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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., University of Western. Ontario, 1966 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR; THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Biochemistry Faculty of Medicine We accept this thesis as conforming to the required standard October, 1970 University of B r i t i s h Columbia In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I ag ree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y pu rposes may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l no t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada - i -ABSTRACT Histones are the basic proteins complexed with MA i n the chromosomes of eukaryotic organisms. A previously un-described histone (histone T.) was discovered i n chromatin prepared from rainbow trout (Salmo g a i r d n e r i i ) testes. Histone T was p u r i f i e d by s e l e c t i v e extraction and ion-exchange chromatography on CM-cellulose. The homogeneity of t h i s protein was examined by polyacrylamide disc gel electrophoresis, SDS disc gel electrophoresis, urea starch gel electrophoresis and g e l f i l t r a t i o n chromatography. Histone T was homogeneous as judged by the above d i f f e r -ent c r i t e r i a . The molecular weight was found to be 14,500 by the mobility of formylated or acetylated histone T o n SDS g e l electrophoresis. The amino acid composition and the N-terminal amino acid' were determined. Peptide maps of the t r y p t i c peptides of histone T! and the major histones i n d i -cated that histone T i s not a degradation product of the major histones. Details of the mechanism of i n i t i a t i o n of protein syn-thesis i n eukaryotes have only recently been discovered. Protamines are the small, highly basic proteins complexed with DNA i n the mature sperm c e l l s of most higher animals. The synthesis of protamine i n trout testes was studied. C e l l suspensions prepared from trout testes at the prota-mine stage of d i f f e r e n t i a t i o n were incubated i n v i t r o and - i i -were found to incorporate 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 into the N-terminal sequence, Met-Pro-Arg..., methionine residue i s removed from protamine aft e r chain completion. Enzymatic a c t i v i t y capable of cleaving the dipeptide, Met-Pro, was found i n t e s t i s c e l l f r a c t i o n s . The amino group of methionine incorporated into ribosome-bound nascent protamine was not blocked. Methionine i n -corporation was 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 indicates a r o l e for methio-nine i n the i n i t i a t i o n of protamine biosynthesis i n the trout, a eukaryote. of protamine. Pulse-chase experiments revealed that the - i i i -ACKNOWLEDGMENT The author wishes to express appreciation to Prof. G.H. Dixon for his excellent guidance and encouragement during the course of this work. Appreciation i s also expressed to Drs. C-L. Hew, J e r g i l , B5» Malchy, K. Marushige, M. Sung and Mr. A. Louie, P. Candido and.J. Durgo f o r exchange of ideas and technical assistance. The Medical Research Council of Canada i s thanked . for providing a Fellowship for the period 1967-70 to the author. The taxpayers of 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 of the University. - i v -DEDICATION ELIZABETH, JACQUELINE AND JEFFREY - V -TABLE OE CONTENTS ABSTRACT ., i ACKNOWLEDGMENT i i i DEDICATION: i v LIST OF TABLES x LIST OF' FIGURES x i i PART ONE INTRODUCTION 1 Early Studies on Chromosomes 1 Extraction and P u r i f i c a t i o n of Histones . . . . 3 Characteristics of Histones . 4 Studies on Chromatin 6 Repressors . . 9 Histones as Repressors 11 "Masking" Hypothesis . . . . . . . 11 Chromosomal RNA . . . . . 12 Structure of Histones 13 Proposed DNA-binding S i t e i n Histone TV . . . . 15 MATERIALS AND METHODS 21 Chemicals . . . . . 21 Abbreviations . . . . . 21 Source of Trout Testes 22 Preparation-of Chromatin 22 Maleylation of Chromatin 23 Dissociation of Histone T by Salt 24 - v i -Acid Extraction of Chromatin 24 Gel Exclusion Chromatography . . 25 Ion-exchange Chromatography 25 Polyacrylamide Disc Gel Electrophoresis . . . 26 Molecular Weight Determination by SDS Gel Electrophoresis 26 Amino Acid Analysis 27 Tryptic Fingerprints 28 Ni-terminus Determination 30 RESULTS AND. DISCUSSION, 32 Maleylation of Chromatin 32 Dissociation of Histone T: by Sa l t 37 Extraction of Chromatin with \\4> TCA 37 P u r i f i c a t i o n of Histone T 37 Molecular Weight Determination by SDS Gel Electrophoresis 37 Amino Acid Composition 47 Comparative Peptide Maps 47 N-terminus Determination . 52 Relative Amount of Histone T i n Testis Chromatin at Various Stages of Maturation . . 54 Histone T i n Tissues Other Than Testis . . . . 57 Conclusion 57 BIBLIOGRAPHY FOR PART ONE 60 - v i i -PART TWO INTRODUCTION 66 H i s t o r i c a l Development of Knowledge of Protein Synthesis . . . . 66 Information f o r Protein Synthesis 70 Ribosomes ' 75 I n i t i a t i o n of Protein Synthesis i n Pro-karyotes 80 F-Met-tRNAf 90 Elongation 93 Termination 100 Eukaryotic I n i t i a t i o n . 102 Protamine Biosynthesis 107 MATERIALS AND METHODS 110 Chemicals 110 Abbreviations 110 Source of Trout Testes . 111 Incubation of C e l l Suspensions 112 Extraction of Protamine 113 Starch Gel Electrophoresis 113 Ion-exchange Chromatography . 115 Edman Degradation 115 Thin-layer Chromatography of PTH Amino Acids . 115 High Voltage Paper Electrophoresis 116 Autoradiography 116 Counting Paper Electrophoretograms 117 0 - v i i i -Formylation 1 1 7 Deformylation . 119 Synthesis of Labelled Marker Peptides . . . . 119 Carboxypeptidase B Digests 123 Pulse-Chase Experiments 123 Inhibitor Studies 127 Nascent Peptides 128 Enzyme Assays 132 RESULTS AND DISCUSSION 136 Incorporation of Intact 14c-methyl-methionine into Protamine . . . 136 Methionine i s N-terminal HO Structure of Methionine Peptide of Protamine 143 Generality of Methionine Incorporation . . . 147 Transient Nature of Methionine Incorporation 1 5 1 Nascent Protamine a) Puromycin-released Nascent Peptides . . 159 b) Ribosome-bound Nascent Peptides . . . . 162 S e n s i t i v i t y of Methionine Incorporation 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 Incorporation in t o Protamine . . 171 Enzyme Assays a) Deacylase A c t i v i t y 174 b) Methionine Aminopeptidase A c t i v i t y . . . 179 - i x -Oonclusion 181 BIBLIOGRAPHY FOR. PART TWO 185 - X -LIST OF TABLES PART..ONE Page 1. Nomenclature and Characteristics of the Major Histones 5 2 . Chemical Composition of Varied Chromatins . 7 3. M o b i l i t i e s of Coloured Markers on High Voltage Paper Electrophoresis 29 4 . Dissociation of Chromosomal Proteins by Maleylation 36 5 . Molecular Weights of Trout Testis Histones T and TTbo as Estimated by Mobil i t y During Electrophoresis on Polyacrylamide Gels i n the Presence of 0.1$ SDS 45 6 . Amino Acid Analyses of Histone T . . . . . . 48 PART TWO 1 . E. c o l i Ribosomes 76 2 . Properties of E. c o l i I n i t i a t i o n Factors . . 86 3 . Nomenclature and Characteristics of Elongation Factors 94 4 . Edman Degradation of -methionine Labelled Protamine 141 5 . Pulse-Chase Experiment B 157 6 . Edman Degradation of Ribosome-bound methionine Labelled Nascent Peptides . . . . 169 7 . E f f e c t of Aminopterin on 35s-methionine - x i -Incorporation into Protamine . 172 8. Deacylase A c t i v i t y 176 9 . Properties of E. c o l i K-acetyl-ornithinase 177 10. Methionine-removing A c t i v i t y 180 11. Properties of an Aminopeptidase on Ribosomes of E. c o l i 182 - x i i -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. Polyacrylamide Disc Gel Electrophoresis of Chromosomal Proteins Released by Chemical Modification by Maleic Anhydride 34 3(a). Gel F i l t r a t i o n of Histones on. a Column, of Bio-Gel P-10 (5.5X140 cm) Eluted" with 0.0.1 IT HOI 38 3(b). Polyacrylamide Disc Gel Electrophoresis . . 38 4(a). Ion-exchange Chromatography of 5$ TCA Extrac-table Chromosomal Proteins on CM-cellulose . 39 4(b). Polyacrylamide Disc Gel Electrophoresis . . 39 5. Rechromatography of Histone T on a Column of CM-52 (1 .8X50 cm) .40 6. Electrophoresis on Polyacrylamide Gels i n the Presence of 0.1$ SDS 42 7. Electrophoresis on Polyacrylamide Gels in. the Presence of 0.1$ SDS 43 8. Mobility-of Standard Proteins Versus the Logarithm of t h e i r Molecular Weights . . . . 44 9. Spectrophotometric Scan of Histone T- . . . . 49 10. Tryptic Fingerprints Stained with Cadmium Ninhydrin . 50 11. Tryptic Fingerprints Showing Arginine-- x i i i -containing Peptides . 51 12. Thin-layer Chromatography of Dansylated R e s i -dues of Histone T, and Standard MS Amino Acids 53 13. Disc Gel Electrophoresis of Acid Extracts of Trout Testis Chromatin at Various Stages Dur-ing Natural Maturation 55 14. A Plot of the Relative Amount of Histone T per mg; of DNA i n Chromatin Prepared from Trout Testis at Various Stages of Natural Matura-t i o n 56 15. Disc Gel Electrophoresis of Acid Extracts of Chromatin from Different Trout Tissues and! Two Guinea Pig Tissues . 58 PART TWO 1 . Formation of I n i t i a t i o n Complexes i n E^ - •„ c o l i 85 2. Mechanism of Elongation, i n Rat Liver . . . . 95 3. Mechanism of Elongation i n E. c o l i 96 4. Sequences of Protamine from Salmo g a i r d n e r i i . . . . . . . . 108 5. Counting Electrophoretograms . 118 6. Autoradiogram of Electrophoretogram of Reac-t i o n Products i n the Synthesis of Met- 1 4C-Pro 121 7. HOmethyl-methionine Incorporation into - x i v -Trout Testis Nuclear Basic Proteins . . . . 137 8. Electrophoretogram of HQ-methyl-methionine Labelled Protamine on a Starch-urea-aluminum lactate Gel 1 3 9 9 . Thin-layer Chromatography of the Reaction. Products of Edman Degradation of 35S-methionine Labelled. Protamine 142 1 0 . Electrophoresis at pH 4 .38 of the Products of Dansylation of 35s-methionine Labelled Protamine . 144 1 1 . Autoradiogram of Electrophoretogram (pH 3 . 6 ) of Enzyme Digests of 35s-methionine Labelled Protamine 145 1 2 . Autoradiogram of Electrophoretogram (pH 3 . 6 ) of Enzyme Digests of Chemically Formylated 35s-methionine Labelled Protamine . . . . . 148 1 3 . Re-electrophoresis at pH 1 . 9 of Peptide A f and the Marker Peptide, P-14-C-Met-Pro-Arg, from Pig. 1 2 149 1 4(a). Autoradiogram of Electrophoretogram (pH 6 . 5 ) of Peptide B f .from Pig. 1 2 150 1 4 ( b ) . Autoradiogram of Descending Chromatogram of Peptide B f from Pig. 1 4(a) 150 1 5 . Ion-exchange Chromatography of 35s-methionine Labelled Protamine on CM-cellulose 152 16 . Ion-exchange Chromatography of Alkaline Phos-- X V -phatase Treated 35s-methionine Labelled Pro-tamine on CM-cellulose 153 17. Pulse-Chase Experiment A 155 18. Pulse-Chase Experiment B 156 19. Autoradiogram of Electrophoretogram (pE 3.6) of 35s-methionine Labelled Products i n the Cytoplasm of Testis C e l l s Incubated with Puromycin 160 20. Re-electrophoresis at pHC6.5 of 'Peptides' from Figs. 19 & 21 161 21. Autoradiogram of Electrophoretogram (jpJi 3.6) of Hc-formate Labelled Products i n the Cyto-plasm of Testis C e l l s Incubated with Puro-mycin 163 22. 35s_me-thion±ne Labelled Ribosomes F r a c t i o n -ated on. Sucrose Density Gradients 164; 23(a). Electrophoresis at pH 3.6 of Trypsin-C.pB; Digests of 35s-methionine Labelled. Nascent Peptides from Disome Region 167 23(b). Re-electrophoresis (pH 1.9) of the Peptides from F i g . 23(a) 167 24. E f f e c t of Inhibitors on 35s-methionine In-corporation into Protamine 170 25. Assay for Testis Deacylase a ) E_35s-Met as Substrate 175 b) Ac-35s-Met as Substrate 175 PART ONE INITIAL CHARACTERIZATION" OE A NEW HISTONE (HISTONE T) FROM TROUT: TESTIS -1 -UTRODUQTION During the decade 1880-1890, the chromosomes of c e l l n u c l e i were i d e n t i f i e d and? investigated by h i s t o l o g i s t s . By 1900 the behaviour of 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 well appreciated and in. 1903» W.S. Sutton gave the f i r s t modern int e r p r e t a t i o n of the re l a t i o n s h i p between genes and chromosomes with a c y t o l o g i c a l explanation of se-gregation and independent assortment. Thus, a concrete basis was established f o r the phenomena of inheritance as described by the Austrian monk, Gregor Johann Mendel, i n a paper i n 1866. The h i s t o l o g i c a l description of the regular and precise duplication and d i s t r i b u t i o n of chromosomes during ordinary c e l l d i v i s i o n plus the separation of paired chromosomes at meiosis a l l correlated well with Mendel's observations and strongly suggested that chromosomes are the c a r r i e r s of genes. The fine structure of chromosomes could not be studied u n t i l the advent of the electron microscope (EM). In the l i g h t microscope, the fundamental element of chromosomes i s the thread-like chromonema; EM reveals that chromonemata are act u a l l y bundles of very fine fibres or single fibres which are c o i l e d and supercoiled. These fibres are 250 & i n d i a -meter but dissociate into two 100 $. f i b r i l s i n EDTA; diges-t i o n of the f i b r i l B with pronase releases one DHA duplex from each. The 100 £ f i b r i l B are c o i l e d and supercoiled; removal of histones causes loss of supercoiling and replace-ment of histones restores supercoiling (1). Chemical studies of the c e l l nucleus began with a paper i n 1871 by F r i e d r i c h Miescher, a student of Hoppe-Seyler. He extracted n u c l e i of pus c e l l s with a l k a l i and fractionated the extract by a simple acid p r e c i p i t a t i o n step. The phos-phate-rich p r e c i p i t a t e , now known to be DNA, seemed to be c h a r a c t e r i s t i c of a l l n u c l e i that he examined and thus the name "nuclein" was assigned to i t . Miescher went on to d i s -cover protamine i n salmon spermatozoa but concluded that i t was a simple nitrogen-rich base. His work was continued by Albrecht Kossel who discovered the histones, rediscovered 1 protamine and did s t r u c t u r a l studies on the nucleic acids (2). The nucleic acid bases and the basic amino acids of h i s -tones and protamine were his p r i n c i p a l i n t e r e s t s . He f e l t that histones and protamine both arose from amino acids released from muscle protein as i t wasted away during the spawning migration of salmonids. The histones of the unripe t e s t i s c e l l n u c l e i were believed to be s i m p l i f i e d during the r i p e n -ing process and d i r e c t l y converted to the protamine of mature sperm c e l l s . The term "nucleohistone" was given i n 1892 by. L i l i e n f e l d (3) to the material extracted from leukocytes or minced thymus with d i s t i l l e d water and pr e c i p i t a b l e i n acetic acid. A f t e r Kossel's book i n 1928 (Ref.4), l i t t l e work was done on histones f o r the next 25 years. Renewed int e r e s t i n t h i s area came with the suggestion by Stedman and Stedman i n 1950 (Ref.5) that gene a c t i v i t y i s suppressed during d i f f e r e n t i a -- 3 -t i o n audi that this might be done by histones. In V i t r o studies (6-8) have shown that DM coinplexed with histones has a r e -duced capacity to serve as a template f o r RNA polymerase com-pared to pure DNA. S i m i l a r l y , removal of histones from chro-matin by exposure to high i o n i c strength or a c i d i c solutions increases the template a c t i v i t y of the r e s i d u a l chromatin ( 9 ) . These r e l a t i v e l y crude experiments suggest that histones can indeed repress t r a n s c r i p t i o n of DNA but the exact mechanism by which t h i s comes about i n vivo remains a great gap i n current knowledge. Biochemical studies were l i m i t e d u n t i l recently by d i f -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 histones; proteolysis during extraction and c r o s s - l i n k i n g of histones by disulphide bonds between cysteine residues both gave r i s e to a large number of components upon electrophoresis and thus to the idea that histones might be very heterogeneous. Large-scale f r a c t i o n a t i o n by sel e c t i v e extraction and p r e c i p i t a t i o n was developed 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 c o l -umns of Amberlite IRC-50 was f i r s t used by Rasmussen, Murray and Luck ( 13 ) ; t h i s method has been used extensively by Cole and co-workers (14) for studies of histone T ( f o r nomencla-ture, see Table 1) with good r e s u l t s but the other histone fractions are cross-contaminated. Sung and Dixon. (15) have been able to p u r i f y each of the major histones by gel f i l t r a t i o n on long columns of Bio-Gel -4-P-10 eluted with 0.01 N! HOI. This method can. he combined with another such as ion-exchange chromatography to obtain r e l a t i v e l y large amounts of i n d i v i d u a l pure histones. Bailey and Dixon (unpublished r e s u l t s ) have devised an e f f i c i e n t method f o r large-scale p u r i f i c a t i o n of histones T and TTb-|. The method involves acid extraction of t o t a l histone from chromatin, adsorbtion and washing at low s a l t concentration on carboxymethyl c e l l u l o s e (CM-cellulose) columns to remove nucleic acids, non-histone proteins and histone T, treatment with cyanogen bromide (w/w i n 75$ formic acid at R.T. f o r 20 h) and, f i n a l l y , gel f i l t r a t i o n on Bio-Gel P-10 i n 0.01 Hi HOI. Cyanogen bromide cleaves the methionine-containing h i s -tones (Tfbp. 111 and T 7 ) into fragments small enough to be retarded during gel f i l t r a t i o n and thereby separated from the other histones (T and! TTb-| ). Table 1 summarizes three of the common nomenclature sys-tems for histones and gives 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 (13) w i l l be followed i n this t h e s i s . One may note that a l l the histones are r e l a t i v e l y small basic proteins which would exist as polycations at physiological pH. In most tissues, the h i s -tone content of basic residues (25-30 molest lys i n e + a r g i n -ine) i s s u f f i c i e n t to neutralize a l l of the negatively charg-ed phosphates of DNA (see Table 2). The t o t a l number of h i s -tone fractions i s quite l i m i t e d i n a l l organisms studied and, furthermore, the stucture of i n d i v i d u a l histone fractions - 5 -TABLE 1 Nomenclature and Characteristics of the Major Histones* Rasmussen, Murray and Johns and Moles$ Class Luck (13) Butler (10) M.W. Lys+Arg Termini (60.61 ) Lysine-r i c h la Tb f1 21,000 28 Frblocked (Ref.62) (Ref.64) C:lysine S l i g h t l y l y s i n e -r i c h TTb-, l i b 2 f2a2 1 4 , 0 0 0 * * 23 Frblocked (Ref.59) C:lysine f2b 13,774 28 K: proline (Ref.63) (Ref.63) C:lysine Arginine-r i c h TTT 1V f3 14,000** 21 BT:alanine (Ref.59) C:alanine f2a1 11 ,282 25 N: Ac-Ser (Ref.39) (Ref.39) C:glycine * Calf thymus ** Estimated from behaviour on e l e c t r o -phoresis and gel f i l t r a t i o n -6-seems to be s i m i l a r regardless of the origin? the l a t t e r f a c t w i l l be discussed i n more d e t a i l l a t e r . The N-termini are l i m i t e d to proline, alanine or a r e s i -due blocked by an acetylfrgr/oup; proline and blocked residues i n t h i s p o s i t i o n have been postulated to serve as protection for the protein against degradation by aminopeptidases (16). This may be p a r t i c u l a r l y important f o r histones since they are believed to lack t e r t i a r y structure (17) and t h e i r N-termini would not be protected by f o l d i n g as i n globular pro-teins. The term "chromatin" refers to the extended form of chromosomes present i n the c e l l nucleus between c e l l d i v i -sions and t h i s i s the form of the chromosomes i n which DNA r e p l i c a t i o n and DNA-dependent RNA synthesis are believed to occur. Several groups (18-21 ) have developed si m i l a r methods for i s o l a t i o n of chromatin from c e l l n u c l e i i n order to per-mit chemical and physical studies to le a r n something about i t s organization. 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 al.