Sigma and s i m i -  seem t o f u n c t i o n i n the  synthesis at r e s t r i c t e d regions  Thus, sigma allows t r a n s c r i p t i o n o f the  on  "pre-  e a r l y " genes of phage T4 (R f.41 ); one of these p r e - e a r l y e  genes codes f o r a p h a g e - s p e c i f i c f a c t o r , s i g m a ^ 4 , which d i s -  - 7 1 -  places the host sigma f a c t o r from the polymerase. The i s then capable  enzyme  of t r a n s c r i b i n g the " e a r l y " genes of T4 (42).  ceases t o t r a n s c r i b e E. c o l i DNA C l u s t e r s of 10-50  but  pyrimidines  on the same s t r a n d of  DNA  have been i m p l i c a t e d as s e r v i n g as b i n d i n g s i t e s f o r RNA  poly-  merase f o r i n i t i a t i o n of t r a n s c r i p t i o n ( 4 3 ) . The r a t e of  ini-  t i a t i o n at v a r i o u s genes i n v i v o i s known to v a r y b l y w i t h ribosomal RNA  considera-  (rRNA) and tRNA chains being  t e d most f r e q u e n t l y (44). The  initia-  "promoter" i s the most d i s t a l  element before the s t r u c t u r a l genes i n the l a c operon thus may  be the s i t e at which RNA  f a c t , d e l e t i o n s extending l a c DNA  polymerase binds  and  (45); i n  i n t o the promoter r e g i o n a b o l i s h  t r a n s c r i p t i o n (46). The  l a c r e p r e s s o r a c t s by  bind-  i n g at the promoter s i t e and p r e v e n t i n g i n i t i a t i o n of  RNA  s y n t h e s i s (47); chains i n i t i a t e d b e f o r e a d d i t i o n o f the r e p r e s s o r are completed. C o n t r o l of the r a t e o f i n i t i a t i o n  of  t r a n s c r i p t i o n at v a r i o u s operons i s o b v i o u s l y c r u c i a l s i n c e the average propagation the same, being 15-26  r a t e of a l l RNA  chains i n E. c o l i i s  n u c l e o t i d e s per second at 29 °C  Thus, d i f f e r e n c e s i n t o t a l s y n t h e s i s o f RNA f l e c t d i f f e r e n c e s i n r a t e s of RNA  (Ref.48).  f r a c t i o n s must r e -  initiation.  c h a i n t e r m i n a t i o n has r e c e n t l y been found to r e q u i r e  a p r o t e i n f a c t o r , rho, which seems to f u n c t i o n i n r e c o g n i t i o n of a t e r m i n a t i o n s i g n a l i n the DNA the absence o f rho, RNA  sequence ( 4 9 ) .  In  chains are l o n g e r than they should  and remain i n a complex with DNA and RNA :  polymerase; t h i s  appears to occur because t e r m i n a t i o n s i g n a l s are not  recog-  be  -72-  nized. There i s c o n s i d e r a b l e  evidence t h a t c o u p l i n g o f t r a n s -  l a t i o n w i t h t r a n s c r i p t i o n i s the r u l e i n E. c o l i w i t h r i b o somes b i n d i n g to RNA chains  a f t e r they ,have been i n i t i a -  ted, (50,51). T h i s c o u p l i n g may be e s s e n t i a l f o r mRNA f u n c t i o n , in. view of r e c e n t work i n d i c a t i n g t h a t mRNA i s r a p i d l y degraded i n the 5 " to 3' d i r e c t i o n i n the absence o f t r a n s l a t i o n (52,53). T r a n s l a t i o n c o u l d p r o t e c t the mRNA from a t t a c k by nucleases  i n the cytoplasm s i n c e s t u d i e s have shown t h a t  each ribosome s h i e l d up t o 35 n u c l e o t i d e s nucleases  are added t o the system (54»55). The enzyme(s) r e -  s p o n s i b l e f o r degradation  o f mRNA i n v i v o have not been i d e n -  t i f i e d d e f i n i t e l y but r e c e n t work (290) H. i *  1  of a mRNA when, the  has i m p l i c a t e d RNase  E. c o l i ; the l a t t e r enzyme degrades RNA in. the 5 ' to 3' 1  d i r e c t i o n w i t h r e l e a s e o f 5' n u c l e o t i d e s  and, i n t e r e s t i n g l y ,  t h i s enzyme r e q u i r e s the presence o f the e l o n g a t i o n f a c t o r s f o r a c t i v i t y ("elongation  f a c t o r s " w i l l be d i s c u s s e d  later  in. t h i s s e c t i o n ) . The c o n t r o l o f a c t i v i t y o f mRNA-degrading enzymes i s o f obvious importance i n determining  the  lifetime  of a mRNA molecule; the presence o f s i g n i f i c a n t secondary s t r u c t u r e i n a mRNA might p r o t e c t i t from degradation and thus a l l o w l o n g - l i v e d mRNA. Genetic  p o l a r i t y due t o nonsense codons i n a p o l y c i s -  t r o n i c mRNA c o u l d be due t o e n d o n u c l e o l y t i c  a t t a c k i n the  r e g i o n o f a c i s t r o n d i s t a l t o a nonsense codon s i n c e t h i s r e g i o n i s not t r a n s l a t e d and hence not p r o t e c t e d by the  -73-  s h i e l d i n g e f f e c t o f ribosomes. An exdnucleasescould then f o l l o w the polymerase the  i n the 5 ' t o 3' d i r e c t i o n and degrade  remainder o f t h e c i s t r o n s b e f o r e they could be t r a n s -  l a t e d . I n f a c t , two groups have evidence t h a t the amount o f mRNA d i s t a l  t o a nonsense codon i s decreased due t o a c c e l e r -  ated d e g r a d a t i o n (53,56). Studies by Oheng on human amnion c e l l s  (57) gave the  f i r s t demonstration o f mRNA i n a eukaryote soon a f t e r the d i s c o v e r y o f DNA-dependent RNA polymerase  i n mammalian l i v e r  and b a c t e r i a (58-60). The p r o p o s a l s t h a t e u k a r y o t i c mRNA's might be more s t a b l e than b a c t e r i a l (61) o r t h a t they might be "masked" (62) were confirmed i n experiments on p r o t e i n s y n t h e s i s i n f e r t i l i z e d sea u r c h i n eggs. Gross and C o u s i n eau (63,64) showed t h a t f e r t i l i z a t i o n l e d t o i n c r e a s e d p r o t e i n s y n t h e s i s even i n the presence o f actinomycin D a t l e v e l s i n h i b i t i n g RNA s y n t h e s i s almost completely. F u r t h e r experiments gave evidence f o r t h e presence o f masked RNA which i s somehow a c t i v a t e d by f e r t i l i z a t i o n (65-67). Thus, the  majority of protein synthesis after f e r t i l i z a t i o n  util-  i z e s maternal mRNA a l r e a d y p r e s e n t i n t h e u n f e r t i l i z e d egg. In the e r y t h r o p o i e t i c system, RNA s y n t h e s i s ceases upon t r a n s f o r m a t i o n from e r y t h r o b l a s t s t o normoblasts a l t h o u g h hemoglobin s y n t h e s i s s t a r t s l a t e r (68,69); the hemoglobin mRNA must be present i n an i n a c t i v e form d u r i n g t h e i n t e r v a l . RNase i n s e r i s i t i v e ^ r i b o s o m a l aggregates have been found i n pre-cleavage eggs o f A s c a r i s l u m b r i c o i d e s and could! he  -74-  activated f o r protein synthesis  and rendered s e n s i t i v e to  RNase by b r i e f exposure to t r y p s i n (70). S i m i l a r f i n d i n g s have been r e p o r t e d i n house f l i e s  (71 ). Monroy et a l . (72)  found evidence of a t r y p s i n s e n s i t i v e i n h i b i t o r of p r o t e i n s y n t h e s i s present  on ribosomes o f u n f e r t i l i z e d sea u r c h i n  eggs; they hypothesized hydrolyzed  t h a t the i n h i b i t o r y p r o t e i n ( s ) i s  by a protease  a c t i v a t e d by the i o n i c changes  which occur i n eggs a f t e r f e r t i l i z a t i o n .  I f confirmed, t h i s  i s an important example of c o n t r o l of p r o t e i n s y n t h e s i s  at  the t r a n s l a t i o n a l l e v e l . C o n t r o l of mRNA s y n t h e s i s i n eukaryotes by means of a sigma f a c t o r type of agent has not been demonstrated  but  m u l t i p l e forms of RNA  (73).  polymerase have been d e s c r i b e d  Sea u r c h i n embryos have 3 d i s t i n c t RNA  polymerase  activities  r e s o l v e d on DEAE-Sephadex while r a t l i v e r n u c l e i have 2 a c t i v i t i e s ; these f r a c t i o n s do not seem t o be i n t e r c o n v e r t i b l e . There seems t o be no s p e c i f i c i t y of the components f o r d i f f e r e n t s i t e s on DNA  i n v i t r o but one  component i n b o t h  t i s s u e s appears to be l o c a l i z e d i n the n u c l e o l i i n v i v o t h e i r f u n c t i o n s may are present  be q u i t e d i s t i n c t . The  i n the nucleoplasm. The  other  and  components  sea u r c h i n n u c l e o l a r  enzyme i n c r e a s e s during development from the b l a s t u l a t o g a s t r u l a stage; t h i s c o r r e l a t e s w e l l w i t h the known i n c r e a s e i n rRNA s y n t h e s i s at the g a s t r u l a stage (74). The has not been e l i m i n a t e d yet t h a t these RNA ities  possibility  polymerase  activ-  are r e l a t e d by a d d i t i o n of s i g m a - l i k e f a c t o r ( s ) t o a  -75-  common " c o r e " enzyme.  RIBOSOMES With t h e establishment o f t h e v i t a l r o l e o f ribosomes i n p r o t e i n s y n t h e s i s (17,75-77), groups soon began t o f i n d r a p i d l y sedimenting aggregates  o f ribosomes both i n v i t r o  i n b a c t e r i a l c e l l - f r e e systems (78,79) and i n v i v o i n r a b b i t reticulocytes  ( 8 0 ) . Warner, Knopf and R i c h (80) proved t h a t  hemoglobin chains a r e s y n t h e s i z e d on a 170 S aggregate o f f i v e 70 S ribosomes and mRNA; they proposed  t h a t each r i b o -  some bears a growing p o l y p e p t i d e c h a i n and t r a v e l s along t h e mRNA from one end t o the o t h e r w i t h new ribosomes e n t e r i n g at  one end while others f a l l  completed  o f f a t t h e other end w i t h t h e i r  p r o t e i n s . T h i s was the f i r s t  assignment o f an a c t i v e  r o l e f o r ribosomes i n p r o t e i n s y n t h e s i s . The d i r e c t i o n o f movement o f the ribosome was shown t o be 5' t o 3' (Ref.81 ) and t h e p o l y p e p t i d e c h a i n was found t o be i n i t i a t e d a t the amino terminus,(82). A summary o f the components o f E . c o l i ribosomes w i t h some o f t h e i r c h a r a c t e r i s t i c s i s g i v e n i n Table 1. There i s o b v i o u s l y a complex.structure  a s s o c i a t e d w i t h ribosomes and  t r a n s l a t i o n o f mRNA i n t o p r o t e i n must t h e r e f o r e r e q u i r e the f u n c t i o n s a s s o c i a t e d w i t h each component. 70 S ribosomes b e a r i n g no nascent p e p t i d e s , as a f t e r r e l e a s e w i t h puromycin, d i s s o c i a t e i n t o 30 S and 50 S s u b u n i t s when t h e M g  + +  c o n c e n t r a t i o n i s lowered  t o 1 mM or l e s s ; how-  -76  TABLE 1 E. c o l i Ribosomes  Subunit  30 S  rRNA S value  16 S  Number o f r-proteins  M.W.  0.53X10 (Ref.127) 6  20 (Ref.97)  Functions associated w i t h subunit .  B i n d i n g o f mRNA and i n i t i a t o r tRNA Proteins sensitive to s t r e p t o m y c i n , spectinomycin and temperature S i t e of ribosomal ambiguity mutation At l e a s t two p r o teins required f o r f i d e l i t y of translation  50 S  23 S 5 S  1.0X10° (Ref. 127) 36,000 (Ref.92)  34 (Ref.97)  Site of peptidyl transferase S i t e of p r o t e i n responsible f o r s e n s i t i v i t y to erythromycin  ever, i f nascent peptide Mg  i s present,  as w i t h EDTA i s r e q u i r e d  + +  d r a s t i c lowering of  ( 8 3 ) . The presence o f pep-  t i d y l tRNA on the 70 S ribosome must s t a b i l i z e t h e i n t e r a c t i o n of the, ribosomal s u b u n i t s . The reasons f o r t h e need f o r two subunits  w i l l be d i s c u s s e d below.(see  There are t h r e e rRNA components present i n the 30 S subunit subunit.  "Initiation").  w i t h a 16 S RNA  and both a 5 S and a 23 S RNA i n the 50 S  The two l a r g e r RNA's have a s m a l l amount o f pseudo-  u r i d y l a t e and methylated n u c l e o t i d e s e i t h e r on the base o r on t h e 2  1  w i t h t h e methyl group  hydroxyl  group o f the r i b o s e  moiety; the 5 S RNA has a higher G-0 content and l a c k s t h e unusual n u c l e o t i d e s . O l i g o n u c l e o t i d e maps of t h e 16 S and 23 S RNA's r e v e a l marked d i f f e r e n c e s o v e r a l l (84) and i n t h e s i t e s of methylation  (85). P h y s i c a l s t u d i e s (86-90) have  shown t h a t up t o 60-75% of t h e n u c l e o t i d e s  o f 23 S RNA are  i n v o l v e d i n base p a i r i n g which f o r c e s the RNA c h a i n t o f o l d c o n s i d e r a b l y i n t o a r a t h e r compact s t r u c t u r e . P a r t i a l s e quence a n a l y s i s of the 16 S RNA (91) has r e v e a l e d n o t o n l y h a i r p i n s but a l s o loops  j o i n e d by double stranded  regions  as i n tRNA. The 5 S RNA from E. c o l i (92) and e u k a r y o t i c cells  (93,94) has been sequenced. The E. c o l i 5 S RNA can  be d i v i d e d (on paper) i n t o two h a l v e s w i t h homology suggesting  considerable  an o r i g i n by gene d u p l i c a t i o n . P h y s i c a l  s t u d i e s r e v e a l ' t h a t 60-80% o f t h e n u c l e o t i d e s  o f 5 S RNA  are i n v o l v e d i n base p a i r s (95,96). At l e a s t twenty ribosomal p r o t e i n s  (r-proteins) are  -78-  present on the 30 S subunit and 34 on the 50 S subunit Seventy percent of these p r o t e i n s have a m o l e c u l a r  (97).  weight  between 10,000 and 20,000. S u r p r i s i n g l y , a l l 20 p r o t e i n s can be d i s s o c i a t e d from the 30 S s u b u n i t at h i g h i o n i c s t r e n g t h and then added back w i t h a good y i e l d of p a r t i c l e s a c t i v e i n p r o t e i n s y n t h e s i s ( 9 8 ) . Some f u n c t i o n s have been assigned t o r - p r o t e i n s by means of r e c o n s t i t u t i o n e x p e r i ments and through the study of r i b o s o m a l mutants.. Thus, p r o t e i n s r e s p o n s i b l e f o r s e n s i t i v i t y to s t r e p t o m y c i n spectinomycin  (99,100),  (101) and h i g h temperature (102) have been  l o c a l i z e d to the 30 S s u b u n i t . The s i t e of a mutation  30 S subunit i s a l s o the  causing misreading  (103) and at l e a s t  p r o t e i n s r e q u i r e d f o r f i d e l i t y of t r a n s l a t i o n (104).  two  The  50 S s u b u n i t i s the s i t e o f p e p t i d y l t r a n s f e r a s e , the enzyme which c a t a l y z e s peptide bond f o r m a t i o n (105,106) and  the  s i t e of a mutation g i v i n g r i s e to r e s i s t a n c e to e r y t h r o mycin  (107).  The  30 S s u b u n i t has a s i t e f o r b i n d i n g mRNA, a s i t e  f o r i n i t i a t o r tRNA and at l e a s t p a r t of the s i t e f o r aminoa c y l tRNA. The l a t t e r two s i t e s are r e f e r r e d to as the P s i t e ( f o r p e p t i d y l or i n i t i a t o r tRNA) and the A s i t e ( f o r aminoa c y l tRNA) (Ref.128,129). In b a c t e r i a , each s p e c i e s of rRNA i s independently t h e s i z e d and m o d i f i e d ; the maturation  syn-  process i n v o l v e s  mainly m e t h y l a t i o n and c o n v e r s i o n of u r i d y l a t e r e s i d u e s to pseudouridylate  (108). Newly s y n t h e s i z e d rRNA i s combined  -79-  w i t h r - p r o t e i n s and t h e r e f o r e sediments more r a p i d l y f r e e rRNA; a t l e a s t two i n t e r m e d i a t e s  than  can be i s o l a t e d which  are i n v o l v e d i n the f o r m a t i o n o f each s u b u n i t . There i s some evidence  t o support  i n i t s unmodified  the i n t e r e s t i n g i d e a t h a t rRNA,  form, serves as mRNA f o r the s y n t h e s i s o f  r - p r o t e i n s . Thus, nascent cell-free  probably  rRNA can serve as a messenger i n a  system and the p r o t e i n products  coelectrophorese  with r - p r o t e i n s (109). Mature rRNA acts as a messenger i n a cell-free  system i f neomycin i s added or i f i t s secondary s t r u c -  t u r e i s destroyed by h e a t i n g (110). Each rRNA has o n l y enough i n f o r m a t i o n t o code f o r about 20% o f the r - p r o t e i n s a s s o c i a ted w i t h i t ; t h e r e f o r e , e i t h e r the genes f o r rRNA are m u l t i ple  and d i f f e r e n t or e l s e the other r - p r o t e i n s must be coded  by another  s e r i e s of genes. The former p o s s i b i l i t y seems un-  l i k e l y s i n c e o n l y one s p e c i e s o f each rRNA i s found  in a  g i v e n organism o r o r g a n e l l e . In eukaryotes,  rRNA i s s y n t h e s i z e d i n the n u c l e o l u s as  a l a r g e 45 S p r e c u r s o r (111-114). While i n the n u c l e o l u s , the rRNA i s methylated  (115) and g r a d u a l l y degraded t o an  18 S and 28 S component plus fragments which are d i s c a r d e d (116,  117).  During  o r j u s t a f t e r i t s s y n t h e s i s , the 45 S RNA  becomes a s s o c i a t e d w i t h p r o t e i n i n an 80 S p a r t i c l e which gives r i s e t o the 60 S s u b u n i t and r e l e a s e s the 18 S RNA which r a p i d l y enters the cytoplasm  t o form the 40 S subunit  by a s s o c i a t i n g w i t h i t s complement o f r - p r o t e i n s (116-118). The  5 S RNA i s t r a n s c r i b e d on n o n - n u c l e o l a r DNA (119). The  -80-  s i t e o f r - p r o t e i n s y n t h e s i s i s not known hut l a b e l l e d r p r o t e i n s are found i n the n u c l e o l u s a f t e r s h o r t pulses and appear t o a s s o c i a t e w i t h rRNA i n the n u c l e o l u s . S i n c e B i r n s t i e l et a l . (120) showed t h a t Xenopus l a e v i s has s e v e r a l hundred copies of the gene f o r rRNA per c e l l , s i m i l a r f i n d i n g s have been r e p o r t e d i n D r o s o p h l l a and HeLa c e l l s  (121)  (122). The need f o r t h i s "gene a m p l i f i c a t i o n "  i s i l l u s t r a t e d by HeLa c e l l s which must make 1 0? new somes per c e l l g e n e r a t i o n of 18-24  hours. Assuming  ribo-  t h e r e are  400 rRNA genes per . c e l l (122), each gene must be transcribed). 20 times per minute; t r a n s c r i p t i o n o f a complete rRNA gene takes 2£ minutes  (123) and t h e r e f o r e , about f i f t y 45 S  RNA  molecules must be t r a n s c r i b e d s i m u l t a n e o u s l y on each gene. The simultaneous t r a n s c r i p t i o n o f a gene by s e v e r a l RNA  poly-  merase ,molecules has been observed i n the e l e c t r o n microscope (124). Amphibian  oocytes perform a remarkable r e p l i c a t i o n  of rRNA genes r e s u l t i n g i n the f o r m a t i o n of up.to 1000-nucleo-r li  per nucleus (125,126); the oocyte s y n t h e s i z e s enormous  numbers of ribosomes which are s u f f i c i e n t f o r the d e v e l o p ment o f the embryo up t o g a s t r u l a t i o n .  INITIATION  OP PROTEIN BIOSYNTHESIS  As each ribosome a s s o c i a t e s w i t h a mRNA molecule t h e r e must be a s p e c i f i c i n t e r a c t i o n t o ensure t h a t p r o t e i n s y n t h e sis  i s begun at the c o r r e c t codon; o t h e r w i s e , nonsense  would be s y n t h e s i z e d and the c e l l c o u l d not f u n c t i o n .  proteins  -81 -  The presence o f a s p e c i a l tRNA i n v o l v e d s o l e l y i n the i n i t i a t i o n o f p r o t e i n s y n t h e s i s i n b a c t e r i a l systems was d i s c o v e r e d by Marcker and Sanger (130). T h i s tRNA, N-Formylmethionyl-tRNAf (F-Met-tRNA ), i s now known t o i n i t i a t e a l l f  b a c t e r i a l p r o t e i n s (131-134) and probably a l l p r o t e i n s i n m i t o c h o n d r i a (135,136), c h l o r o p l a s t s (137) and blue-green algae (138). E. c o l i has two methionine a c c e p t i n g  tRNA's,  tRNAf ^ and t R N A ^ . These are b o t h charged by the same 6  et  methionyl-tRNA synthetase (139)  but o n l y methionine a t t a c h e d  t o tRNAj> t can be f o r m y l a t e d by a t r a n s f o r m y l a s e which r e e  quires N - F o r m y l t e t r a h y d r o f o l a t e as the formyl donor (140, 10  ;  141). Both o f these tRNA's respond t o the codon AUG but F-MettRNAf a l s o responds t o GUG;  t h i s degeneracy i n r e a d i n g of  the f i r s t l e t t e r o f a codon i s unique t o tRNAjp*. The codons AUG and GUG serve as i n i t i a t o r codons i n v i t r o (140,142) but o n l y AUG has so f a r been shown t o operate i n v i v o as d e t e r mined i n RNA  phage R17 and  (143).  