(22). The mass r a t i o s r e l a t i v e to MA of histones, non-histone pro-teins and RNA are given f o r several chromatin preparations; the a c t i v i t y of the chromatin as a template f o r RNA polymer-ase i s given f o r each case but does not correlate with the content of any of the three components mentioned above. Most chromatin preparations have a histone:MA mass r a t i o close to unity; the average content of 25-30 molest of basic r e s i -•7-TABLE 2 Chemical Compositions of Varied Chromatins -(22) Mass Ratios** Source of Chromatin Pea embryo axis Pea vegetative bud Pea growing cotyledon Rat l i v e r Rat ascites tumour Human HeLa c e l l s Cow thymus Sea urchin b i a s t u l a Sea urchin pluteus Non-histone Template* DNA Histone Protein RNA A c t i v i t y 1.03 1.30 0.76 1.00 1.16 1.02 1.14 1.04 0.86 0.29 0.10 0.36 0.67 1.00 0.71 0.33 0.48 1.04 0.26 0.11 0.13 0.04 0.13 0.09 0.01 0.04 0.08 12$ 6 32 20 10 10 15 10 20 * Relative to MA (deproteinized) ** Relative to MA as 1 - 8 -dues can then be calculated as being s u f f i c i e n t to ne u t r a l -ize a l l the DNA phosphates as mentioned e a r l i e r . Isolated chromatin contains bound RNA polymerase and cata-lyzes the synthesis of RNA from the four ribonucleotide t r i -phosphates. The enzyme has been p u r i f i e d from the chromatin of pea plants (7) and various mammalian tissues (23). The a c t i v i t y of the endogenous enzyme i s r e l a t i v e l y low but chro-matin can serve as a template f o r synthesis of RNA by added enzyme; reaction mixtures with excess E. c o l i RNA polymerase can synthesize RNA i n amounts up to f i f t y times that of the template DNA supplied. RNA synthesized i n t h i s manner can serve as a template for protein synthesis with products r e -presentative of the tissue used to prepare the chromatin (24). A l l chromatins show r e s t r i c t i o n of template a c t i v i t y f o r RNA synthesis compared to deproteinized DNA from the same source; f o r example, Table 2 shows that l i v e r chromatin has a template a c t i v i t y 20% that of l i v e r DNA. The template ac-t i v i t y of a given chromatin preparation has been found to be proportional to the a c t i v i t y of the tissue of o r i g i n i n RNA synthesis; t h i s suggests that the i n vivo properties of chromatin are preserved i n v i t r o . The RNA transcribed from chromatin i n v i t r o has a base composition d i f f e r e n t from that transcribed on deproteinized DNA ( 9 ) , i n d i c a t i n g that the same DNA sequences are not transcribed i n both eases. Paul and G-ilmour (25) used the technique of hybridization to show that a r e s t r i c t e d portion of DNA i n chromatin i s transcribed by RNA polymerase; i n the case of thymus chromatin they -9-found template a c t i v i t y 7$ that using deproteinized DNA. In further experiments (26), involving competition h y b r i d i -zation, these authors showed that the portion of MA a v a i l a -ble for t r a n s c r i p t i o n i n chromatin from various tissues i s not the same. They also established that the RNA t r a n s c r i b -ed i n v i t r o on chromatin i s i d e n t i c a l to that transcribed i n vivo i n the same tis s u e . This i s good additional e v i -dence that chromatin maintains i t s o r i g i n a l properties af-t e r I s o l a t i o n and assay i n v i t r o . Development has been defined as the synthesis of a s p e c i f i c protein at 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 could 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 part of development. This means that the c e l l must be able to s p e c i f i c a l l y activate those genes required at a c e r t a i n time and inactivate or keep inactivated those genes which are not required; thus, a reticulocyte employs i t s hemoglobin genes but not i t s myosin gene(s). This a b i l i t y of c e l l s has long intrigued 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 de t a i l s of the mechanisms probably i n -volved have been discovered. Examples are the r o l e of i n -ducers during development and the a b i l i t y of hormones to stimulate synthesis of s p e c i f i c proteins including enzymes and s t r u c t u r a l proteins. The complete series of steps i n -volved i n the action of inducers or hormones are not known but i n the case of the l a t t e r , increases i n both the kinds and amounts of RNA synthesized i s one 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 defined as the agents responsible fo r the s p e c i f i c and direct i n a c t i v a t i o n of a gene or family 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 yet i n eukaryotes but a discussion of some of t h e i r expected features might be useful. I f one accepts that eukaryotic c e l l s have about 5X10-* genes (28) and that each repressor controls as many as 10 genes (e.g. f o r the enzymes i n a metabolic pathway), then there would5 be the order of 5X10^ ind i v i d u a l repressors. Eukaryotic c e l l s contain about 5X10 1 2 daltons of DNA and thus about 1 0 1 0 base pa i r s ; to be unique, a sequence would have to be 16 base pairs long. A r e -pressor must then recognize a DNA sequence at l e a s t t h i s long and the recognition should be highly s p e c i f i c . This could : be accomplished by a protein or a nucleic acid but there are obvious problems with either suggestion. RNA has the desired a b i l i t y by v i r t u e of base pai r i n g but th i s requires either separation of DNA strands or formation of a t r i p l e x i n t h i s region. There might be problems with removal of an RNA r e -pressor since the minimum of 16 base pairs would give a s t a -ble complex. On the other hand, a protein repressor might be removed a f t e r undergoing a hormone or other "inducer" pro-duced conformational change. One wonders i f there could have evolved 50,000 i n d i v i d u a l proteins each capable of recogniz-ing a d i f f e r e n t DNA sequence; recognition of a single base pair change i n a sequence of 16 or more base pairs i s asking - 1 1 -a l o t from a protein. Of course, the repression mechanism may not involve these proposed repressors. Histones appear to lack the requirements of being the sole repressors i n eukaryotes. There are almost c e r t a i n l y fewer than 15 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 tissue or species v a r i a t i o n i n numbers or types of histones. Histones show only l i m i t e d recogni-t i o n of DM with histone T preferring regions r i c h i n A-T base pairs and histones 111 and TY preferring G-C regions (30) . At high i o n i c strength, polylysine binds p r e f e r e n t i a l -l y to A-T pairs and polyarginine to G-0 pairs (31) . The s i g -nificance of these interactions seems l i m i t e d i n view of the fact that each histone f r a c t i o n can completely repress DNA i n v i t r o (32). Bonner and co-workers (22,24) found by several methods that s e l e c t i v e removal of histones from chromatin increases i t s template a c t i v i t y to almost that of deproteinized DNA. The simplest i n t e r p r e t a t i o n of the above fa c t i s that h i s -tones are the sole repressors 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 possible to separate the effects of the methods used to remove the histones from t h e i r removal per se, since both operations probably cause drastic changes i n the organization of the chromatin. I f the r e s u l t s above are v a l i d , then the non-histone proteins remaining i n the chromatin cannot alone r e s t r i c t t r a n s c r i p t i o n . The "masking" hypothesis (33) requires the protection -12-of c e r t a i n genes, perhaps by non-histone proteins, while the remaining genes are repressed or masked by the histones. Thus histones would act i n a r e l a t i v e l y nonspecific manner but s t i l l an essential one. Involvement of the non-histone pro-teins i n the s p e c i f i c i t y of repression i s implied by the work of Paul and Gilmour (34). These authors reconstituted chromatin from i t s components and then assayed by competi-t i o n hybridization the RNA product produced on these tem-plates under the action of RNA polymerase. They found that the non-histone proteins are essential i n the r e c o n s t i t u -t i o n of a chromatin with the same.properties as the o r i -g i n a l chromatin. There i s some evidence f o r ft. RNA component i n chromatin which may be covalently bound to a chromosomal protein,(35) and seems to be required 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 (36, 37). This chromosomal RNA (cRNA) i s 40-60 nucleotides long by endgroup analysis and contains 8-10 moles$ of dihydro-uridylate or dihydrpthymidylate depending on the species. cRNA can be prepared by disso l v i n g p u r i f i e d chromatin i n 4 M CsCl and centrifuging at highspeed; a p e l l i c l e forms at the top of the tube and contains RNA and protein. The cRNA i s recovered from the p e l l i c l e by phenol extraction a f t e r pronase digestion. Huang (38) found that p r i o r to pronase digestion, cRNA i s associated with a protein i n a complex stable to high s a l t and agents capable of disrupting H-bonds; this implies -13-but does not prove the presence of a covalent bond between cRNA and protein. Pronase digestion and a l k a l i n e hydrolysis of the cRNA:protein complex released a small peptide bound to dihydrouridylate; acid hydrolysis of t h i s " l i n k e r " pro-duced beta-alanine and serine. Huang therefore concluded that cRNA i s li n k e d to a protein by a peptide bond between a serine residue and ureidopropionic acid, the product of ring-opening of dihydrouridylate. Bekhor, Kung and Bonner (36) showed that cRNA hybrid-izes to native DNA from the same species i n the presence of 5 M urea at 1 °0 and that a mRNA preparation from the same source does not hybridize to DNA under these conditions. I f the work above can be confirmed with the cRNA-protein com-plex characterized more completely, one would anticipate a very important role f o r the complex i n the control 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 i n amino acid composition, terminal residues and electrophore-t i c mobility. Histone TV from pea seedlings and c a l f thymus have been sequenced (39) and d i f f e r i n sequence at only two positions and even these substitutions are conservative; the only other differences are the number and s i t e s of acetyla-t i o n and methylation of epsilon amino groups of l y s i n e r e s i -dues . Histone TV i s , therefore, the protein whose stucture i s most r i g i d l y conserved during evolution of any yet studied; -14-only with cytochrome c has the sequence of another protein from both a plant (40) and an animal ( 4 1 ) been established and i n that case more than one-third of the residues d i f f e r * The r i g i d s e l e c t i o n required to preserve the 102 residue se-quence of histone T 7 suggests that the entire molecule i s important i n i t s function and any changes must be d e l e t e r i -ous to the organism. The authors state that since the only obvious constant features of DNA from pea seedlings and c a l f thymus are the sugar-phosphate backbone and the double h e l i -cal nature, histone T 7 may be uniquely suited to inter a c t with the h e l i c a l backbone of DNA; both DNAs contain the same 4 bases, of course, but these must be present i n the same sequences very seldomly. This histone i s also i n t e r e s t -ing because of the d i s t r i b u t i o n of basic and hydrophobic residues; 17 of the 27 basic amino acids are i n the N-ter-minal h a l f of the molecule and 5 of the 6 aromatic amino acids are i n the G-terminal h a l f . This asymmetric " d i s t r i -bution has been taken to indicate a DNA-binding s i t e i n the N-terminal h a l f and a hydrophobic binding s i t e i n the C-terminal h a l f possibly f o r i n t e r a c t i o n with other proteins (42). Bustin and Cole (43) cleaved rabbit thymus histone T at i t s single tyrosine residue with N-bromosuccinimide; amino acid analyses and endgroup determinations of the whole histone and the two fragments were done to 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 acid analysis) had 2 3 moles$ basic r e s i -dues and the C-terminal fragment (M.W. about 15,000 from amino acid analysis) had 3 1 molest basic residues. On the basis of t h i s rather unimpressive asymmetry, the authors suggested a DNA-binding s i t e i n the C-terminal fragment; since t h i s region i s 2.5 times larger than the N-terminal piece, they may well be correct. Cole and co-workers ( 4 4 - 4 7 ) have fractionated histone T from various sources into as many as 5 components by i o n -exchange chromatography on Amberlite IRC-50; these compo-nents are a l l s i m i l a r i n s i z e and amino ac i d composition. One of the p u r i f i e d components of histone T from rabbit thy-mus was compared with whole histone T from the same source by performing the N-bromosuccinimide cleavage and peptide mapping of the fragments. The N-terminal fragments showed several differences whilst the C-terminal fragments were nearly i d e n t i c a l . This suggests that histone T has a v a r i -able N-terminal region and a constant C-terminal region; t h i s would be analogous to the i n d i v i d u a l chains of immunoglobu-l i n s . Sequence analysis of histone T components should give the f i n a l answer to the nature of the heterogeneity of h i s -tone T and, hopefully, some insight into i t s function. Supporting evidence f o r the presence of DNA-binding s i t e s i n histones comes from the work of Dixon and co-workers. At the stage of spermatogenesis i n trout t e s t i s when histones are replaced by protamine, there i s a marked phosphorylation -16-of histones which occurs independently of protein synthe-s i s (48,49). The si t e s of phosphorylation i n the in d i v i d u a l histones were investigated and recently these studies were extended by Sung and Dixon (15,50). With the possible excep-t i o n of histone TTbp, there appears to be a single s i t e of phosphorylation i n each of the histones. The s i t e i n histone T i s i n the C-terminal fragment a f t e r N-bromosuccinimide cleavage and i s i n the sequence Lys-Ser(P04 )-Pro-kLys; the C-terminal fragment was the region suggested by Bustin and Cole as a DNA-binding s i t e (43). In histones 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; th i s evidence indicates a region of homology between these histones and. also l o c a l -izes the s i t e of phosphorylation i n histone TV i n the half of the molecule r i c h i n basic residues (42). Sung and Dixon (15,50) have proposed a model for a DNA-binding s i t e i n the N-terminal 18 residues of histone T7 . They assumed that t h i s region i s i n the alpha-helical con-formation when associated with DNA and arranged the 18 r e s i -dues i n the form of 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 circum-ference of the wheel represents the polypeptide backbone of a right-handed alpha-helical protein as viewed from the C-terminus; the projections are the amino acid sidechains of consecutive residues spaced at 100° interv a l s since there are 3.6 residues per turn i n an alpha-helix. The serine residue F i g . 1 H e l i c a l Wheel Arrangement of the N-terminal 18 Residues of Histone T7 HELICAL WHEEL OF RESIDUES 1 - 1 8 OF HISTONE IV Note: This i l l u s t r a t i o n was taken from the work of Sung and Dixon (50). Consecutive residues are numbered s t a r t i n g from the N-terminus as #1. The l e f t side of the figure shows the histone modified only by acetylation of the N-terminal serine; the r i g h t side shows complete mo-d i f i c a t i o n a f t e r phosphorylation and acetylation. Fur-ther explanation i s given i n the text. -18-which i s phosphorylated i n vivo (48) i s situated on the same side of the h e l i x as a c l u s t e r of four l y s i n e s ; the sides adjacent to the cluster above are occupied by an arc of 4 glycines and an arc i n which 4 out of 6 residues are glycine. A molecular model of the sequence was constructed as a right-handed alpha-helix and was found to f i t neatly into the major groove of a model of double h e l i c a l MA i n the B form. The 4 amino groups of the sidechains of lysines 5, 8, 12 and 16 and the guanidino group of arginine 17 a l l make close contact with 5 consecutive phosphate groups on the same DNA strand; arginine 3 interacts with a phosphate group on the opposite MA strand. The predominance of g l y -cine residues i n the arcs adjacent to the lysine-serine arc allows the histone sequence to f i t completely within the ma-jor groove. At the time of histone replacement i n trout t e s t i s , there i s also extensive acetylation of epsilon amino groups of lysine residues i n the N-terminal region of histone TV (Oandido and Dixon, i n preparation). I f the lysine-serine arc i s completely modified by acetylation and phosphoryla-t i o n , the net charge of the histone up to position 18 w i l l change from +6 to +0.3, assuming a charge of -1.7 on the serine phosphate at physiological pH. The model of Sung and Dixon i s supported by the recent work of Wagner (80). One of the assumptions of the model i s that the N-terminal region i s a c t u a l l y i n the alpha-helical - 1 9 -conformation i n association with DNA; the n e u t r a l i z a t i o n of charge on the lysines by the DNA phosphates would be required since charge repulsion would d e s t a b i l i z e a h e l i x . Wagner found that c a l f thymus histone TV undergoes a marked confor-mational change i n the presence of polyvinylphosphate (PVP) or DNA as indicated by changes i n the c i r c u l a r dichroism spectrum. The r e s u l t s indicated an increase i n the r a t i o of alpha-helix to random c o i l i n the histone upon i n t e r a c t i o n with either polymer. The actual increase i n h e l i c a l content was estimated to be 10-23%; t h i s would involve 10-23 of the 102 residues i n the protein and f i t s n i c e l y with the 18 r e -sidue binding s i t e of the model. Evidently, histone T does not undergo a conformational change when associated with DNA (81); one would then expect a d i f f e r e n t binding s i t e for t h i s histone and the sequence data suggest that t h i s i s so (15). This s i t e must be i n the proper conformation before associating with DNA or else the change i s too small to be detected by the methods used. If the homology between histones TTb-j and TV extends as fa r as 18 residues without s i g n i f i c a n t change, then the mo-^  del would apply to histone TTb-j as we l l . Thus, at l e a s t two of the histones have a s i t e suited f o r binding to DNA and modification of t h i s region by acetylation and phosphory-l a t i o n may allow reversal of binding. Structural studies on histones have therefore given clues to the basis of t h e i r be-haviour i n chromatin and further studies w i l l undoubtedly aid i n determining t h e i r function. -20-Part One of this thesis w i l l describe the work done on a histone, t e n t a t i v e l y named histone I, discovered in. trout t e s t i s chromatin; t h i s histone has not been described pre-viously-. Histone T was found to be s i m i l a r to histone TTbg i n s i z e , N-terminal residue and amino acid composition. How-ever, s t r u c t u r a l studies of the new histone revealed that i t can not be a degradation product of any of the major h i s -tones . -21-MATERIALS AMD METHODS  Chemicals and Abbreviations a) Chemicals Commonly used chemicals were obtained commercially and were of reagent grade. Bio-Gel P-10 and CM-cellulose were obtained from Bio-Rad Laboratories; CM-52 and chromatogra-phy paper (3 MM) from Whatman; and porcine trypsin from Novo Industri. b) Abbreviations Most abbreviations are those recommended by the Journal 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 per weight v/v: volume per volume Ac: acetyl A-T: adenine-thymine base pair G-C: guanine-cytosine base pair EDTA: ethylenediaminetatraacetate TRIS: tris(hydroxymethyl)aminomethane TCA: t r i c h l o r a c e t i c acid N-terminus: amino terminus C-terminus: carboxyl terminus - 2 2 -DNSC1: 1-dimethylaminonaphthalene-5-sulphonyl chloride DNS: dansyl Trout Testis Naturally maturing rainbow?trout t e s t i s (Salmo g a i r d n e r i i ) was kindly donated by Mr. and Mrs. Hans Lehmann of the Sun Valley Trout Farm at Mission, B.C. Testes were transported (1 h) on i c e , washed and frozen at -80 °C u n t i l the time of use. Experimental Methods 1. Preparation of Chromatin Chromatin was i s o l a t e d from trout t e s t i s by the method of Marushige and Bonner (9) with a l l steps performed at 4 °C. Frozen testes were p a r t i a l l y thawed and homogenized i n a Waring blendor with 3 volumes of saline-EDTA (0.075 M NaCl, 0.024 M EDTA, pH 8.0) at highspeed f o r 2 minutes, f i l t e r e d through several layers of washed cheesecloth and centrifuged at 1000 g f o r 10 minutes. The nuclear p e l l e t was washed once by resuspending i n saline-EDTA and centrifugation and then lysed i n 0.01 M THIS buffer (pH 8.0). Chromatin was sedimented at 10,000 g f o r 10 minutes and washed once with the same buffer. Sheared chromatin was prepared by homo-genizing chromatin i n 0.01 M phosphate buffer (pH 8.0) or i n 0.01 M TRIS buffer (pH 8.0) at highspeed i n the Waring blendor f o r 2 minutes and then centrifuging at 10,000 g for 15 minutes a f t e r which the sheared chromatin remains i n the supernatant. In some experiments, chromatin was further -23-p u r i f i e d by layering 6 ml of sheared chromatin over 25 ml of 1.7 M sucrose underlayed with 4 ml of 2 M sucrose. The upper one-third of the tube was gently mixed and then centrifuged at 22,000 rpm for 2 h i n the SW-27 rotor of a Spinco L2-65 preparative ultracentrifuge. The chromatin p e l l e t was recov-ered by c a r e f u l l y aspirating the supernatant. 