A major c h a r a c t e r i s t i c of the i n i t i a t o r tRNA i s t h a t Met-tRNAf,  even when not f o r m y l a t e d , donates methionine ex-  c l u s i v e l y i n t o the N - t e r m i n a l p o s i t i o n of newly s y n t h e s i z e d p r o t e i n s whereas Met-tRNAjj donates i t s amino a c i d o n l y to i n t e r n a l p o s i t i o n s i n the p r o t e i n s (144). The i n a b i l i t y o f Met-tRNAf  to i n c o r p o r a t e methionine i n t e r n a l l y i s l i k e l y  due to the f a c t that i t cannot form a complex w i t h the aminoacyl tRNA b i n d i n g f a c t o r T„ (Ref. 145). Met-tRNAju, even  -82-  when f o r m y l a t e d c h e m i c a l l y , i s unable  t o serve as an i n i t i a -  t o r (146). These f a c t s make i t c l e a r t h a t t R M ^  e t  is specially  adapted f o r i t s f u n c t i o n and i n t e r a c t s s p e c i f i c a l l y w i t h a t l e a s t one enzyme ( t r a n s f o r m y l a s e ) and w i t h the ribosome-mRNA complex. A s p e c i a l i n t e r a c t i o n w i t h the ribosome i s a l s o i n d i c a t e d by the r a p i d r e a c t i o n o f F-Met-tRNAf w i t h puromycin (147), a r e a c t i o n o c c u r r i n g only w i t h s u b s t i t u t e d aminoacyltRNA's present i n the P s i t e o f the ribosome. I n i t i a t i o n cannot occur s i m p l y by s e l e c t i n g the AUG  first  codon adjacent t o the 5' end o f the mRNA s i n c e i t i s  known t h a t i n i t i a t i o n occurs s i m u l t a n e o u s l y a t a l l o f the c i s t r o n s on a p o l y c i s t r o n i c mRNA i n v i t r o (148-150). S i n c e there must be s e v e r a l AUG and GUG codons i n t e r n a l l y i n any l a r g e mRNA, t h e r e must be some mechanism f o r s e l e c t i n g  the  c o r r e c t ones. This c o u l d be done by having s p e c i f i c sequences around the i n i t i a t o r codons but not the i n t e r n a l or o u t - o f phase codons. R e c o g n i t i o n o f these s p e c i f i c sequences might w e l l r e q u i r e s p e c i a l f a c t o r s and, i n f a c t , a r o l e f o r s e v e r a l p r o t e i n f a c t o r s i n i n i t i a t i o n has been found by s e v e r a l groups u s i n g b a c t e r i a l systems (151-153). Three d i s t i n c t f a c t o r s have been recovered by washing ribosomes a t h i g h i o n i c s t r e n g t h such as w i t h 1 M NR4CI f o l l o w e d by ion-exchange chromatography  on D E A E - c e l l u l o s e (154-156). These f a c t o r s have been des-  i g n a t e d as f 1, f 2 and f 3 and t h e i r f u n c t i o n s w i l l be d i s c u s s e d below. U n t i l f a i r l y r e c e n t l y i t was thought  t h a t 70 S ribosomes  -83-  were d i r e c t l y i n v o l v e d i n the i n i t i a t i o n mechanism. S e v e r a l l i n e s o f evidence make i t c l e a r now t h a t the 30 S subunit i s i n v o l v e d i n the f i r s t steps o f i n i t i a t i o n . Thus, Greenshpan and Revel (157) found t h a t phage T4 mRNA binds t o the 30 S s u b u n i t i n the presence o f i n i t i a t i o n f a c t o r f 2 ( i - f a c t o r f 2 ) ; t h i s r e a c t i o n was shown t o be independent  o f the presence o f  anyitRNA. Others found t h a t f o r m a t i o n o f a complex o f 30 S s u b u n i t :mRNA:i*-Met-tRNA  f  occurs and i s an o b l i g a t o r y r e q u i r e -  ment f o r p r o t e i n s y n t h e s i s , ( 1 5 8 , 1 5 9 ) . A continuous exchange o f r i b o s o m a l s u b u n i t s d u r i n g p r o t e i n s y n t h e s i s has been w e l l documented (158-161) and suggests t h a t the 70 S ribosome d i s s o c i a t e s i n t o s u b u n i t s a t the end o f t r a n s l a t i o n o f a c i s t r o n ; the s u b u n i t s then r a n domize and recombine d u r i n g i n i t i a t i o n o f t r a n s l a t i o n o f another mRNA. A p r o t e i n f a c t o r r e q u i r e d f o r the d i s s o c i a t i o n has been recovered i n the 1 M NH4CI wash o f the 30 S s u b u n i t (162) and i s a c t i v e o n l y on the " r u n o f f " ribosomes  which are  f r e e from the peptidyl-tRNA and mRNA (163). T h i s f a c t o r has r e c e n t l y been shown t o probably be i d e n t i c a l w i t h i - f a c t o r f 3 (Ref.164) and i s s t i m u l a t e d by a d d i t i o n o f ATP or GTP. Ribosomes washed t o remove i - f a c t o r s are a c t i v e i n p r o t e i n synthesis at high M g  + +  concentration i n a c e l l - f r e e  system w i t h s y n t h e t i c p o l y r i b o n u c l e o t i d e s , GTP, aminoacyltRNA's and e l o n g a t i o n f a c t o r s . However, these ribosomes  will  not f a i t h f u l l y t r a n s l a t e n a t u r a l mRNA. I n i t i a t i o n o f p r o t e i n s y n t h e s i s w i t h F-Met-tRNAf r e q u i r e s the i - f a c t o r s  (165)  and.  -84-  GTP  (166-168). G u t h r i e and Nomura (159) showed t h a t a 30 S  i-complex would o n l y form w i t h F-Met-tRNAf i n the presence of i - f a c t o r s and would not form w i t h any other  aminoacyl-tRNA.  A summary o f the known f e a t u r e s o f E. c o l i i - f a c t o r s i s g i v e n i n Table 2 and F i g . 1. Both f1 and f 2 are r e q u i r e d f o r b i n d i n g o f F-Met-tRNAf t o ribosomes i n the presence o f the t r i n u c l e o t i d e s AUG o r GUG and GTP (165); these f a c t o r s a r e a l s o r e q u i r e d f o r the t r a n s l a t i o n o f p o l y U a t low Mg*  +  and  in. the presence of N-Acetyl-phenylalanyl-tRNA (Ac-Phe-tRNA) (169). I n a d d i t i o n t o f1 and f 2 , f 3 i s r e q u i r e d f o r the t r a n s l a t i o n o f n a t u r a l mRNA (154,155,169,170).  f 3 may thus  be n e c e s s a r y f o r i n t e r a c t i o n w i t h a s p e c i a l r e g i o n around the i n i t i a t o r codons i n n a t u r a l mRNA. A l l three f a c t o r s are r e q u i r e d f o r maximal i n c o r p o r a t i o n o f methionine from F-MettRNAf i n t o p r o t e i n and f o r maximal s y n t h e s i s o f a c t i v e l y s o zyme i n an E. c o l i c e l l - f r e e system primed by phage T4 mRNA (171). The i - f a c t o r s are probably r e l e a s e d from the ribosome a f t e r i n i t i a t i o n o f p r o t e i n s y n t h e s i s ; they can o n l y be recovered from n a t i v e 30 S s u b u n i t s and not from the 50 S s u b u n i t , 70 S ribosomes or polysomes  (172,173). I - f a c t o r s  are not found i n the HSS and t h e r e f o r e must be p r e s e n t i n l i m i t i n g amounts which combine r a p i d l y w i t h any f r e e 30 S s u b u n i t s . A f t e r f o r m a t i o n o f the 30 S i-complex, a 50 S subunit sis  j o i n s t o form a 70 S complex  a c t i v e i n p r o t e i n synthe-  (174); Thach e t a l . (175) showed t h a t t r i t i u m l a b e l l e d  -85-  Formation o f I n i t i a t i o n Complexes i n E . c o l i  1) 70 S ribosome reaches a t e r m i n a t o r codon and i n the presence o f a p r o t e i n d i s s o c i a t i o n f a c t o r d i s s o c i a t e s i n t o two s u b u n i t s , 2) 3 0 S + mRNA + f 2 •+- f 3  (162)  50 S and 30 S. >  (30 S:mRNA)  >  (30 S i-complex)  >  (70 S a c t i v e complex) .+  3) F-Met-tRNAf + f1 + GTP — + (30 S:mRNA) 4)  50 S + ( 3 0 S i-complex)  GDP  Note: GTP may be r e q u i r e d  + P i + free i - f a c t o r s  f o r t r a n s l o c a t i o n of  F-Met-tRNAf t o the r i b o s o m a l P s i t e t h e r e i s evidence that i t binds at the A s i t e  (205).  since  initially  -86-  TABLE 2 P r o p e r t i e s o f E. c o l i Initiation  Factor  f1  M.W.  9000 (Ref.175)  Factors  Oharacteristics  Binds t o 30 S s u b u n i t i n the presence o f f 2 , AUG, GTP and FrMet-tRETA . f  f2  70,000 (Ref.175)  Heat s e n s i t i v e . E s s e n t i a l SH g r o u p ( s ) . In the presence o f GTP, i s p r o t e c t e d from SH reagents (199)., GTPase a c t i v i t y i n the presence of ribosomal s u b u n i t s ; may actua l l y c o n s i s t o f two components (200). Can complex w i t h GTP.  f3  29,000 (Ref.201)  Can be assayed by a b i l i t y t o b i n d t o n a t u r a l mRNA or A p U p G ( p A ) w i t h the complex r e t a i n e d on a M i l l i p o r e f i l t e r (202). 40  Required f o r the t r a n s l a t i o n of n a t u r a l mRNA (201 ). Has a ribosome-dependent GTPase activity resistant to fusidic a c i d (203). May g i v e t r a n s l a t i o n a l c o n t r o l by v a r i a b l e a f f i n i t y f o r d i f f e r ent sequences around i n i t i a t o r codons (155).  -87-  f1  i s r e l e a s e d upon f o r m a t i o n o f the 70 S complex  and, as  mentioned above, the other i - f a c t o r s are p r o b a b l y r e l e a s e d together with f 1 . In the absence o f i - f a c t o r s n e i t h e r F-Met-tRNA nor f  mRNA w i l l bind t o ribosomes (153). R e v e l e t a l . (154) showed by EM t h a t f 2 s t i m u l a t e s attachment o f ribosomes t o nascent RNA  being s y n t h e s i z e d on phage T4 DNA. Thus, i - f a c t o r s are  i n v o l v e d i n promoting i n t e r a c t i o n between ribosomes and b o t h mRNA and the i n i t i a t o r tRNA. O l a r k (176) found that phage f 2 RNA s t i m u l a t e d b i n d i n g of P-Met-tRNAf t o ribosomes much b e t t e r a t 37 °C than a t , 25 °C and proposed t h a t the d i f f e r e n c e was due t o p a r t i a l m e l t i n g of the secondary s t r u c t u r e o f the f 2 RNA. D i s r u p t i o n of H-bonds could expose i n i t i a t o r codons which are b u r i e d i n double s t r a n d e d regions and are n o r m a l l y unable t o i n t e r a c t w i t h i - f a c t o r s , F-Met-tRNA  f  and 30 S s u b u n i t s . The work o f  S t e i t z (55) on the sequences around the i n i t i a t o r  codons i n  R17 RNA confirms the presence o f secondary s t r u c t u r e around the  codons. She incubated R17 RNA w i t h ribosomes, i - f a c t o r s ,  F-Met-tRNAf and GTP which thus a l l o w e d ribosomes t o b i n d t o i n i t i a t o r s i t e s but no c h a i n e l o n g a t i o n c o u l d occur i n the absence of e l o n g a t i o n f a c t o r s . The R17 RNA:ribosome was  complex  d i g e s t e d w i t h p a n c r e a t i c RNase t o degrade exposed r e g i o n s  of v i r a l RNA not s h i e l d e d by the ribosomes ( 5 4 ) . Three f r a g ments p r o t e c t e d by the ribosomes were i s o l a t e d and sequenced. In a l l three c i s t r o n s , the i n i t i a t o r codon was AUG and t o t h e  -88-  left  o f each AUG codon and i n phase w i t h i t was an UGA  t e r m i n a t o r codon present i n the i n t e r c i s t r o n a l r e g i o n . These UGA codons c o u l d be p a r t o f r e c o g n i t i o n s i t e s f o r i n i t i a t i o n or c o u l d be reinforcement  t e r m i n a t o r codons; t h e l a t t e r  p o s s i b i l i t y seems u n l i k e l y s i n c e t h e r e i s no apparent need for is  a t e r m i n a t o r codon b e f o r e t h e f i r s t c i s t r o n and y e t t h e r e one. The AUG codon f o r i n i t i a t i o n of t h e coat p r o t e i n  was found a t the end of a h a i r p i n l o o p i n which 11 out o f 12 p o s s i b l e base p a i r s are made; t h e other two i n i t i a t o r  codons  were found t o be exposed i n r e g i o n s where no base p a i r i n g i s p o s s i b l e . Most i n t e r e s t i n g was the d i s c o v e r y t h a t n o n - i n i t i a t o r AUG sequences a r e b u r i e d i n b a s e - p a i r e d r e g i o n s and t h e r e f o r e are u n a v a i l a b l e f o r i n i t i a t i o n . Thus, the i n c r e a s e d b i n d i n g of F-Met-tRNAf  t o phage f 2 RNA a t h i g h e r temperatures ob-  served by C l a r k ( 1 7 6 ) and mentioned above, c o u l d be due t o m e l t i n g o f regions and exposure of n o n - i n i t i a t o r  AUG.,sequences.  H i n d l e y and S t a p l e s (143) sequenced around one of the i n i t i a t o r codons i n phage Q^RNA; they found the AUG i n i t i a tor  codon f o r t h e coat p r o t e i n t o be s i t u a t e d a t the end o f  a looped s t r u c t u r e with 5 base p a i r s i n the stem. More work i s r e q u i r e d t o determine i f the exposed p o s i t i o n o f i n i t i a tor  codons i s a g e n e r a l r u l e . D i f f e r e n c e s i n t h e a f f i n i t i e s  of i - f a c t o r s f o r d i f f e r e n t sequences around i n i t i a t o r  codons  c o u l d provide a mechanism f o r t r a n s l a t i o n a l c o n t r o l o f t h e rate of protein synthesis. Lodish (177,178)  d i s c o v e r e d t h a t ribosomes from B a c i l l u s  -89-  stearothermophilus can o n l y t r a n s l a t e one of the three c i s - t r o n s of phage f 2 RNA;  t h i s was  the c i s t r o n f o r the matura-  t i o n p r o t e i n (A p r o t e i n ) , the product s y n t h e s i z e d i n the l e a s t amount by the normal host of the phage, E. c o l i . s p e c i f i c i t y of ribosomes from B a c i l l u s was  The  stearothermophilus  found to be a p r o p e r t y of the 30 S s u b u n i t by p r e p a r i n g  h y b r i d ribosomes w i t h v a r i o u s combinations  of s u b u n i t s from  both b a c t e r i a . Thus, E. c o l i 30 S and. B a c i l l u s p h i l u s 50 S subunits w i l l  stearothermo-  t r a n s l a t e a l l three c i s t r o n s i n  the c e l l - f r e e system employed; the source of supernatant f a c t o r s or i - f a c t o r s made no d i f f e r e n c e . T h i s s u r p r i s i n g  dis-  covery i m p l i e s t h a t the 30 S s u b u n i t i s i n v o l v e d i n r e c o g n i t i o n of n a t u r a l mRNA's and,  at l e a s t i n the case above, can  d i s c r i m i n a t e a g a i n s t f o r e i g n mRNA. One  c o u l d argue t h a t  B a c i l l u s stearothermophilus i s an unusual  organism s i n c e  i t grows w e l l at 65 °C and t h a t i t s 30 S ribosomal s u b u n i t s have evolved i n such a way  t h a t they cannot  interact with  at l e a s t some heterologous mRNA's. N e v e r t h e l e s s , Lodish's c  observations can e x p l a i n h o s t - s p e c i f i c i t y of phage f2 and the phenomenon o f r i b o s o m a l s p e c i f i c i t y may eral  be of more gen-  importance., When E. c o l i i n f e c t e d w i t h phage M12  w i t h phage T4, r e p l i c a t i o n of M12 b l o c k was  i s superinfected  i s prevented  (179); the  l o c a l i z e d to an e f f e c t of T4 on p r o t e i n s y n t h e s i s  d i r e c t e d by M12  RNA  (180). During T4 i n f e c t i o n of E.  coli.  the host ribosomes are m o d i f i e d by a d d i t i o n o f s e v e r a l T4-  -90-  specific  p r o t e i n s t o both ribosomal subunits  i n f e c t i o n begins (181 )-. Hsu and Weiss (182) modified  soon a f t e r t h e showed t h a t T4-  E . c o l i ribosomes db n o t t r a n s l a t e phage MS2 RNA  or even E. c o l i mRNA n e a r l y as e f f i c i e n t l y as normal E. c o l i ribosomes; however, m o d i f i e d  and normal ribosomes t r a n s l a t e  T4 mRNA e q u a l l y w e l l . The T4 " r e s t r i c t i o n f a c t o r s " c o u l d be washed from the m o d i f i e d  ribosomes by 2 M NH^Cl and a d d i t i o n  of i - f a c t o r s from normal ribosomes r e s t o r e d the a b i l i t y o f the washed ribosomes t o t r a n s l a t e mRNA's other than T4 RNA (183,184). T h i s i n t e r e s t i n g o b s e r v a t i o n present  means t h a t f a c t o r s  i n t h e ribosomal wash are capable o f determining  the i n t e r a c t i o n o f ribosomes w i t h n a t u r a l mRNA's. This i s a c l e a r example o f t r a n s n a t i o n a l c o n t r o l and c o u l d be o f widespread importance.  F-Met-tRNA  f  I n i t i a t i o n of protein synthesis  i n a l l 70 S ribosomal  systems i n v o l v e s F-Met-tRNAf (185). The presence o f two methionine a c c e p t i n g  tRNA's i n E. c o l i was known before  the d i f f e r e n c e s i n function;were d i s c o v e r e d The  absolute  (186,187).  s p e c i f i c i t y o f i - f a c t o r s f o r F-Met-tRNAf i n  the b i n d i n g r e a c t i o n w i t h i n i t i a t o r codons and GTP (146) i n d i c a t e s t h a t both the blocked one  amino group and a t l e a s t  s i t e on the tRNA must be r e c o g n i z e d .  Both Met-tRNA's  have been sequenced (188,189) but the s t r u c t u r e s do not r e a d i l y r e v e a l a p o s s i b l e r e c o g n i t i o n s i t e f o r the t r a n s -  -91-  formylase or i - f a c t o r s . The i n a c t i v i t y of unformylated Met-tRNAf i n the b i n d i n g assay c o u l d be due t o a r e q u i r e d conformational change i n the tRNA o c c u r r i n g upon f o r m y l a t i o n o f t h e amino group of methionine;  i t i s known t h a t  a c y l a t i o n can cause a c o n f o r m a t i o n a l change i n aminoacyltRNA (190)., F-Met-tRNA  f  must a l s o be r e c o g n i z e d by aminoacyl-tRNA  deacylase, an enzyme t h a t h y d r o l y z e s any other blocked aminoa c y l -tRNA s i n c l u d i n g F-Met-tRN/^ (191 ). 1  As one might expect, E. c o l i d e p l e t e d o f f o l a t e i s rendered d e f i c i e n t s p e c i f i c a l l y i n i t s c a p a c i t y t o i n i t i a t e p r o t e i n s y n t h e s i s (291 ). However, i n s t u d i e s on f o l a t e depleted Streptococcus faecium. Pine, Gordon and Sarimo (292) found evidence t h a t t h i s organism  can r e a d i l y i n i t i a t e  pro-  t e i n s w i t h or without formylated Met-tRNA . When grown i n f  f o l a t e - d e f i c i e n t medium and i n t h e presence  o f the f o l a t e  a n t a g o n i s t , trimethoprim, Streptococcus faecium growing w i t h methionine  continues  i n c o r p o r a t i o n i n t o tRNA and p r o t e i n  N - t e r m i n i at u n a l t e r e d l e v e l s but completely without l a t i o n . The authors proposed  formy-  t h a t L a c t o b a c t e r i a c e a e and  mammalian c e l l s have a f o l a t e - i n d e p e n d e n t pathway o f p r o t e i n i n i t i a t i o n s i n c e both types of c e l l s can grow i n d e f i n i t e l y i n the absence of f o l a t e as l o n g as products b o l i s m such as thymidine  of one-carbon meta-  are p r o v i d e d .  The p r o p e r t i e s o f Met-tRNA's were s t u d i e d u s i n g a l l 64 r i b o t r i n u c l e o t i d e s and v a r i o u s p o l y r i b o n u c l e o t i d e s (142). At  -92-  h i g h Mg  and  i n the absence of i - f a c t o r s , F-Met-tRNAf bound  t o the codons AUG, GCG,  UGG,  UCG  GUG  or AGG.  and UUG  and  Met-tRNA  o n l y s l i g h t l y to AOG,  bound to AUG  and GUG.  OUG,  The  bind-  m i n g of F-Met-tRNAf shows a constant  requirement f o r G i n the  t h i r d p o s i t i o n but i n the three codons which gave s i g n i f i cant b i n d i n g , there i s ambiguity i n the r e a d i n g n u c l e o t i d e . The  of the  first  b i n d i n g to the t h r e e codons d i d not change  w i t h a d d i t i o n of i - f a c t o r s but i n experiments w i t h p o l y r i b o n u c l e o t i d e s , o n l y AUG  and GUG  Assuming t h a t F-Met-tRNA t i o n , then the misreading due  f  c o u l d serve as  initiators.  i s a s i n g l e homogeneous  prepara-  of the f i r s t n u c l e o t i d e must be  to the s p e c i a l i n t e r a c t i o n s of t h i s tRNA w i t h  and the 30 S s u b u n i t . GUA  was  i-factors  found to serve as a weak i n i t i a -  t o r which i m p l i e s t h i r d l e t t e r ambiguity as i n o r d i n a r y generacy Waller  de-  (192). (193)  found t h a t most E. c o l i p r o t e i n s have  methionine, a l a n i n e , s e r i n e or t h r e o n i n e r e s i d u e ; i n B. s u b t i l i s . 80$ found to have N-terminal vious by 1965  as the  N-terminal  of the bulk p r o t e i n s were  alanine  (194). I t was  therefore  ob-  t h a t there must be a mechanism f o r removal of  the formyl group and  q u i t e o f t e n the methionine as w e l l from  the N-terminus of newly s y n t h e s i z e d deformylase a c t i v i t y was  proteins i n bacteria. A  soon found (195-197) which removed  the formyl group from peptides much f a s t e r than a c e t y l groups; the enzyme was  found to be i n a c t i v e a g a i n s t e i t h e r a c y l group  on f r e e methionine. The  E. c o l i deformylase i s q u i t e l a b i l e ,  -93i  being r a p i d l y i n a c t i v a t e d a t 37 °C i n the presence mereaptoethanol  o f 5 mM  (196); t h i s probably e x p l a i n s the ease w i t h  which formylated peptides can be r e c o v e r e d from E. c o l i  cell-  f r e e systems. The B. s u b t i l i s enzyme i s a s s o c i a t e d w i t h the ribosomes and can be washed o f f w i t h 0.5 M NH4CI; t h i s e n zyme i s s t a b l e t o t h i o l s The methionine  (197).  