2. Maleylation Sheared chromatin was prepared i n 0.01 M phosphate buf-f e r (pH 8.0) and diluted to 1 mg/ml as estimated by absorp-t i o n at 260 nm (1 mg chromatin DNA/ml has an A26O = 20). The chromatin solution was s t i r r e d magnetically at 4 and f i n e l y powdered maleic anhydride was slowly added and the pH was maintained at 8.0 by addition of 0.1 N NaOH with a micropipette. The end of the reaction was indicated by a constant pH after adding a l l the 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 reac-ti o n mix was then centrifuged at 22,00.0 rpm for 2 h i n the SW-27 rotor; the supernatant then contained any proteins released by maleylation. Chromatin could be removed quickly at the end of the reaction by adding acetic acid to lower the pH to 4.5 which causes the chromatin t o aggregate, and i t can then be sedimented by centrifugation at 10,000 g-for 1Q minutes. After either method, the supernatant was then d i a -lyzed against 0.1 M acetic acid at 37 °C for four days or 50 °C for two days i n order to hydrolyze the a c i d - l a b i l e maleyl groups. Control histone samples showed no detectable -24-degradation a f t e r this treatment when electrophoresed on polyacrylamide disc gels. 3. S a l t Dissociation 1 .5 M NaCl i n 0.01 M TRIS (pH 8.0) was added slowly with vigorous s t i r r i n g to sheared chromatin u n t i l the s a l t con-centration was 0.3 M. The sol u t i o n was then adjusted to pH 4.5 with addition of acetic acid and aggregated chromatin sedimented at 10,000 g f o r 10 minutes. The supernatant was dialyzed against 0.1 M acetic acid at 4 °C and l y o p h i l i z e d . 4 . Acid Extraction a) 5$ TOA Chromatin was homogenized at lowspeed i n a Waring blendor while 10 or 20$ TCA was slowly added u n t i l a f i n a l concentration of 5$ TCA was reached (v/v); s t i r r i n g was con-tinued f o r another minute. The extract was then centrifuged twice at 20,000 g f o r 20 minutes. The supernatant was then adjusted to 20$ i n TCA by addition of s o l i d TCA with s t i r -r ing and a heavy white p r e c i p i t a t e formed immediately. After standing at 4 °C f o r 1 h, the precipitated protein was r e -covered by centrifuging at 10,000 g f o r 20 minutes. The pe l l e t was washed once or twice with ether and dried i n vacuo. b) 0.2 M H 2S0 4 Chromatin was extracted by homogenizing with 0.2 M H 2S0 4 i n the Waring blendor f o r 2 minutes at highspeed f o l -lowed by centrifugation at 10,000 g f o r 20 minutes.twice. -25-The supernatant was saved, 3-4 volumes of cold 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 precipitate was recovered by centrifugation, washed once with ethanol and dried i n vacuo. 5. Gel Exclusion Chromatography Histones were fractionated by chromatography on Bio-Gel P-10 or Sephadex G-100 eluting with 0.01 N HC1 and monitor^ ing the A230 o f the eluate. 6. Ion-exchange Chromatography CMkiellulose r e s i n was treated as follows: s t i r r e d i n 0.5 N NaOH for 30 minutes, washed with d i s t i l l e d water u n t i l neutral, s t i r r e d i n 0.5 N HC1 f o r 30 minutes and washed u n t i l neutral. The HC1 step was repeated and then the r e s i n was s t i r r e d i n 1-2 M L i C l for 10 minutes, allowed to s e t t l e f o r 10-15 minutes, decanted, the L i C l step repeated 3-4 times and the column poured. The column was washed overnight with 0.15 M L i C l i n 0.01 M l i t h i u m acetate pH 5 . 0 . The 5% TCA extractable proteins were dissolved i n 0.01 M l i t h i u m acetate pH 5 . 0 and applied to the CM-cellulose c o l -umn (3X30 cm). The column was washed with 50-100 ml of s t a r t i n g buffer and then a l i n e a r gradient of L i C l was s t a r t -ed. The gradient was generated from 1000 ml each of 0.15 M and 0.75 M L i C l i n 0.01 M l i t h i u m acetate pH 5 . 0 . The flow rate was about 100 ml/h and the A 2 ^ Q of the eluate was moni-tored. Salt concentration i n every f i f t h f r a c t i o n was measured -26-with a conductivity meter making reference to standard s o l u -tions of lithium chloride. OM-52 r e s i n was obtained i n a precycled and preswollen form but fines were removed by suspending the r e s i n i n 1-2 M LiOl and allowing the larger p a r t i c l e s to s e t t l e f o r about 30 minutes. A column was poured and subsequent steps were as above except for s a l t concentration and the flow rate was much slower. 7. Polyacrylamide Disc Gel Electrophoresis The method of Bonner et al.(18 ) was followed f o r f r a c -tionating histones on polyacrylamide disc gels. Modifica-tions included the use of longer gels (7 cm) and use of methyl green to judge the length of electrophoresis; the l a t t e r dye was found to migrate s l i g h t l y f a s t e r than prota-mine. The power supply was adjusted to 4-5 milliamperes per gel and the potential was usually 25 volts/cm. Gels were stained i n 1$ Amido Black 1GB i n destaining solu t i o n (7$ acetic acid i n 40$ ethanol) f o r at lea s t 1 h and de-stained with several changes of the destaining solution. Protein samples were dissolved at 1 mg/ml i n 6 M urea and 0 . 0 1 - 0 . 0 3 ml was applied to each gel a f t e r the apparatus was completely arranged and f i l l e d with buffer. 8. Molecular Weight Estimation by SDS Gel Electrophoresis The method of Weber and Osborn (52) was followed. Sam-ples and standards were dissolved i n 6 M urea i n d i a l y s i s -27-buffer (0.01 M phosphate buffer pH 7.0, 0.1$ SDS and 0.1$ 2-mereaptoethanol) at 0.5 mg/ml and incubated at 37 °C for 1 h. 10$ or 15$ gels were prepared and placed i n the appara-tus with buffer; 0.01-0.05 ml of each sample was mixed with 0.005 ml concentrated 2-mercaptoethanol and 0.005 ml 0.05$ bromphenol blue and applied to the appropriate g e l by under-laying the buffer. Electrophoresis was at about 5 volts/cm fo r about 5 h or u n t i l the bromphenol blue marker had a l -most reached the end of the gels. Gels were then stained i n 0.25$ Ooomassie B r i l l i a n t Blue i n 10$ acetic acid i n 50$ methanol overnight and destained with several changes of 7.5$ acetic acid i n 5$ methanol. The distance migrated by the bromphenol blue and the gel length were measured before staining; a f t e r staining, the gel length and distance mi-grated by the protein sample were measured. The mobility of the sample was calculated by the following formula: distance of protein length of gel before migration s t a i n i n g M o b i l i t y = X : '— distance of dye length of gel a f t e r migration destaining This formula allows for s l i g h t differences i n the potential across gels and f o r swelling or shrinking during staining and destaining. 8. Amino Acid Analysis Samples were hydrolyzed i n 0.5 ml of 6.If HC1 i n evacuat-- 2 8 -ed sealed glass tubes at 110 0 f o r various, times and then dried i n vacuo over NaOH. After 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 buffer (pH 2.2), aliquots were applied to the long or short coluntns of a Beckman Model 1200 amino acid analyzer using the 2 h procedure (53). A few samples were analyzed by the single column method of Devenyi (54). 9. Tryptic Fingerprints Samples were digested with porcine t r y p s i n (Novo Indus-t r i ) with an enzyme:substrate r a t i o of 1:25 (w/w) for 4 h at 37 °0 i n 0.2 M NH4HC03 (pH 8.0). After 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 -solved i n pH 6.5 buffer (pyridine/acetic acid/water, 100: 4:900, v/v) and aliquots equal to 0.5 A-230 vaii-^a o f Protein were applied to Whatman 3 MM paper. Electrophoresis was at about 40 volts/cm for about 1 h, the exact time being de-termined by the migration of coloured reference dyes whose ch a r a c t e r i s t i c s are outlined i n Table 3. The paper was dried and the s t r i p with the sample peptides was cut out and sewn into a new sheet of Whatman 3 MM paper. Descending chroma-tography was then performed f o r 20 h with the solvent sys-tem butanol/water/acetic acid/formic acid, 45:15:15:1* v/v. The sheets were dried and stained with cadmium ninhydrin (55) or phenanthrene quinone (56). The l a t t e r procedure involves a f r e s h l y prepared spray consisting of equal v o l -umes of 0.02$ phenanthrene quinone i n ethanol and 10$ NaOH I - 2 9 -TABLE 3 M o b i l i t i e s of Coloured Markers on High Voltage Paper Electrophoresis Dye pH 2.1 pH 3.6 pH 6.5 DHP l y s i n e Ser u= 0.56 u= 0 u= 0 DNP Agmatine Ser Lys Asp u= 1.1 u= 0.67 u= -0.57 Xylene Cyanol PF Ser Lys Asp u= -0.3 u= -0.43 u= 0.35 Orange G Lys Asp u= -0.86 u= 0.92 Methyl Green Ser Lys Asp u= 2.0 u= 1.05 u= -0.91 Crystal V i o l e t Ser Lys Asp u= 0.82 u= 0.46 u= -0.37 -30-i n 60% ethanol; the chromatogram i s sprayed, dried and exa-mined under UV ill u m i n a t i o n . Arginine-containing peptides fluoresce strongly and may be c i r c l e d and photographed with a Polaroid camera with a UV f i l t e r as described under "N-terminus determination" below. The same chromatogram may be stained with cadmium ninhydrin aft e r washing with ethanol 3 times and once with 5% acetic acid i n acetone. 10. N-terminus Determination The dansylation method of Gray (57) was used to i d e n t i -f y the N-terminus of histone T. One mg of the protein was dissolved i n 0.05 ml of 0.1 M NaHCO^, l y o p h i l i z e d to remove NH3, redissolved i n d i s t i l l e d water and the pH checked with litmus paper to assure a pH close to 8 . 0 . DNS-01 was d i s -solved at 50 mg/ml i n acetone just before use and 0.05 ml was added with mixing to the sample; the tube was sealed and incubated at 37 °C for 3 h. A pr e c i p i t a t e formed during the reaction and was saved; the supernatant was desalted by gel f i l t r a t i o n on a column of Sephadex G-10 (1X15 cm) eluted with 0.01 N HC1 and then l y o p h i l i z e d . The pr e c i p i t a t e and supernatant fractions were both hydrolyzed i n 6 N H01 at 110 °0 f o r 8 h i n sealed, evacuated glass tubes. After dry-ing i n vacuo over NaOH, the samples were redissolved i n 0.025 ml of 1 M NH4OH and 0.005 ml applied to each pf two t h i n -layer plates. These plates had been prepared by spreading a s l u r r y of s i l i c a gel G (25 g i n 50 ml water) i n a layer 0.25 mm thick on glass plates (20X20 cm) with the aid of a Desaga spreader. The plates were a i r dried and heated to 100 GC f o r 15 minutes just before use. The solvent systems employed were those described by Black and Dixon (58); non-polar DNS derivatives are separated i n solvent system 1 (chloroform/methanol/acetic a c i d , 95:10:1, v/v) and polar derivatives are resolved i n solvent system 2 (n-propanol/ concentrated NH4OR", 80:20, v/v). The f i r s t system requires about 1£ h and the second about 2-2% h to give adequate separation. After drying, the plates were examined under UV i l l u m i n a t i o n and photographed immediately with a Polaroid Model 160 camera with a copy lens and UV f i l t e r and Polar-oid Land Picture R o l l Type 47 (3000 speed). -32-RESULTS AND DISCUSSION The maleylation of chromatin was studied to determine whether chemical modification of histones i s alone s u f f i -cient to promote t h e i r d i s s o c i a t i o n from DNA i n analogy to the postulated role of the i n vivo modifications of h i s -tones by s p e c i f i c enzymes. These i n vivo modifications i n -clude phosphorylation of s e r y l (48,66) and threonyl (66) groups, acetylation of epsilon- (67) and alpha-amino groups (68,69) and methylation of epsilon-amino groups (70,71 ) or guanidino groups (72). These modifications are believed to be important during periods of gene a c t i v a t i o n (73,74) and at the time of histone replacement by protamine as discussed i n the "Introduction". Phosphorylation i n t r o -duces about 1.7 negative charges per serine or threonine modified whilst acetylation removes the single p o s i t i v e charge of amino groups. Methylation may introduce up to three methyl groups on the epsilon-amino group of l y s i n e but does not change the charge; methylation increases the bulk of the sidechain and the b a s i c i t y of the nitrogen. The l a t t e r would not be s i g n i f i c a n t at physiological pH but the former may be very important i n i t s influence on the i n t e r -action of histones with other molecules. Maleic anhydride was chosen for chemical modification since t h i s reagent reacts r a p i d l y with uncharged amino groups; modification of a l y s i n e sidechain by t h i s means w i l l change i t s charge from +1 to -1. Since histones con-t a i n 10-25 moles% of l y s i n e , complete modification by maleic anhydride would reduce the net charge by 20-50 units per molecule of 100 residues. By use of r e l a t i v e l y mild condi-tions and perhaps i s o t o p i c a l l y l a b e l l e d maleic anhydride i t might prove possible to determine the s i t e s and numbers of lysines which must be modified to allow a histone 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 trout t e s t i s was prepared and maleylated as described i n "Experi-mental Methods". Maleic anhydride was used at 10 or 20 times molar excess calculated on the basis of 1 mg of histone per mg of chromatin DNA and an average histone content of 15 moles% of l y s i n e . The control samples were processed iden-t i c a l l y but either without maleic anhydride or with sodium maleate instead. Proteins released from chromatin by the treatment above were demaleylated and electrophoresed on polyacrylamide disc gels with the r e s u l t shown i n Pig. 2 . The major protein released by maleylation (gels 3 and 4) i s a r a p i d l y migrating band with twice the mobility of h i s -tone T. Lesser amounts of histones T, 1Tb 1 and TTb 2 are also released. The fa s t component had been noted previously as a minor f r a c t i o n of trout histones but was not further s t u -died at that time. A histone component migrating i n t h i s p o sition has not been described i n other organisms and i t w i l l be referred to below as histone T ( f o r t r o u t ) . The complete absence of histones 111 and ?T7 i n the - 3 4 -F i g . 2 Polyacrylamide disc gel electrophoresis of chromosomal pro-teins released by chemical modification by maleic anhydride. - I 1 2 3 4 5 6 7 8 Migration was from top to bottom and the i d e n t i t y of the bands i s indicated by the l i n e s and numbers at the l e f t . The samples are numbered at the bottom and include: 1 and 5, t o t a l trout t e s t i s histones; 2, no additions; 3 and 4, ten and twenty times molar excess of maleic anhydride; 6-8, ten, twenty and f i f t y times molar excess of sodium maleate. 111d i s the dimer of histone 111 due to disulphide formation between cysteine residues (Note: 1 cysteine residue per molecule of histone 111 i n t r o u t ) . -35-dissociated material suggests that they are either unavail-able to the maleic anhydride or else chemical modification of amino groups alone i s not s u f f i c i e n t to dissociate them. In addition, 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 effect of maley-l a t i o n would be l e s s than i n the other histones. The f a c i l e d i s s o c i a t i o n of histone T would thus imply 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 , this i s true. Gels 2 and 6-8 i n F i g . 2 show that histones are not dissociated from chromatin under the experimental conditions i n the ab-sence of maleic anhydride. The actual amounts of protein dissociated from chromatin by the experimental conditions was measured and i s given i n Table 4. These figures confirm that d i s s o c i a t i o n of histones i s due to maleylation and not due to the s a l t produced upon hydrolysis of excess ma-l e i c anhydride and n e u t r a l i z a t i o n with NaOH. Although i t i s i n t e r e s t i n g to note that maleylation i s s u f f i c i e n t to dissociate c e r t a i n histones, the mechanism probably involves extensive and random modification of l y -sines; also, the modification causes a large charge (+1 to -1 ) and s i z e change of the l y s i n e sidechains. As discussed i n the "Introduction", i n vivo histone modification i s characterized by r e s t r i c t i o n to c e r t a i n s i t e s i n these pro-tei n s . Because of t h e i r l i m i t a t i o n s , the chemical modifica-t i o n studies were discontinued and the new histone (histone T) was further characterized. -36-TABLE 4 Dissociation of Chromosomal Proteins by Maleylation Treatment Mg Protein Released* No additions 1.0 Sodium maleate 2:1 1.0 10:1 0.8 20:1 0.9 50:1 1.0 Maleic anhydride 2:1 0.9 10:1 2.3 20:1 2.5 50:1 8.7 * Mg of protein released i n t o the supernatant and measured by the Lowry method (65). -37-In addition to i t s release upon lim i t e d maleylation, histone T could be extracted from chromatin by 0,3 M NaCl at pH 4.5 b u t " i t was found that extraction by 5% TCA (75) was simpler and offered the advantage of avoiding proteo-l y t i c degradation during the process. Extraction with 5% TCA was found to remove histones T and T as well as some protamine; a l l of the protein i n the extract could be pre-c i p i t a t e d by addition of s o l i d TCA to a concentration of 20% (w/v). Further p u r i f i c a t i o n was done either on Bio-Gel P-10 i n 0.01 N HC1 as i n F i g . 3 or on OM-cellulose eluted with a l i n e a r gradient of L i C l as i n F i g . 4. Highly p u r i -f i e d histone T elutes as a s i n g l e peak afte r histone T on Bio-Gel P-10 or before histone T on CM-cellulose. In Octo-ber stage n a t u r a l l y maturing trout t e s t i s , histone T was found to form about 0.5% of the t o t a l histone. Rechromato-graphy of histone T on CM-52 (microgranular CM-cellulose,, Whatman) i s shown, i n F i g . 5 and reveals a single peak at 230 nm with a shoulder on the ascending limb; no d i f f e r - ' ences could be found by amino acid analysis or peptide mapping i n aliquots taken from either side of the peak. The molecular weight of histone T was estimated by the method of Weber and Osborn (52) which involves measur-ing the mobility of the sample together with standard pro-teins of known molecular weight during electrophoresis on polyacrylamide gels i n the presence of the detergent, sod-ium dodecyl sulphate (SDS). Since histones are quite basic -38-F i g . 3 a) Gel f i l t r a t i o n of histones on a column of Bio-Gel P-10 (5.5XHO cm) eluted with 0.01 I f HC1. B I O - G E L P - I O ( 5 - 5 X I 4 0 c m J ELUTE WITH 6 Ol N H C L IO 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 I 0 0 110 I 2 0 F R A C T I O N NO V 0 L « l 4 m l . b) Polyacrylamide disc gel electrophoresis 1 2 3 Samples are: 1, t o t a l trout t e s t i s histones; 2, peak A from Pig. 3a; and 3» peak B from Pig. 3a. -39-f i g . 4 a) Ion-exchange chromatography of 5$ TCA extractable chromosomal proteins on CM-cellulose. FRACTION NO-The column (3X30 cm) was eluted with a l i n e a r gradient of L i C l . b) Polyacrylamide disc gel electrophoresis 1 2 3 Samples are: 1, t o -t a l trout t e s t i s h i s -tones ; 2, peak A from Pig. 4a; and 3, peak B from Pig. 4a. Pig. 5 Rechromatography of histone T on a column of CM-52 (1.8X50 cm) I-5-l I O -o IO CM < 0-5-0O .Q--D-..•-B-B-2 0 4 0 6 0 8 0 I 0 0 FRACTION NO--0-5 -0-3 - O l o Li >-cr < 120 The column was eluted with a l i n e a r gradient of L i C l generated from 300 ml each of 0.2 M and 0.45 M L i C l i n 0.01 M l i t h i u m acetate pH 5.0. -4(1 and might behave anomalously, aliquots of histone T were either formylated by the mixed anhydride method of Sheehan and Yang (76) or acetylated as described i n "Experimental Methods" i n Part 2 of t h i s t h e s i s . Either of the above modifications would block a l l of the free amino groups of a histone and markedly reduce i t s net charge at pH 7, the pHi at which the SDS gels are electrophoresed. It was ob-served that unmodified histones are insoluble i n the SDS buffer unless 6 M urea i s present; however, the modified histones are soluble even i n the absence of urea. Typical gels are shown i n Pig. 6 and Pig. 7. A graph of mobility versus the logarithm of the molecular weight i s shown i n Pig. 8 and a summary of the r e s u l t s i s given i n Table 5. Untreated histone T gave a molecular weight of 16,400 whereas formylated histone T gave 14,500 and acety-la t e d preparations gave H , 1 ° Q . Acetylated histone TTbo gave a value of 14 ,400 which i s only 4$ higher than the value assigned from the sequence data ( 6 3 ) . The SDS method therefore seems to be suitable for estimation of the mole-cular weights of histones i f they are modified to reduce t h e i r net charge. Weber and Osborn (52) did a careful study on f o r t y pro-teins of known molecular weight using the SDS method. They found that 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 point or amino acid composition and depended s o l e l y on the molecular weight. The results were excellent not only for -42-*±g. 6 Electrophoresis on polyacrylamide gels i n the presence of 0 .1$ SDS according to Weber and Osborn (52). 1 2 3 4 5 6 7 8 The samples are: 1, untreated histone T; 2, formylated histone T; 3, myoglobin (M.W. 17,200); 4 , pepsin (M.W. 35,000); 5, ribonuclease (M.W. 13,700); 6, lysozyme (M.W. 14,300); 7, try p s i n (M.W. 23 , 3 0 0 ) ; and 8, chymo-trypsinogen (M.W. 25,700). There are some impurities i n the myoglobin sample and the ribonuclease sample. The chymotrypsinogen has p a r t i a l l y autolyzed. These are 15$ gels. - 4 3 -* i g . 7 Electrophoresis on polyacrylamide gels i n the presence of 0.1% SDS according to Weber and Osborn (52). II »; — I 2 Samples are: 1, pepsin and lysozyme; 2, ribonuclease and chymotrypsinogen; 3 , myoglobin; 4 , t r y p s i n ; 5, acetylated histone T; 6, formylated histone T; 7, acetylated histone Tib?. There i s a slow contaminant i n pepsin and the trypsin i s p a r t i a l l y autolyzed. There i s a trace of histone T i n the histone TTbo sam-ple. These are 15% gels. -44-F i g . 8 M o b i l i t y of standard proteins versus the logarithm of t h e i r molecular weights i 1 1 1 1 i i r 0-2 0-4 0-6 0-8 MOBILITY The mobility of thei standard proteins was measured and plotted against t h e i r established molecular weights as reported by Weber and Osborn (52). The points above represent: 1, pepsin; 2, chymotrypsinogen; 3, t r y p s i n ; 4, myoglobin; 5, ribonuclease; and 6, lysozyme. -45-TAB1E 5 Molecular weights of trout t e s t i s histones T and TTb2 as estimated by mobility during electrophoresis on polyacryla-mide gels i n the presence of 0.1% SDS. Histone Apparent M.W. M.W. corrected fo r mass of modi-fying groups I. 1 Unmodified 16,400 i i Pormylated 15 , 3 0 0 i i i Acetylated 15 , 3 0 0 16,400 14,500 14,100 H b 2 15,300 14,400* * This value f o r histone 11 bo i s only 4% higher than that from the sequence data ( 6 3 ) . - 4 6 -globular proteins but also for highly h e l i c a l , rodrshaped molecules such as myosin and tropomyosin. For 37 proteins over the molecular weight range 10,000 to 70,000 the maxi-mal deviation was l e s s than 10$ and usually less than t h i s . No serious deviations Were found. Dunker and Rueckert (77) studied 24 proteins on SDS gels and the e f f e c t of chemical modification upon mobility. They found that 10$ gels gave good results over the molecular weight range 10,000 to 100,000 daltons with an accuracy within 5-6$ pf the accepted values f o r most proteins. Ribonuclease, a basic protein, gave the only serious deviation by behaving 21$ too l a r g e i however, cytochrome c i s also a basic protein and did not give a serious deviation. Lysozyme was chemically modified to carry 8 extra positive charges, 8 extra neutral groups or 8 extra negative charges; the change i n mobility with a charge change of 16 units was only 12$. The deviation of ribonuclease was found to be probably related to folding of the protein since a l k y l a t i o n or disulphide interchange reduced the deviation to less than 10$; the unfolded pro-t e i n probably can bind more SDS and therefore migrate f a s -t e r . I t seems safe to assume no problems i n f o l d i n g with histone T which has no disulphide bonds so that the mole-cular weight must be close to 14,500. This figure i s con-s i s t e n t with the behaviour of histone T during gel f i l t r a -t i o n on Sephadex G-100 or on Bio-Gel P-10 or P-60 since i t elutes with histones TT which have molecular weights close • - 4 7 -to 14,000. Amino acid analyses were performed on samples of p u r i -f i e d M s tone T hydrolyzed for various times with the res u l t s summarized i n Table 6. The number of residues was calculated assuming a molecular weight close to 14,500 and an i n t e g r a l number of aspartic acid residues. The main features of the analyses are the large amounts of ly s i n e and alanine which account f o r 50% of the residues i n the protein and also the complete absence of aromatic or sulphur-containing amino acids. A spectrophotometric scan (Pig. 9) of a solution of histone T showed no absorbance i n the 260-300 nm range and thus confirmed the absence of aromatics including trypto-phan., Analyses of the basic amino acids performed on a long basic 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 histone T could be as high as +37 at pH 7 or as low as +21 i f none of the aci d i c s exist as amides; a r e l a t i v e l y high charge seems l i k e l y i n view of the rapid migration of histone T on polyacrylamide gels at pH 4. Tryptic fingerprints of histone T were compared with those of histones T, TTb1 and TTW with the res u l t s shown i n Pigs. 10 and 11. After careful comparison, 13 of the 27 peptides of histone T were found to be unique to i t and these are marked i n Pig. 10; the remaining peptides could not be distinguished from some present i n other histones. Arginine-containing peptides were located by the phenan--48-TABLE 6 Amino Acid Analyses of Histone T Moles Percent j r 0 < o f residues Residue 21 h 42 h 70 h f o r M.W. = Lysine 23.1 23.4 22.1 27.2 Histidine 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 Cysteine G 0 0 0 Glycine 7.4 6.8 7.3 9.1 Alanine 25 .4 25.5 26 .3 31.2 Valine 3.4 3.9 4 . 0 5 .0 Methionine 0 0 0 0 Isoleucine 0 0 0 0 Leucine 1.2 1.4 1.3 2 . 0 Tyrosine 0 0 0 0 Phenylalanine G 0 0 0 Total 122 - 4 9 -* i g . 9 Spectrophotometry Scan of Histone T WAVELENGTH nm Histone TI was dissolved i n water at about 0.5 mg/ml and scanned i n a Unicam spectrophotometer (Model S P 800 B). - 5 0 -Pig. 10 Tryptic f i n g e r p r i n t s stained with cadmium ninhydrin. H I S T O N E T H I S T O N E I H I S T O N E S I I Electrophoresis was at pH 6.5 i n the horizontal plane, the o r i g i n being marked with an rt0M. Chromatography was perform-ed v e r t i c a l l y . Peptides shaded i n black were positive for arginine by the phenanthrene quinone test (see Pig. 11). Peptides unique to histone T are marked with a horizontal dash. -51 -F i g . 11 Tryptic fingerprints showing arginine-containing peptides. Chromatograms were sprayed with phenanthrene quinone (56), dried and photographed under UV il l u m i n a t i o n . The fluorescent spots are the peptides containing arg-i n i n e . These fingerprints were prepared i d e n t i c a l l y to those i n Pig. 10 and as outlined i n section #9 of "Methods". HISTONE I HISTONE T HISTONES H -52-threne quinone t e s t (56) as shown i n Pig. 11 i n which the positive peptides are revealed as fluorescent spots. The presence of 9 arginine peptides i n histone T i s consistent with the number of arginine residues determined by amino acid analysis and assuming a molecular weight of 14,500; the l a t t e r method indicated 9.1 arginine residues (see Table 6). These fingerprints also eliminate the p o s s i b i l -i t y that histone T i s a degradation product of histones T, 11b.j or 11b 2. Histone TV i s too small to give r i s e to h i s -tone T by degradation and both the former and histone 111 lack s u f f i c i e n t l y s i n e f o r t h i s . On the basis of 27 l y s i n e and 9 arginine residues i n histone T one would expect up to 37 peptides on a t r y p t i c f i n g e r p r i n t . The presence of only 27 peptides on the fingerprints performed could be due to several reasons including incomplete f r a c t i o n a t i o n of peptides by the f i n -gerprint method, re s i s t a n t cleavages or repeated sequences. The presence of 14 peptides i n histone T t r y p t i c digests that are indistinguishable from those i n the other histones examined may indicate p a r t i a l homology between histone T and the others. This i s not s u r p r i s i n g i n view of the homology between portions of histones TTb1 and TV as discussed i n the "Introduction". The N-terminus of histone T was investigated using the dansylation method of Gray (57) with the r e s u l t s shown i n Pig. 12. The thin-layer plates reveal DNS-Pro afte r the Pig. 12. Thin-layer chromatography of dansylated residues of histone T and standard DNS amino acids. The plates were photographed under UV i l l u m i n a t i o n . Samples include: 1, 4, 7 and 11, DNS-Val; 2, 6 and 9, DNS-Pro; 3 and 10, mixture of mono- and di-DNS-Lys; 5, p r e c i p i t a t e from dan-s y l a t i o n of histone T; 8, soluble por t i o n a f t e r dansylation of histone T; and 12, DNS-Arg. SYSTEM I SYSTEM 2 I 2 3 4 5 6 7 8 9 I O I I I 2 -5:4-f i r s t solvent system and a large amount of mono-DNS-Lys a f t e r the second solvent system i n both the prec i p i t a t e and the soluble portion a f t e r dansylation of histone T. The N-terminus must therefore be proline and the mono-DNS-Lys arises from the large number of 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, using hydrolysis times of 20-24 h, were negative probably because of the known l a b i l i -t y of DNS-Pro to acid hydrolysis. Positive r e s u l t s were obtained a f t e r hydrolysis times of 6-8 h. Histone T thus resembles histone TTbg i n i t s N-terminus, si z e and l y s i n e : arginine r a t i o ( 3 : 1 ) . Chromosomal basic proteins were extracted from trout t e s t i s at d i f f e r e n t months of natural maturation and elec-trophoresed on polyacrylamide gels with the res u l t s shown i n Pig. 13. The major histones and histone T-are a l l pre-sent at a l l stages examined since the testes always contain some immature c e l l s . The r e l a t i v e amount of histone T per unit weight of DNA at the various stages was measured by scanning the gels i n Pig. 13 with the aid of a Jbyce-Loebl Chromoscan with an integrator; the amount of DNA from which each sample was extracted was measured by the Burton modifi-cation of the Dische method ( 7 9 ) . The r e s u l t s i n Pig. 14 indicate that the r e l a t i v e amount of histone T reaches a maximum i n natural maturing t e s t i s at 7 weeks (early Octo-ber) and declines as protamine replacement occurs. These results are'similar to the observation of Marushige and F i g . 13 Disc gel electrophoresis of acid extracts of trout t e s t i s chromatin at various stages during natural maturation. 0-2 M H 2 S0 4 5% TCA • I i i | TOTAL HISTONE AUG SEPT OCT NOV DEC Chromatin was f i r s t extracted with 5% TCA and then with 0 . 2 M H2SO4 as described i n "Methods". The d i r e c t i o n of electrophoresis was from r i g h t to l e f t . -56-F i g . H A plot of the r e l a t i v e amount of histone T per mg of DM i n chromatin prepared from trout t e s t i s at various stages of natural maturation. The gels shown i n Pig. 13 were scanned using a Joyce-Loebl Ohromoscan with an. integrator; the amount of DNA i n the chromatin samples was mea-sured by the Burton modification of the Dische method (79). Dixon (78) that the mass r a t i o of histone:DUA reaches a maximum of 1.4 before protamine appears and declines l a t e r with i n d i v i d u a l histone fractions decreasing at d i f f e r e n t rates. This increase i n histone:DNA r a t i o during early ma-turation i s s u r p r i s i n g since i t implies either synthesis of histones independently of DNA synthesis or else degradation of DNA. 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 synthesis has been shown to be t i g h t l y coupled to DNA synthesis (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 at present. Pig. 15 shows that histone T i s present i n other trout tissues including l i v e r , heart and spleen and therefore i s not t i s s u e - s p e c i f i c . The only other species examined was the guinea pig and a component with the mobility of histone T i s present i n the testes of t h i s animal. The only t i s s u e -s p e c i f i c and s p e c i e s - s p e c i f i c histone known i s the l y s i n e -r i c h , s e r i n e - r i c h histone present i n the nucleated erythro-cytes of several birds (86). Conclusion A previously undescribed histone f r a c t i o n has been p u r i -f i e d from trout t e s t i s chromatin and p a r t i a l l y characterized. This protein, designated histone T, has a molecular weight of about 14,500 and has an unusual amino acid composition even f o r a histone. F i f t y percent of the residues are either lys i n e or alanine and sulphur-containing and aromatic amino acids are t o t a l l y absent. Histone T i s a minor component of -58-Fi g . 15 Disc gel electrophoresis of acid extracts of chromatin from d i f f e r e n t trout tissues and two guinea pig tissues. 0-2 M H 2 S0 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 extracted with 5$ TCA and then with 0.2 M H2SO4. The d i r e c t i o n of electrophoresis was from r i g h t to l e f t . MG.P. M refers to guinea pig. trout t e s t i s histones, forming about 0 . 5 % of the t o t a l at the October stage of natural maturation. This histone i s r e a d i l y displaced from i t s association with DNA i n chroma-t i n either by chemical modification with 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 histone i n highly p u r i f i e d chromatin elim-inates the p o s s i b i l i t y that i t originates elsewhere i n the c e l l . Experimental evidence has been described that rules out the p o s s i b i l i t y that histone T might be a degradation product of the major histones. Histone T i s present i n maximal amount at a stage before rapid histone removal occurs; a degradation product would be maximal during the time of histone removal. Peptide maps revealed several pep-tides unique to histone T; a degradation product could only have one or two unique peptides. Preliminary experiments have shown incorporation of into histone T at the time when protamine i s replacing the histones but the s i t e of phosphorylation has not been examined i n more d e t a i l . iVom the success of the sequence studies on histone T T (Ref.39), one anticipates that se-quence work on histone T both around the s i t e ( s ) of phos-phorylation and the complete sequence w i l l give additional clues to the mechanism of action of these chromosomal com-ponents. BIBLIOGRAPHY 1. E d i t o r i a l note, Nature, 223, 1202 (1969). 2. 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Hardin, J.A., Einem, G.E., and Lindsay, D.T., J . C e l l B i o l . , 32, 709 (1967). 83. Gurley, L.R., and Hardin, J.M., Arch.Biochem.Biophys., 128, 285 (1968). 84. Robbins, E., and Borun, T.W., Proc. US Nat.Acad.Sci., 57, 409 (1967). 85. Spalding, J . , Kajiwara, K., and Mueller, G.C, Proc. US Nat.Acad.Sci., 56, 1535 (1966). 86. Neelin, J.M., and Butler, 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 in t e r e s t i n protein "biosynthesis i n recent years i s e a s i l y explained by the importance of these molecules i n the functioning of a l l b i o l o g i c a l systems. The complement of pro-teins i n a c e l l provides much of the basis for the appearance and behaviour of the c e l l , that i s , the phenotype or state of d i f f e r e n t i a t i o n . This i s l a r g e l y a r e f l e c t i o n of the a b i l i t y of proteins to in t e r a c t i n a highly s p e c i f i c manner with other molecules^including other proteins. Examples of such i n t e r a c -tions are the many enzymes with a great v a r i e t y of substrates, ; subunits of enzymes, subunits of structures such as microtu-bules and viruses, and repressor proteins with s p e c i f i c DNA sequences. Proteins perform key s t r u c t u r a l and regulatory roles i n the c e l l and t h e i r biosynthesis must be c a r e f u l l y controlled 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 early 1930's showed that proteins are well-defined chemi-ca l e n t i t i e s and hence amenable to chemical and physical ana-l y s i s . Sanger's work on i n s u l i n (1) revealed for the f i r s t time the complete sequence of amino acids i n a protein; the complexity of the sequence c l e a r l y eliminated older ideas that proteins might consist of simple repeating amino acid se-quences (2). As recently as 1951 some thought that protein synthesis might proceed by reversal of proteolysis (3) or by - 6 7 -the action of transpeptidases on either amino acids or pep-tides ( 4 ) . Others, however, raised objections to these pro-posals since the equilibrium p o s i t i o n with proteases i s f a r to the side of hydrolysis ( 5 , 6 ) and these enzymes are not ab-so l u t e l y s p e c i f i c i n distinguishing between the sidechains of amino acids which are s i m i l a r ( 7 ) . C l e a r l y missing was an understanding of the r e l a t i o n -ship between protein structure and genetic information. Beadle hinted at such a re l a t i o n s h i p i n his "one gene, - one enzyme" hypothesis (8) but did not make clea r the nature of the r e l a -tionship; this was obviously due to the lack of knowledge about the chemical basis for genes at this time. The "sequence" hypo-thesis was probably f i r s t stated by Dounce (9) i n 1952; he postulated that amino acids were ordered by s p e c i f i c i n t e r -action with a nucleic acid template and then polymerized into a protein. With the publication of the structure of DM i n 1953 (Ref.10) much intere s t i n genetic coding was aroused and Gramow (11) proposed that the amino acids f i t into pockets on DNA formed by the base pairs i n a s p e c i f i c manner determined by stereochemical r u l e s . Thus,people were beginning to think of the genetic information f o r protein biosynthesis as being stored i n the base sequence of a nucleic acid. A hint as to the nature of the template was given as early as 1941 i n the work of Caspersson (12,13) and Brachet (14) who both found a good c o r r e l a t i o n between the amount of ribonucleoprotein and the rate of protein synthesis i n various c e l l s . -68-The discovery of H Q by Ruben and Kamen (15) followed by the preparation of amino acids l a b e l l e d with t h i s isotope (16) gave a great aid to those studying protein biosynthesis. The systems used progressed from whole animals to i n v i t r o studies on organs, tissue s l i c e s , c e l l suspensions and c e l l -free systems. The early c e l l - f r e e systems were prepared from tissues such as r a t l i v e r since there were problems i n remov-ing 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. Sieckevitz and Zamecnik (17) found that protein synthesis i n a c e l l - f r e e system from rat l i v e r required simultaneous oxidative phos-phorylation by mitochondria present i n the homogenate and that the microsomes were most active i n the synthesis. This confirm-ed e a r l i e r work (18) i n which dinitrophenol (DNP) was found to i n h i b i t protein synthesis i n mammalian c e l l s and DNP was a l -ready known to dissociate oxidative phosphorylation from r e s -p i r a t i o n (19). Lipmann (20) and Kalckar (21) had both postu-lated i n 1941 that phosphorylated derivatives of amino acids were required f o r protein biosynthesis. Zamecnik (22) found that ATP or an ATP-generating system could replace the need for active mitochondria i n a c e l l - f r e e system; th i s eliminated the p o s s i b i l i t y that mitochondrial products other than ATP were required i n his system. Hoagland (23) studied the mechanism of amino acid a c t i v a t i o n by ATP by looking for ^ 2P-pyrophosphate exchange into ATP i n the pre-sence of amino acids and HpH'5" enzymes. Exchange occurred and was shown to be dependent on the presence of an amino -69-acid. The formation of an aminoacyl adenylate was implied by the formation of aminoacyl hydroxamates upon addition of hydroxylamine and was confirmed by the synthesis of 1-leucyl-AMP (24); the l a t t e r compound, i n the presence of pyrophos-phate and "pH 5" enzymes, led to the synthesis of ATP. The fate of the activated aminoacyl-AMP derivatives was unknown. Holley (25) demonstrated an RNase s e n s i t i v e step between a c t i v a t i o n of amino acids and t h e i r incorporation into protein. In the same year, Zamecnik et a l . (26) reported the discovery of 1^C-leucine t i g h t l y coupled to an RNA com-ponent present i n the highspeed supernatant (HSS); this RNA f r a c t i o n contained what i s now known as transfer RNA (tRNA). Hoagland et al.