r e s i d u e a t the N.-terminus o f newly s y n -  t h e s i z e d b a c t e r i a l p r o t e i n s i s probably removed by the a c t i o n of  a s p e c i f i c aminopeptidase.  d e s c r i b e d an aminopeptidase  Matheson and Dick (198) have  a c t i v i t y on the ribosomes o f  E. c o l i which has the s p e c i f i c i t y one would expect t o a c count f o r the known N - t e r m i n i o f E. c o l i p r o t e i n s ; t h i s enzyme w i l l be d i s c u s s e d i n more d e t a i l  later.  ELONGATION During e l o n g a t i o n the ribosome moves along the mRNA i n the 5' t o 3' d i r e c t i o n i n steps o f three n u c l e o t i d e s each. P r o t e i n f a c t o r s r e q u i r e d f o r e l o n g a t i o n can be i s o l a t e d from the HSS and form 2-3$ o f the p r o t e i n i n t h i s  fraction  i n growing E. c o l i c e l l s (204). A summary o f the nomenclature and some of the p r o p e r t i e s o f the e l o n g a t i o n f a c t o r s from d i f f e r e n t sources i s g i v e n i n Table 3. An o u t l i n e o f the steps i n which the f a c t o r s f u n c t i o n i s given, i n P i g . 2 (rat  l i v e r ) and P i g . 3 ( E . c o l i ) . Nathans and Lipmann (213) were the f i r s t t o i s o l a t e  e l o n g a t i o n f a c t o r s and d i d so u s i n g the HSS from E. c o l i .  -94-  TABLE 3 Nomenclature and C h a r a c t e r i s t i c s of E l o n g a t i o n F a c t o r s  E. c o l i  Bacillus stearothermophilus  Ts  S1  Rabbit R e t i c u l o c y t es TF-1  Rat . Liver TF-1  MW 186,000 3 subunits (Ref.207)  Tii  I n h i b i t e d by fusidic acid (Ref.212)  MW 84,000 4 subunits (Ref.206)  B i n d i n g aminoacyl-tRNA t o the A s i t e of ribosomes.  Same as above.  S3  S2  Function  TF-2  Translocation of p e p t i d y l MW 65,000 tRNA from s i t e MW 70,000 S e n s i t i v e A to s i t e P (Ref.208) to SH a - w i t h movement Very s e n s i of ribosome on gents ( t i v e t o SH reagents and Ref.209) mRNA by one codbn a t a time. cycloheximide (Ref.209) TF-2  Note: s " i n Ts r e f e r s t o s t a b i l i t y o f the f a c t o r M  n  u  M  in. Tu r e f e r s t o i t s i n s t a b i l i t y  TF-1 i s t r a n s f e r a s e 1 (aminoacyl-tRNA b i n d i n g enzyme) TF-2 i s t r a n s f e r a s e 2 ( t r a n s l o c a s e )  -95-  Fig. 2 Mechanism of E l o n g a t i o n i n Rat L i v e r  1 ) aa-tRNA + TF-1 + GTP  9>  (aa-tRNA:TF-1 :GTP) Complex 1  2) Complex 1 + Ribosome :mRNA  >  TF-1 + GDP + P i + Ribosome: aa-tRNA (A s i t e )  3) Peptidyl-tRNA  (P s i t e ) +  aa-tRNA (A s i t e )  —»  P e p t i d y l - t R N A * (A s i t e )  ^ P e p t i d y l t r a n s f e r a s e (60 S s u b u n i t )  41) Peptidyl-tRNA* (A s i t e ) +  P e p t i d y l - t R N A * (P s i t e ) +  GTP  GDP + P i + r e l e a s e o f d i s I  charged tRNA  TF-2  Note: aa-tRNA = aminoacyl-tRNA * one amino a c i d l o n g e r than p r e v i o u s l y  -96-  Fig. 3 Mechanism of E l o n g a t i o n i n E. c o l i Mg  + +  1 ) Ts + Tu + GTP  >  (Ts:Tu:GTP) Complex 1  Mg —L-,. > NH + +  2) Complex 1 + aa-tRNA  Ts + (Tu:GTP:aa-tRNA)  4  3) Complex ,2 + Ribosome:mRNA  >  Complex 2 (Ribosome:mRNA:aa-tRNA)  +  GDP:Tu + P i 4) (Ribosome:mRNA:aa-tRNA: peptidyl-tRNA)  (Ribosome:mRNA:peptidyl^  tRNA*)  Peptidyl transferase (50 S s u b u n i t ) 5) Peptidyl-tRNA* (A s i t e ) +  Peptidyl-tRNA* (P s i t e ) + y  GTP ^  release o f discharged tRNA + GDP + P i  Note: * one amino a c i d l o n g e r Ts may be r e q u i r e d t o d i s s o c i a t e GDP:Tu. (Ref.211)  -97-  These f a c t o r s were e v e n t u a l l y r e s o l v e d i n t o three f r a c t i o n s c a l l e d T s , Tu and G (Ref.214,215); a l l three have been p u r i f i e d and: pure G has been c r y s t a l l i z e d  (206,216,217).  Ts and Tu a r e required' f o r b i n d i n g 1  o f the c o r r e c t i n -  coming aminoacyl-tRNA to the r i b o s o m a l A s i t e i n a r e a c t i o n r e q u i r i n g GTP. If- peptidyl-tRNA o r F-Met-tRNA the P s i t e , peptide  bond f o r m a t i o n  f  i s present i n  occurs by t r a n s f e r of the  a c y l group a t the P s i t e t o the amino group o f t h e aminoacyltRNA a t the A s i t e . .Formation o f t h e peptide  bond i s c a t a -  l y z e d by p e p t i d y l t r a n s f e r a s e , an enzyme a c t i v i t y present the 50 S subunit formation  (105,106,218). The energy f o r peptide  on  bond  i s s u p p l i e d by the r e l a t i v e l y h i g h energy e s t e r  bond between the tRNA and t h e p e p t i d y l moiety.(219). Waterson, Beaud and Lengyel (211) have r e c e n t l y s t u d i e d the f u n c t i o n o f e l o n g a t i o n f a c t o r S1 i n B'. s t e a r o t h e r m o p h i l us which i s e q u i v a l e n t r a t h e r than being  t o Ts i n E . c o l i . They found t h a t  d i r e c t l y i n v o l v e d i n the b i n d i n g o f amino-  a c y l -tRNA, S1 i s r e q u i r e d f o r d i s s o c i a t i o n o f t h e GDP:S3 complex r e l e a s e d a f t e r b i n d i n g  o f aminoacyl-tRNA t o the  ribosome as i n t h e f o l l o w i n g scheme: 1 ) S3 + GTP + Phe-tRNA  >  (S3:GTP:Phe-tRNA) Complex 1  2) Complex 1 + Ribosome:Poly U:.< , Ac-Phe-tRNA  GDP:S3 + P i + Ribosome: *  P o l y U;:Ac-diPhe-tRNA  ( S i t e A)  -98-  3) Ribosome:Poly  Ribosome:poly U:Ac-  U:Ac-diPhe-tRNA  ( S i t e A) + GTP  S2  diPhe-tRNA  ( S i t e P) +  discharged tRNA + GDP + P i GDP + S3  4) GDP:S3 S1  R e a c t i o n 2) above i n v o l v e s both b i n d i n g o f aminoacyl-tRNA at the A s i t e and peptide bond f o r m a t i o n c a t a l y z e d by p e p t i dyl transferase. GDPCP, an analogue o f GTP t h a t cannot be h y d r o l y z e d between the beta and gamma phosphates, can r e p l a c e GTP i n the b i n d i n g r e a c t i o n f o r aminoacyl-tRNA but no p e p t i d e bond i s formed (220,221). Since GTP i s n o t r e q u i r e d f o r p e p t i d e bond formation per se (105,106,218), the energy from GTP must be used i n a r e a c t i o n which binds aminoacyl-tRNA a t s i t e A i n a s p e c i f i c r e l a t i o n s h i p w i t h t h e ribosome which i s n e c e s s a r y f o r peptide bond  formation.  A f t e r peptide bond f o r m a t i o n , peptidyl-tRNA the A s i t e and discharged tRNA a t the P s i t e .  remains in.  Translocase  ( f a c t o r G or S2) c a t a l y z e s t r a n s l o c a t i o n i n a r e a c t i o n ;  whereby peptidyl-tRNA  s h i f t s t o the P s i t e and. the d i s -  charged tRNA i s r e l e a s e d (219,222,217) and the ribosome moves along the mRNA by 3 n u c l e o t i d e s i n the 5' t o 3' d i r e c t i o n . A s p e c i f i c i n h i b i t o r o f t r a n s l o c a s e i s the s t e r o i d , a n t i b i o t i c , f u s i d i c a c i d (212$. S e v e r a l models f o r the mechanism of t r a n s l o c a t i o n have been proposed r e c e n t l y (223,  -99-  224)  but w i l l not be d i s c u s s e d S c h r e i e r andi N o l l (225)  here.  found evidence t h a t a 60 S  complex i s formed i n an E. c o l i c e l l - f r e e system by a c t i o n of ribosome s u b u n i t s ,  p o l y U and uncharged  the complex s h i f t s to 70 S upon a d d i t i o n of the Tu:GTP:Phe-tRNA w i t h r e l e a s e o f Tu, GDP cluded t h a t the energy of GTP r e a c t i o n causing  and  inter-  tRNA  ;  E n e  complex,  P i . They con-  i s required for d r i v i n g a  a l a r g e conformational  change of the r i b o -  some to a more compact form which sediments f a s t e r . The  au-  t h o r s a l s o found evidence f o r a t h i r d s i t e on the ribosome formed d u r i n g the s h i f t from 60 S to 70 S; the data c a t e d t h a t two  molecules of Phe-tRNA were present  indi-  on each  70 S ribosome. Eukaryotic  e l o n g a t i o n f a c t o r s have been s t u d i e d i n the  r e t i c u l o c y t e system (226,227,207,208), i n r a t l i v e r 229) two  and  i n yeast  (230). These systems a p p a r e n t l y  f a c t o r s w i t h t r a n s f e r a s e 1 analogous to Ts and  2 3 2 ) and  transferase 2 equivalent  (228,  o n l y have Tu  (231»  to f a c t o r G (233-235). I t  i s i n t e r e s t i n g to note t h a t d i p h t h e r i a t o x i n , a p r o t e i n of m o l e c u l a r weight 65,000, acts by s p e c i f i c a l l y eukaryotic  t r a n s f e r a s e 2 ( t r a n s l o c a s e ) ( R e f . 2 3 6 ) . Guinea  pigs are k i l l e d by as l i t t l e widespread n e c r o s i s  as 0.05  ug o f the t o x i n w i t h  in. many organs. The  found to c a t a l y z e the f o l l o w i n g unusual TE-2  inhibiting  + NAD  +  >  t o x i n has  been  reactioni(237)*  ADPR:TP-2 + n i c o t i n a m i d e  +  H  +  -100-  The above r e a c t i o n can be m o d i f i e d w i t h t h e use o f l a b e l l e d NAD  +  0-  ( l a b e l l e d i n the adenosine m o i e t y ) as an assay  f o r t r a n s f e r a s e 2; as l i t t l e  as 5-10 ng can be d e t e c t e d  (238).  P e p t i d y l t r a n s f e r a s e , t h e 50 S subunit enzyme r e s p o n s i b l e f o r p e p t i d e bond f o r m a t i o n , i s dormant i n t h e absence of 30 S s u b u n i t s but can be a c t i v a t e d by low M.W. a l c o h o l s (239). T h i s enzyme appears  t o i n t e r a c t w i t h the CpCpA-peptide  and OpA-amino a c i d m o i e t i e s o f the two s u b s t r a t e s bound t o the ribosome a t the P and A s i t e s r e s p e c t i v e l y . A s i m i l a r enzyme has been found i n eukaryotes  (233) and i s present on  the 60 S s u b u n i t (240). The b a c t e r i a l enzyme i s s p e c i f i c a l l y i n h i b i t e d by t h e a n t i b i o t i c s chloramphenicol, l i n c o m y c i n , s t r e p t o g r a m i n A, g o u g e r o t i n and sparsomycin.  The e u k a r y o t i c  enzyme i s n o t i n h i b i t e d by t h e a n t i b i o t i c s above but i s i n h i b i t e d by the a n t i b i o t i c , anisomycin  (240). These s t u d i e s  have t h e r e f o r e h e l p e d e x p l a i n t h e s e l e c t i v e a c t i o n of a n t i b i o t i c s on b a c t e r i a and not on e u k a r y o t i c c e l l s or v i c e versa.  TERMINATION The codons UAA, UAG and UGA code f o r no amino a c i d s normally and are r e f e r r e d t o as "nonsense" or " t e r m i n a t o r " codons. When, a ribosomal:peptidyl-tRNA  complex reaches a  t e r m i n a t o r codon on a mRNA, some mechanism must b r i n g about h y d r o l y s i s o f t h e e s t e r bond between t h e p e p t i d y l and tRNA m o i e t i e s . This would then a l l o w the completed  p r o t e i n t o be  -101-  released. Capecchi (241)  s t u d i e d the r e l e a s e of the N-terminal  hexapeptide of coat p r o t e i n of phage R17 t a t i o n a t the seventh codon (CAG  mutated to UAG).  ceeded i n i s o l a t i n g a p r o t e i n f a c t o r , R, E. c o l i and  found t h a t R was  w i t h an amber He  mu-  suc-  from the HSS  of  r e q u i r e d f o r the r e l e a s e  of  the hexapeptide from a p u r i f i e d peptidyl-tRNA:ribosome complex. Oaskey et a l . (242)  used a d i f f e r e n t assay f o r r e l e a s e  f a c t o r s . F-Met-tRNAf was  bound t o ribosomes i n the  of the t r i n u c l e o t i d e AUG;  presence  then a t e r m i n a t o r t r i n u c l e o t i d e  and  t e s t p r o t e i n were added and  was  measured. With t h i s assay t h e y were able t o r e s o l v e R  i n t o two  components, R1  but not UGA UAG  and R2;  whereas R2 was  the r e l e a s e o f f r e e F-Met  R1  responded to UAG  a c t i v e w i t h UGA  (Ref.243). A t h i r d f a c t o r , S, has  the r a t e of f o r m a t i o n ribosome:factor  and UAA  and but  been, found to  or s t a b i l i t y of the t e r m i n a t o r  UAA not  increase codon:  R complex (244).  P e p t i d y l t r a n s f e r a s e may  be i n v o l v e d i n r e l e a s e s i n c e  i n h i b i t o r s of t h i s enzyme a l s o i n h i b i t r e l e a s e . The sence of the t e r m i n a t o r  complex may  f e r a s e t o a c t as a hydrolase  and  pre-  allow p e p t i d y l t r a n s -  thus break the e s t e r bond  h o l d i n g the completed p r o t e i n to the l a s t tRNA. Capecchi and K l e i n (245) R1  and R2  used a n t i s e r a to  purified  to t e s t t h e i r r o l e i n r e l e a s e of completed p r o -  t e i n s i n a c e l l - f r e e system d i r e c t e d by R17  RNA.  Their r e -  -102-  s u l t s i n d i c a t e d t h a t these f a c t o r s are r e q u i r e d f o r r e l e a s e of completed p r o t e i n s and t h a t e i t h e r f a c t o r c o u l d promote r e l e a s e of e i t h e r the coat p r o t e i n or the r e p l i c a s e ;  this  i m p l i e s t h a t both cis.trons terminate w i t h UAA s i n c e t h i s i s the o n l y one o f t h e t h r e e t e r m i n a t o r codons r e c o g n i z e d by both R1 and R2. N i c h o l s (246) sequenced the p o r t i o n o f R17 RNA a t the end o f the coat p r o t e i n c i s t r o n and found two c o n s e c u t i v e t e r m i n a t o r codons, UAAUAG.. T h i s may mean t h a t , a t l e a s t i n some systems, two terminators are r e q u i r e d t o ensure t h a t r e l e a s e occurs between c i s t r o n s even i f a suppressor tRNA i s p r e s e n t . The UAA codon i s c o n s i s t e n t w i t h the r e s u l t s o f Oapecchi and K l e i n mentioned above.  EUKARYOTIC INITIATION The mechanism o f i n i t i a t i o n i n e u k a r y o t i c c e l l s was unknown u n t i l v e r y r e c e n t l y and n o t a l l o f t h e d e t a i l s are s e t t l e d y e t . Two major s p e c i e s o f methionine are present i n t h e cytoplasm rat  liver  a c c e p t i n g tRNA  o f guinea p i g l i v e r  (247),  (185) and yeast (248) but there i s no d e t e c t a b l e  t r a n s f o r m y l a s e i n t h e cytoplasm  of these c e l l s . These tRNA's  w i l l be r e f e r r e d t o as Caskey, Beaudet and N i r e n b e r g pig  (249) found t h a t g u i n e a  l i v e r Met-tRNA^* binds t o ribosomes i n response t o  e i t h e r AUG" or GUG. whereas Met-tRNA,^ binds o n l y t o AUG. S u r p r i s i n g l y , p u r i f i e d E. c o l i t r a n s f o r m y l a s e was found t o  -103-  r e a d i l y formylate was  Met-tRNA^* from g u i n e a p i g l i v e r  i n a c t i v e a g a i n s t Met-tRNA * (24-7). U s i n g the m  c i n assay of Leder and Bursztyn. (250), i t was  but  purqmy-  shown t h a t  Met-tRNA^.* binds a t the P s i t e on the ribosome s i n c e  the  bulk of i t r e a c t s r a p i d l y w i t h puromycin. A l l of these r e a c t i o n s of Met-tENA^* are e x a c t l y analogous to those w i t h b a c t e r i a l F-Met-tRNAf or Met-tRNA . Some s t u c t u r a l f e a t u r e s f  of e u k a r y o t i c  tRNA^** must have been preserved  during  evol-  u t i o n from lower forms. S t r u c t u r a l d i f f e r e n c e s between the two  i n i t i a t o r tRNA's have been found (251 ) but t h i s i s not  s u r p r i s i n g i n view of the l a r g e gap b a c t e r i a on any  evolutionary  f  primed w i t h phage f 2 RNA Recently, the two  and  scale.  Formylated y e a s t Met-tRNA * was i n i t i a t i n g protein synthesis  between eukaryotes  found to be capable of  i n an E. c o l i c e l l - f r e e system  (248).  Smith and Marcker (252)  separated  and  studied  Met-tRNA's from mouse a s c i t e s tumour c e l l s , mouse  l i v e r and y e a s t . They found t h a t Met-tRNA * f  incorporates  methionine e x c l u s i v e l y i n t o the N-terminal p o s i t i o n s o f newly s y n t h e s i z e d  p r o t e i n s i n . a c e l l - f r e e system from  mouse a s c i t e s tumour c e l l s primed w i t h s y n t h e t i c p o l y r i b o n u c l e o t i d e s . Met-tRNAjjj*, i n c o n t r a s t , i n c o r p o r a t e s only i n t o i n t e r n a l p o s i t i o n s i n the new  p r o t e i n s . This i s  v e r y good evidence t h a t Met-tRNAf* can i n i t i a t e but  methionine  proteins  does not prove t h a t t h i s i s happening i n v i v o . In f a c t ,  when the c e l l - f r e e system was  primed w i t h a n a t u r a l mRNA  -104-  (encephalomyocarditis v i r u s RNA), s i g n i f i c a n t  incorpora-  t i o n o f methionine from Met-tRNA^* i n t o v i r a l  proteins  c o u l d not be demonstrated. The authors suggested t h a t i n c o r p o r a t e d methionine might be removed r a p i d l y from the N-terminus by a s p e c i f i c aminopeptidase o r the methionine might not be i n c o r p o r a t e d ; i n t h e l a t t e r case, the MettRNAf,* would f u n c t i o n t o s e l e c t the proper r e a d i n g frame on the mRNA. Brown and Smith (253) e s t a b l i s h e d t h a t  formylated  Met-tRNAf* was a c t i v e i n an E. c o l i c e l l - f r e e system but not  i n one d e r i v e d from a s c i t e s c e l l s . This i m p l i e d t h a t  formylated Met-tRNA * cannot i n t e r a c t w i t h 80 S ribosomes f  and i s c o n s i s t e n t w i t h the absence o f t r a n s f o r m y l a s e  activ-  ++ i t y i n the cytoplasm o f e u k a r y o t i c c e l l s . A t low Mg  only  p o l y r i b o n u c l e o t i d e s w i t h an AUG codon were t r a n s l a t e d i n the a s c i t e s c e l l - f r e e system. AUG- codons a t or near the 5'  end  of the s y n t h e t i c mRNA's always i n t e r a c t e d w i t h Met-tRNA^* and not w i t h Met-tRNA . S i m i l a r l y , GUG a t or near the 5  1  m#  end always i n t e r a c t e d w i t h M e t - t R N A ^ and not w i t h Val-tRNA. Stewart et a l . (254) s e l e c t e d 42 mutants  o f baker's yeast  l a c k i n g isocytochrome c; n i n e r e v e r t a n t s were i s o l a t e d and examined. Some o f the r e v e r t a n t s were found t o have one or two a d d i t i o n a l r e s i d u e s a t the N-terminus; the a d d i t i o n a l r e s i d u e s i n c l u d e d M e t - I l e u , Met-Leu, Met-Arg and Y a l . The authors concluded t h a t the normal mRNA f o r isocytochrome c must have an AUG. codon before t h e codon f o r t h e normal  -105-  N.-terminal r e s i d u e and AUG  codon had  t h a t i n the o r i g i n a l mutants  changed to Aug  the  ( l i e u ) , QUG; (Leu), AGG  (Arg)  or GUG! ( V a l ) . They a l s o concluded t h a t the  revertants  must have a r i s e n by mutations g i v i n g an AUG  codon to  the  l e f t of the o r i g i n a l i n i t i a t o r codon. I f these i n t e r p r e t a t i o n s are c o r r e c t , AUG  must serve  as an i n i t i a t o r codon.  i n the mRNA f o r yeast isocytochrome c; the authors d i d not mention the p o s s i b i l i t y t h a t t h i s p r o t e i n could be a m i t o c h o n d r i a l product. Liew, H a s l e t t and A l l f r e y (255)  have found evidence  that Ac-Ser-tRNA i s i n v o l v e d i n the i n i t i a t i o n of of h i s t o n e Tf  in. r e g e n e r a t i n g  r a t l i v e r . This p r o t e i n  the N-terminal sequence A c - S e r - G l y - A r g . . . had  found e a r l i e r t h a t l i t t l e  synthesis  ( 2 5 6 ) . The  or no a c e t y l a t i o n of  sis  i n h i b i t e d by puromycin. However, the ^0-serine  of ^H-acetate and  1  newly s y n t h e s i z e d  histone  synthe-  incorporation,  i n t o Ac-Ser from d i g e s t s  IV was  case of n u c l e a r h i s t o n e W , was  synthe-  the a c e t y l a t i o n which d i d occur d u r i n g h i s t o n e was  q u i t e low;  corporated  of  a l s o , i n the  the i n c o r p o r a t i o n of  acetate  not n e a r l y as s e n s i t i v e as s e r i n e to i n h i b i t i o n  puromycin. The  authors  the  N-terminal s e r i n e occurs i n the absence of h i s t o n e sis;  has  l a t t e r suggests t h a t the acetate  by  can be i n -  independently of p r o t e i n s y n t h e s i s . Both l a b e l s  were found in. a 4 S RNA  and  d i g e s t i o n of the l a t t e r w i t h  p a n c r e a t i c RNase r e l e a s e d a product w i t h the expected b i l i t y of % - a c e t y l - H c - s e r y l - a d e n o s i n e  upon h i g h  mo-  voltage  -106-  e l e c t r o p h o r e s i s . T h i s was taken t o i n d i c a t e the presence o f Ac-Ser-tRNA i n r e g e n e r a t i n g tRNA f o r h i s t o n e  r a t l i v e r a c t i n g as an i n i t i a t o r  I V s y n t h e s i s . S i n c e t h i s represents  quite  a departure from a mechanism i n v o l v i n g M e t - t R N A ^ m o r e evidence i s r e q u i r e d before  t h i s proposal  M i l l e r and Schweet (257) sis  can be accepted.  