(27 ) showed the important r o l e of aminoacyl-tRNA i n protein synthesis; Zachau, Acs and Lipmann (28) found that the carboxyl group of the amino acid moiety i s linked by an ester bond to the 2' or 3 1 hydroxyl group of the ribose moiety of adenosine at the V terminus of the tRNA. The f i r s t p u r i f i c a t i o n of an a c t i v a t i n g enzyme (29) l e d to the discovery that the same enzyme i s responsible both f o r the a c t i v a t i o n of an amino acid and i t s coupling to the correct tRNA. Crick's adaptor hypothesis (30) stated that tRNA's were adaptor molecules f o r carrying amino acids to the correct pos-i t i o n on a hypothetical RNA template. Discovery of ayRNA f r a c -t i o n with the expected properties of a template for protein synthesis was made by several groups ( 3 1 - 3 3 ) ; t h i s f r a c t i o n i s now termed "messenger RNA" or mRNA. The assignment of co-- 7 0 -dons for each amino acid was the climax of this work ( 3 4 -3 6 ) . With the development of a c e l l - f r e e system from E. c o l i ( 3 7 ) , b a c t e r i a l protein synthesis has been studied extensive-l y and i s now understood i n more d e t a i l than eukaryotic pro-t e i n synthesis. Some of the known steps of protein synthesis i n b a c t e r i a l systems w i l l be discussed below and eukaryotic systems w i l l be referred 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, with the exception of RNA phage and RNA viruses, carry t h e i r genetic information i n the form of DNA. Protein synthesis takes place on RNA templates which are usually copied from DNA by the enzyme(s), DNA-dependent RNA polymerase. The E. c o l i enzyme has been p u r i f i e d ( 3 8 ) and consists of 5 d i f f e r e n t subunits with a t o t a l molecular weight of about 3 3 0 , 0 0 0 . One of the subunits of the E. c o l i enzyme, referred to as sigma factor, i s dissociated from the enzyme a f t e r i n i t i a -t i o n of RNA synthesis on a DNA template ( 3 9 ) . Sigma and s i m i -l a r factors i n other bacteria ( 4 0 ) seem to function i n the correct i n i t i a t i o n of RNA synthesis at r e s t r i c t e d regions on a DNA template. Thus, sigma allows t r a n s c r i p t i o n of the "pre-early" genes of phage T4 (R ef.41 ); one of these pre-early genes codes f o r a phage-specific f a c t o r , sigma^4, which d i s -- 7 1 -places the host sigma factor from the polymerase. The enzyme i s then capable of transcribing the "early" genes of T4 but ceases to transcribe E. c o l i DNA ( 4 2 ) . Clusters of 10-50 pyrimidines on the same strand of DNA have been implicated as serving as binding s i t e s for RNA poly-merase for i n i t i a t i o n of t r a n s c r i p t i o n (43). The rate of i n i -t i a t i o n at various genes i n vivo i s known to vary considera-bly with ribosomal RNA (rRNA) and tRNA chains being i n i t i a -ted most frequently (44). The "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 and thus may be the s i t e at which RNA polymerase binds (45); i n f a c t , deletions extending into the promoter region abolish l a c DNA t r a n s c r i p t i o n (46). The l a c repressor acts by bind-ing at the promoter s i t e and preventing i n i t i a t i o n of RNA synthesis (47); chains i n i t i a t e d before addition of the r e -pressor are completed. Control of the rate of 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 various operons i s obviously c r u c i a l since the average propagation rate of a l l RNA chains i n E. c o l i i s the same, being 15-26 nucleotides per second at 29 °C (Ref.48). Thus, differences i n t o t a l synthesis of RNA fractions must r e -f l e c t differences i n rates of i n i t i a t i o n . RNA chain termination has recently been found to require a protein factor, rho, which seems to function i n recogni-t i o n of a termination signal i n the DNA sequence (49). In the absence of rho, RNA chains are longer than they should be and remain i n a complex with :DNA and RNA polymerase; t h i s appears to occur because termination signals are not recog--72-nized. There i s considerable evidence that coupling of trans-l a t i o n with t r a n s c r i p t i o n i s the rule i n E. c o l i with r i b o -somes binding to RNA chains a f t e r they ,have been i n i t i a -ted, (50,51). This coupling may be es s e n t i a l for mRNA function, in. view of recent work i n d i c a t i n g that mRNA i s r a p i d l y de-graded i n the 5" to 3' d i r e c t i o n i n the absence of t r a n s l a -t i o n (52,53). Translation could protect the mRNA from attack by nucleases i n the cytoplasm since studies have shown that each ribosome s h i e l d up to 35 nucleotides of a mRNA when, the nucleases are added to the system (54»55). The enzyme(s) r e -sponsible f o r degradation of mRNA i n vivo have not been iden-t i f i e d d e f i n i t e l y but recent work (290) has implicated RNase H. i * 1 E. c o l i ; the l a t t e r enzyme degrades RNA in. the 51' to 3' dir e c t i o n with release of 5' nucleotides and, i n t e r e s t i n g l y , this enzyme requires the presence of the elongation factors for a c t i v i t y ("elongation f a c t o r s " w i l l be discussed l a t e r in. this s e ction). The control of a c t i v i t y of mRNA-degrading enzymes i s of obvious importance i n determining the l i f e t i m e of a mRNA molecule; the presence of s i g n i f i c a n t secondary structure i n a mRNA might protect i t from degradation and thus allow long-lived mRNA. Genetic p o l a r i t y due to nonsense codons i n a p o l y c i s -tronic mRNA could be due to endonucleolytic attack i n the region of a c i s t r o n d i s t a l to a nonsense codon since t h i s region i s not translated and hence not protected by the -73-shielding effect of ribosomes. An exdnucleasescould then follow the polymerase i n the 5 ' t o 3' di r e c t i o n and degrade the remainder of the cistrons before they could be trans-l a t e d . In f a c t , two groups have evidence that the amount of mRNA d i s t a l to a nonsense codon i s decreased due to acceler-ated degradation (53,56). Studies by Oheng on human amnion c e l l s (57) gave the f i r s t demonstration of mRNA i n a eukaryote soon a f t e r the discovery of DNA-dependent RNA polymerase i n mammalian l i v e r and bacteria (58-60). The proposals that eukaryotic mRNA's might be more stable than b a c t e r i a l (61) or that they might be "masked" (62) were confirmed i n experiments on protein synthesis i n f e r t i l i z e d sea urchin eggs. Gross and Cousin-eau (63,64) showed that f e r t i l i z a t i o n l e d to increased pro-t e i n synthesis even i n the presence of actinomycin D at levels i n h i b i t i n g RNA synthesis almost completely. Further experiments gave evidence for the presence of masked RNA which i s somehow activated 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 u t i l -izes maternal mRNA already present i n the u n f e r t i l i z e d egg. In the erythropoietic system, RNA synthesis ceases upon transformation from erythroblasts to normoblasts although hemoglobin synthesis starts l a t e r (68,69); the hemoglobin mRNA must be present i n an inac t i v e form during the i n t e r v a l . RNase inserisitive^ribosomal aggregates have been found i n pre-cleavage eggs of Ascaris lumbricoides and could! he -74-activated for protein synthesis and rendered sensitive to RNase by b r i e f exposure to t r y p s i n (70). Similar findings have been reported i n house f l i e s (71 ). Monroy et a l . (72) found evidence of a trypsin s e n s i t i v e i n h i b i t o r of protein synthesis present on ribosomes of u n f e r t i l i z e d sea urchin eggs; they hypothesized that the i n h i b i t o r y protein(s) i s hydrolyzed by a protease activated by the i o n i c changes which occur i n eggs after 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 control of protein synthesis at the t r a n s l a t i o n a l l e v e l . Control of mRNA synthesis i n eukaryotes by means of a sigma factor type of agent has not been demonstrated but multiple forms of RNA polymerase have been described (73). Sea urchin embryos have 3 d i s t i n c t RNA polymerase a c t i v i t i e s resolved 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 fractions do not seem to be i n t e r c o n v e r t i -ble. There seems to be no s p e c i f i c i t y of the components for 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 both tissues 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 vivo and t h e i r functions may be quite d i s t i n c t . The other components are present i n the nucleoplasm. The sea urchin nucleolar enzyme increases during development from the b l a s t u l a t o gastrula stage; t h i s correlates well with the known increase i n rRNA synthesis at the gastrula stage (74). The p o s s i b i l i t y has not been eliminated yet that these RNA polymerase a c t i v -i t i e s are related by addition of sigma-like f a c t o r ( s ) to a -75-common "core" enzyme. RIBOSOMES With the establishment of the v i t a l r o l e of ribosomes i n protein synthesis (17,75-77), groups soon began to f i n d r a p i d l y sedimenting aggregates of ribosomes both i n v i t r o i n bacterial c e l l - f r e e systems (78,79) and i n vivo i n rabbit reticulocytes (80). Warner, Knopf and Rich (80) proved that hemoglobin chains are synthesized on a 170 S aggregate of f i v e 70 S ribosomes and mRNA; they proposed that each r i b o -some bears a growing polypeptide chain and travels along the mRNA from one end to the other with new ribosomes entering at one end while others f a l l o ff at the other end with t h e i r completed proteins. This was the f i r s t assignment of an active r o l e f o r ribosomes i n protein synthesis. The d i r e c t i o n of movement of the ribosome was shown to be 5' to 3' (Ref.81 ) and the polypeptide chain was found to be i n i t i a t e d at the amino terminus,(82). A summary of the components of E. c o l i ribosomes with some of t h e i r c h a r a c t e r i s t i c s i s given i n Table 1. There i s obviously a complex.structure associated with ribosomes and tra n s l a t i o n of mRNA into protein must therefore require the functions associated with each component. 70 S ribosomes bearing no nascent peptides, as af t e r r e -lease with puromycin, dissociate into 30 S and 50 S subunits when the Mg + + concentration i s lowered to 1 mM or l e s s ; how--76 TABLE 1 E. c o l i Ribosomes rRNA Subunit S value M.W. Number of r-proteins Functions associated with subunit . 30 S 16 S 0.53X106 20 Binding of mRNA and (Ref.127) (Ref.97) i n i t i a t o r tRNA Proteins sensitive to streptomycin, spectinomycin and temperature S i t e of ribosomal ambiguity mutation At l e a s t two pro-teins required for f i d e l i t y of trans-l a t i o n 50 S 23 S 5 S 1.0X10° (Ref. 127) 36,000 (Ref.92) 34 (Ref.97) S i t e of peptidyl transferase S i t e of protein responsible for s e n s i t i v i t y to erythromycin ever, i f nascent peptide i s present, d r a s t i c lowering of Mg + + as with EDTA i s required (83). The presence of pep-t i d y l tRNA on the 70 S ribosome must s t a b i l i z e the i n t e r a c -t i o n of the, ribosomal subunits. The reasons for the need f o r two subunits w i l l be discussed below.(see " I n i t i a t i o n " ) . There are three rRNA components present with a 16 S RNA i n the 30 S subunit and both a 5 S and a 23 S RNA i n the 50 S subunit. The two larger RNA's have a small amount of pseudo-urid y l a t e and methylated nucleotides with the methyl group either on the base or on the 2 1 hydroxyl group of the ribose moiety; the 5 S RNA has a higher G-0 content and lacks the unusual nucleotides. Oligonucleotide maps of the 16 S and 23 S RNA's reveal marked differences o v e r a l l (84) and i n the sit e s of methylation (85). Physical studies (86-90) have shown that up to 60-75% of the nucleotides of 23 S RNA are involved i n base pairing which forces the RNA chain to f o l d considerably into a rather compact structure. P a r t i a l se-quence analysis of the 16 S RNA (91) has revealed not only hairpins but also loops joined by double stranded regions as i n tRNA. The 5 S RNA from E. c o l i (92) and eukaryotic c e l l s (93,94) has been sequenced. The E. c o l i 5 S RNA can be divided (on paper) into two halves with considerable homology suggesting an o r i g i n by gene duplication. Physical studies reveal' that 60-80% of the nucleotides of 5 S RNA are involved i n base pairs (95,96). At l e a s t twenty ribosomal proteins (r-proteins) are -78-present on the 30 S subunit and 34 on the 50 S subunit (97). Seventy percent of these proteins have a molecular weight between 10,000 and 20,000. Surprisingly, a l l 20 proteins can be dissociated from the 30 S subunit at high i o n i c strength and then added back with a good y i e l d of p a r t i c l e s active i n protein synthesis (98). Some functions have been assigned to r-proteins by means of re c o n s t i t u t i o n experi-ments and through the study of ribosomal mutants.. Thus, proteins responsible for s e n s i t i v i t y to streptomycin (99,100), spectinomycin (101) and high temperature (102) have been l o c a l i z e d to the 30 S subunit. The 30 S subunit i s also the s i t e of a mutation causing misreading (103) and at l e a s t two proteins required f o r f i d e l i t y of t r a n s l a t i o n (104). The 50 S subunit i s the s i t e of peptidyl transferase, the enzyme which catalyzes peptide bond formation (105,106) and the s i t e of a mutation giving r i s e to resistance to erythro-mycin (107). The 30 S subunit has a s i t e for binding 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 part of the s i t e for amino-acyl tRNA. The l a t t e r two s i t e s are referred to as the P s i t e (for peptidyl or i n i t i a t o r tRNA) and the A s i t e (for amino-acyl tRNA) (Ref.128,129). In bacteria, each species of rRNA i s independently syn-thesized and modified; the maturation process involves mainly methylation and conversion of u r i d y l a t e residues to pseudouridylate (108). Newly synthesized rRNA i s combined - 7 9 -with r-proteins and therefore sediments more r a p i d l y than free rRNA; at least two intermediates can be i s o l a t e d which are involved i n the formation of each subunit. There i s some evidence to support the i n t e r e s t i n g idea that rRNA, probably i n i t s unmodified form, serves as mRNA for the synthesis of r-proteins. Thus, nascent rRNA can serve as a messenger i n a c e l l - f r e e system and the protein products coelectrophorese with r-proteins (109). Mature rRNA acts as a messenger i n a c e l l - f r e e system i f neomycin i s added or i f i t s secondary s t r u c -ture i s destroyed by heating (110). Each rRNA has only enough information to code f o r about 20% of the r-proteins associa-ted with i t ; therefore, either the genes for rRNA are m u l t i -ple and d i f f e r e n t or else the other r-proteins must be coded by another series of genes. The former p o s s i b i l i t y seems un-l i k e l y since only one species of each rRNA i s found i n a given organism or organelle. In eukaryotes, rRNA i s synthesized i n the nucleolus as a large 45 S precursor (111-114). While i n the nucleolus, the rRNA i s methylated (115) and gradually degraded to an 18 S and 28 S component plus fragments which are discarded (116, 117). During or just a f t e r i t s synthesis, the 45 S RNA becomes associated with protein i n an 80 S p a r t i c l e which gives r i s e to the 60 S subunit and releases the 18 S RNA which r a p i d l y enters the cytoplasm to form the 40 S subunit by associating with i t s complement of r-proteins (116-118). The 5 S RNA i s transcribed on non-nucleolar DNA (119). The -80-s i t e of r-protein synthesis i s not known hut l a b e l l e d r -proteins are found i n the nucleolus a f t e r short pulses and appear to associate with rRNA i n the nucleolus. Since B i r n s t i e l et a l . (120) showed that Xenopus laevis has several hundred copies of the gene f o r rRNA per c e l l , s i m i l a r findings have been reported i n Drosophlla (121) and HeLa c e l l s (122). The need f o r this "gene amplification" 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 r i b o -somes per c e l l generation of 18-24 hours. Assuming there are 400 rRNA genes per .cell (122), each gene must be transcribed). 