found t h a t hemoglobin s y n t h e -  i n a r e t i c u l o c y t e c e l l - f r e e system u s i n g washed r i b o -  somes was s t i m u l a t e d  by a d d i t i o n o f t h e wash f r a c t i o n ; i n  the absence o f t h e wash f r a c t i o n , t h e N-terminal v a l i n e o f hemoglobin chains  remained u n l a b e l l e d . They a l s o showed  t h a t t h e wash f r a c t i o n lowered t h e M g d i r e c t e d polyphenylalanine  synthesis  These f i n d i n g s were confirmed (258)  + +  optimum f o r p o l y U  from 10 mM t o 5 mM. and t h e s u g g e s t i o n  made t h a t t h e wash f r a c t i o n contained  i-factors.similar to  those i n b a c t e r i a l systems. R e t i c u l o c y t e i - f a c t o r s have s i n c e been f r a c t i o n a t e d i n t o 3 components l a b e l l e d M1, M2 and M3 (Ref.259). M1 binds Ac-Phe-tRNA t o r e t i c u l o c y t e ribosomes. M2 i s r e q u i r e d f o r i n i t i a t i o n o f both p o l y U and n a t u r a l mRNA d i r e c t e d p r o t e i n s y n t h e s i s . M3 i s o n l y necessary f o r i n i t i a t i o n  on n a t u r a l  mRNA's and may be analogous,to b a c t e r i a l i - f a c t o r f 3 . Heywood (260)  studied formation  of i n i t i a t i o n  complexes  w i t h the mRNA f o r myosin from c h i c k muscle. The muscle  cell  i - f a c t o r s were found t o be n e c e s s a r y f o r b i n d i n g the myosin mRNA t o c h i c k r e t i c u l o c y t e ribosomes. This suggests t h a t s p e c i f i c i - f a c t o r s are i n v o l v e d i n r e c o g n i t i o n o f n a t u r a l  -107-  mRNA's i n eukaryotes and seems analogous to a s i m i l a r phenomenon i n b a c t e r i a l systems (183,184). A l s o ,  this  work g i v e s another mechanism f o r t r a n s l a t i o n a l c o n t r o l o f p r o t e i n s y n t h e s i s which c o u l d be o f g e n e r a l This summary o f p r o t e i n s y n t h e s i s has complexity  and how  importances  shown some of i t s  knowledge o f the mechanisms has  explained  such phenomena as the mechanism of a c t i o n of a n t i b i o t i c s and r e g u l a t i o n of p r o t e i n s y n t h e s i s .  PROTAMINE BIOSYNTHESIS Only a b r i e f summary w i l l be g i v e n here as d e t a i l e d accounts have been g i v e n r e c e n t l y (293,294). Protamines are the h i g h l y b a s i c p r o t e i n s bound to  DNA  i n the mature sperm c e l l s of most higher animals. The  three  major components o f protamine i n the rainbow t r o u t (Salmo g a i r d n e r i i ) have been sequenced by Ando and Watanabe  (261)  w i t h the r e s u l t s shown i n F i g . 4. Of the 32-33 r e s i d u e s , 22 are a r g i n i n e and  these occur  i n blocks  of up to s i x con-  s e c u t i v e r e s i d u e s . There are o n l y s i x other amino a c i d s sent and  these are a l l n e u t r a l s ; there are no  sulphur-containing  pre-  aromatic or  amino a c i d s .  Protamine s y n t h e s i s takes p l a c e at the spermatid o f d i f f e r e n t i a t i o n (262,264). T h i s s y n t h e s i s occurs cytoplasmic  21-  stage on  120 S disomes c o n s i s t i n g o f 77 S ribosomes  presumably, mRNA (265,266). The  s y n t h e s i s was  found to  extremely s e n s i t i v e t o i n h i b i t i o n by cycloheximide  and  and, be  -108-  Fig. 4 Sequences o f Protamines from Salmo g a i r d n e r i i *  1A Pro-Arg^-S er-S er-S e r - A r g - P r o - V a l - A r g ^ - P r o - A r g - V a l - S er-Argg• 2  Gly-Gly-Arg  4  Pro-Argg-Ser-Ser-Ser-Arg-Pro-Ileu-Arg4-Pro-Arg2-Val-Ser-Arg5Gly-Gly-Arg  4  2 Pro-Arg^-Ser-Ser-Ser-Arg-Pro-Val-Arg4-Ala-Arg -Val-Ser-Arg62  Gly^Gly-Arg  * Ando and Watanabe  (Ref.261)  4  -109-  and was  l e s s s e n s i t i v e t o puromycin (264). In the  of actinomycin D, protamine  presence  s y n t h e s i s continues u n i n h i b i t e d  f o r s e v e r a l hours i m p l y i n g t h a t the mRNA i s q u i t e s t a b l e . Newly s y n t h e s i z e d protamine  i s phosphorylated  on i t s  s e r y l r e s i d u e s (267,268) p r o b a b l y due t o the a c t i o n of the protamine  k i n a s e s t u d i e d by J e r g i l and Dixon  (269).  protamine  i s r a p i d l y t r a n s p o r t e d i n t o the nucleus  New  and  thence i n t o the chromatin where i t i s dephosphorylated  and  r e p l a c e s the h i s t o n e s and a c i d i c p r o t e i n s i n a s s o c i a t i o n w i t h the DNA  (268,270). This replacement  process i s a s s o c i a -  t e d w i t h condensation of chromatin and a decrease i n temp l a t e a c t i v i t y when assayed w i t h the E. c o l i RNA in vitro  polymerase  (270).  I f a s s o c i a t i o n of protamine  w i t h DNA  represses that r e -  g i o n , then the spermatid must c a r e f u l l y c o n t r o l the r e p l a c e ment i n order t o prevent premature r e p r e s s i o n of gene f u n c t i o n s r e q u i r e d l a t e r . Thus, the replacement  process  cannot  be j u s t a k i n d o f c a t i o n exchange. The blocks o f a r g i n i n e i n protamine s u i t e d as b i n d i n g s i t e s t o DNA protamine  appear t o be w e l l  but i t i s not c l e a r t h a t  serves any f u n c t i o n other than a l l o w i n g a l a r g e  amount of a p o l y a n i o n (DNA)  to pack i n t o the s m a l l volume  of the sperm head; there i s about 2 metres of DNA volume of 20 c u b i c microns.  in a  MATERIALS AND METHODS Chemicals a)  and A b b r e v i a t i o n s  Chemicals Common chemicals were o b t a i n e d commercially  and were  o f reagent grade. P h e n y l i s o t h i o c y a n a t e and N-ethyl morphol i n e were purchased  from Eastman and were r e d i s t i l l e d  use. Anhydrous t r i f l u o r a c e t i c  before  a c i d was from Matheson, C o l e -  man and B e l l . Whatman 3 MM paper was used 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 and chromatography. P y r i d i n e was r e d i s t i l l e d a f t e r r e f l u x i n g with ninhydrin. t-butyloxycarbonyl-methionylN-hydroxysuccinimide  e s t e r was obtained from Mann Research  L a b o r a t o r i e s . Kodak "Royal Blue" X-ray f i l m was used f o r autoradiography.  Carboxypeptidase  B was from Worthington.  b) A b b r e v i a t i o n s RNase: r i b o n u c l e a s e tRNA: t r a n s f e r RNA mRNA: messenger RNA rRNA: ribosomal RNA EDTA: e t h y l e n e d i a m i n e t e t r a a c e t a t e M.W.:  m o l e c u l a r weight  EM: e l e c t r o n microscope i-factors: i n i t i a t i o n factors SH groups: s u l f h y d r y l groups NAD : n i c o t i n a m i d e adenine d i n u c l e o t i d e +  -111-  PITC: p h e n y i i s o t h i o c y a n a t e TMKS: TRIS-HC1, G.05 M; magnesium a c e t a t e , 0.005 M; potassium c h l o r i d e , 0.025 M; and s u c r o s e , 0.25 M. The pHiwas u s u a l l y 7.6 hut i n some experiments was  6.8  TMK: TMKS minus the sucrose TEA: t r i f l u o r a c e t i c  acid  Experimental Methods 1 . Fish A l l of the experiments here were w i t h t e s t e s from the rainbow t r o u t  (Salmo g a i r d n e r i i ) . During the p e r i o d o f  n a t u r a l m a t u r a t i o n , August t o December, t e s t e s were o b t a i n ed from t h e Sun V a l l e y t r o u t farm a t M i s s i o n , B.C. through the c o o p e r a t i o n o f Mr. and Mrs. Hans Lehmann. The t e s t e s were t r a n s p o r t e d t o the l a b o r a t o r y i n i c e and used immedia t e l y i n experiments. A l t e r n a t i v e l y , n a t u r a l l y maturing t r o u t were brought t o t h e l a b o r a t o r y and kept i n a q u a r i a f o r 2-3 weeks d u r i n g which they were used f o r experiments. For the p e r i o d January t o J u l y , t r o u t 1 —2 years o l d were kept i n a q u a r i a a t 10-12 °0 and i n j e c t e d t w i c e weekly w i t h 0.1 ml o f crude salmon p i t u i t a r y e x t r a c t  (Oncorhynchus  tshawytscha) c o n t a i n i n g gonadotrophic a c t i v i t y (271). Such i n j e c t i o n s induce complete m a t u r a t i o n of t h e t e s t e s i n about 10-12 weeks. Induced f i s h were g i v e n an e x t r a i n j e c t i o n 24 hours b e f o r e any experiments.  -112-  2. I n c u b a t i o n of C e l l  Suspensions  P i s h were a n a e s t h e s i z e d i n a d i l u t e s o l u t i o n o f T r i c a i n e Methanesulfonate  ( E t h y l m-aminobenzoate Methanesul-  f o n a t e ) obtained from F r a s e r M e d i c a l S u p p l i e s , Vancouver. The t e s t e s were removed and washed i n a beaker o f Hank's balanced s a l t s o l u t i o n a t 4 °C. T e s t i s weight was recorded' and then t h e blood v e s s e l s were i n c i s e d t o a l l o w most o f the blood t o escape. The t e s t e s were s c i s s o r - m i n c e d i n 4 volumes of Hank's s o l u t i o n and then homogenized by hand i n a P o t t e r - E l v e h j e m tube w i t h a T e f l o n p e s t l e . The c e l l  sus-  pension was f i l t e r e d through 2-3 l a y e r s o f pre-washed c h e e s e c l o t h t o remove c o n n e c t i v e t i s s u e and l a r g e clumps o f c e l l s . The c e l l s were u s u a l l y washed once by sedimenting a t 1000 g f o r 10 minutes and then resuspending i n f r e s h Hank's solution. Hank's s o l u t i o n was always m o d i f i e d by o m i t t i n g NaHCO^ and adding TRIS-HC1 t o 0.01 M and pH 7.35; t h i s gave a s o l u t i o n w i t h constant pH i n c o n t r a s t t o the s t a n d a r d Hank's s o l u t i o n i n which t h e pH i n c r e a s e s as C 0  2  escapes.  Amino  a c i d s , except f o r those t o be i n c l u d e d i n l a b e l l e d form, were added from concentrated s t o c k s o l u t i o n s t o g i v e a f i n a l c o n c e n t r a t i o n i d e n t i c a l t o t h e amino a c i d s i n t i s s u e c u l t u r e medium "199" (Ref.295). F o r i n c u b a t i o n s over 1 hour, vitamins were added a t the c o n c e n t r a t i o n used i n Waymouth's medium (296). Incubations were done by adding the c e l l suspension t o  -113-  a g l a s s v e s s e l and shaking i n a g y r a t o r y water bath  (New  Brunswick) a t 20, 18 or 15 °0; a l l r e c e n t experiments  were  done at 15 °C s i n c e f i s h enzymes might be l a b i l e at h i g h e r temperatures. Rapid s t i r r i n g i s r e q u i r e d t o prevent the c e l l s from clumping. A f t e r an i n c u b a t i o n , the bulk of the f r e e i s o t o p e was  removed by sedimenting the c e l l s at 1000  g  f o r 10 minutes and d i s c a r d i n g the supernatant. 3. E x t r a c t i o n o f  Protamine  Whole c e l l s , n u c l e i or chromatin were prepared e x t r a c t e d w i t h 0.2  N HOI  or 0.2  and  M H2SO4 as d e s c r i b e d i n  Part 1 "Methods". The crude a c i d e x t r a c t a b l e p r o t e i n s were u s u a l l y p r e f r a c t i o n a t e d by adsorbing on a column of c e l l u l o s e e q u i l i b r a t e d w i t h 0.2 a c e t a t e pH 5.0.  The column was  CM-  M L i C l i n 0.0,1 M l i t h i u m t h e n washed w i t h 0.2 M L i O l ,  0.7 M L i O l and water and f i n a l l y e l u t e d w i t h 0.2 N HOI remove the t i g h t l y bound protamine.which  was  to  then l y o p h i l -  i z e d . The L i O l washes removed n u c l e i c a c i d s , nonhistone t e i n s and the bulk of the h i s t o n e s . Pure protamine was  propre-  pared by chromatography o f the l y o p h i l i z e d f r a c t i o n above on a column of B i o - G e l P-10  e l u t e d w i t h 0.2 M a c e t i c  acid  (267). Small amounts o f the crude a c i d e x t r a c t a b l e p r o t e i n s c o u l d be d i r e c t l y a p p l i e d t o B i o - G e l P-10 t i o n o f protamine 4.  w i t h good s e p a r a -  from the other p r o t e i n s .  S t a r c h Gel E l e c t r o p h o r e s i s The method o f Sung and Smithies was  f o l l o w e d w i t h some  -114-  modifications  (272). 100 g of s t a r c h and  120 g o f u r e a were  added t o 400 ml of aluminum l a c t a t e b u f f e r (pH 3.1 ) i n a l a r g e Erlenmeyer  f l a s k and heated w i t h constant shaking i n  a b o i l i n g water bath f o r 5-8 minutes. was  then poured  The s t a r c h s o l u t i o n  i n t o the g e l t r a y and a s m a l l amount  was  poured around the s l o t - f o r m e r b e f o r e a p p l y i n g the l i d to prevent a i r bubbles  near the s l o t s . The f i n a l g e l composi-  t i o n i s 4 M u r e a and about 0.02 E l e c t r o p h o r e s i s was  M aluminum l a c t a t e at pHI3.4.  v e r t i c a l l y downwards at about 8 v o l t s / c m  f o r about 10 h; the exact l e n g t h o f the e l e c t r o p h o r e s i s was determined migrates  by the m i g r a t i o n o f a methyl green.marker which  j u s t ahead o f protamine  The g e l was  under these c o n d i t i o n s .  removed from the t r a y and t r i s e c t e d ; the  outer s l i c e s were s t a i n e d i n 200 ml o f Amido Black 1% a c e t i c a c i d  (0.125%, w/v)  10B;in  t o which 0.6 ml o f 1 M c o b a l t  n i t r a t e was  added j u s t before use. A f t e r s t a i n i n g a t l e a s t  45 minutes,  the s t a i n was  d r a i n e d and r e p l a c e d by 0.5  M  HgSO^; i n l e s s than 10 minutes the background fades t o a grey c o l o u r arid the p r o t e i n bands s t a i n b l a c k or b l u e . T h i s s t a i n was  found t o be 100 times as s e n s i t i v e as o r d i n a r y  s t a i n i n g w i t h Amido Black The middle s l i c e was  (272). r e s l i c e d t r a n s v e r s e l y at 3 mm  v a l s and these p i e c e s were p l a c e d i n s c i n t i l l a t i o n w i t h 0.75  ml of NCS  inter-  vials  s o l u b i l i z e r o v e r n i g h t . 7.5 ml of t o l u -  ene s c i n t i l l a t i o n f l u i d was i n g a t room temperature  added t o each and a f t e r s t a n d -  f o r 2-4  h the samples were  counted.  -115-  5.  Ion-Exchange Chromatography Protamine components were p a r t i a l l y r e s o l v e d by i o n -  exchange chromatography on C M - c e l l u l o s e u s i n g the methods d e s c r i b e d i n P a r t 1 except t h a t the g r a d i e n t o f l i t h i u m c h l o r i d e was from G . 7 to 1 . 3 M. 6 . Edman Degradation The method o f Edman ( 2 7 3 ) as m o d i f i e d by Hew ( 2 7 4 ) was f o l l o w e d . The p r o t e i n sample ( l e s s than G . 1 micromoles) was d i s s o l v e d i n 0.1) ml o f 50% aqueous p y r i d i n e (v/v) i n a s m a l l g l a s s tube. Then 0 . 1 ml o f PITC s o l u t i o n (1 ml o f r e d i s t i l l e d N-ethyl morpholine p l u s 0 . 1 ml o f PITC) was added w i t h mixing f o l l o w e d by degassing  w i t h n i t r o g e n . The tube was  s e a l e d and i n c u b a t e d a t 37 °C f o r 2 - | - 3 h. The r e a c t i o n mix was e x t r a c t e d 3 times w i t h 0 . 5 ml o f benzene and l y o p h i l i z e d . The r e s i d u e was d i s s o l v e d i n 0 . 2 ml o f anhydrous TFA, f l u s h e d w i t h n i t r o g e n and incubated mix  a t 37 °C f o r 1 h. The  was d r i e d i n vacuo, r e d i s s o l v e d i n 0;5o>ml water and  e x t r a c t e d 4 times w i t h 1 ml p o r t i o n s o f e t h y l a c e t a t e . A l i quots o f both phases were d r i e d i n s c i n t i l l a t i o n  vials,  r e d i s s o l v e d and counted i n Bray's s o l u t i o n ( 2 7 5 ) . 7.  Thin-Layer  Chromatography  For i d e n t i f i c a t i o n o f the r e s i d u e removed by Edman deg r a d a t i o n , an a l i q u o t of the e t h y l acetate phase was d r i e d and t r e a t e d w i t h 1 W HC1 a t 8 0 °C f o r 15 minutes t o convert the t h i a z o l i n o n e s t o the more s t a b l e t h i o h y d a n t o i n d e r i v a -  -116-  t i v e s . The sample was  then d r i e d , r e d i s s o l v e d i n 90$  acetic  a c i d and a l i q u o t s a p p l i e d t o a t h i n - l a y e r sheet of s i l i c a gel  w i t h f l u o r e s c e n t i n d i c a t o r (Eastman chromogram sheet  6060). Standard PTH amino a c i d s were s p o t t e d and the sheet was  developed  i n the s o l v e n t system,  heptane:n-butanol:  75$ f o r m i c a c i d (100:60:18, v/v) f o r 2£ h. A f t e r d r y i n g , the sheet was  photographed  under UV i l l u m i n a t i o n u s i n g a  P o l a r o i d camera as d e s c r i b e d i n Methods o f P a r t 1. The was  then cut at 0.5  scintillation  sheet  em i n t e r v a l s and counted i n toluene  fluid.  8. High Voltage Paper E l e c t r o p h o r e s i s Whatman 3 MM systems: pH 1.9 v/v);  pH 3.6  and pH 6.5  paper was  used w i t h the f o l l o w i n g b u f f e r  ( a c e t i c a c i d : f o r m i c acid:water, 8:2:90,  ( p y r i d i n e : a c e t i c acid:water, 1:10:89, v / v ) ;  ( p y r i d i n e : a c e t i c acid:water, 100:4:900, v / v ) .  The l e n g t h of e l e c t r o p h o r e s i s was  judged by the m i g r a t i o n  of c o l o u r e d dyes as d i s c u s s e d i n Methods o f P a r t 1. 9.  Autoradiography A f t e r thorough d r y i n g , paper  electrophoretograms were  l a b e l l e d w i t h r a d i o a c t i v e i n k and p l a c e d i n l i g h t - t i g h t j a c k e t s w i t h a sheet of Kodak "Royal Blue" X-ray  film;  weights were p l a c e d on the j a c k e t to ensure good c o n t a c t between the paper and the f i l m . A f t e r adequate  exposure,  approximately 24 h per 20,000 CPM  t  was  of  5 5  S  developed w i t h Kodak X-ray r e a g e n t s .  or  1 4  -  C f  n  e  f  i  l  m  -117-  10.  Counting Paper  Electrophoretograms  In pases where the sample c o n s i s t e d of l e s s than 5000 CPM  of 55s or H Q ,  an a l t e r n a t i v e method to  was used. As shown i n F i g . 5 ,  a p o r t i o n of the s t r i p pf  paper c o n t a i n i n g the sample peptides was at 1 cm or 0.5  autoradiography  cut t r a n s v e r s e l y  i n c h i n t e r v a l s , placed i n s c i n t i l l a t i o n v i a l s  w i t h 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 and counted. or 35>S +,  ne  e f f i c i e n c y of c o u n t i n g on paper was  5 0 - 6 0 $ of  that f o r an i d e n t i c a l sample d i s s o l v e d i n Bray's t i o n f l u i d . Thus, a few hundred CPM trophoresed and counted  For  scintilla-  o f sample c o u l d be  elec-  a c c u r a t e l y without the v e r y l o n g  ex-  posure p e r i o d r e q u i r e d f o r autoradiography. S i n c e o n l y a p o r t i o n of the sample s t r i p was  counted,  the remainder o f  the sample peptides c o u l d be l o c a t e d , cut out and phoresed  or s u b j e c t e d to descending  re-electro-  chromatography to con-  f i r m i d e n t i t y w i t h marker p e p t i d e s . The marker peptides c o u l d be l o c a t e d by autoradiography  overnight i f 2 5 , 0 0 0  CPM  or more was a p p l i e d . 11,  Formylation Samples were formylated.by  the method of Sheehan and  Yang ( 2 7 6 ) . The d r i e d sample was 98$ formic a c i d at 10-12  d i s s o l v e d i n 4 volumes o f  °C; t h r e e a l i q u o t s o f a c e t i c  an-  h y d r i d e , 1 volume each, were added w i t h mixing at 5 minute i n t e r v a l s and the r e a c t i o n was  allowed to continue f o r 1 h  a f t e r the l a s t a d d i t i o n . The mix was a dry formylated sample.  lyophilized leaving  -118-  Fig. 5 Counting Electrophoretograms  0  Cut out & count  Apply markers here  Origin Sample a p p l i e d here; can r e r u n p o r t i o n to r i g h t of d o t t e d lines ©  -119-  1 2 . Deforraylation Anhydrous 1 N HOI  i n methanol was  prepared  by d r i p p i n g  concentrated  HOI  i n t o concentrated ^ S O ^  r e l e a s e d HOI  gas  to bubble through methanol at 4 °C;  methanol was  t i t r a t e d and d i l u t e d t o 1 N HC1  d e f o r m y l a t i o n , a d r i e d sample was  and a l l o w i n g the the  content.  For  d i s s o l v e d i n 0 . 5 - 1 . 0 ml  o f the HC1 i n methanol, s e a l e d and incubated at 50 °C f o r 1 h. The mix  was  then d r i e d i n vacuo. Since t h i s method can  cause e s t e r i f i c a t i o n of c a r b o x y l groups by methanol, r e c e n t experiments were done u s i n g the method of Adams and (132)  Capecchi  i n which the sample i s t r e a t e d w i t h aqueous 1 N  at 100 °C f o r 10 minutes. This method was  HC1  found to g i v e  95% d e f o r m y l a t i o n and l e s s than 5% peptide bond h y d r o l y s i s  (132). 13.  Synthesis o f L a b e l l e d Marker a) M e t - C - P r o and 14  Peptides  F-Met- C-Pro U  25 m i c r o c u r i e s of 4 c _ p r o l i n e ( u n i f o r m l y l a b e l l e d , 1  265 mCi/mMole, Amersham) \wasr e d i s s o l v e d i n 0.2  d r i e d i n a s m a l l tube  ml of methylene c h l o r i d e . 25 mg  t-butyloxycarbonyl-methionyl-N-hydroxysuccinimide (Mann Research L a b o r a t o r i e s ) was tube was  and of  ester  added w i t h mixing and  the  s e a l e d and incuba.ted at 37 °C o v e r n i g h t . In the  morning, the sample was ethanol, 0.2  dry and t h e r e f o r e 0 . 4  ml of methylene c h l o r i d e and 1 mg  were added and i n c u b a t i o n was  continued  ml of 50% of HaHCO^  at 37 °C f o r 30 min-  utes a c c o r d i n g to Anderson, Zimmerman and C a l l a h a n  (277).  -120-  The sample was  d r i e d , r e d i s s o l v e d i n 1 ml of anhydrous  TFA  and incubated at 37 °0 f o r 1 h« A f t e r d r y i n g a g a i n , the sample was  r e d i s s o l v e d and e l e c t r o p h o r e s e d at pH 3.6  an autoradiogram  performed  and  w i t h the r e s u l t shown i n P i g . 6.  The band corresponding to M e t - 4 c - P r o was 1  e l u t e d from  paper w i t h 30$ a c e t i c a c i d and a t o t a l of 9.4X10  6  the  CPM  was  recovered f o r a y i e l d of 37$, the y i e l d c o u l d have been doubled i f h y d r o l y s i s w i t h TPA had removed a l l of the t - b u t y l o x y c a r b o n y l groups from the d i p e p t i d e . An a l i q u o t o f M e t - 4 c - P r o was 1  f o r m y l a t e d as d e s c r i b e d  e a r l i e r to produce P-Met- ^C-Pro. 1  b) C - M e t - P r o - A r g and F- 4c-Met-Pro-Arg 1  H  I t was  decided to prepare these peptides by coup-  l i n g ^.-methionine 1  to u n l a b e l l e d protamine.  T h i s c o u l d be  done without b l o c k i n g any groups on protamine s i n c e the secondary  amino group of the N-terminal p r o l i n e i s the o n l y  group i n protamine which i s s i g n i f i c a n t l y r e a c t i v e w i t h the a c t i v a t e d e s t e r employed below. Protamine propionate  was  prepared by a p p l y i n g 30 mg of protamine sulphate t o a QAE-Sephadex column (1X10 cm) i n the 0BT the column w i t h 0.2  N p r o p i o n i c a c i d . The  form and  eluting  e l u a t e was  then  l y o p h i l i z e d . T h i s conversion of protamine from the sulphate t o the propionate was  an attempt  to make i t more s o l u b l e  i n the s o l v e n t system d e s c r i b e d below. 10 m i c r o c u r i e s o f Ho-methionine ( u n i f o r m l y l a b e l l e d , 233 mCi/mMole, Amersham) was  formylated and coupled to  -121-  Fig. 6 Autoradiogram o f Electrophoretogram  of Reaction  Products  i n the Synthesis o f Met- *C-Pro 1  ©  0  Note: 0, o r i g i n ; 1, t - b u t y l o x y c a r b o n y l - m e t h i o n y l 1  4(3-proline; 2, f r e e 4 C - p r o l i n e ; 3, Met- 4(31  Pro. Band #3 was e l u t e d w i t h 30$ a c e t i c E l e c t r o p h o r e s i s was at pH 3.6.  1  acid.  -122-  F-hydroxysuccinimide by d i s s o l v i n g 2 mg o f t h e l a t t e r i n 0.2 ml o f dioxane and adding i t t o the F- -C-Met; then 1 mg 14  of d i c y c l o h e x y l c a r b o d i i m i d e was added and the mix was i n cubated a t 15 °0 f o r 15 minutes and o v e r n i g h t a t 4 °0. The supernatant was a s p i r a t e d from t h e c r y s t a l s o f d i c y c l o h e x y l urea and d r i e d . The r e s i d u e , c o n t a i n i n g t h e N-hydroxysuccinimide e s t e r o f F- -C-Met, was d i s s o l v e d i n 0.7 ml o f 95% 14  e t h a n o l . 30 mg o f protamine  propionate i n 0.7 ml o f water  and 1 mg o f NaHOO^ vwas added t o t h e e s t e r s o l u t i o n and mixed. A cloudy mixture r e s u l t e d which was p a r t i a l l y  clear-  ed by a d d i t i o n o f 0.2 ml o f dioxane. The mix was shaken a t 20 ° C f o r 22 h and then d i l u t e d 5 times w i t h water and a d sorbed t o a column o f Amberlite IRC-50 i n t h e H  +  form. The  column was then washed w i t h water t o remove s a l t and excess reagents and f i n a l l y the t i g h t l y bound protamine was e l u t e d with 0.2 N HC1 and l y o p h i l i z e d . The protamine  was then i n -  cubated i n 0.2 M ethylenediamine, pH 10.0 a t 37 °G: f o r 30 minutes t o d e - e s t e r i f y any s e r y l r e s i d u e s t h a t might have r e a c t e d w i t h the F- ^O-methionyl-Ff-hydroxysuccinimide 1  ester.  The i n c u b a t i o n mix was then d r i e d , r e d i s s o l v e d and chromatographed  on Sephadex G-10 i n 0.2 M a c e t i c a c i d ; the v o i d  1  f r a c t i o n was l y o p h i l i z e d and c o n t a i n e d 680,000 0PM f o r a y i e l d of 7%. The low y i e l d was p r o b a b l y due t o t h e i n s o l u b i l i t y o f the r e a c t a n t s but enough r e a c t i o n d i d occur t o g i v e a u s e f u l product. The product was d i g e s t e d w i t h p o r c i n e t r y p s i n (1:20,  -123-  w/w)  i n 0.2  M M^HOO^ at 37 °C f o r 4 h and d r i e d i n vacuo.  A p o r t i o n was  t r e a t e d w i t h 1 IT HOI i n methanol  at 50  f o r 1 h and d r i e d . Since the N"-terminal sequence  °G  of a l l  major t r o u t protamine components i s Pro-Arg-Arg...  (261  ),  the above procedures gave the t r y p t i c peptides F- 4c-Met1  Pro-Arg and Hc-Met-Pro-Arg. 14.  Carboxypeptidase B D i g e s t s Vigorous d i g e s t i o n w i t h carboxypeptidase B was  t o c l e a v e the Pro-Arg bond i n the t r i p e p t i d e ,  found  Met-Pro-Arg.  S u i t a b l e c o n d i t i o n s were a n enzyme:substrate r a t i o o f 1:1 (w/w)  and i n c u b a t i o n i n 0.2 M FEMO b u f f e r (0.2  morpholine i n water a d j u s t e d t o pH 8.5 at 37 °C f o r 6-8 15.  Pulse-Chase  M N-ethyl  with acetic acid)  h. Experiments  E a r l y experiments i n v o l v e d i n c u b a t i n g t e s t i s  cells  from protamine-stage t r o u t w i t h 35s-methionine and - a r g i n i n e f o r 30-60 minutes, washing the c e l l s and c o n t i n u i n g the i n c u b a t i o n i n f r e s h medium w i t h u n l a b e l l e d amino a c i d s . At i n t e r v a l s , a l i q u o t s of c e l l s were removed and f r o z e n i n a dry i c e bath. Protamine was  p u r i f i e d from  eaoh sample and the s p e c i f i c a c t i v i t y of both l a b e l s  was  measured. In these experiments i t was not p o s s i b l e t o show c o n c l u s i v e l y a l o s s of methionine from protamine duri n g the "chase" p e r i o d ; i n f a c t , the s p e c i f i c a c t i v i t y o f the methionine l a b e l i n protamine continued t o i n c r e a s e f o r  -124  about 30 minutes e a r l y i n the chase p e r i o d . T h i s i n d i c a t e d t h a t the c e l l s had b u i l t up an i n t r a c e l l u l a r pool o f l a b e l l e d methionine t h a t c o u l d not be r a p i d l y d i l u t e d w i t h exogenous u n l a b e l l e d methionine. Success i n demonstrating the removal o f methionine from newly s y n t h e s i z e d protamine r e q u i r e d s h o r t e r l a b e l l i n g p e r i o d s o f 5-10 minutes a t 17 °0, sedimenting the c e l l s and resuspending i n f r e s h medium w i t h u n l a b e l l e d amino a c i d s . I n c u b a t i o n f o r the chase p e r i o d was c o n t i n u e d a t 17 °C. F u r t h e r d e t a i l s o f two experiments w i l l be d i s c u s s e d . Experiment A A c e l l s u s p e n s i o n from 15 g o f n a t u r a l l y maturing t r o u t t e s t i s was prepared. The s u s p e n s i o n was d i v i d e d i n h a l f ; 4 m i c r o c u r i e s / m l o f 35s-methionine  (573 mCi/mMole,  Amersham)  was added t o one h a l f and 4 m i c r o c u r i e s / m l o f ^Hr-arginine (1.2 Ci/mMole, New England N u c l e a r ) was added t o the other h a l f . I n c u b a t i o n was at 18 °C f o r 10 minutes, the c e l l s were then washed, resuspended i n Hank's s o l u t i o n made 1 mM i n u n l a b e l l e d methionine and a r g i n i n e and i n c u b a t i o n was cont i n u e d a t 18 °C f o r 2 h. Equal a l i q u o t s o f c e l l s were r e moved a t i n t e r v a l s s t a r t i n g a t t h e beginning o f the chase p e r i o d . These were f r o z e n and l a t e r a l l were processed by homogenizing  i n saline-EDTA ( 0 . 0 7 5 M NaCl, 0 . 0 2 4 M EDTA,  pH 8.0) and n u c l e i were p e l l e t e d at 3000 g f o r 10 minutes. The n u c l e a r p e l l e t s were e x t r a c t e d t w i c e w i t h 0.2 N HC1 a t 4 °0 by homogenizing w i t h a TRI-R t i s s u e homogenizer  (TRI-R  -125-  Instruments,  New  Y o r k ) . The a c i d e x t r a c t s were n e u t r a l i z e d  w i t h LiOH 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 at 13,000 g f o r 15 minutes. The supernatants were to i n d i v i d u a l CM-cellulose columns ( 1 . 5 X 5 cm)  adsorbed  equilibrated  w i t h 0.2 M L i O l . The columns were then washed w i t h 0.2 f o l l o w e d by 0 . 7 5 M L i O l u n t i l no l a b e l was  M  present i n the  e l u a t e . The columns were then washed w i t h water or 0.01  M  l i t h i u m a c e t a t e to remove most o f the L i O l from the column and f i n a l l y the t i g h t l y bound protamine was 0.2  N HC1  eluted with  and l y o p h i l i z e d . The protamine samples were then  f r e e of a l l f r e e l a b e l l e d amino a c i d s and a l l other prot e i n s except a s m a l l amount of h i s t o n e s ; the l a t t e r were removed by chromatography of each sample on B i o - G e l columns (2.6X15 cm) e l u t e d w i t h 0.2 M a c e t i c a c i d . protamine peaks were l y o p h i l i z e d i n d i v i d u a l l y and  P-10 The  redissolv-  ed i n d i s t i l l e d water; t h e i r absorbance at 230 nm was sured and a l i q u o t s were counted  i n Bray's  mea-  solution.  Experiment B A c e l l suspension was  prepared from hormonally  induced  t r o u t t e s t e s . The c e l l c o n c e n t r a t i o n i n the suspension measured w i t h the use o f a hemocytometer and the was  suspension  d i l u t e d to 10^ c e l l s / m l i n Hank's s o l u t i o n . A 1.6  a l i q u o t of c e l l s was  me  (690  ml  incubated i n a small g l a s s tube at  17 °C f o r 6 minutes i n the presence 35s_ thionine  was  o f 100 m i c r o c u r i e s o f  mCi/mMole, Amersham) and  100  microcur-  i e s of ^H-arginine (1.85 Oi/mMole, Amersham). At 6 minutes,  -126-  0.1 ml o f c e l l s was  removed and i n j e c t e d i n t o 5 ml of c o l d  TCA-tungstate (10% TCA,  0.25%  sodium t u n g s t a t e , pH 2, Ref.  265). The remainder o f the c e l l s were washed b y c e n t r i f u g i n g at 1000 g f o r 10 minutes and resuspending i n 3 ml o f f r e s h medium c o n t a i n i n g a l l 20 u n l a b e l l e d amino a c i d s . I n c u b a t i o n was  continued at 17 °C and 0.2 ml a l i q u o t s o f c e l l s  were removed a t i n t e r v a l s s t a r t i n g immediately a f t e r r e s u s pension. Cycloheximide was added at 60 minutes o f the chase t o stop any f u r t h e r protamine s y n t h e s i s even though i t was known t h a t t h i s was  probably too l a t e t o make any  differ-  ence i n the apparent r a t e o f methionine removal. T h i s i n h i b i t o r was  not added e a r l i e r i n the experiment s i n c e i t  might have a c t u a l l y i n t e r f e r e d w i t h methionine removal; c y cloheximide i n h i b i t s  e u k a r y o t i c t r a n s f e r a s e 2 by r e a c t i o n  at i t s e s s e n t i a l SH group and might t h e r e f o r e i n t e r f e r e w i t h the p u t a t i v e methionine aminopeptidase (281 ). I n r e t r o s p e c t , the cycloheximide a d d i t i o n was not n e c e s s a r y as v i r t u a l l y a l l o f the methionine had been removed from p r o tamine by 30 minutes o f the chase. The method o f A. Louie and G.H.  Dixon ( i n p r e p a r a t i o n )  f o r c o l l e c t i n g s m a l l numbers o f c e l l s and e x t r a c t i n g , sepa r a t i n g and a s s a y i n g the r a d i o a c t i v i t y i n t h e i r b a s i c p r o t e i n s was  employed  as f o l l o w s : 2X10*7 c e l l s were f i l t e r e d  onto g l a s s f i b r e f i l t e r s  and washed w i t h 40 ml of c o l d  10% TCA-tungstate and 10 ml of c o l d 95% e t h a n o l . The s e c -  -127-  t i o n s o f the f i l t e r s  b e a r i n g the c e l l s were c u t out ( 4 X 8 mm)  and p l a c e d i n the s l o t s o f a starch-urea-aluminum l a c t a t e gel  w i t h 0.05 ml o f 0 . 4 N H C 1 and e l e c t r o p h o r e s i s was  formed a t 8 volts/cm f o r 1 0 h a t 4 ° C s l i c e d , s t a i n e d and counted  The g e l was  per-  then  as d e s c r i b e d e a r l i e r i n t h i s  section. 16. I n h i b i t o r Studies A c e l l suspension was prepared maturing  from 1 4 g of n a t u r a l l y  t r o u t t e s t i s and d i v i d e d i n t o 12 equal  aliquots.  Each was i n c u b a t e d at 4 °C f o r 3 0 minutes i n the p r e sence o f v a r i o u s c o n c e n t r a t i o n s o f cycloheximide  or c h l o r -  amphenicol w i t h c o n t r o l samples r e c e i v i n g no a d d i t i o n s . -methionine  ( 5 7 3 mCi/mMole, Amersham) was then added t o  each sample a t 3 m i c r o c u r i e s / m l and i n c u b a t i o n continued a t 18 °C f o r 1 h. The c e l l s were then c h i l l e d , washed and f r o zen i n a d r y i c e bath. C e l l p e l l e t s were homogenized i n TMKS u s i n g the TRI-R t i s s u e homogenizer at h i g h speed f o r 2 minutes and n u c l e i were then sedimented at 3 0 0 0 g f o r 1 0 minutes. The n u c l e a r p e l l e t s were e x t r a c t e d by homogenizing i n c o l d 0 . 2 M H 2 S O 4 f o l l o w e d by c e n t r i f u g a t i o n a t 1 3 , 0 0 0 g for  15 minutes; b a s i c p r o t e i n s were p r e c i p i t a t e d by a d d i -  t i o n o f 4 volumes of 9 5 % ethanol t o the supernatants  above  and s t a n d i n g a t - 2 0 °G o v e r n i g h t . The p r e c i p i t a t e s were c o l l e c t e d by c e n t r i f u g a t i o n , r e d i s s o l v e d i n 0 . 0 1 M l i t h i u m a c e t a t e and adsorbed columns ( 1 . 5 X 5  i n d i v i d u a l l y t o a s e r i e s of C M - c e l l u l o s e  cm) i n the H  +  form. The columns were washed  -128-  w i t h 0.75  M L i O l u n t i l no r a d i o a c t i v i t y remained i n t h e  e l u a t e and then washed w i t h water and f i n a l l y the hound protamine  was  e l u t e d w i t h 0.2  N HC1 and  tightly  lyophilized.  The samples were r e d i s s o l v e d i n d i s t i l l e d water and  the  absorbance at 230 nm measured: a l i q u o t s were counted i n Bray's  solution.  A s i m i l a r experiment  t o the above was  done t o t e s t  the e f f e c t of aminopterin ( o b t a i n e d from the Drug  Develop-  ment Branch, Cancer Chemotherapy, N a t i o n a l I n s t i t u t e o f H e a l t h , Bethesda, Maryland) on protamine c e l l suspension was  synthesis. A  prepared from 12 g of n a t u r a l l y matur-  i n g t r o u t t e s t i s and d i v i d e d i n t o 6 equal a l i q u o t s . Each was  i n c u b a t e d at 18 °C f o r 30 minutes i n the presence  of  v a r i o u s c o n c e n t r a t i o n s o f aminopterin w i t h c o n t r o l s r e c e i v i n g no a d d i t i o n s , ^ s ^ e - f c h i o n i n e (500 was  mCi/mMole, Amersham)  then added t o each sample at 5 m i c r o c u r i e s / m l and continued at 18 °C f o r 1 h. The  b a t i o n was  incu-  c e l l s were then  c h i l l e d , washed and f r o z e n i n a dry i c e bath. F u r t h e r p r o c e s s i n g was 17.  i d e n t i c a l t o t h a t i n the experiment  above.  Nascent Peptides a) Puromycin-rel eased Nascent Peptides 15 g of n a t u r a l l y maturing  stage) was  u t e s . The  (October  used to prepare a c e l l suspension. H a l f of the  suspension was methionine  trout testis  (500  i n c u b a t e d w i t h 3 m i c r o c u r i e s / m l of  35s-  mCi/mMole, Amersham) at 18 °C f o r 10 min-  other h a l f was  incubated with 4 microcuries/ml  -129-  o f 1 C-formate (45.8 mOi/mMole, sodium s a l t , Amersham) a t 4  18 °0 f o r 10 minutes. Puromycin was then added t o both samp l e s a t a c o n c e n t r a t i o n o f 5X10""^ M and i n c u b a t i o n was  con-  t i n u e d a t 18 °0 f o r 1 h. The c e l l s were c h i l l e d , washed and f r o z e n . The c e l l p e l l e t s were homogenized i n TMKS and the homogenates c e n t r i f u g e d a t 20,000 g f o r 15 minutes. The supernatants were p l a c e d i n tubes, underlayed w i t h 6 ml o f 2 M s u c r o s e i n TMK and c e n t r i f u g e d a t 22,000 rpm f o r 10 h i n the SW-27 r o t o r o f the Spinco L2-65 u l t r a c e n t r i f u g e . The upper two-thirds o f the supernatants were saved. Columns (1X5 cm) o f Dowex-50 i n the H  +  form and QAE-  Sephadex i n the 0H~ form were prepared. The pH o f the supernatants was a d j u s t e d t o 2 and they were adsorbed on the Dowex-50 columns. These columns were then washed w i t h 0.01 N HG1 and the e l u a t e s were saved and pH a d j u s t e d t o 8 w i t h NaOH. The l a t t e r were adsorbed t o the QAE-Sephadex columns and washed w i t h water. The Dowex-50 columns were f i n a l l y e l u t e d w i t h 4 M; p y r i d i n e and the e l u a t e s were d i l u t e d and l y o p h i l i z e d . The QAE-Sephadex columns were e l u t e d w i t h 2 M. a c e t i c a c i d and the e l u a t e s were d i l u t e d and l y o p h i l i z e d . The 4 samples were then d i g e s t e d w i t h p o r c i n e t r y p s i n or t r y p s i n f o l l o w e d by carboxypeptidase B'. The d i g e s t s were e l e c t r o p h o r e s e d a t pHf. 3.6 and an autoradiogram performed. phoresed  C e r t a i n l a b e l l e d spots were c u t out and r e - e l e c t r o -  a t pH 6.5 and an autoradiogram  performed. Marker  peptides were i n c l u d e d i n both e l e c t r o p h e r e t o g r a m s .  -130-  b) Ribosome-bound Nascent Peptides A cell  suspension from 12 g o f hormonally  t r o u t t e s t i s was  induced  incubated at 15 °C f o r 20 minutes w i t h  20 m i c r o c u r i e s / m l of 35s-methionine (690 mCi/mMole, Amersham). At 15 minutes cycloheximide was  added to 2X10""4 M  i n order t o a r r e s t nascent p e p t i d e s on the ribosomes d u r i n g the subsequent s t e p s ; otherwise, the chains might be comp l e t e d and r e l e a s e d and i n the absence of f u r t h e r  initia-  t i o n , l i t t l e nascent p r o t e i n would remain on the At 20 minutes,  ribosomes.  the c e l l s were washed: and then homogenized  i n m o d i f i e d TMKS s o l u t i o n CpH 6.8)  c o n t a i n i n g 2X10" " c y c l o 4  heximide u s i n g the TRI-R t i s s u e homogenizer at medium speed for  1 minute. The homogenate was  10 minutes; X-100 TMK  the supernatant was  c e n t r i f u g e d at 15,000 g f o r a d j u s t e d t o 0.5$  i n Triton  by a d d i t i o n of a 20$ s o l u t i o n of the detergent i n  and r e c e n t r i f u g e d at 15,000 g f o r 20 minutes.  natant was  The  i n 13  l a y e r e d over 2 ml of 30$ sucrose i n TMK  tubes and c e n t r i f u g e d a t 60,000 rpm r o t o r f o r 2^ h a t 4 0 . The G  gently aspirat-  ed and the ribosome p e l l e t s washed g e n t l y w i t h  TMK.  i n 6 ml of TMK  a Pasteur p i p e t t e and any clumps were removed by g a t i o n at 13,000 g f o r 10 minutes.  