20 times per minute; t r a n s c r i p t i o n of a complete rRNA gene takes 2£ minutes (123) and therefore, about f i f t y 45 S RNA molecules must be transcribed simultaneously on each gene. The simultaneous t r a n s c r i p t i o n of a gene by several RNA poly-merase ,molecules has been observed i n the electron 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 formation of up.to 1000-nucleo-r l i per nucleus (125,126); the oocyte synthesizes enormous numbers of ribosomes which are s u f f i c i e n t for the develop-ment of the embryo up to ga s t r u l a t i o n . INITIATION OP PROTEIN BIOSYNTHESIS As each ribosome associates with a mRNA molecule there must be a s p e c i f i c i n t e r a c t i o n to ensure that protein synthe-s i s i s begun at the correct codon; otherwise, nonsense proteins would be synthesized and the c e l l could not function. -81 -The presence of a s p e c i a l tRNA involved s o l e l y i n the i n i t i a t i o n of protein synthesis i n b a c t e r i a l systems was discovered by Marcker and Sanger (130). This tRNA, N-Formyl-methionyl-tRNAf (F-Met-tRNA f), i s now known to i n i t i a t e a l l b a c t e r i a l proteins (131-134) and probably a l l proteins i n mitochondria (135,136), chloroplasts (137) and blue-green algae (138). E. c o l i has two methionine accepting tRNA's, tRNAf 6^ and tRNA^ e t. These are both charged by the same methionyl-tRNA synthetase (139) but only methionine attached to tRNAj>et can be formylated by a transformylase which r e -quires N 1 0-Formyltetrahydrofolate as the formyl donor (140,; 141). Both of these tRNA's respond to the codon AUG but F-Met-tRNAf also responds to GUG; th i s degeneracy i n reading of the f i r s t l e t t e r of a codon i s unique to 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 only AUG has so f a r been shown to operate i n vivo as deter-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 that Met-tRNAf, even when not formylated, donates methionine ex-c l u s i v e l y into the N-terminal p o s i t i o n of newly synthesized proteins whereas Met-tRNAjj donates i t s amino acid only to int e r n a l positions i n the proteins (144). The i n a b i l i t y of Met-tRNAf to incorporate 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 with the aminoacyl tRNA binding factor T„ (Ref. 145). Met-tRNAju, even - 8 2 -when formylated chemically, i s unable to serve as an i n i t i a -tor (146). These facts make i t c l e a r that t R M ^ e t i s s p e c i a l l y adapted f o r i t s function and interacts s p e c i f i c a l l y with at le a s t one enzyme (transformylase) and with the ribosome-mRNA complex. A s p e c i a l i n t e r a c t i o n with the ribosome i s also i n d i -cated by the rapid reaction of F-Met-tRNAf with puromycin (147), a reaction occurring only with substituted aminoacyl-tRNA's present i n the P s i t e of the ribosome. I n i t i a t i o n cannot occur simply by sel e c t i n g the f i r s t AUG codon adjacent to the 5' end of the mRNA since i t i s known that i n i t i a t i o n occurs simultaneously at a l l of the cistrons on a pol y c i s t r o n i c mRNA i n v i t r o (148-150). Since there must be several AUG and GUG codons i n t e r n a l l y i n any large mRNA, there must be some mechanism fo r s e l e c t i n g the correct ones. This could 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 out-of-phase codons. Recognition of these s p e c i f i c sequences might well require s p e c i a l factors and, i n f a c t , a r o l e for several protein factors i n i n i t i a t i o n has been found by several groups using b a c t e r i a l systems (151-153). Three d i s t i n c t factors have been recovered by washing ribosomes at high i o n i c strength such as with 1 M NR4CI followed by ion-exchange chromatogra-phy on DEAE-cellulose (154-156). These factors have been des-ignated as f 1, f2 and f3 and t h e i r functions w i l l be discussed below. U n t i l f a i r l y recently i t was thought that 70 S ribosomes -83-were d i r e c t l y involved i n the i n i t i a t i o n mechanism. Several l i n e s of evidence make i t clear now that the 30 S subunit i s involved i n the f i r s t steps of i n i t i a t i o n . Thus, Greenshpan and Revel (157) found that phage T4 mRNA binds to the 30 S subunit i n the presence of i n i t i a t i o n factor f2 ( i - f a c t o r f 2 ) ; this reaction was shown to be independent of the presence of anyitRNA. Others found that formation of a complex of 30 S subunit :mRNA:i*-Met-tRNAf occurs and i s an obligatory require-ment fo r protein synthesis,(158,159). A continuous exchange of ribosomal subunits during pro-t e i n synthesis has been well documented (158-161) and sug-gests that the 70 S ribosome dissociates into subunits at the end of t r a n s l a t i o n of a c i s t r o n ; the subunits then ran-domize and recombine during i n i t i a t i o n of t r a n s l a t i o n of another mRNA. A protein factor required 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 of the 30 S subunit (162) and i s active only on the "runoff" ribosomes which are free from the peptidyl-tRNA and mRNA (163). This factor has recently been shown to probably be i d e n t i c a l with i - f a c t o r f3 (Ref.164) and i s stimulated by addition of ATP or GTP. Ribosomes washed to remove i - f a c t o r s are active i n pro-t e i n synthesis at high Mg + + concentration i n a c e l l - f r e e system with synthetic polyribonucleotides, GTP, aminoacyl-tRNA's and elongation f a c t o r s . However, these ribosomes w i l l not f a i t h f u l l y translate natural mRNA. I n i t i a t i o n of protein synthesis with F-Met-tRNAf requires the i - f a c t o r s (165) and. -84-GTP (166-168). Guthrie and Nomura (159) showed that a 30 S i-complex would only form with F-Met-tRNAf i n the presence of i - f a c t o r s and would not form with any other aminoacyl-tRNA. A summary of the known features of E. c o l i i - f a c t o r s i s given i n Table 2 and F i g . 1. Both f1 and f2 are required f o r binding of F-Met-tRNAf to ribosomes i n the presence of the trinu c l e o t i d e s AUG or GUG and GTP (165); these factors are also required f o r the t r a n s l a t i o n of poly U at low Mg*+ and in. the presence of N-Acetyl-phenylalanyl-tRNA (Ac-Phe-tRNA) (169). In addition to f1 and f2, f3 i s required f o r the t r a n s l a t i o n of natural mRNA (154,155,169,170). f3 may thus be necessary f o r i n t e r a c t i o n with a sp e c i a l region around the i n i t i a t o r codons i n natural mRNA. A l l three factors are required f o r maximal incorporation of methionine from F-Met-tRNAf into protein and for maximal synthesis of active 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 released from the ribosome afte r i n i t i a t i o n of protein synthesis; they can only be recovered from native 30 S subunits and not from the 50 S subunit, 70 S ribosomes or polysomes (172,173). I-factors are not found i n the HSS and therefore must be present i n l i m i t i n g amounts which combine r a p i d l y with any free 30 S subunits. After formation of the 30 S i-complex, a 50 S sub-unit joins to form a 70 S complex active i n protein synthe-s i s (174); Thach et al. (175) showed that t r i t i u m l a b e l l e d - 8 5 -Formation of I n i t i a t i o n Complexes i n E. c o l i 1) 70 S ribosome reaches a terminator codon and i n the presence of a protein d i s s o c i a t i o n factor (162) dissociates into two subunits, 50 S and 30 S. 2) 30 S + mRNA + f2 •+- f3 > (30 S:mRNA) 3) F-Met-tRNAf + f1 + GTP — > (30 S i-complex) + (30 S:mRNA) 4 ) 50 S + ( 3 0 S i-complex) > (70 S active complex) .+ GDP + P i + free i - f a c t o r s Note: GTP may be required f o r translocation of F-Met-tRNAf to the ribosomal P s i t e since there i s evidence that i t binds i n i t i a l l y at the A s i t e (205). -86-TABLE 2 Properties of E. c o l i I n i t i a t i o n Factors Factor M.W. Oharacteristics f1 9000 Binds to 30 S subunit i n the (Ref.175) presence of f2, AUG, GTP and FrMet-tRETAf. f2 70,000 Heat s e n s i t i v e . (Ref.175) Essential SH group(s). In the presence of GTP, i s protected from SH reagents (199)., GTPase a c t i v i t y i n the presence of ribosomal subunits; may actu-a l l y consist of two components (200). Can complex with GTP. f3 29,000 Can be assayed by a b i l i t y to (Ref.201) bind to natural mRNA or ApUpG(pA) 4 0 with the complex retained on a M i l l i p o r e f i l -t e r (202). Required f o r the t r a n s l a t i o n of natural mRNA (201 ). Has a ribosome-dependent GTPase a c t i v i t y r e s i s t a n t to f u s i d i c acid (203). May give t r a n s l a t i o n a l control by variable a f f i n i t y for d i f f e r -ent sequences around i n i t i a t o r codons (155). -87-f1 i s released upon formation of the 70 S complex and, as mentioned above, the other i - f a c t o r s are probably released together with f 1 . In the absence of i- f a c t o r s neither F-Met-tRNAf nor mRNA w i l l bind to ribosomes (153). Revel et al. (154) showed by EM that f2 stimulates attachment of ribosomes to nascent RNA being synthesized on phage T4 DNA. Thus, i - f a c t o r s are involved i n promoting i n t e r a c t i o n between ribosomes and both mRNA and the i n i t i a t o r tRNA. Olark (176) found that phage f2 RNA stimulated binding of P-Met-tRNAf to ribosomes much better at 37 °C than at , 25 °C and proposed that the difference was due to p a r t i a l melting of the secondary structure of the f2 RNA. Disruption of H-bonds could expose i n i t i a t o r codons which are buried i n double stranded regions and are normally unable to int e r a c t with i - f a c t o r s , F-Met-tRNAf and 30 S subunits. The work of Ste 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 of secondary structure around the codons. She incubated R17 RNA with ribosomes, i - f a c t o r s , F-Met-tRNAf and GTP which thus allowed ribosomes to bind to i n i t i a t o r s i t e s but no chain elongation could occur i n the absence of elongation f a c t o r s . The R17 RNA:ribosome complex was digested with pancreatic RNase to degrade exposed regions of v i r a l RNA not shielded by the ribosomes (54). Three f r a g -ments protected 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 to the -88-l e f t of each AUG codon and i n phase with i t was an UGA terminator codon present i n the i n t e r c i s t r o n a l region. These UGA codons could be part of recognition s i t e s f o r i n i t i a t i o n or could be reinforcement terminator codons; the 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 since there i s no apparent need fo r a terminator codon before the f i r s t c i s t r o n and yet there i s one. The AUG codon f o r i n i t i a t i o n of the coat protein was found at the end of a hai r p i n loop i n which 11 out of 12 possible base pairs are made; the other two i n i t i a t o r codons were found to be exposed i n regions where no base pairing i s possible. Most i n t e r e s t i n g was the discovery that n o n - i n i t i a t o r AUG sequences are buried i n base-paired regions and therefore are unavailable f o r i n i t i a t i o n . Thus, the increased binding of F-Met-tRNAf to phage f 2 RNA at higher temperatures ob-served by Clark (176) and mentioned above, could be due to melting of regions and exposure of n o n - i n i t i a t o r AUG.,sequences. Hindley and Staples (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 for the coat protein to be situated at the end of a looped structure with 5 base pairs i n the stem. More work i s required to determine i f the exposed position of i n i t i a -tor codons i s a general r u l e . Differences i n the 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 could provide a mechanism for t r a n s l a t i o n a l control of the rate of protein synthesis. Lodish (177,178) discovered that ribosomes from B a c i l l u s -89-stearothermophilus can only translate one of the three cis--trons of phage f2 RNA; this was the c i s t r o n f o r the matura-t i o n protein (A protein), the product synthesized i n the l e a s t amount by the normal host of the phage, E. c o l i . The s p e c i f i c i t y of ribosomes from B a c i l l u s stearothermophilus was found to be a property of the 30 S subunit by preparing hybrid ribosomes with various combinations of subunits from both bacteria. Thus, E. c o l i 30 S and. B a c i l l u s stearothermo- philus 50 S subunits w i l l translate a l l three cistrons i n the c e l l - f r e e system employed; the source of supernatant factors or i - f a c t o r s made no difference. This surprising d i s -covery implies that the 30 S subunit i s involved i n recogni-ti o n of natural mRNA's and, at l e a s t i n the case above, can discriminate against foreign mRNA. One could argue that Bacillus stearothermophilus i s an unusual organism since i t grows well at 65 °C and that i t s 30 S ribosomal subunits have evolved i n such a way that they cannot interact with at l e a s t some heterologous mRNA's. Nevertheless, Lodish's c observations can explain h o s t - s p e c i f i c i t y of phage f2 and the phenomenon of ribosomal s p e c i f i c i t y may be of more gen-e r a l importance., When E. c o l i infected with phage M12 i s superinfected with phage T4, r e p l i c a t i o n of M12 i s prevented (179); the block was l o c a l i z e d to an effect of T4 on protein synthesis directed by M12 RNA (180). During T4 i n f e c t i o n of E. c o l i . the host ribosomes are modified by addition of several T4--90-s p e c i f i c proteins to both ribosomal subunits soon a f t e r the in f e c t i o n begins (181 )-. Hsu and Weiss (182) showed that T4-modified E. c o l i ribosomes db not translate phage MS2 RNA or even E. c o l i mRNA nearly as e f f i c i e n t l y as normal E. c o l i ribosomes; however, modified and normal ribosomes translate T4 mRNA equally w e l l . The T4 " r e s t r i c t i o n f actors" could be washed from the modified ribosomes by 2 M NH^Cl and addition of i - f a c t o r s from normal ribosomes restored the a b i l i t y of the washed ribosomes to translate mRNA's other than T4 RNA (183,184). This i n t e r e s t i n g observation means that factors present i n the ribosomal wash are capable of determining the i n t e r a c t i o n of ribosomes with natural mRNA's. This i s a clear example of transnational control and could be of wide-spread importance. F-Met-tRNAf I n i t i a t i o n of protein synthesis i n a l l 70 S ribosomal systems involves F-Met-tRNAf (185). The presence of two methionine accepting tRNA's i n E. c o l i was known before the differences i n function;were discovered (186,187). The absolute s p e c i f i c i t y of i - f a c t o r s for F-Met-tRNAf i n the binding reaction with i n i t i a t o r codons and GTP (146) indicates that both the blocked amino group and at le a s t one s i t e on the tRNA must be recognized. Both Met-tRNA's have been sequenced (188,189) but the structures do not re a d i l y reveal a possible recognition s i t e f o r the trans--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 binding assay could be due to a required conformational change i n the tRNA occurring upon formyla-t i o n of the amino group of methionine; i t i s known that acylation can cause a conformational change i n aminoacyl-tRNA (190)., F-Met-tRNAf must also be recognized by aminoacyl-tRNA deacylase, an enzyme that hydrolyzes any other blocked amino-acyl -tRNA1 s including F-Met-tRN/^ (191 ). As one might expect, E. c o l i depleted of fo l a t e i s rendered de f i c i e n t s p e c i f i c a l l y i n i t s capacity to i n i t i a t e protein synthesis (291 ). However, i n studies on f o l a t e -depleted Streptococcus faecium. Pine, Gordon and Sarimo (292) found evidence that this organism can r e a d i l y i n i t i a t e pro-teins with or without formylated Met-tRNAf. When grown i n f o l a t e - d e f i c i e n t medium and i n the presence of the fo l a t e antagonist, trimethoprim, Streptococcus faecium continues growing with methionine incorporation into tRNA and protein N-termini at unaltered l e v e l s but completely without formy-l a t i o n . The authors proposed that Lactobacteriaceae and mammalian c e l l s have a folate-independent pathway of protein i n i t i a t i o n since 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 long as products of one-carbon meta-bolism such as thymidine are provided. The properties of Met-tRNA's were studied using a l l 64 ribo t r i n u c l e o t i d e s and various polyribonucleotides (142). At -92-high Mg and i n the absence of i - f a c t o r s , F-Met-tRNAf bound to the codons AUG, GUG and UUG and only s l i g h t l y to AOG, OUG, GCG, UGG, UCG or AGG. Met-tRNA bound to AUG and GUG. The bind-m ing of F-Met-tRNAf shows a constant requirement for G i n the t h i r d position but i n the three codons which gave s i g n i f i -cant binding, there i s ambiguity i n the reading of the f i r s t nucleotide. The binding to the three codons did not change with addition of i - f a c t o r s but i n experiments with polyribo-nucleotides, only AUG and GUG could serve as i n i t i a t o r s . Assuming that F-Met-tRNAf i s a s i n g l e homogeneous prepara-t i o n , then the misreading of the f i r s t nucleotide must be due to the s p e c i a l interactions of t h i s tRNA with i - f a c t o r s and the 30 S subunit. GUA was found to serve as a weak i n i t i a -t or which implies t h i r d l e t t e r ambiguity as i n ordinary de-generacy (192). Waller (193) found that most E. c o l i proteins have methionine, alanine, serine or threonine as the N-terminal residue; i n B. s u b t i l i s . 80$ of the bulk proteins were found to have N-terminal alanine (194). It was therefore ob-vious by 1965 that there must be a mechanism for removal of the formyl group and quite often the methionine as well from the N-terminus of newly synthesized proteins i n bacteria. A deformylase a c t i v i t y was soon found (195-197) which removed the formyl group from peptides much faster than acetyl groups; the enzyme was found to be inactive against either acyl group on free methionine. The E. c o l i deformylase i s quite l a b i l e , -93-i being r a p i d l y inactivated at 37 °C i n the presence of 5 mM mereaptoethanol (196); this probably explains the ease with which formylated peptides can be recovered from E. c o l i c e l l -free systems. The B. s u b t i l i s enzyme i s associated with the ribosomes and can be washed o f f with 0.5 M NH4CI; this en-zyme i s stable to t h i o l s (197). The methionine residue at the N.-terminus of newly syn-thesized b a c t e r i a l proteins i s probably removed by the action of a s p e c i f i c aminopeptidase. Matheson and Dick (198) have described an aminopeptidase a c t i v i t y on the ribosomes of E. c o l i which has the s p e c i f i c i t y one would expect to ac-count f o r the known N-termini of E. c o l i proteins; this enzyme w i l l be discussed i n more d e t a i l l a t e r . ELONGATION During elongation the ribosome moves along the mRNA i n the 5' to 3' d i r e c t i o n i n steps of three nucleotides each. Protein factors required for elongation can be i s o l a t e d from the HSS and form 2-3$ of the protein i n this f r a c t i o n i n growing E. c o l i c e l l s (204). A summary of the nomencla-ture and some of the properties of the elongation factors from d i f f e r e n t sources i s given i n Table 3. An outline of the steps i n which the factors function i s given, i n Pig. 2 (rat l i v e r ) and Pig. 3 (E. c o l i ) . Nathans and Lipmann (213) were the f i r s t to i s o l a t e elongation factors and did so using the HSS from E. c o l i . -94-TABLE 3 Nomenclature and Characteristics of Elongation Factors B a c i l l u s  E. c o l i stearothermophilus Rabbit Rat . Reticulocyt es Liver Function Ts S1 TF-1 MW 186,000 3 subunits (Ref.207) TF-1 Binding amino-acyl-tRNA to the A s i t e of ribosomes. Tii S3 Same as above. MW 84,000 4 subunits (Ref.206) S2 Inhibited by f u s i d i c acid (Ref.212) TF-2 MW 70,000 (Ref.208) Very s e n s i -t i v e to SH reagents and cycloheximide (Ref.209) TF-2 MW 65,000 Sensitive to SH a-gents ( Ref.209) Translocation of p e p t i d y l -tRNA from s i t e A to s i t e P with movement of ribosome on mRNA by one co-dbn at a time. Note: M s " i n Ts refers to s t a b i l i t y of the factor n u M in. Tu refers to i t s i n s t a b i l i t y TF-1 i s transferase 1 (aminoacyl-tRNA binding enzyme) TF-2 i s transferase 2 (translocase) -95-F i g . 2 Mechanism of Elongation i n Rat Li 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 ) + — » Peptidyl-tRNA* (A s i t e ) aa-tRNA (A s i t e ) ^ Peptidyl transferase (60 S subunit) 41) Peptidyl-tRNA* (A s i t e ) + Peptidyl-tRNA* (P s i t e ) + GTP GDP + P i + release of d i s -I charged tRNA TF-2 Note: aa-tRNA = aminoacyl-tRNA * one amino acid longer than previously -96-F i g . 3 Mechanism of Elongation i n E. c o l i Mg + + 1 ) Ts + Tu + GTP > (Ts:Tu:GTP) Complex 1 Mg + + 2) Complex 1 + aa-tRNA — L - , . > Ts + (Tu:GTP:aa-tRNA) Complex 2 NH 4 3) Complex ,2 + Ribosome:mRNA > (Ribosome:mRNA:aa-tRNA) + GDP:Tu + P i 4) (Ribosome:mRNA:aa-tRNA: (Ribosome:mRNA:peptidyl-peptidyl-tRNA) ^ tRNA*) Peptidyl transferase (50 S subunit) 5) Peptidyl-tRNA* (A s i t e ) + Peptidyl-tRNA* (P s i t e ) + GTP y release of discharged ^ tRNA + GDP + P i Note: * one amino acid longer Ts may be required to dissociate GDP:Tu. (Ref.211) -97-These factors were eventually resolved into three fractions c a l l e d Ts, 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 are required'1 f o r binding of the correct i n -coming aminoacyl-tRNA to the ribosomal A s i t e i n a reaction requiring GTP. If- peptidyl-tRNA or F-Met-tRNAf i s present i n the P s i t e , peptide bond formation occurs by transfer of the acyl group at the P s i t e to the amino group of the aminoacyl-tRNA at the A s i t e . .Formation of the peptide bond i s cata-lyzed by peptidyl transferase, an enzyme a c t i v i t y present on the 50 S subunit (105,106,218). The energy for peptide bond formation i s supplied by the r e l a t i v e l y high energy ester bond between the tRNA and the peptidyl moiety.(219). Waterson, Beaud and Lengyel (211) have recently studied the function of elongation f a c t o r S1 i n B'. stearothermophil-us which i s equivalent to Ts i n E. c o l i . They found that rather than being d i r e c t l y involved i n the binding of amino-acyl -tRNA, S1 i s required f o r d i s s o c i a t i o n of the GDP:S3 complex released a f t e r binding of aminoacyl-tRNA to the ribosome as i n the following scheme: 1 ) S3 + GTP + Phe-tRNA > (S3:GTP:Phe-tRNA) Complex 1 2) Complex 1 + Ribosome:Poly U:.