by use  of  centrifu-  The supernatant was d i -  v i d e d i n t o t h r e e 2 ml a l i q u o t s and l a y e r e d over each o f t h r e e tubes c o n t a i n i n g l i n e a r g r a d i e n t s of sucrose 10 t o 30$ i n TMK;  ml  i n the Spinco L2-65  supernatant was  The ribosomes were resuspended  super-  from  the g r a d i e n t s had been formed u s i n g a  -131-  Beckman d e n s i t y g r a d i e n t former  (DGF-IM-3). The tubes were  c e n t r i f u g e d a t 25,000 rpm i n the Spinco SW-27 r o t o r f o r 3-J- h and stopped without b r a k i n g . The tubes were punctured a t the bottom and 1 ml f r a c t i o n s c o l l e c t e d . The absorbance a t 254 nm o f each f r a c t i o n was measured and 0.2 ml a l i q u o t s were counted  i n Bray's  solution.-  The sucrose g r a d i e n t f r a c t i o n s were pooled i n groups from the monosome and polysome r e g i o n s , d i l u t e d w i t h TMK CpHi6.8) and c e n t r i f u g e d a t 25,500 rpm i n the SW-27 r o t o r f o r 6 h a t 4 ° C The supernatants were decanted  and t h e  ribosome p e l l e t s t r e a t e d as f o l l o w s : i , Monosomes and disomes were suspended i n 1 ml o f 0.2 M NH4HOO3 and d i g e s t e d w i t h 0.05 mg o f p a n c r e a t i c RNase a t 37 °C f o r 15 minutes and then l y o p h i l i z e d . The r e s i d u e was t r e a t e d w i t h 0.2 M ethylenediamine, pH 10 a t 37 °0 f o r 15 minutes t o h y d r o l y z e nascent p e p t i d e s from adenosine;  this  i s a m o d i f i c a t i o n o f the method o f Acs and Lipmann (297). The pH was then adjusted" t o 5 . O l w i t h - a c e t i c - a c i d and some 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 . The s u pernatant was d r i e d and d i g e s t e d w i t h 0.05 mg o f p o r c i n e t r y p s i n i n 0.25 ml o f 0.2 M NH HC0 4  5  a t 37 °G f o r 3 h. One-  t h i r d o f t h e d i g e s t was f u r t h e r t r e a t e d w i t h 0.1 mg o f CpBi at 37 °0 f o r 4 h. The d i g e s t s were pooled, d r i e d ,  redissolv-  ed and e l e c t r o p h o r e s e d at pH 3.6 t o g e t h e r w i t h marker pept i d e s . An autoradiogram  was performed  t o l o c a t e the r a d i o -  a c t i v e marker p e p t i d e s and t h e sample s t r i p s were c u t a t  -132-  0.5  i n c h i n t e r v a l s and counted i n 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 . As o u t l i n e d i n F i g . 5 ,  the remainder of the sample  peptide r e g i o n s were cut out, sewn i n t o a new sheet o f paper and r e - e l e c t r o p h o r e s e d a t pH 1.9 The paper was  w i t h marker p e p t i d e s .  a g a i n cut and counted as  above.  i i Ribosomes from 3 r e g i o n s o f the s u c r o s e g r a d i e n t were i n c u b a t e d i n 0 . 5 ml of 1 M NH^OH at 37 °0 f o r 30 minutes t o h y d r o l y z e nascent p e p t i d e s from tRNA. TCA was  then  added t o 10% and,, a f t e r s t a n d i n g at 4 °C f o r 1 h, the tubes were c e n t r i f u g e d at 1 3 , 0 0 0 g f o r 10 minutes. The supernatants were e x t r a c t e d 3 times w i t h e t h e r t o remove TCA and then l y o p h i l i z e d . The r e s i d u e s , c o n t a i n i n g 35s-methionine  labelled  nascent p e p t i d e s , were submitted t o Edman d e g r a d a t i o n as d e s c r i b e d e a r l i e r . A l i q u o t s of e t h y l a c e t a t e and aqueous phases were counted i n Bray's s o l u t i o n . 18.  Enzyme Assays Trout t e s t i s was  assayed f o r enzyme a c t i v i t i e s capable  of removing methionine from protamine and the d i p e p t i d e , Met-Pro; t e s t i s e x t r a c t s were a l s o assayed f o r the a b i l i t y to remove a c y l groups from methionine and the d i p e p t i d e , Met-Pro. Enzyme e x t r a c t s were prepared from f r o z e n October t e s tes which had been s t o r e d at - 8 0 °G.  The t e s t e s were p a r -  t i a l l y thawed and then homogenized in. TMKS c o n t a i n i n g I O  -4-  C l e l a n d ' s reagent ( d i t h i o t h r e i t o l ) i n a Waring b l e n d o r at h i g h speed f o r 2 minutes. N u c l e i were p e l l e t e d at 1500 g  M  -133-  f o r 10 minutes, m i t o c h o n d r i a a t 20,000 g f o r 30 minutes and ribosomes a t 25,000 rpm i n t h e SW-27 r o t o r f o r 4 h . N u c l e i and m i t o c h o n d r i a were l y s e d i n TMK (pH 7.0) and centrifuged  a t 20,000 g f o r 20 minutes, s a v i n g  the superna-  t a n t . Ribosomes were washed w i t h 1 M NH4CI i n TMK and  then c e n t r i f u g e d  overnight  a t 26,000 rpm i n t h e SW-27 r o t o r f o r  6 h, s a v i n g the supernatant. A l l e x t r a c t s were p r e c i p i t a t e d at 90% s a t u r a t i o n w i t h ammonium s u l p h a t e ;  the p r e c i p i t a t e s  were c o l l e c t e d by c e n t r i f u g a t i o n , r e d i s s o l v e d i n TMK (pH 7.0) and  dialyzed against  p r e c i p i t a t e d during The  the same b u f f e r a t 4 °C. M a t e r i a l which d i a l y s i s was removed by c e n t r i f u g a t i o n .  e x t r a c t , volumes were each measured and then d i v i d e d i n t o  s e v e r a l small batches; some were f r o z e n a t -80 °0 and some were s t o r e d a t 4 °0 and used w i t h i n a week, a) Assay f o r methionine removing enzyme i  ^ S n n e t h i o n i n e l a b e l l e d protamine was p u r i f i e d  from t e s t i s c e l l s incubated w i t h t h i s l a b e l as d e s c r i b e d i n s e c t i o n #3 above. 4 c - a r g i n i n e 1  l a b e l l e d protamine was  prepared i n the same f a s h i o n . The n u c l e a r postmitochondrial  l y s a t e and t h e  supernatant f r a c t i o n s o f t e s t i s  extracts  were assayed as f o l l o w s : 0.01 ml o f l a b e l l e d protamine i n water, 0.02 ml o f 2 M F a C l i n water, 0.1 ml o f e x t r a c t and TMK up t o a t o t a l volume o f 0.2 ml were mixed and i n c u b a t e d at 15 °0 f o r 40 minutes. R e a c t i o n was stopped by c h i l l i n g and  a d d i t i o n o f 0.02 ml o f 3.4 N, HC1. A 0.1 ml a l i q u o t o f  each r e a c t i o n mix was a p p l i e d t o f i l t e r paper d i s c s which  -134-  were plunged i n c o l d TCA-tungstate  (10$ TCA,  0.25$ sodium  t u n g s t a t e , pH 2 ) , washed w i t h the same, b o i l e d f o r 1 minute i n the same, washed a g a i n and then washed w i t h 95$ e t h anol and f i n a l l y w i t h e t h e r . The d r i e d d i s c s were counted i n toluene s c i n t i l l a t i o n  fluid.  i i Met- 4c-Pro was 1  t i o n #13  above. Assay was  enzyme e x t r a c t ( i n TMK,  prepared as d e s c r i b e d i n s e c -  done by i n c u b a t i n g 0.05  pH 7.0) w i t h 0.01  ml o f  ml of l a b e l l e d  d i p e p t i d e i n water at 16 °C f o r 45 minutes. R e a c t i o n was stopped by adding 0.01  ml of 1 N KOH  and c h i l l i n g . The sam-  ples were then a p p l i e d t o paper and e l e c t r o p h o r e s e d a t pH 3.6  and 40 v o l t s / c m f o r 20-40 minutes. R e a c t i o n products  were l o c a t e d by autoradiography, cut out and counted i n toluene s c i n t i l l a t i o n  fluid,  b) Assay f o r "Deformylase" A c y l a t e d amino a c i d s were prepared by mixing a s m a l l amount o f l a b e l l e d amino a c i d w i t h a l a r g e amount o f the same u n l a b e l l e d amino a c i d and then performing the a c y l a t i o n r e a c t i o n . Thus, 20 micromoles amino a c i d plus approximately 0.01 led  o f an u n l a b e l l e d  micromoles  of unlabel-  amino a c i d were d r i e d i n a tube and r e d i s s o l v e d i n  0.4 ml of 0.2 M NEMO b u f f e r (pH 8.5). Then, 0.05 the a p p r o p r i a t e a c i d anhydride was  added and r e a c t i o n a l l o w -  ed at 20 °C f o r 1 h. To ensure complete each of r e d i s t i l l e d  ml of  a c y l a t i o n , 0.05  p y r i d i n e ( c o n c e n t r a t e d ) and 0.05  ml  ml o f  the a c i d anhydride were added i n t h a t order and the tubes  -135were shaken at 20 °0 o v e r n i g h t .  Each tube was  then e x t r a c t -  ed 3 times w i t h 5 ml p o r t i o n s of ether to remove the excess reagents;  b u t y r i c , v a l e r i c and hexanoic a c i d s a l l formed  emulsions during the r e a c t i o n and had t o be removed by  the  ether e x t r a c t i o n s s i n c e they c o u l d not be l y o p h i l i z e d .  The  aqueous phase were then 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 5 ml of TMK  (pH 7.0).  Thus, each p r e p a r a t i o n was  4X10~  5  M in  acy-  l a t e d amino a c i d . Assay was TMK  and  by i n c u b a t i n g 0.025 ml enzyme e x t r a c t , 0.025 ml  0.025 ml o f 4X10"3 M s u b s t r a t e at 20 °0 f o r 30 min-  u t e s . R e a c t i o n was  stopped by c h i l l i n g and  ml o f 1 N KOH.  samples were t h e n a p p l i e d t o paper  The  electrophoresed  at pH 6.5  and 40 volts/cm  a d d i t i o n of  w i t h KOH  s c i n t i l l a t i o n f l u i d . The  and  f o r 30 minutes.  Products were l o c a t e d by autoradiography, cut out and ed i n toluene  0.01  count-  r e a c t i o n s were stopped  because the a c y l a t e d s u b s t r a t e s would be n e u t r a l  i f a c i d was  used and might c o - p r e c i p i t a t e w i t h the protein,  i n the i n c u b a t i o n  mix.  -136-  RESULTS AFP DISCUSSION I n c o r p o r a t i o n of Methionine A c e l l s u s p e n s i o n was maturing t r o u t t e s t i s  prepared from 25 g o f n a t u r a l l y  (December s t a g e ) and was i n c u b a t e d  w i t h 1 m i c r o c u r i e / m l o f ^-c-methyl-methionine 1  New  (11 mCi/mMole,  England N u c l e a r ) a t 20 °C f o r 2 h. The c e l l s were then  washed, homogenized i n saline-EDTA and n u c l e i p e l l e t e d at 3000 g f o r 10 minutes. The n u c l e a r p e l l e t was twice w i t h 0.2 M B^SO^  extracted  and the a c i d - s o l u b l e p r o t e i n s p r e -  c i p i t a t e d w i t h a d d i t i o n o f 4 volumes o f 95$ e t h a n o l and s t a n d i n g at -20 °C o v e r n i g h t . The p r e c i p i t a t e was  redissolv-  ed and chromatographed on a column of B i o - G e l P-10  (5X 45  e l u t e d w i t h 0.2 M a c e t i c a c i d . The absorbance at 230 nm  cm)  of  each f r a c t i o n was measured and a l i q u o t s were counted i n Bray's s o l u t i o n . The r e s u l t s are shown i n P i g . 7 and cate t h a t methionine was  indi-  i n c o r p o r a t e d i n t o both the h i s t o n e s  (peak 1) and protamine (peak 2 ) . This r e s u l t was  surprising  s i n c e r o u t i n e amino a c i d a n a l y s e s had not r e v e a l e d the p r e sence o f any methionine i n protamine. To ensure t h a t the l a b e l l e d methionine was  actually i n  protamine and not i n some other p r o t e i n e l u t i n g a t the same p o s i t i o n on the B i o - G e l P-10 ( P i g . 7) was  column, an a l i q u o t o f peak 2  e l e c t r o p h o r e s e d w i t h an u n l a b e l l e d  marker on a starch-urea-aluminum f o r 12 h. The g e l was  protamine  l a c t a t e g e l at 6 volts/cm  s l i c e d , s t a i n e d and counted as des-  c r i b e d i n s e c t i o n #4 of "Methods" except t h a t the s m a l l  - 1 3 7 -  *ig.  7  ^ ^C-methyl-methionine  Incorporation  Into Trout T e s t i s Nuclear B a s i c P r o t e i n s  Note: Polyacrylamide d i s c g e l e l e c t r o p h o r e s i s o f peak 1 r e v e a l e d h i s t o n e s and peak 2 had only protamine.  -138-  g e l s l i c e s were d i s s o l v e d i n 0.4 hydroxide and counted i n Bray's Cab-O-Sil  ml o f tetraethylammonium s o l u t i o n c o n t a i n i n g 3$  (w/v). The r e s u l t s shown i n F i g . 8 i n d i c a t e t h a t  a l l of the r a d i o a c t i v i t y c o e l e c t r o p h o r e s e d w i t h the u n l a b e l l e d protamine  or j u s t behind i t ; newly s y n t h e s i z e d p r o -  tamine i s phosphorylated wards the cathode dephosphorylated  (267,268) and migrates slower t o -  at pH 3.4, protamine  the pH of the g e l , than o l d e r  (269) which i s the predominant  form i n the marker. The g e l s l i c e s here were p r o b a b l y too wide (0.7 cm)  t o r e v e a l s e p a r a t i o n of phosphorylated  tamine components: as observed r e c e n t l y by Louie and (unpublished r e s u l t s ) . However, the r e s u l t s here t h a t the l a b e l l e d methionine  was  proDixon  indicate  incorporated into prota-  mine . S i n c e the methyl group of methionine  r e a d i l y enters the  1-carbon p o o l , the ^0-methyl l a b e l c o u l d have entered p r o 1  tamine as s e r i n e or as a methyl group on a r e s i d u e such as a r g i n i n e (278). A c c o r d i n g l y , an a l i q u o t o f peak 2 ( F i g . 7) was  h y d r o l y z e d w i t h 6 N! HC1  s u l t i n g mixture pH 1.9  at 110 °C f o r 22 h and the r e -  of f r e e amino a c i d s was  and 60 volts/cm f o r 45 minutes.  s t r i p was  e l e c t r o p h o r e s e d at P a r t of the sample  s t a i n e d w i t h n i n h y d r i n together w i t h the r e g i o n  c o n t a i n i n g standard amino a c i d s and the remainder sample r e g i o n was  cut at 0.5  of the  i n c h i n t e r v a l s and counted;.in.  toluene s c i n t i l l a t i o n f l u i d . A l l of the r a d i o a c t i v i t y  co-  e l e c t r o p h o r e s e d w i t h the u n l a b e l l e d methionine marker. T h i s  -139-  Fig. 8 Electrophoresis of ^c-methyl-methionine Labelled 1  Protamine on a Starch-urea-aluminum l a c t a t e g e l  1500-  1000-  500-  DISTANCE  cm  0  Note: 0, o r i g i n ; P, p o s i t i o n of unlabelled protamine marker on stained g e l s l i c e s . The middle gel s l i c e was cut at 0.7 cm i n t e r v a l s for counting.  -140-  experiment was repeated  a f t e r performic a c i d o x i d a t i o n o f  the a c i d h y d r o l y s a t e and t h e r a d i o a c t i v i t y  coelectrophores-  ed w i t h an u n l a b e l l e d methionine sulphone marker at pH 1 . 9 . These r e s u l t s showed t h a t the k<-methyl 1/  l  a  ^  e  l  w  a  s  all  i n c o r p o r a t e d i n t o protamine as i n t a c t methionine.  Methionine i s N-terminal The p o s s i b i l i t y t h a t methionine was i n v o l v e d i n the i n i t i a t i o n o f protamine b i o s y n t h e s i s was obvious a l experiments were done t o t r y t o c o n f i r m  and s e v e r -  this.  To determine whether t h e i n c o r p o r a t e d methionine was N-terminal, Edman degradation  and d a n s y l a t i o n were perform-  ed on p u r i f i e d 35s-methionine l a b e l l e d protamine s y n t h e s i z e d i n aasuspension  o f t r o u t t e s t i s c e l l s . Table 4 shows t h a t  76-89% o f the 35s -methionine l a b e l can be recovered  i n the  e t h y l a c e t a t e phase a f t e r one Edman degradation. To conf i r m t h a t a l l t h e l a b e l i n the e t h y l a c e t a t e phase was PTH-35s-methionine (the p h e n y l t h i o h y d a n t o i n  derivative of  35s-methionine), an a l i q u o t o f the e t h y l a c e t a t e phase was d r i e d and t r e a t e d as i n s e c t i o n #7 o f "Methods". T h i s i n volved c o n v e r s i o n o f the t h i a z o l i n o n e d e r i v a t i v e t o the more s t a b l e P T H - d e r i v a t i v e  f o l l o w e d by t h i n - l a y e r chromato-  graphy on s i l i c a g e l w i t h standard PTH amino a c i d s and -the s o l v e n t system, heptane:n-butanol:75% formic a c i d  (100:60:18,  v / v ) . Sections o f the t h i n - l a y e r p l a t e were counted and the r e s u l t s i n P i g . 9 i n d i c a t e t h a t a l l o f the r a d i o a c t i v i t y  -141-  TABLE 4 Edman. Degradation o f 35s-methionine L a b e l l e d Protamine  Experiment  Aqueous Phase  Ethyl Acetate Phase  % PTH^Met  1  1,660*  14,080*  89%  2  5,568  17,561  76  3  5,214  13,523  72  * CPM i n phase  -142-  *ig. 9 T h i n - l a y e r Chromatography of the R e a c t i o n Edman Degradation of 35s-methionine  t  0  2  3  4  DISTANCE  5  Products o f  L a b e l l e d Protamine  6  7  8  9  cm  Note: Chromatography was f o r 2\  h i n the solvent  system, heptane:n-butanol:75$ formic  acid  (100:60:18, v / v ) . Markers were l o c a t e d by t h e i r quenching of the f l u o r e s c e n t  silica  g e l under UV i l l u m i n a t i o n : 1, PTH-methionine* sulphoxide;  2, PTH-methionine. 0, o r i g i n .  -143-  co-chromatographed w i t h the -unlabelled  PTH-methionine  marker. Thus, a l l of the counts i n the e t h y l a c e t a t e phase must have come from the N-terminus o f protamine. D a n s y l a t i o n was performed by the method o f Gray  (279)  and a f t e r a c i d h y d r o l y s i s and e l e c t r o p h o r e s i s a t pH  4.38,  the  electropherogram was  the  DNS  examined w i t h UV l i g h t to l o c a t e  amino a c i d markers. The sample s t r i p was cut at 0 . 5  i n c h i n t e r v a l s and counted i n 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 w i t h the r e s u l t s shown i n F i g . 1 0 . As can be seen, about 70$ of the 35s-methionine i n protamine was r e c o v e r e d as d a n s y l - 3 5 s - m e t h i o n i n e . T h i s confirmed the r e s u l t s u s i n g the  Edman d e g r a d a t i o n and i n d i c a t e d t h a t most, i f not a l l ,  of the methionine i n c o r p o r a t e d i n protamine i s present at the  N-terminus. Protamine i s i n s o l u b l e i n the s o l v e n t s  employed i n these N-terminal methods and one would  there-  f o r e not expect q u a n t i t a t i v e y i e l d s of the t e r m i n a l amino a c i d under these c o n d i t i o n s .  S t r u c t u r e of t h e Methionine P e p t i d e When 35s-methionine-protamine s y n t h e s i z e d i n t e s t i s c e l l s i s d i g e s t e d w i t h t r y p s i n and the r e s u l t i n g peptides e l e c t r o p h o r e s e d a t pH 3 . 6 ,  a s i n g l e b a s i c p e p t i d e , A, i s  seen on the autoradiogram as i l l u s t r a t e d  i n Fig.  11, A  t r a c e of a p e p t i d e t h a t i s o n l y s l i g h t l y b a s i c and which was  o r i g i n a l l y thought t o be f o r m y l a t e d (280)  has been  seen o c c a s i o n a l l y but both i t s a n d the major p e p t i d e  change  -144-  Fig.  10  E l e c t r o p h o r e s i s a t pH 4.38  of the products of d a n s y l a t i o n  of 35s-methionine l a b e l l e d  protamine  ELECTROPHORESIS  pH 4 3 8  o DNS-MET . DNS-MET SO,  UJ  O  I 30,000  20,000  —T  €  1  1  4  1  I  2  I  I  0  1  1  2  1  4  DISTANCE  1  1  6  1  1  1  8  I  1  10  (INCHES)  Note: Upper frame, an a l i q u o t o f 35s-methionine protamine was  dansylated, hydrolyzed i n a c i d  and e l e c t r o p h o r e s e d at pH 4.38 Lower frame, f r e e  (Ref.279).  35s-methionine.  -145-  F i g . 11 Autoradiogram o f Electrophoretogram  (pH 3.6)  of  Enzyme Digests of 35s-methionine L a b e l l e d Protamine  0 MP  V  t  B  0 : O r i g i n , A: T r y p t i c p e p t i d e , B: Product  of a c t i o n  of CpB on peptide A, MP: Met- 4c-Pro marker p e p t i d e . 1  -146-  m o b i l i t y a f t e r a c e t y l a t i o n and t h e r e f o r e n e i t h e r can  be  f o r m y l a t e d ; the minor peptide i s p o s s i b l y the r e s u l t  of  h y d r o l y s i s at the Pro-Arg  bond by a contaminant o f t r y p s i n  under the vigorous d i g e s t i o n c o n d i t i o n s used. F i g . . 1 1 shows t h a t f u r t h e r d i g e s t i o n of p e p t i d e A w i t h CpB y i e l d s a l a b e l l e d p e p t i d e , B, which c o e l e c t r o p h o r e s e s w i t h the Met-  1  ^c.-Pro  marker; some i n t a c t peptide A remains a f t e r CpB s i n c e the Pro-Arg  bond i s attacked o n l y s l o w l y by t h i s enzyme. These  r e s u l t s suggest Pro-Arg.  a t e n t a t i v e sequence f o r p e p t i d e A of  Met-  In a d d i t i o n , since t r y p s i n alone y i e l d s a s i n g l e  l a b e l l e d p e p t i d e , a l l of the methionine enters o n l y  one  sequence i n protamine and taken w i t h the N-terminal  data,  the c o n c l u s i o n i s t h a t t h i s must be the N-terminal  tryptic  peptide. Chemical d e f o r m y l a t i o n of the F- 4-C-Met-Pro-Arg marker 1  by treatment #12  w i t h anhydrous HC1  i n methanol as i n s e c t i o n  of "Methods" gave a product which s t r e a k e d d u r i n g subse-  quent e l e c t r o p h o r e s i s at pH 3 . 6 . approach was  T h e r e f o r e , an a l t e r n a t i v e  taken whereby 35s-methionine protamine b i o -  s y n t h e t i c a l l y l a b e l l e d by t e s t i s c e l l s was mylated  (276)  chemically f o r -  i n order to compare the l a b e l l e d t r y p t i c pep-  t i d e from i t w i t h s y n t h e t i c formylated marker&peptides. Thus, the c h e m i c a l l y f o r m y l a t e d , b i o s y n t h e t i c a l l y l a b e l l e d  35  s  -methionine protamine was  d i g e s t e d w i t h t r y p s i n and  p o r t i o n f u r t h e r d i g e s t e d w i t h CpB. e l e c t r o p h o r e s e d at pH 3 . 6  a  The d i g e s t s were then  and an autoradiogram  i s shown i n  -147-  Pig. 12.  T r y p s i n alone y i e l d e d a weakly b a s i c p e p t i d e ,  from b i o s y n t h e t i c a l l y l a b e l l e d and protamine which c o e l e c t r o p h p r e s e d Arg marker. T h i s was pH 1.9  confirmed  as shown i n P i g . 13.  The  chemically  Af,  formylated  w i t h the P - ^ o - M e t - P r o -  by r e - e l e c t r o p h o r e s i s at P- C-Met-Pro-Arg marker 14  had been through s e v e r a l procedures as o u t l i n e d e a r l i e r and  e x i s t e d m a i n l y i n the sulphoxide  s l i g h t l y slower at pH 1.9  form which migrates  than the reduced form which i s  the major form i n the b i o s y n t h e t i c a l l y l a b e l l e d protamine. However, peptide Af and the reduced p o r t i o n of the marker have i d e n t i c a l m o b i l i t i e s . T r y p s i n f o l l o w e d by CpB gave a l a b e l l e d p e p t i d e , Bf, which c o e l e c t r o p h o r e s e s 1 4  C - P r o marker at pH. 3.6  s i s at pH 6.5 (Pig. H h ) . corporated  (Pig. Ha)  ( P i g . 12)  w i t h the P-Met-  and upon r e - e l e c t r o p h o r e -  and upon descending chromatography  I t i s c l e a r , t h e r e f o r e , t h a t methionine i s i n i n t o protamine i n the N-terminal  sequence  Met-  Pro-Arg... during the b i o s y n t h e s i s of protamine i n t r o u t spermatid  cells.  G e n e r a l i t y of Methionine  Incorporation  The p o s s i b i l i t y remained t h a t methionine was corporated  only i n -  i n t o a minor component of protamine r a t h e r than  i n t o a l l components as would be r e q u i r e d i f i t played  a  g e n e r a l r o l e i n the i n i t i a t i o n of s y n t h e s i s of protamine. A c e l l suspension  was  t h e r e f o r e incubated w i t h 35s-methio-  nine and the protamine was  p a r t i a l l y p u r i f i e d and  then  -148-  Pig.  12  Autoradiogram of Electrophoretogram Enzyme Digests of C h e m i c a l l y  (pH 3 . 6 )  of  Pormylated  35s-methionine Protamine  A : f  T r y p t i c peptide, B :  on peptide A , f  4,  Product  f  CpB  Markers: 2, F-Met-HC-Ero; '» Met-HC-Pro  F- C-Met-Pro-Arg; 5 , 14  of a c t i o n of  1  *C-Met-Pro-Arg.  -149-  13  Fig. R e - e l e c t r o p h o r e s i s at pH 1.9  o f peptide A  f  and the marker  p e p t i d e , P-Hc-Met-Pro-Arg, from P i g . 12.  e :  I 0  A,  ©  Note: Af, t r y p t i c peptide o f b i o s y n t h e t i c a l l y c h e m i c a l l y formylated 4,  35s_ +,hionine me  labelled,  protaminei  F-Hc-Met-Pro-Arg marker p e p t i d e . 0,  origin.  -150-  P i g . 14 a) Autoradiogram of Electrophoretogram (pH 6.5) of Peptide B  f  from P i g . 12.  b) Autoradiogram of Descending Chromatogram of Peptide B^ from above ( 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, v/v, f o r 16 h ) .  • 4  #-§  <  -151-  chromatographed on a C M - c e l l u l o s e  column e l u t e d w i t h  l i n e a r g r a d i e n t of L i C l i n order to achieve p a r t i a l  a separa-  t i o n of the protamine components as p r e v i o u s l y d e s c r i b e d ( 2 6 5 ) . The  e l u t i o n diagram i n P i g . 15 shows i n c o r p o r a t i o n  of the l a b e l across the o p t i c a l d e n s i t y p r o f i l e w i t h s i x d i s t i n c t peaks of r a d i o a c t i v i t y . As mentioned p r e v i o u s l y , newly s y n t h e s i z e d protamine i s phosphorylated  on i t s s e r y l  r e s i d u e s and t h i s m o d i f i c a t i o n a f f e c t s the e l u t i o n b e h a v i our on C M - c e l l u l o s e  (268). Therefore,  o f 35s_methionine protamine was  another p r e p a r a t i o n  d i g e s t e d w i t h E. c o l i a l k a -  l i n e phosphatase (Worthington) and  chromatographed on a  column of C M - c e l l u l o s e under s l i g h t l y d i f f e r e n t from those used i n P i g . 1 5 . l a b e l was  still  conditions  As can be seen i n P i g . 1 6 ,  the  i n c o r p o r a t e d across the o p t i c a l d e n s i t y  pro-  f i l e w i t h 3 major peaks and 3 or 4 minor peaks. C l e a r l y then, methionine i s i n c o r p o r a t e d i n t o s e v e r a l protamine components and must be o f g e n e r a l importance i n t h e i r b i o s y n t h e s i s .  T r a n s i e n t Nature of Methionine  Incorporation  In b a c t e r i a l systems where the r o l e of N-formyl-methionine i s w e l l e s t a b l i s h e d i n p r o t e i n c h a i n i n i t i a t i o n the N-terminal completion.  methionine i s u s u a l l y removed a f t e r  To t e s t f o r methionine removal i n the  system, c e l l suspensions were pulsed w i t h l a b e l l e d  (185),  chain testis arginine  and methionine and then chased i n f r e s h medium w i t h u n l a b e l l e d amino acids as d e s c r i b e d i n s e c t i o n #15  of "Methods".  -152-  Fig.  15  Ionr-Exchange Chromatography  of  35s-methionine L a b e l l e d Protamine on C M - c e l l u l o s e  C M C 2 - 9 x ae C M -  LINEAR  FRACTION  GRADIENT  LiCI  NO-  Note: C M - c e l l u l o s e column (2.5X26 cm) 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 o f L i C I generated from 600 ml each o f 0.6 and 1.3 M L i C I i n 0.01 l i t h i u m a c e t a t e (pH 5.0).  M  -153-  Fig. lion-exchange  Chromatography  16  o f A l k a l i n e Phosphatase  Treated  -methionine L a b e l l e d Protamine on C M - c e l l u l o s e .  Note: C M - c e l l u l o s e column  (2X30 cm) e l u t e d w i t h  l i n e a r g r a d i e n t o f L i C I generated from 500 ml each o f 0.6  and 1.4 M L i C I i n 0.01  M  l i t h i u m a c e t a t e (pH 5.0). F r a c t i o n volumes about 10 ml each. 25 mg o f l a b e l l e d p r o t a mine d i g e s t e d w i t h 0.25 mg E. c o l i  alkaline  phosphatase f o r 2 h a t 37 ° C in. 0.2 M NH4HCO3.  -154-  Fig.  17 shows the r e s u l t s of Experiment A i n whieh the  s p e c i f i c a c t i v i t i e s of b o t h l a b e l s i n protamine were f o l l o w ed f o r 2 h a f t e r a 1G minUte p u l s e . The i n c o r p o r a t e d  argin-  ine  i s s t a b l e d u r i n g the chase i n d i c a t i n g t h a t protamine i s  not  degraded a f t e r i t s s y n t h e s i s ; however, the s p e c i f i c a c -  t i v i t y o f methionine (35s)  f a l l s d u r i n g the chase by about  50% at 2 h. Even a f t e r a 10 minute p u l s e one may note t h a t the  s p e c i f i c a c t i v i t y of methionine i n protamine c o n t i n u e s  to r i s e f o r the f i r s t 30 minutes o f the chase. Fig.  18 and T a b l e 5 show the r e s u l t s o f Experiment  i n which a 6 minute p u l s e was  B  f o l l o w e d by a 5% h chase. Bas-  i c p r o t e i n s were f r a c t i o n a t e d on a starch-urea-aluminum l a c t a t e g e l and the g e l s l i c e s were counted f o r 35s-methionine and  - a r g i n i n e by counting i n two channels 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 ( c o u n t i n g at A-JQ and H^Q, standard 35s-methionine sample  A  o v e r l a p p e d i n t o the lower  channel to the e x t e n t o f 2 7 . 3 $ o f the upper channel counts whereas no 3 - a r g i n i n e counts e n t e r e d the upper c h a n n e l ) . H  On the s t a i n e d g e l s l i c e s , mature dephospho-protamine  mi-  g r a t e d 1 3 . 0 - 1 5 . 0 cm from the o r i g i n and "nascent" p r o t a mine, 1 0 . 0 - 1 3 . 0 cm. F i g . 18 shows a p l o t o f the r a t i o o f 35s-methionine t o 3H^arginine i n g e l s l i c e s a t v a r i o u s times d u r i n g the experiment. In. frames A - 0 , one may  see s e v e r a l  peaks mainly in. the r e g i o n behind protamine where the newl'y s y n t h e s i z e d , phospho-protamines  migrate. The peaks i n the  r e g i o n from 7 . 0 - 1 0 . 0 cm are probably h i g h l y p h o s p h o r y l a t e d  -155-  17 Pulse-Chase Experiment A  I  "600  CD <  i  X  - 4 0 0 to  < - 2 0 0 CL  CO  60  r 80  T I M E (min)  Rote: The pulse was from minus 10 minutes t o time 0. S p e c i f i c a c t i v i t y i s i n CPM per A-230 of protamine.  1  -156-  Fig.  18  Pulse-Chase Experiment  B  P  DISTANCE FROM ORIGIN (cm)  Frames r e p r e s e n t : A, end of 6 minute p u l s e ; B, time 0 a f t e r resuspending c e l l s  f o r chase;  C-H,,  10, 30, 60, 120, 240 and 330 minutes o f chase. P, p o s i t i o n of u n l a b e l l e d protamine marker on stained g e l s l i c e s .  -157-  TABLE 5 Pulse-Chase Experiment B:  Total 3H-Arg CPM  Time End  of pulse  Total 35s,Met 0PM  GPM 35s-Met per 1000 0PM 3H-Arg  2931  5883  2010  0  1450  2843  1960  10  1573  1736  1100  30  3070  435  .142  60  5739  316  55  120  4182  826  197  240  8625  222  26  330  4773  784  164  Minutes a f t e r resuspension:  Note: T o t a l CPM r e f e r s t o t h e t o t a l r a d i o a c t i v i t y i n the g e l s l i c e s from 9 . 8 - 1 3 . 7 cm. V a r i a b i l i t y i n t o t a l CPM o f 3R^arginine i s proba b l y due t o the d i f f i c u l t y i n w e t t i n g the g l a s s f i b r e d i s c s i n the g e l s l o t s and thus a v a r i a b l e amount o f b a s i c p r o t e i n s i n the s l o t s .  i s extracted  by the a c i d  -158-  protamine components which have been observed i n t h i s r e g i o n i n r e c e n t experiments o f L o u i e and Dixon ( u n p u b l i s h e d results). By 30 minutes  of the chase, both the Met:Arg  ratio  ( F i g . 18, frame D) and the a b s o l u t e amount o f methionine i n the protamine r e g i o n (Table 5) had f a l l e n markedly. This r a p i d removal of methionine from newly s y n t h e s i z e d p r o t a mine must mean t h a t the c e l l s used i n t h i s experiment  had  v e r y a c t i v e methionine aminopeptidase a c t i v i t y . The  cells  were from hormonally induced t r o u t t e s t i s i n which  matura-  t i o n i s a c c e l e r a t e d and t h i s may  e x p l a i n the r a p i d removal,,  of methionine. The f a s t e r apparent r a t e o f removal . ; > during:;^ the p e r i o d 10-30 minutes as compared w i t h the p e r i o d minutes may  0-10  be due t o c o n t i n u e d i n c o r p o r a t i o n o f 35  S  -methionine from c e l l pools d u r i n g the e a r l y chase p e r i o d . T h i s c o u l d be prevented by e a r l y a d d i t i o n of cycloheximide a t the b e g i n n i n g bf the chase. Experiment A ( F i g . 17) measured the r a t e o f methionine removal i n protamine present i n chromatin whereas experiment B measured the removal from w h o l e - c e l l protamine; the l a t t e r would i n c l u d e newly s y n t h e s i z e d protamine not y e t t r a n s p o r t e d i n t o the chromatin. Thus, experiment A shows t h a t methionine can be removed from completed protamine even i n chromatin! s i n c e t h i s experiment measured methionine removal' o n l y i n chromatin, t h i s may  e x p l a i n the slower r a t e o f r e -  moval than i n experiment Bi Cytoplasmic protamine.may have  -159-  i t s N-terminal methionine removed f a s t e r than protamine i n chromatin; as w i l l be shown l a t e r , enzyme a c t i v i t y capable of c l e a v i n g the Met-Pro bond i s predominantly i n the c y t o plasm a l t h o u g h there i s s i g n i f i c a n t a c t i v i t y i n the n u c l e u s .  Nascent Protamine a) Puromycin R e l e a s e d Nascent P e p t i d e s As d e s c r i b e d i n s e c t i o n #17  of "Methods' , t e s t i s 1  suspensions were i n c u b a t e d w i t h 4C-formate or 1  35s-methio-  nine f o r 1 0 minutes, puromycin was added t o 5 X 1 0 ~ i n c u b a t i o n was  cell  4  M and  c o n t i n u e d f o r 1 h. S i n c e puromycin causes  premature r e l e a s e of nascent p r o t e i n c h a i n s by a c t i n g as an analogue o f the 3 ' was  terminus o f aminoacyl-tRNA  (298), i t  f e l t t h a t t h i s would be a means to determine i f the  methionine i n c o r p o r a t e d a t the N-terminus  of protamine i s  f o r m y l a t e d . Thus, l a b e l l e d products i n the s o l u b l e cytoplasm from the above i n c u b a t i o n s were f r a c t i o n a t e d on ion-exchange columns, d i g e s t e d w i t h t r y p s i n or t r y p s i n and GpB,,  electro-  phoresed w i t h marker p e p t i d e s and autoradiograms performed. F i g . 19 shows the r e s u l t s o f e l e c t r o p h o r e s i s at pHi 3 . 6 of the 35s-methionine l a b e l l e d products o f the above p r o cedures. 'Peptide'A has the m o b i l i t y of Met-Pro but was not f u r t h e r i n v e s t i g a t e d . 'Peptide' B. migrated c l o s e to the F^Met14(j-Pro marker a t t h i s pH but when e l u t e d from the paper and r e - e l e c t r o p h o r e s e d at pH 6 . 5 ,  the r e s u l t i n F i g . 20  i n d i c a t e s t h a t 'peptide' B i s not i d e n t i c a l w i t h F - M e t - 0 1 4  -160-  Fig.  19  Autoradiogram o f Electrophoretogram (pH 3.6) o f  35s_  methionine L a b e l l e d Products i n the Cytoplasm o f T e s t i s C e l l s Incubated w i t h Puromycin.  Note: 0, o r i g i n ; 1, F - 4 c - M e t marker; 2, 1  P-Met- 4-C-Pro 1  marker; 3, Met-14-C-Pro marker. The 6 samples a r e , left  to r i g h t : Dowex-50 r e t a i n e d m a t e r i a l  - control,  t r y p s i n and t r y p s i n - C p B d i g e s t s ; and QAE-Sephadex retained material  - c o n t r o l , t r y p s i n and t r y p s i n -  CpB d i g e s t s . The 3 'peptides'  t o the l e f t  e l u t e d and r e - e l e c t r o p h o r e s e d  a t pH 6.5  of B were  ( P i g . 20).  -161-  Pig. 20 a t pH 6 . 5  Re-electrophoresis  of ' p e p t i d e s  f  f  •  I  I  I  Note: 0 , o r i g i n ; 1,  1  from P i g s . 19 & 21  -2  P-Met- C-Pro marker; 14  2 , P- -C-Met marker. Samples a r e : B, 14  5 5  S-  methionine l a b e l l e d m a t e r i a l from P i g . 1 9 ; C-E, Pig.  l4  C - f o r m a t e l a b e l l e d m a t e r i a l from 21.  -162-  Pro. The t r a c e of l a b e l l e d m a t e r i a l from the  'peptide' B  1  sample and m i g r a t i n g c l o s e to the P-Met- 4-0-Pro marker i n 1  P i g . 20 separated from the l a t t e r upon descending  chromato-  graphy. P i g . 21 shows the H o - f o r m a t e  l a b e l l e d products o f the  experiment above a f t e 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 3.6.  pH  'Peptides' 0 and D have the m o b i l i t y of the P - M e t - H © -  Pro marker and  'peptide' E has the m o b i l i t y o f P- 4-0-Met 1  marker; however, when r e - e l e c t r o p h o r e s e d at pH 6.5  as shown  i n P i g . 20, none o f the sample 'peptides' c o e l e c t r o p h o r e s e w i t h the marker p e p t i d e or amino a c i d . The r e s u l t s above s t r o n g l y suggest at the Ni-terminus  of nascent  t h a t the  methionine  protamine i s never f o r m y l a t e d  as i t i s i n b a c t e r i a l systems as d i s c u s s e d ; e a r l i e r ; - The only other p o s s i b i l i t y i s t h a t the formyl group i s removed very soon a f t e r c h a i n i n i t i a t i o n and v e r y r a p i d l y . b) Ribosome Bound Nascent Peptides T e s t i s c e l l s from hormonally  induced t r o u t at the  protamine stage of maturation were i n c u b a t e d w i t h methionine  (20 m i c r o c u r i e s / m l ) f o r 20 minutes at 15  w i t h a d d i t i o n of cycloheximide  t o 2X10"  4  35s_ °C  M a f t e r 15 minutes  of the i n c u b a t i o n i n order t o a r r e s t nascent  p r o t e i n chains  on the ribosomes. Ribosomes were then i s o l a t e d and  fraction-  ated on 10-30$ sucrose g r a d i e n t s as d e s c r i b e d i n s e c t i o n #17 of "Methods". One may  of the g r a d i e n t s i s shown i n P i g . 22.  see t h a t methionine  i s incorporated g e n e r a l l y across  One  -163-  P i g . 21 Autoradiogram  of Electrophoretogram (pH 3.6) o f  formate L a b e l l e d Products i n the Cytoplasm  1 4  C-  of T e s t i s  C e l l s Incubated w i t h Puromycin.  I Note: 0, o r i g i n ; 1, P-Met- 4-C-Pro marker; 2, P-Ho-Met 1  marker; 3, Met- 4-C-Pro marker. The 6 samples a r e , 1  l e f t t o r i g h t : Dowex-50 r e t a i n e d m a t e r i a l - c o n t r o l , t r y p s i n and trypsin-CpB d i g e s t s ; QAE-Sephadex r e t a i n ed m a t e r i a l - c o n t r o l , t r y p s i n and t r y p s i n - C p B d i g e s t s The 2 'peptides' t o t h e r i g h t o f C and the 3 t o the left  of I) and E were e l u t e d and r e r u n ( P i g . 2 0 ) .  -164-  F i g . 22 35s-methionine L a b e l l e d Ribosomes F r a c t i o n a t e d on Sucrose D e n s i t y G r a d i e n t s  BOTTOM  TOP  FRACTION NO.  F r a c t i o n s 1 5 - 2 7 were pooled f o r r e l e a s e o f nascent protamine.  Fractions 1-7,  8-13  and 1 4 - 2 0 from two  other g r a d i e n t s were pooled f o r r e l e a s e of nascent peptides and Edman d e g r a d a t i o n .  -165-  the polysome r e g i o n w i t h the s p e c i f i c a c t i v i t y i n c r e a s i n g w i t h polysome s i z e ; t h a t i s , CPM.of 35s-methionine per A254 i n c r e a s e s towards the bottom of the g r a d i e n t . The p a t t e r n i s s i m i l a r t o t h a t observed f o r a r g i n i n e i n c o r p o r a t i o n a t t h e November stage o f n a t u r a l m a t u r a t i o n  (266). Some of the  i n c r e a s e i n s p e c i f i c a c t i v i t y i n h i g h e r polysomes c o u l d be due  t o t h e presence of nascent p r o t e i n s w i t h i n t e r n a l meth-  i o n i n e r e s i d u e s ; data s u p p o r t i n g t h i s hypothesis presented  below. Another reason  f o r the h i g h e r  w i l l be  specific  a c t i v i t y of h i g h e r polysomes c o u l d be the n o n s p e c i f i c b i n d i n g o f newly s y n t h e s i z e d p r o t e i n s i n c l u d i n g protamine t o polysomes a f t e r completion  o f s y n t h e s i s and r e l e a s e ; i f  t h i s were the case, one would have t o p o s t u l a t e .that the higher polysomes are capable  o f b i n d i n g more p r o t e i n per  ribosome than lower polysomes. Unpublished work of G-ilmour, Louie and Dixon has r e v e a l e d the presence o f newly  synthe-  s i z e d protamine r i g h t across the ribosome p r o f i l e as d e t e r mined by e x t r a c t i o n of v a r i o u s r e g i o n s and e l e c t r o p h o r e s i s on starch-urea-aluminum l a c t a t e g e l s . The presence of p r o tamine across t h e ribosome p r o f i l e and e x t r a c t a b l e w i t h  acid  i m p l i e s t h a t newly s y n t h e s i z e d protamine i s b i n d i n g nons p e c i f i c a l l y ; p r i o r t o c h a i n completion  and r e l e a s e , p r o -  tamine would not e x t r a c t i n a c i d as i t would s t i l l  be attach-  ed t o tRNA. I f protamine were s y n t h e s i z e d on polysomes h i g h er than disomes, one would expect a s p e c i f i c a c t i v i t y no higher than t h a t on monosomes s i n c e one expects o n l y one  -166-  nascent  p r o t e i n per ribosome.  The monosome and disome peaks from one g r a d i e n t were pooled, d i l u t e d and p e l l e t e d by c e n t r i f u g a t i o n ; nascent peptides were r e l e a s e d by d i g e s t i o n w i t h p a n c r e a t i c RFase f o l l o w e d by m i l d a l k a l i n e h y d r o l y s i s . The r e l e a s e d peptides were d i g e s t e d w i t h t r y p s i n and a p o r t i o n was f u r t h e r d i g e s t ed w i t h OpB. The pooled d i g e s t s were e l e c t r o p h o r e s e d a t pH 3.6 w i t h marker p e p t i d e s ; t h e markers were l o c a t e d by autor a d i o g r a p h y and the sample p e p t i d e s by c u t t i n g out p a r t o f the sample s t r i p and counting as d e s c r i b e d i n "Methods". The r e s u l t s shown i n P i g . 23(a) suggested f r e e methionine,  the presence o f  Met-Pro and Met-Pro-Arg. T h i s was c o n f i r m -  ed by c u t t i n g out the remainder of the sample peptides above and r e - e l e c t r o p h o r e s i s a t pH 1 . 9 w i t h the r e s u l t shown i n P i g . 