< , GDP:S3 + P i + Ribosome: Ac-Phe-tRNA * Poly U;:Ac-diPhe-tRNA (Site A) -98-3) Ribosome:Poly U:Ac-diPhe-tRNA (Site A) + GTP Ribosome:poly U:Ac-diPhe-tRNA (Site P) + S2 discharged tRNA + GDP + P i 4) GDP:S3 GDP + S3 S1 Reaction 2) above involves both binding of aminoacyl-tRNA at the A s i t e and peptide bond formation catalyzed by p e p t i -dyl transferase. GDPCP, an analogue of GTP that cannot be hydrolyzed be-tween the beta and gamma phosphates, can replace GTP i n the binding reaction f o r aminoacyl-tRNA but no peptide bond i s formed (220,221). Since GTP i s not required for peptide bond formation per se (105,106,218), the energy from GTP must be used i n a reaction which binds aminoacyl-tRNA at 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 with the ribosome which i s necess-ary f o r peptide bond formation. After peptide bond formation, peptidyl-tRNA remains in. the A s i t e and discharged tRNA at the P s i t e . Translocase (factor G; or S2) catalyzes translocation i n a reaction whereby peptidyl-tRNA s h i f t s to the P s i t e and. the d i s -charged tRNA i s released (219,222,217) and the ribosome moves along the mRNA by 3 nucleotides i n the 5' to 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 of translocase i s the steroid, a n t i b i o t i c , f u s i d i c acid (212$. Several models f o r the me-chanism of translocation have been proposed recently (223, -99-224) but w i l l not be discussed here. Schreier andi N o l l (225) found evidence that 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 i n t e r -action of ribosome subunits, poly U and uncharged tRNA E n e; the complex s h i f t s to 70 S upon addition of the complex, Tu:GTP:Phe-tRNA with release of Tu, GDP and P i . They con-cluded that the energy of GTP i s required for d r i v i n g a reaction causing a large conformational change of the r i b o -some to a more compact form which sediments f a s t e r . The au-thors also found evidence for a t h i r d s i t e on the ribosome formed during the s h i f t from 60 S to 70 S; the data i n d i -cated that two molecules of Phe-tRNA were present on each 70 S ribosome. Eukaryotic elongation factors have been studied i n the re t i c u l o c y t e system (226,227,207,208), i n rat l i v e r (228, 229) and i n yeast (230). These systems apparently only have two factors with transferase 1 analogous to Ts and Tu (231» 2 3 2 ) and transferase 2 equivalent 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 that diphtheria toxin, a protein of molecular weight 65,000, acts by s p e c i f i c a l l y i n h i b i t i n g eukaryotic transferase 2 (translocase) (Ref.2 3 6 ) . Guinea pigs are k i l l e d by as l i t t l e as 0.05 ug of the toxin with widespread necrosis in. many organs. The toxin has been found to catalyze the following unusual reaction i(237)* TE-2 + NAD+ > ADPR:TP-2 + nicotinamide + H + -100-The above reaction can be modified with the use of 0-l a b e l l e d NAD+ ( l a b e l l e d i n the adenosine moiety) as an assay for transferase 2; as l i t t l e as 5-10 ng can be detected (238). Peptidyl transferase, the 50 S subunit enzyme respon-s i b l e f o r peptide bond formation, i s dormant i n the absence of 30 S subunits but can be activated by low M.W. alcohols (239). This enzyme appears to int e r a c t with the CpCpA-peptide and OpA-amino acid moieties of the two substrates bound to the ribosome at the P and A si t e s respectively. A s i m i l a r enzyme has been found i n eukaryotes (233) and i s present on the 60 S subunit (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 the a n t i b i o t i c s chloramphenicol, lincomycin, streptogramin A, gougerotin and sparsomycin. The eukaryotic enzyme i s not i n h i b i t e d by the a n t i b i o t i c s above but i s in h i b i t e d by the a n t i b i o t i c , anisomycin (240). These studies have therefore helped explain the s e l e c t i v e action of a n t i -b i o t i c s on bacteria and not on eukaryotic c e l l s or v i c e -versa. TERMINATION The codons UAA, UAG and UGA code f o r no amino acids normally and are referred to as "nonsense" or "terminator" codons. When, a ribosomal:peptidyl-tRNA complex reaches a terminator codon on a mRNA, some mechanism must bring about hydrolysis of the ester bond between the peptidyl and tRNA moieties. This would then allow the completed protein to be -101-released. Capecchi (241) studied the release of the N-terminal hexapeptide of coat protein of phage R17 with an amber mu-t a t i o n at the seventh codon (CAG mutated to UAG). He suc-ceeded i n i s o l a t i n g a protein f a c t o r , R, from the HSS of E. c o l i and found that R was required for the release of the hexapeptide from a p u r i f i e d peptidyl-tRNA:ribosome com-plex. Oaskey et al. (242) used a d i f f e r e n t assay f o r release f a c t o r s . F-Met-tRNAf was bound to ribosomes i n the presence of the t r i n u c l e o t i d e AUG; then a terminator t r i n u c l e o t i d e and test protein were added and the release of free F-Met was measured. With th i s assay they were able to resolve R into two components, R1 and R2; R1 responded to UAG and UAA but not UGA whereas R2 was active with UGA and UAA but not UAG (Ref.243). A t h i r d f a c t o r , S, has been, found to increase the rate of formation or s t a b i l i t y of the terminator codon: ribosome:factor R complex (244). Peptidyl transferase may be involved i n release since i n h i b i t o r s of t h i s enzyme also i n h i b i t release. The pre-sence of the terminator complex may allow peptidyl trans-ferase to act as a hydrolase and thus break the ester bond holding the completed protein to the l a s t tRNA. Capecchi and K l e i n (245) used antisera to p u r i f i e d R1 and R2 to test t h e i r r o l e i n release of completed pro-teins i n a c e l l - f r e e system directed by R17 RNA. Their r e --102-su l t s indicated that these factors are required f o r release of completed proteins and that either factor could promote release of either the coat protein or the r e p l i c a s e ; this implies that both cis.trons terminate with UAA since this i s the only one of the three terminator codons recognized by both R1 and R2. Nichols (246) sequenced the portion of R17 RNA at the end of the coat protein c i s t r o n and found two consecutive terminator codons, UAAUAG.. This may mean that, at l e a s t i n some systems, two terminators are required to ensure that release occurs between cistrons even i f a suppressor tRNA i s present. The UAA codon i s consistent with the r e -sults of Oapecchi and K l e i n mentioned above. EUKARYOTIC INITIATION The mechanism of i n i t i a t i o n i n eukaryotic c e l l s was unknown u n t i l very recently and not a l l of the det a i l s are s e t t l e d yet. Two major species of methionine accepting tRNA are present i n the cytoplasm of guinea pig l i v e r (247), ra t l i v e r (185) and yeast (248) but there i s no detectable transformylase i n the cytoplasm of these c e l l s . These tRNA's w i l l be referred to as Caskey, Beaudet and Nirenberg (249) found that guinea pig l i v e r Met-tRNA^* binds to ribosomes i n response to either AUG" or GUG. whereas Met-tRNA,^ binds only to AUG. Surprisingly, p u r i f i e d E. c o l i transformylase was found to -103-r e a d i l y formylate Met-tRNA^* from guinea pig l i v e r but was inactive against Met-tRNAm* (24-7). Using the purqmy-c i n assay of Leder and Bursztyn. (250), i t was shown that Met-tRNA^.* binds at the P s i t e on the ribosome since the bulk of i t reacts r a p i d l y with puromycin. A l l of these reactions of Met-tENA^* are exactly analogous to those with b a c t e r i a l F-Met-tRNAf or Met-tRNAf. Some stu c t u r a l features of eukaryotic tRNA^** must have been preserved during e v o l -ution from lower forms. Structural differences 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 su r p r i s i n g i n view of the large gap between eukaryotes and bacteria on any evolutionary scale. Formylated yeast Met-tRNA f* was found to be capable of i n i t i a t i n g protein synthesis i n an E. c o l i c e l l - f r e e system primed with phage f2 RNA (248). Recently, Smith and Marcker (252) separated and studied the two Met-tRNA's from mouse ascites tumour c e l l s , mouse l i v e r and yeast. They found that Met-tRNA f* incorporates methionine exclu s i v e l y into the N-terminal positions of newly synthesized proteins in. a c e l l - f r e e system from mouse ascites tumour c e l l s primed with synthetic p o l y r i b o -nucleotides. Met-tRNAjjj*, i n contrast, incorporates methionine only into i n t e r n a l positions i n the new proteins. This i s very good evidence that Met-tRNAf* can i n i t i a t e proteins but does not prove that this i s happening i n vivo. In f a c t , when the c e l l - f r e e system was primed with a natural mRNA -104-(encephalomyocarditis virus RNA), s i g n i f i c a n t incorpora-t i o n of methionine from Met-tRNA^* into v i r a l proteins could not be demonstrated. The authors suggested that i n -corporated 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 or the methionine might not be incorporated; i n the l a t t e r case, the Met-tRNAf,* would function to sele c t the proper reading frame on the mRNA. Brown and Smith (253) established that formylated Met-tRNAf* was active i n an E. c o l i c e l l - f r e e system but not i n one derived from ascites c e l l s . This implied that formylated Met-tRNA f* cannot i n t e r a c t with 80 S ribosomes and i s consistent with the absence of transformylase a c t i v -++ i t y i n the cytoplasm of eukaryotic c e l l s . At low Mg only polyribonucleotides with an AUG codon were translated i n the ascites c e l l - f r e e system. AUG- codons at or near the 5' end of the synthetic mRNA's always interacted with Met-tRNA^* and not with Met-tRNA m #. S i m i l a r l y , GUG at or near the 5 1 end always interacted with Met-tRNA^ and not with Val-tRNA. Stewart et al. (254) selected 42 mutants of baker's yeast lacking isocytochrome c; nine revertants were i s o l a t e d and examined. Some of the revertants were found to have one or two additional residues at the N-terminus; the additional residues included Met-Ileu, Met-Leu, Met-Arg and Yal. The authors concluded that the normal mRNA fo r isocytochrome c must have an AUG. codon before the codon for the normal - 1 0 5 -N.-terminal residue and that i n the o r i g i n a l mutants the AUG codon had changed to Aug ( l i e u ) , QUG; (Leu), AGG (Arg) or GUG! (Val). They also concluded that the revertants must have arisen by mutations giving 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 -tations are correct, AUG must serve as an i n i t i a t o r codon. i n the mRNA for yeast isocytochrome c; the authors did not mention the p o s s i b i l i t y that t h i s protein could be a mito-chondrial product. Liew, Haslett and A l l f r e y (255) have found evidence that Ac-Ser-tRNA i s involved i n the i n i t i a t i o n of synthesis of histone Tf in. regenerating r a t l i v e r . This protein has the N-terminal sequence Ac-Ser-Gly-Arg.. . (256) . The authors had found e a r l i e r that l i t t l e or no acetylation of the N-terminal serine occurs i n the absence of histone synthe-s i s ; the acetylation which did occur during histone synthe-s i s was i n h i b i t e d by puromycin. However, the incorporation, of ^H-acetate and 1 ^ 0-serine into Ac-Ser from digests of newly synthesized histone IV was quite low; also, i n the case of nuclear histone W , the incorporation of acetate was not nearly as sensitive as serine to i n h i b i t i o n by puromycin. The l a t t e r suggests that the acetate can be i n -corporated independently of protein synthesis. Both labels were found in. a 4 S RNA and digestion of the l a t t e r with pancreatic RNase released a product with the expected mo-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 high voltage -106-electrophoresis. This was taken to indicate the presence of Ac-Ser-tRNA i n regenerating rat l i v e r acting as an i n i t i a t o r tRNA fo r histone IV synthesis. Since this represents quite a departure from a mechanism involving Met-tRNA^more evidence i s required before t h i s proposal can be accepted. M i l l e r and Schweet (257) found that hemoglobin synthe-s i s i n a r e t i c u l o c y t e c e l l - f r e e system using washed r i b o -somes was stimulated by addition of the wash f r a c t i o n ; i n the absence of the wash f r a c t i o n , the N-terminal valine of hemoglobin chains remained unlabelled. They also showed that the wash f r a c t i o n lowered the Mg + + optimum f o r poly U directed polyphenylalanine synthesis from 10 mM to 5 mM. These findings were confirmed (258) and the suggestion made that the wash f r a c t i o n contained i - f a c t o r s . s i m i l a r to those i n b a c t e r i a l systems. Reticulocyte i - f a c t o r s have since been fractionated into 3 components l a b e l l e d M1, M2 and M3 (Ref.259). M1 binds Ac-Phe-tRNA to re t i c u l o c y t e ribosomes. M2 i s required f o r i n i t i a t i o n of both poly U and natural mRNA directed protein synthesis. M3 i s only necessary f o r i n i t i a t i o n on natural 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 with the mRNA f o r myosin from chick muscle. The muscle c e l l i - f a c t o r s were found to be necessary for binding the myosin mRNA to chick r e t i c u l o c y t e ribosomes. This suggests that s p e c i f i c i - f a c t o r s are involved i n recognition of natural -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). Also, this work gives another mechanism for t r a n s l a t i o n a l control of protein synthesis which could be of general importances This summary of protein synthesis has shown some of i t s complexity and how knowledge of the mechanisms has explained such phenomena as the mechanism of action of a n t i b i o t i c s and regulation of protein synthesis. PROTAMINE BIOSYNTHESIS Only a b r i e f summary w i l l be given here as detailed accounts have been given recently (293,294). Protamines are the highly basic proteins bound to DNA i n the mature sperm c e l l s of most higher animals. The three major components of protamine i n the rainbow trout (Salmo  g a i r d n e r i i ) have been sequenced by Ando and Watanabe (261) with the r e s u l t s shown i n F i g . 4. Of the 32-33 residues, 21-22 are arginine and these occur i n blocks of up to s i x con-secutive residues. There are only s i x other amino acids pre-sent and these are a l l neutrals; there are no aromatic or sulphur-containing amino acids. Protamine synthesis takes place at the spermatid stage of d i f f e r e n t i a t i o n (262,264). This synthesis occurs on cytoplasmic 120 S disomes consisting of 77 S ribosomes and, presumably, mRNA (265,266). The synthesis was found to be extremely sen s i t i v e to i n h i b i t i o n by cycloheximide and -108-F i g . 4 Sequences of Protamines from Salmo g a i r d n e r i i * 1A Pro-Arg^-S er-S er-S er-Arg-Pro-Val-Arg^-Pro-Arg 2-Val-S er-Argg• Gly-Gly-Arg 4 Pro-Argg-Ser-Ser-Ser-Arg-Pro-Ileu-Arg4-Pro-Arg2-Val-Ser-Arg5-Gly-Gly-Arg 4 2 Pro-Arg^-Ser-Ser-Ser-Arg-Pro-Val-Arg4-Ala-Arg 2-Val-Ser-Arg6-Gly^Gly-Arg 4 * Ando and Watanabe (Ref.261) - 109-and was less s e n s i t i v e to puromycin (264). In the presence of actinomycin D, protamine synthesis continues uninhibited for several hours implying that the mRNA i s quite stable. Newly synthesized protamine i s phosphorylated on i t s se r y l residues (267,268) probably due to the action of the protamine kinase studied by J e r g i l and Dixon (269). New protamine i s r a p i d l y transported into the nucleus and thence into the chromatin where i t i s dephosphorylated and replaces the histones and a c i d i c proteins i n association with the DNA (268,270). This replacement process i s associa-ted with condensation of chromatin and a decrease i n tem-plate a c t i v i t y when assayed with the E. c o l i RNA polymerase i n v i t r o (270). If association of protamine with DNA represses that r e -gion, then the spermatid must c a r e f u l l y control the replace-ment i n order to prevent premature repression of gene func-tions required l a t e r . Thus, the replacement process cannot be just a kind of cation exchange. The blocks of arginine i n protamine appear to be well suited as binding s i t e s to DNA but i t i s not clear that protamine serves any function other than allowing a large amount of a polyanion (DNA) to pack into the small volume of the sperm head; there i s about 2 metres of DNA i n a volume of 20 cubic microns. MATERIALS AND METHODS Chemicals and Abbreviations a) Chemicals Common chemicals were obtained commercially and were of reagent grade. Phenylisothiocyanate and N-ethyl morpho-l i n e were purchased from Eastman and were r e d i s t i l l e d before use. Anhydrous t r i f l u o r a c e t i c a c i d was from Matheson, Cole-man and B e l l . Whatman 3 MM paper was used f o r high voltage electrophoresis and chromatography. Pyridine 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-methionyl-N-hydroxysuccinimide ester was obtained from Mann Research Laboratories. Kodak "Royal Blue" X-ray f i l m was used for autoradiography. Carboxypeptidase B was from Worthington. b) Abbreviations RNase: ribonuclease tRNA: transfer RNA mRNA: messenger RNA rRNA: ribosomal RNA EDTA: ethylenediaminetetraacetate M.W.: molecular weight EM: electron microscope i - f a c t o r s : i n i t i a t i o n factors SH groups: sul f h y d r y l groups NAD+: nicotinamide adenine dinucleotide -111-PITC: phenyiisothiocyanate TMKS: TRIS-HC1, G.05 M; magnesium acetate, 0.005 M; potassium chloride, 0.025 M; and sucrose, 0.25 M. The pHiwas usually 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 . F i s h A l l of the experiments here were with testes from the rainbow trout (Salmo g a i r d n e r i i ) . During the period of natural maturation, August to December, testes were obtain-ed from the Sun Valley trout farm at Mission, B.C. through the cooperation of Mr. and Mrs. Hans Lehmann. The testes were transported to the laboratory i n ice and used immedi-at 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 trout were brought to the laboratory and kept i n aquaria for 2-3 weeks during which they were used f o r experiments. For the period January to July, trout 1 —2 years old were kept i n aquaria at 10-12 °0 and injected twice weekly with 0.1 ml of crude salmon p i t u i t a r y extract (Oncorhynchus  tshawytscha) containing gonadotrophic a c t i v i t y (271). Such injections induce complete maturation of the testes i n about 10-12 weeks. Induced f i s h were given an extra i n j e c t i o n 24 hours before any experiments. -112-2. Incubation of C e l l Suspensions Pish were anaesthesized i n a d i l u t e s o l u t i o n of T r i -caine Methanesulfonate (Ethyl m-aminobenzoate Methanesul-fonate) obtained from Fraser Medical Supplies, Vancouver. The testes were removed and washed i n a beaker of Hank's balanced s a l t s o l u t i o n at 4 °C. Testis weight was recorded' and then the blood vessels were in c i s e d to allow most of the blood to escape. The testes were scissor-minced i n 4 volumes of Hank's solut i o n and then homogenized by hand i n a Potter-Elvehjem tube with a Teflon pestle. The c e l l sus-pension was f i l t e r e d through 2-3 layers of pre-washed cheesecloth to remove connective tissue and large clumps of c e l l s . The c e l l s were usually washed once by sedimenting at 1000 g f o r 10 minutes and then resuspending i n fr e s h Hank's soluti o n . Hank's s o l u t i o n was always modified by omitting NaHCO^ and adding TRIS-HC1 to 0.01 M and pH 7.35; th i s gave a soluti o n with constant pH i n contrast to the standard Hank's solution i n which the pH increases as C0 2 escapes. Amino acids, except f o r those to be included i n l a b e l l e d form, were added from concentrated stock solutions to give a f i n a l concentration i d e n t i c a l to the amino acids i n ti s s u e culture medium "199" (Ref.295). For incubations over 1 hour, vitamins were added at the concentration used i n Waymouth's medium (296). Incubations were done by adding the c e l l suspension to -113-a glass vessel and shaking i n a gyratory water bath (New Brunswick) at 20, 18 or 15 °0; a l l recent experiments were done at 15 °C since f i s h enzymes might be l a b i l e at higher temperatures. Rapid s t i r r i n g i s required to prevent the c e l l s from clumping. After an incubation, the bulk of the free isotope was removed by sedimenting the c e l l s at 1000 g for 10 minutes and discarding the supernatant. 