2 3 ( b ) . V i r t u a l l y a l l o f t h e methionine l a b e l on the monosomes and disomes was t h e r e f o r e present as f r e e methion i n e , Met-Pro or Met-Pro-Arg; t h e r e was o n l y a t r a c e o f l a b e l i n t h e a c i d i c r e g i o n i n P i g . 23(a) where t h e f o r m y l a ted marker peptides  migrate.  S i n c e the ribosomes were washed t o remove any f r e e methionine p r i o r t o r e l e a s e o f nascent p e p t i d e s , t h e p r e sence o f f r e e methionine i n t h e m a t e r i a l r e l e a s e d from monosomes and disomes i s i n t e r e s t i n g ; one p o s s i b i l i t y i s t h a t i t might have been present as 35s-.Met-tRNA * on the monof  somes as p a r t of an i n i t i a t i o n complex. I n b a c t e r i a l systems, the formyl group on P-Met-tRNAf i s not a t t a c k e d by the de-  -167-  F i g . 23 a) E l e c t r o p h o r e s i s a t pH 3 . 6 of t r y p s i n - C p B  d i g e s t s of  35s-methionine l a b e l l e d nascent peptides  N  FMP  FM  from disome r e g i o n .  MPA  MP  o a CD  a  300 •  UJ  200  in Q.  I00  O  0  j-n-\ 4  3  2  n 1  0  1  DISTANCE FROM ORIGIN  b) R e - e l e c t r o p h o r e s i s  (pH 1 . 9 )  2  3  0  4  (inches)  o f the peptides  ®  I20-  above.  ®  LU 80 CO  40-  IO DISTANCE  20  LL  30  0  (cm)  Note: FM, F-HG>Met; FMP, F-Met- 4-G-Pro; Ni, n e u t r a l marker 1  ( e p s i l o n - D N P - l y s i n e ) ; MP and 1 , M e t - 0 - P r o ; 2 , H  Met; MPA and 3 ,  l4  G-Met-Pro-Arg.  5 5  S-  -168-  formylase and the above treatment of F-Met; thus the presence  would l e a d to the r e l e a s e  of Met  and the absence of F-Met  i n the m a t e r i a l r e l e a s e d from t e s t i s ribosomes t h a t the methionine  suggests  i s never f o r m y l a t e d i n the t e s t i s  sy-  stem. The other two sucrose g r a d i e n t s of 35s-methionine l a b e l led 8-13  ribosomes were processed as f o l l o w s . F r a c t i o n s and 14-20  were pooled, d i l u t e d w i t h TMK  and  1-7,  pelleted  by c e n t r i f u g a t i o n . Nascent peptides were r e l e a s e d by  hydro-  l y s i s w i t h 1 M NH^OH at 37 °0 f o r 30 minutes and e x t r a c t e d as d e s c r i b e d i n s e c t i o n #17 t e r i a l was  of "Methods". The r e l e a s e d  ma-  s u b j e c t e d to one Edman degradation w i t h the r e -  s u l t s shown i n Table 6. V i r t u a l l y a l l o f the methionine in. the disome r e g i o n r e a c t e d and t h e r e f o r e must be or f r e e . A lower p r o p o r t i o n of the methionine polysomes r e a c t e d ; t h i s suggests methionine  N-terminal  i n the h i g h e r  t h a t some of the l a b e l l e d  i s i n t e r n a l i n nascent p r o t e i n s on the ribosomes  i n t h i s r e g i o n . However, the l a r g e p r o p o r t i o n of N-terminal methionine  i n the higher polysomes may mean t h a t p r o t e i n s  other than protamine are a l s o i n i t i a t e d w i t h methionine the t r o u t t e s t i s  in  system.  Inhibitors Fig.  24 shows the s e n s i t i v i t y of methionine  incorpora-  t i o n i n t o protamine t o i n h i b i t i o n . b y cycloheximide chloramphenicol. The extreme s e n s i t i v i t y to  and  cycloheximide  -169-  ; TABLE 6 Edman D e g r a d a t i o n o f Ribosome-Bound -methionine L a b e l l e d Nascent Peptides  Region on gradient (fractions)  CPM: i n aqueous phase  CPM' i n e t h y l a c e t a t e phase  %- o f 35s-Met t h a t i s N.-terminal ,  1-7*  1848  6278  77%  8-13  1065  3256  75  14-20  1091  9617  90  * Assuming t h a t a l l the r a d i o a c t i v i t y i n the e t h y l a c e t a t e phase i s from N-termini.; a c t u a l l y , as d i s c u s s e d i n the t e x t , some o f the r a d i o a c t i v i t y must come from f r e e methionine. ** F r a c t i o n 1 i s a t the bottom of the g r a d i e n t .  -170-  *ig. Effect of Inhibitors  24 on 35s-methionine  I n c o r p o r a t i o n i n t o Protamine  Log Molarity Inhibitor  F o t e : S p e c i f i c a c t i v i t y r e f e r s t o CPM  3 5s-Met per A230 °^ protamine. Procedures d e s c r i b e d i n s e c t i o n #16 o f "Methods".  -171-  i s c h a r a c t e r i s t i c of e u k a r y o t i c c y t o p l a s m i c p r o t e i n synthes i s and has been d e s c r i b e d p r e v i o u s l y f o r a r g i n i n e i n c o r p o r a t i o n i n t o protamine (264). Cycloheximide  has been found  t o i n h i b i t both c h a i n i n i t i a t i o n and e l o n g a t i o n (281 ) and i s known t o s p e c i f i c a l l y i n h i b i t  eukaryotic transferase 2  (Ref.209). The g r e a t s e n s i t i v i t y of methionine  incorpora-  t i o n to t h i s i n h i b i t o r r u l e s out a mechanism i n v o l v i n g N-terminal a d d i t i o n of amino a c i d s as d e s c r i b e d i n E. (282,283) and i n r a t l i v e r  coli  (284). The i n s e n s i t i v i t y of p r o -  tamine s y n t h e s i s t o a l l but h i g h l e v e l s of  chloramphenicol  together w i t h work on the i n t r a c e l l u l a r s i t e of protamine s y n t h e s i s (265,266) e l i m i n a t e s m i t o c h o n d r i a as the l o c a t i o n of s y n t h e s i s . As d e s c r i b e d i n s e c t i o n #16 was  of "Methods", a m i n o p t e r i n  t e s t e d f o r i t s e f f e c t on methionine  protamine.  incorporation into  The r e s u l t s g i v e n i n T a b l e 7 i n d i c a t e v e r y  little  i n h i b i t i o n by t h i s agent. S i n c e aminopterin i s known to i n h i b i t e u k a r y o t i c d i h y d r o f o l a t e reductase  (299), f o r m y l a -  t i o n o f Met-tRNAf*, i f i t o c c u r r e d , would be i n h i b i t e d one would then expect i n h i b i t i o n of p r o t e i n i n i t i a t i o n . r e s u l t s w i t h methionine  -formate  The  i n c o r p o r a t i o n i n t o protamine t h e r e -  f o r e imply t h a t f o r m y l a t i o n of the methionine cur or at l e a s t i s not  and  does not  oc-  necessary.  I n c o r p o r a t i o n , i n t o Protamine  C e l l suspensions  incubated w i t h H c - f o r m a t e were found  -172-  TABLE 7 E f f e c t of Aminopterin on 35s-methionine Incorporation, i n t o Protamine  Specific activity* (OPM per A 2 3 0 )  Sample  % of Control  Control  1  1910  2  2160  Aminopterin M  1730  85%  M  1660  82  M  1620  80  10-3 M  1800  88  10"  6  IO"  5  IO " -4  * CPM 35s-Met per A23O of protamine measured as o u t l i n e d , i n s e c t i o n #16 of "Methods".  - 1 7 3 -  to i n c o r p o r a t e incorporated  the l a b e l i n t o protamine but none o f t h e  l a b e l was v o l a t i l e a f t e r a c i d h y d r o l y s i s . I n  f a c t , a c i d h y d r o l y s i s and e l e c t r o p h o r e s i s a t pH 1.9 r e v e a l ed t h a t a l l o f t h e formate l a b e l i n protamine had entered as s e r i n e . The i n c o r p o r a t i o n o f formate i n t o s e r i n e i s p r e sumably due t o t h e presence i n t r o u t t e s t i s o f a hydroxymethyl t r a n s f e r a s e which can s y n t h e s i z e and  s e r i n e from g l y c i n e  -methylene t e t r a h y d r o f o l a t e , the methylene moiety  being  d e r i v e d from formate. T h i s pathway i s o u t l i n e d  Formate  + THFA + ATP  below:  *  FfO-Formyl THFA  »  N5,1O-Methenyl THFA  >  H5-, 10-Methylene THFA + NADP+  >  S e r i n e + THFA  1 N -Formyl  THFA  10  2 E 5 » °-Methenyl THFA + H + NADPH. 1  +  N5,10_Methylene THFA + glycine  3  Note: 1, f o r m y l t e t r a h y d r o f o l a t e methenyltetrahydrofolate  synthetase;  cyclohydrolase; 3 ,  hydroxymethyltetrahydrofolate 4, 1 - s e r i n e  hydroxymethyl  2,  dehydrogenase;  transferase.  Enzyme Assays. P r e l i m i n a r y s t u d i e s were done t o assay f o r enzymes i n t r o u t t e s t e s capable o f removing a c y l groups from methionine or Met-Pro  and f o r enzymes capable o f removing the  -174-  methionine r e s i d u e from protamine or the d i p e p t i d e ,  Met-  Pro. a) Deacylase A c t i v i t y Strong deacylase a c t i v i t y was found i n the h i g h speed supernatant u s i n g the assay d e s c r i b e d i n s e c t i o n #18  of  "Methods". P i g . 25 shows an autoradiogram o f an e l e c t r o phoretogram a t p H ' 6 . 5 of the r e a c t i o n products a f t e r  incu-  b a t i n g t e s t i s e x t r a c t s w i t h e i t h e r F-35s-Met or Ac-35s-Met. The l a b e l l e d product of d e a c y l a t i o n o f e i t h e r s u b s t r a t e i s f r e e 35s-methionine which remains near the o r i g i n d u r i n g e l e c t r o p h o r e s i s a t pH 6 . 5 .  The p r e f e r e n c e of the t e s t i s  de-  a c y l a s e f o r P-Met r a t h e r than Ac-Met i s obvious j u s t by "inspecting these autoradiograms. Table 8 summarizes the p r o p e r t i e s o f the t e s t i s  deacy-  l a s e . The a c t i v i t y i s m a i n l y i n the h i g h speed supernatant and p r e f e r s the formyl group over the other a c y l groups t r i e d . There i s s p e c i f i c i t y f o r the amino a c i d as w e l l s i n c e P - I l e u and even R-formyl-methionine sulphone are i n a c t i v e as s u b s t r a t e s . There i s v e r y l i t t l e a c t i v i t y w i t h P-Met-Pro as s u b s t r a t e . A s i m i l a r a c t i v i t y i s p r e s e n t i n E. c o l i ( 3 0 0 ) and has been a t t r i b u t e d t o the enzyme N - a c e t y l - o r n i t h i n a s e  (N-acetyl-  1 - o r n i t h i n e - amidohydrolase) which i s an enzyme i n v o l v e d i n o r n i t h i n e b i o s y n t h e s i s i n b a c t e r i a (301). T h i s enzyme has a marked s p e c i f i c i t y f o r P-Met as s u b s t r a t e as shown i n Table 9 which i s taken from a paper by Pry and Lamborg  (300).  -175F i g . 25 Assay f o r T e s t i s Deacylase a) F - 3 5  s - M e t  S  ubstrat< ©  M  f  M  f  Note: F, F-35s-Met; Ac, Ac-35s-Met; 1 , c o n t r o l ; 2 , nuclear l y s a t e ; 3 , h i g h speed sup.; 4 , m i t o c h o n d r i a l l y s a t e ; 5 , ribosome wash. 0 ,  b) Ac-35s-Met as s u b s t r a t e  origin.  I n c u b a t i o n was ©  with  0 . 0 5 ml e x t r a c t , 0.01 ml s u b s t r a t e , and TMK up to 0.11 ml a t 18 °0 f o r 1 h.  o  I  •  2  •  3  •,  4  5  • ^  -176-  TABLE. 8 Deacylase 1 ) C e l l Fraction.  Activity , Rate*  Nucleoplasm  580  High speed sup. Ribosome wash Mitochondrial lysate 2) S u b s t r a t e  82  40  1  167  4  Rate** 28.0  Acetyl-Met  12.2  Propionyl-Met  0  Butyryl-Met  0.9  Valeryl-^Iet  1.3  Hexanoyl-Met  0.8  Formyl-Met  0.7  sulphone  Formyl-lieu F-Met-Pro H i g h speed sup.  0 Rate*** 1.5  Nuclear l y s a t e  0  Ribosome wash  0  ** nMoles  13$  3540  Formyl-Met  * nMoles  $ Total  Formyl-Met  d e a c y l a t e d per 30 min per g t e s t i s a t 20 °0  d e a c y l a t e d per 0.025 ml HSS per 30 min a t 20 °0  *** $ d e a c y l a t e d per 0.05 ml e x t r a c t per 45 min at 16 °0  -177-  TABLE 9 P r o p e r t i e s of E. c o l i N - a c e t y l - o r n i t h i n a s e (Ref.3Q0)  A c y l a t e d Amino A c i d 1-methionine  R e l a t i v e * (formyA c t i v i t y -lated) 100  R e l a t i v e * (acetyActivity lated) 53  1-alanine  5.0  5.0  1-valine  1.4  0.1  1-tyrosine  1.5  0  1-leucine  4.0  3.7  1-isoleucine  1.3  1 »-5  d-methionine  -  0.4  * The r a t e s a r e r e l a t i v e t o the r a t e o f h y d r o l y s i s of F-Met s e t a t 100. I n c u b a t i o n a t 25 °C f o r 13 minutes.  -1 7 8 -  The  enzyme cannot be important in. b a c t e r i a l p r o t e i n s y n -  t h e s i s s i n c e mutants d e f i c i e n t i n i t show normal growth and do not have enhanced l e v e l s of F-Met at the, N ^ t e r m i n i o f t h e i r p r o t e i n s (300). With i t s r a p i d h y d r o l y s i s o f F-Met and v e r y slow lysis  hydro-  of F-Met-Pro, the t e s t i s deacylase i s q u i t e the oppos-  i t e t o the b a c t e r i a l deformylase removal  (195-197) r e s p o n s i b l e f o r  of f o r m y l groups from nascent p r o t e i n s . I t thus  seems t h a t the t e s t i s a c t i v i t y must be due to a deacylase not i n v o l v e d d i r e c t l y i n p r o t e i n s y n t h e s i s . P r e v i o u s l y d e s c r i b e d deacylases i n animal t i s s u e s i n clude Aryl-formylamine amidohydrolase  in liver  (302) which  hydrolyzes F - f o r m y l - l - k y n u r e n i n e , A r y l - a c y l a m i n e amidohydrol a s e i n c h i c k k i d n e y (303)  which h y d r o l y z e s F - a c y l - a n i l i d e s ,  1 0 - f o r m y l t e t r a h y d r o f o l a t e amidohydrolase  in liver  (304)  which removes formate from F °-formyl-tetrahydrofolate, 1  F - a c y l a m i n o a c i d amidohydrolase  (305,306) which removes a  wide v a r i e t y o f a c y l groups from amino a c i d s , and a s p a r t a t e amidohydrolase f u l comparison  from k i d n e y (305,306).  F-acyl-  A meaning-  o f the t e s t i s deacylase w i t h any of the above  a c t i v i t i e s i s not p o s s i b l e w i t h the d a t a a v a i l a b l e .  Hog  k i d n e y a c y l a s e T i s q u i t e a c t i v e w i t h Ac-Met as s u b s t r a t e (306) but F-Met was source i s about  not compared; a c y l a s e TT from the same  o n e - t h i r d as a c t i v e w i t h Ac-Met as w i t h i t s  n a t u r a l s u b s t r a t e , Ac-Asp, but F-Met was  not compared. At  present, then, i t i s not p o s s i b l e t o say i f F-Met i s the  -179-  n a t u r a l s u b s t r a t e of the t e s t i s b) Methionine  deacylase.  Aminopeptidase A c t i v i t y  T e s t i s e x t r a c t s were assayed  with  35  s  -methionine  and  - a r g i n i n e protamine b i o s y n t h e t i c a l l y l a b e l l e d i n c e l l suspensions  i n c u b a t e d w i t h the l a b e l s s e p a r a t e l y . The  g i v e n i n Table 10(a) show t h a t t e s t i s crease i n TCA-tungstate  results  e x t r a c t s cause a de-  p r e c i p i t a b l e 35s-methionine p r o t a -  mine counts. However, the e x t r a c t s a l s o giveL-a c o n s i d e r a b l e decrease i n a c i d p r e c i p i t a b l e  1 4  C - a r g i n i n e protamine  counts,  i n d i c a t i n g t h a t t h e r e must be e x t e n s i v e p r o t e o l y s i s of p r o tamine. I t was  decided to d i s c o n t i n u e t h i s type of assay  but i t c o u l d be a u s e f u l assay f o r an attempt a h s p e c i f i c methionine  aminopeptidase.  The l a t t e r would  g i v e a decrease i n ^5s-methionine but not a c i d p r e c i p i t a b l e counts i n  140-arginine  protamine.  M e t - 0 - P r o , prepared as i n s e c t i o n #13 l4  was  to i s o l a t e  of "Methods",  a l s o t r i e d as a s u b s t r a t e i n these s t u d i e s . The  g i v e n i n Table 10(b) r e v e a l the presence  of a c t i v i t y  results capable  of h y d r o l y z i n g t h i s d i p e p t i d e mainly i n the h i g h speed pernatant but s i g n i f i c a n t a c t i v i t y was  su-  a l s o present i n the  n u c l e a r l y s a t e and ribosome wash. As mentioned e a r l i e r , n u c l e a r a c t i v i t y d e t e c t e d here may a l of N-terminal methionine  the  be r e s p o n s i b l e f o r remov-  from any protamine t h a t  escapes  the cytoplasmic enzyme. The t e s t i s enzyme;.has not been a s sayed w i t h other substrates>;such as longer peptides w i t h  -180-  TABLE 1,0 Methionine-removing  Activity  a) Protamine as s u b s t r a t e Enzyme  14c?-Arg Protamine as s u b s t r a t e  35s-Met Protamine as s u b s t r a t e  High speed sup.  41.3$ *  73.2$  Nuclear l y s a t e  14.2  40.9  * $ decrease i n TCA-tungstate p r e c i p i t a b l e counts. Incubations were w i t h 0.1 ml e x t r a c t , 0 . 0 2 ml 2 M NaCl, 0.01 ml s u b s t r a t e i n water, and TMK up t o 0.2 ml f o r 40 min at 15 °0. b) M e t - H c - P r o as s u b s t r a t e Enzyme  Rate*  Relative** activity  $ Total  High speed sup.  73.9  100  57  Nuclear l y s a t e  65.8  50  28  Ribosome wash  53.4  27  15  F o t e : * $ h y d r o l y z e d i n 45 min a t 16 °0 ** C o r r e c t e d f o r volume of e x t r a c t  -181-  methionine i n the Nr-terminal p o s i t i o n ; t h i s type of study should  be done when the enzyme i s p u r i f i e d .  Nevertheless,  i n view of the f a c t t h a t X-Pro bonds are r e s i s t a n t to many proteases w i t h the p o s s i b l e e x c e p t i o n  of  (289), the t e s t i s a c t i v i t y i s unusual and  imidodipeptidase could well  be  i n v o l v e d i n v i v o i n removing methionine from the N-terminus of newly s y n t h e s i z e d  p r o t e i n s . Matheson and Dick (198)  r e c e n t l y d e s c r i b e d some of the p r o p e r t i e s of an dase present  on ribosomes i n E. c o l i and  have  aminopepti-  Table 11  i s taken  from t h e i r paper. With Met-X d i p e p t i d e s , the b a c t e r i a l zyme s t r o n g l y p r e f e r s l e u c i n e or methionine i n the  en-  C-terminal  p o s i t i o n and shows l i t t l e a c t i v i t y a g a i n s t Ala-X or Ser-X d i p e p t i d e s ; the authors s t a t e t h a t the enzyme i s a l s o i n a c t i v e a g a i n s t t r i p e p t i d e s w i t h s e r i n e or a l a n i n e i n the N-terminal p o s i t i o n . These p r o p e r t i e s seem w e l l s u i t e d to account f o r the i n c i d e n c e p r o t e i n from E. c o l i nine and  threonine  of N - t e r m i n a l r e s i d u e s  (193)  i n bulk  where s e r i n e , a l a n i n e , methio-  are the only N - t e r m i n i found i n most  proteins. Unfortunately,  Met-Pro was  not  t r i e d as a sub-  strate . Conclusion Protamine s y n t h e s i s  i n t r o u t t e s t i s has  been shown to  i n v o l v e t r a n s i e n t i n c o r p o r a t i o n of methionine at the terminus o f the newly s y n t h e s i z e d on the ribosomes, i s there any  N-  c h a i n s . At no time, even  evidence t h a t the amino  -182-  TABLE 11 P r o p e r t i e s of an Aminopeptidase on Ribosomes o f E.  C-terminal residue  coli*  N-t erminal r es i due Methionine Alanine Serine  leucine  96**  30  14  methionine  80  18  10  phenylalanine  25  7  5  glycine  25  0  1  valine  1  .3  0  3  serine  13  0  alanine  11  0  0  glycyl-glycine  87  0  0  alanyl-serine  88  glycyl-methionine  155  Gly-Met-Met  165  * From Matheson and D i c k  (198)  ** Rates are g i v e n as a percentage of the r a t e of h y d r o l y s i s o f l e u c y l - l e u c i n e (0.026 micromoles per min per mg  p r o t e i n ) at 30  °C.  -183-  group of the methionine i s The  formylated.  i n c o r p o r a t i o n o f methionine i n t o the s e v e r a l com-  ponents o f protamine at t h e i r N-termini  and the subsequent  removal of methionine from t h i s p o s i t i o n i s s t r o n g presumpt i v e evidence  f o r a.role f o r t h i s residue i n i n i t i a t i o n  of  p r o t e i n s y n t h e s i s i h the t r o u t t e s t i s system. There are s e v e r a l f e a t u r e s o f t h i s system which have probably  aided  a demonstration of methionine i n c o r p o r a t i o n . P i s h t e s t i s matures r e l a t i v e l y synchronously  and t e s t e s may  be  obtained  which c o n t a i n mainly spermatids which are i n the process completely  r e p l a c i n g the h i s t o n e s i n chromosomes by  of  prota-  mine. Thus, protamine i s being s y n t h e s i z e d i n l a r g e amounts and s i n c e methionine i s absent from i n t e r n a l p o s i t i o n s , at l e a s t i n the major components (261 ), the i n c o r p o r a t i o n of methionine a t the N-terminus was The  e a s i l y observed.  s m a l l s i z e of protamine, 32-33 r e s i d u e s l o n g , means  t h a t the e n t i r e molecule can be p r o t e c t e d or " s h i e l d e d " by the ribosome d u r i n g i t s s y n t h e s i s ; R i c h , E i k e n b e r r y Malkin  and  (287,288) e s t a b l i s h e d t h a t ribosomes p r o t e c t n a s -  cent p r o t e i n s up to 35 r e s i d u e s l o n g from e x t e r n a l a t t a c k by p r o t e o l y t i c enzymes. A l s o , as d i s c u s s e d above, the Met-Pro bond may  be at l e a s t p a r t i a l l y r e s i s t a n t to the  aminopepti-  dase r e s p o n s i b l e f o r removing the methionine. 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