3. Extraction of Protamine Whole c e l l s , nuclei or chromatin were prepared and extracted with 0.2 N HOI or 0.2 M H2SO4 as described i n Part 1 "Methods". The crude acid extractable proteins were usually prefractionated by adsorbing on a column of CM-c e l l u l o s e equilibrated with 0.2 M L i C l i n 0.0,1 M l i t h i u m acetate pH 5.0. The column was then washed with 0.2 M L i O l , 0.7 M L i O l and water and f i n a l l y eluted with 0.2 N HOI to remove the t i g h t l y bound protamine.which was then l y o p h i l -ized. The Li O l washes removed nucleic acids, nonhistone pro-teins and the bulk of the histones. Pure protamine was pre-pared by chromatography of 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 Bio-Gel P-10 eluted with 0.2 M acetic acid (267). Small amounts of the crude acid extractable proteins could be d i r e c t l y applied to Bio-Gel P-10 with good separa-t i o n of protamine from the other proteins. 4. Starch Gel Electrophoresis The method of Sung and Smithies was followed with some -114-modifications (272). 100 g of starch and 120 g of urea were added to 400 ml of aluminum l a c t a t e buffer (pH 3.1 ) i n a large Erlenmeyer f l a s k and heated with constant shaking i n a b o i l i n g water bath for 5-8 minutes. The starch s o l u t i o n was then poured into the gel tray and a small amount was poured around the slot-former before applying the l i d to prevent a i r bubbles near the s l o t s . The f i n a l gel composi-t i o n i s 4 M urea and about 0.02 M aluminum l a c t a t e at pHI3.4. Electrophoresis was v e r t i c a l l y downwards at about 8 volts/cm for about 10 h; the exact length of the electrophoresis was determined by the migration of a methyl green.marker which migrates just ahead of protamine under these conditions. The gel was removed from the tray and t r i s e c t e d ; the outer s l i c e s were stained i n 200 ml of Amido Black 10B;in 1% acetic acid (0.125%, w/v) to which 0.6 ml of 1 M cobalt n i t r a t e was added just before use. After s t a i n i n g at l e a s t 45 minutes, the s t a i n was drained and replaced by 0.5 M HgSO^; i n less than 10 minutes the background fades to a grey colour arid the protein bands s t a i n black or blue. This s t a i n was found to be 100 times as sens i t i v e as ordinary staining with Amido Black (272). The middle s l i c e was r e s l i c e d transversely at 3 mm i n t e r -vals and these pieces were placed i n s c i n t i l l a t i o n v i a l s with 0.75 ml of NCS s o l u b i l i z e r overnight. 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 added to each and a f t e r stand-ing at room temperature f o r 2-4 h the samples were counted. - 1 1 5 -5 . Ion-Exchange Chromatography Protamine components were p a r t i a l l y resolved by i o n -exchange chromatography on CM-cellulose using the methods described i n Part 1 except that the gradient of l i t h i u m chloride was from G .7 to 1 . 3 M. 6 . Edman Degradation The method of Edman ( 2 7 3 ) as modified by Hew ( 2 7 4 ) was followed. The protein sample (less than G.1 micromoles) was dissolved i n 0.1) ml of 50% aqueous pyridine (v/v) i n a small glass tube. Then 0 . 1 ml of PITC solution (1 ml of r e d i s t i l -led N-ethyl morpholine plus 0 . 1 ml of PITC) was added with mixing followed by degassing with nitrogen. The tube was sealed and incubated at 37 °C f o r 2 -| -3 h. The reaction mix was extracted 3 times with 0 . 5 ml of benzene and l y o p h i l -ized. The residue was dissolved i n 0 . 2 ml of anhydrous TFA, flushed with nitrogen and incubated at 37 °C f o r 1 h. The mix was dried i n vacuo, redissolved i n 0;5o>ml water and extracted 4 times with 1 ml portions of ethyl acetate. A l i -quots of both phases were dried i n s c i n t i l l a t i o n v i a l s , redissolved 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 of the residue removed by Edman de-gradation, an aliquot of the ethyl acetate phase was dried and treated with 1 W HC1 at 8 0 °C f o r 15 minutes to convert the thiazolinones to the more stable thiohydantoin deriva--116-t i v e s . The sample was then dried, redissolved i n 90$ acetic acid and aliquots applied to a thin-layer sheet of s i l i c a gel with fluorescent indicator (Eastman chromogram sheet 6060). Standard PTH amino acids were spotted and the sheet was developed i n the solvent system, heptane:n-butanol: 75$ formic acid (100:60:18, v/v) f o r 2£ h. After drying, the sheet was photographed under UV i l l u m i n a t i o n using a Polaroid camera as described i n Methods of Part 1. The sheet was then cut at 0.5 em i n t e r v a l s and counted i n toluene s c i n t i l l a t i o n f l u i d . 8. High Voltage Paper Electrophoresis Whatman 3 MM paper was used with the following buffer systems: pH 1.9 (acetic acid:formic acid:water, 8:2:90, v/v); pH 3.6 (pyridine:acetic acid:water, 1:10:89, v/v); and pH 6.5 (pyridine:acetic acid:water, 100:4:900, v/v). The length of electrophoresis was judged by the migration of coloured dyes as discussed i n Methods of Part 1. 9. Autoradiography After thorough drying, paper electrophoretograms were l a b e l l e d with radioactive ink and placed i n l i g h t - t i g h t jackets with a sheet of Kodak "Royal Blue" X-ray f i l m ; weights were placed on the jacket to ensure good contact between the paper and the f i l m . After adequate exposure, approximately 24 h per 20,000 CPM of 5 5 S or 1 4- C f t n e f i l m was developed with Kodak X-ray reagents. - 1 1 7 -10. Counting Paper Electrophoretograms In pases where the sample consisted of l e s s than 5000 CPM of 55s or H Q , an alternative method to autoradiography was used. As shown i n F i g . 5 , a portion of the s t r i p pf paper containing the sample peptides was cut transversely at 1 cm or 0 .5 inch 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 with 5 ml of toluene s c i n t i l l a t i o n f l u i d and counted. For or 35>S +,ne e f f i c i e n c y of counting on paper was 50 -60$ of that for an i d e n t i c a l sample dissolved i n Bray's s c i n t i l l a -t i o n f l u i d . Thus, a few hundred CPM of sample could be e l e c -trophoresed and counted accurately without the very long ex-posure period required for autoradiography. Since only a portion of the sample s t r i p was counted, the remainder of the sample peptides could be located, cut out and r e - e l e c t r o -phoresed or subjected to descending chromatography to con-firm i d e n t i t y with marker peptides. The marker peptides could be located by autoradiography overnight i f 25 ,000 CPM or more was applied. 11, Formylation Samples were formylated.by the method of Sheehan and Yang ( 2 7 6 ) . The dried sample was dissolved i n 4 volumes of 98$ formic acid at 10-12 °C; three aliquots of acetic an-hydride, 1 volume each, were added with mixing at 5 minute int e r v a l s and the reaction was allowed to continue for 1 h after the l a s t addition. The mix was l y o p h i l i z e d leaving a dry formylated sample. - 1 1 8 -F i g . 5 Counting Electrophoretograms 0 Cut out & count Apply markers here Origin Sample applied here; can r e -run portion to r i g h t of dotted l i n e s © -119-12. Deforraylation Anhydrous 1 N HOI i n methanol was prepared by dripping concentrated HOI into concentrated ^SO^ and allowing the released HOI gas to bubble through methanol at 4 °C; the methanol was t i t r a t e d and diluted to 1 N HC1 content. For deformylation, a dried sample was dissolved i n 0 . 5 - 1 . 0 ml of the HC1 i n methanol, sealed and incubated at 50 °C for 1 h. The mix was then dried 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 carboxyl groups by methanol, recent experiments were done using the method of Adams and Capecchi (132) i n which the sample i s treated with aqueous 1 N HC1 at 100 °C for 10 minutes. This method was found to give 95% deformylation and less than 5% peptide bond hydrolysis (132) . 13. Synthesis of Labelled Marker Peptides a) Met- 1 4C-Pro and F-Met- UC-Pro 25 microcuries of 1 4 c_proline (uniformly l a b e l l e d , 265 mCi/mMole, Amersham) \was- dried i n a small tube and redissolved i n 0 .2 ml of methylene chloride. 25 mg of t-butyloxycarbonyl-methionyl-N-hydroxysuccinimide ester (Mann Research Laboratories) was added with mixing and the tube was sealed and incuba.ted at 37 °C overnight. In the morning, the sample was dry and therefore 0 . 4 ml of 50% ethanol, 0 .2 ml of methylene chloride and 1 mg of HaHCO^ were added and incubation was continued at 37 °C f o r 30 min-utes according to Anderson, Zimmerman and Callahan (277) . -120-The sample was dried, redissolved i n 1 ml of anhydrous TFA and incubated at 37 °0 f o r 1 h« After drying again, the sample was redissolved and electrophoresed at pH 3.6 and an autoradiogram performed with the r e s u l t shown i n Pig. 6. The band corresponding to Met - 1 4c-Pro was eluted from the paper with 30$ acetic acid and a t o t a l of 9.4X106 CPM was recovered f o r a y i e l d of 37$, the y i e l d could have been doubled i f hydrolysis with TPA had removed a l l of the t-butyloxycarbonyl groups from the dipeptide. An aliquot of Met - 1 4c-Pro was formylated as described e a r l i e r to produce P-Met-1^C-Pro. b) HC-Met-Pro-Arg and F- 1 4c-Met-Pro-Arg I t was decided to prepare these peptides by coup-l i n g 1^.-methionine to unlabelled protamine. This could be done without blocking any groups on protamine since the secondary amino group of the N-terminal proline i s the only group i n protamine which i s s i g n i f i c a n t l y reactive with the activated ester employed below. Protamine propionate was prepared by applying 30 mg of protamine sulphate to a QAE-Sephadex column (1X10 cm) i n the 0BT form and eluting the column with 0.2 N propionic acid. The eluate was then l y o p h i l i z e d . This conversion of protamine from the sulphate to the propionate was an attempt to make i t more soluble i n the solvent system described below. 10 microcuries of Ho-methionine (uniformly l a b e l l e d , 233 mCi/mMole, Amersham) was formylated and coupled to -121-F i g . 6 Autoradiogram of Electrophoretogram of Reaction Products i n the Synthesis of Met- 1*C-Pro © 0 Note: 0, o r i g i n ; 1, t-butyloxycarbonyl-methionyl-14(3-proline; 2, free 14C-proline; 3, Met-14(3-Pro. Band #3 was eluted with 30$ acetic acid. Electrophoresis was at pH 3.6. - 1 2 2 -F-hydroxysuccinimide by dissolving 2 mg of the l a t t e r i n 0.2 ml of dioxane and adding i t to the F-14-C-Met; then 1 mg of dicyclohexylcarbodiimide was added and the mix was i n -cubated at 15 °0 for 15 minutes and overnight at 4 °0. The supernatant was aspirated from the cr y s t a l s of dicyclohexyl-urea and dried. The residue, containing the N-hydroxysuccin-imide ester of F-14-C-Met, was dissolved i n 0.7 ml of 95% ethanol. 30 mg of protamine propionate i n 0.7 ml of water and 1 mg of NaHOO^ vwas added to the ester s o l u t i o n and mixed. A cloudy mixture resulted which was p a r t i a l l y c l e a r -ed by addition of 0.2 ml of dioxane. The mix was shaken at 20 °C for 22 h and then diluted 5 times with water and ad-sorbed to a column of Amberlite IRC-50 i n the H + form. The column was then washed with water to 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 eluted 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 at 37 °G: for 30 minutes to de-esterify any s e r y l residues that might have reacted with the F- 1 ^O-methionyl-Ff-hydroxysuccinimide ester. The incubation mix was then dried, redissolved and chroma-tographed on Sephadex G-10 i n 0.2 M acetic acid; the void 1 f r a c t i o n was l y o p h i l i z e d and contained 680,000 0PM for a y i e l d of 7%. The low y i e l d was probably due to the i n s o l u -b i l i t y of the reactants but enough reaction did occur to give a useful product. The product was digested with porcine t r y p s i n (1:20, - 1 2 3 -w/w) i n 0.2 M M^HOO^ at 37 °C f o r 4 h and dried i n vacuo. A portion was treated with 1 IT HOI i n methanol at 50 °G f o r 1 h and dried. Since the N"-terminal sequence of a l l major trout protamine components i s Pro-Arg-Arg.. . (261 ), the above procedures gave the t r y p t i c peptides F- 14c-Met-Pro-Arg and Hc-Met-Pro-Arg. 14. Carboxypeptidase B Digests Vigorous digestion with carboxypeptidase B was found to cleave the Pro-Arg bond i n the t r i p e p t i d e , Met-Pro-Arg. Suitable conditions were an enzyme:substrate r a t i o of 1:1 (w/w) and incubation i n 0.2 M FEMO buffer (0.2 M N-ethyl morpholine i n water adjusted to pH 8.5 with acetic acid) at 37 °C for 6-8 h. 15. Pulse-Chase Experiments Early experiments involved incubating t e s t i s c e l l s from protamine-stage trout with 35s-methionine and -arginine f o r 30-60 minutes, washing the c e l l s and con-tinuing the incubation i n fresh medium with unlabelled amino acids. At i n t e r v a l s , aliquots of c e l l s were removed and frozen 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 labels was measured. In these experiments i t was not possible to show conclusively a loss of methionine from protamine dur-ing the "chase" period; i n f a c t , the s p e c i f i c a c t i v i t y of the methionine l a b e l i n protamine continued to increase for -124 about 30 minutes early i n the chase period. This indicated that 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 of l a -belled methionine that could not be r a p i d l y diluted with exogenous unlabelled methionine. Success i n demonstrating the removal of methionine from newly synthesized protamine required shorter l a b e l l i n g periods of 5-10 minutes at 17 °0, sedimenting the c e l l s and resuspending i n fresh medium with unlabelled amino acids. Incubation for the chase period was continued at 17 °C. Further d e t a i l s of two experiments w i l l be discussed. Experiment A A c e l l suspension from 15 g of n a t u r a l l y maturing trout t e s t i s was prepared. The suspension was divided i n h a l f ; 4 microcuries/ml of 35s-methionine (573 mCi/mMole, Amersham) was added to one h a l f and 4 microcuries/ml of ^Hr-arginine (1.2 Ci/mMole, New England Nuclear) was added to the other h a l f . Incubation was at 18 °C f o r 10 minutes, the c e l l s were then washed, resuspended i n Hank's solut i o n made 1 mM i n unlabelled methionine and arginine and incubation was con-tinued at 18 °C f o r 2 h. Equal aliquots of c e l l s were r e -moved at i n t e r v a l s s t a r t i n g at the beginning of the chase period. These were frozen and l a t e r a l l were processed by homogenizing i n saline-EDTA (0 .075 M NaCl, 0.024 M EDTA, pH 8.0) and nu c l e i were pelleted at 3000 g for 10 minutes. The nuclear p e l l e t s were extracted twice with 0.2 N HC1 at 4 °0 by homogenizing with a TRI-R tissue homogenizer (TRI-R -125-Instruments, New York). The acid extracts were neutralized with LiOH and insoluble material removed by centrifugation at 13,000 g f o r 15 minutes. The supernatants were adsorbed to i n d i v i d u a l CM-cellulose columns ( 1 . 5 X 5 cm) equilibrated with 0.2 M L i O l . The columns were then washed with 0.2 M followed by 0 . 7 5 M L i O l u n t i l no l a b e l was present i n the eluate. The columns were then washed with water or 0.01 M lithium acetate to remove most of 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 eluted with 0.2 N HC1 and l y o p h i l i z e d . The protamine samples were then free of a l l free l a b e l l e d amino acids and a l l other pro-teins except a small amount of histones; the l a t t e r were removed by chromatography of each sample on Bio-Gel P-10 columns (2.6X15 cm) eluted with 0.2 M acetic acid. The 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 r e d i s s o l v -ed i n d i s t i l l e d water; t h e i r absorbance at 230 nm was mea-sured and aliquots were counted i n Bray's solution. Experiment B A c e l l suspension was prepared from hormonally induced trout testes. The c e l l concentration i n the suspension was measured with the use of a hemocytometer and the suspension was diluted to 10^ cells/ml i n Hank's so l u t i o n . A 1.6 ml aliquot of c e l l s was incubated i n a small glass tube at 17 °C f o r 6 minutes i n the presence of 100 microcuries of 35s_ m ethionine (690 mCi/mMole, Amersham) and 100 microcur-ies of ^H-arginine (1.85 Oi/mMole, Amersham). At 6 minutes, -126-0.1 ml of c e l l s was removed and injected into 5 ml of cold TCA-tungstate (10% TCA, 0.25% sodium tungstate, pH 2, Ref. 265). The remainder of the c e l l s were washed b y c e n t r i f u g -ing at 1000 g for 10 minutes and resuspending i n 3 ml of fresh medium containing a l l 20 unlabelled amino acids. In-cubation was continued at 17 °C and 0.2 ml aliquots of c e l l s were removed at inter v a l s s t a r t i n g immediately aft e r resus-pension. Cycloheximide was added at 60 minutes of the chase to stop any further protamine synthesis even though i t was known that t h i s was probably too l a t e to make any d i f f e r -ence i n the apparent rate of methionine removal. This i n -h i b i t o r was not added e a r l i e r i n the experiment since i t might have a c t u a l l y interfered with methionine removal; cy-cloheximide i n h i b i t s eukaryotic transferase 2 by reaction at i t s ess e n t i a l SH group and might therefore i n t e r f e r e with the putative methionine aminopeptidase (281 ). In r e -trospect, the cycloheximide addition was not necessary as v i r t u a l l y a l l of the methionine had been removed from pro-tamine by 30 minutes of the chase. The method of A. Louie and G.H. Dixon ( i n preparation) for c o l l e c t i n g small numbers of c e l l s and extracting, sep-arating and assaying the r a d i o a c t i v i t y i n t h e i r basic pro-teins was employed as follows: 2X10*7 c e l l s were f i l t e r e d onto glass f i b r e f i l t e r s and washed with 40 ml of cold 10% TCA-tungstate and 10 ml of cold 95% ethanol. The sec--127-tions of the f i l t e r s bearing the c e l l s were cut out ( 4 X 8 mm) and placed i n the s l o t s of a starch-urea-aluminum l a c t a t e gel with 0.05 ml of 0 . 4 N HC1 and electrophoresis was per-formed at 8 volts/cm for 1 0 h at 4 ° C The gel was then s l i c e d , stained and counted as described e a r l i e r i n t h i s section. 16. Inhibitor Studies A c e l l suspension was prepared from 14 g of na t u r a l l y maturing trout t e s t i s and divided into 12 equal aliquots. Each was incubated at 4 °C f o r 3 0 minutes i n the pre-sence of various concentrations of cycloheximide or chlo r -amphenicol with control samples receiving no additions. -methionine ( 5 7 3 mCi/mMole, Amersham) was then added to each sample at 3 microcuries/ml and incubation continued at 18 °C for 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 dry i c e bath. C e l l p e l l e t s were homogenized i n TMKS using the TRI-R tissue homogenizer at high speed f o r 2 minutes and n u c l e i were then sedimented at 3 0 0 0 g for 1 0 minutes. The nuclear p e l l e t s were extracted by homogenizing i n cold 0 . 2 M H 2 S O 4 followed by centrifugation at 1 3 , 0 0 0 g for 15 minutes; basic proteins were precipitated by addi-t i o n of 4 volumes of 9 5 % ethanol to the supernatants above and standing at - 2 0 °G overnight. The precipitates were colle c t e d by centrifugation, redissolved i n 0 . 0 1 M l i t h i u m acetate and adsorbed i n d i v i d u a l l y to a series of CM-cellulose columns ( 1 . 5 X 5 cm) i n the H + form. The columns were washed - 1 2 8 -with 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 the eluate and then washed with water and f i n a l l y the t i g h t l y hound protamine was eluted with 0.2 N HC1 and l y o p h i l i z e d . The samples were redissolved i n d i s t i l l e d water and the absorbance at 230 nm measured: aliquots were counted i n Bray's s o l u t i o n . A s i m i l a r experiment to the above was done to test the effect of aminopterin (obtained from the Drug Develop-ment Branch, Cancer Chemotherapy, National I n s t i t u t e of Health, Bethesda, Maryland) on protamine synthesis. A c e l l suspension was prepared from 12 g of n a t u r a l l y matur-ing trout t e s t i s and divided into 6 equal aliqu o t s . Each was incubated at 18 °C f o r 30 minutes i n the presence of various concentrations of aminopterin with controls r e c e i v -ing no additions, ^ s^e-fchionine (500 mCi/mMole, Amersham) was then added to each sample at 5 microcuries/ml and in c u -bation was continued at 18 °C f o r 1 h. The c e l l s were then c h i l l e d , washed and frozen i n a dry i c e bath. Further pro-cessing was i d e n t i c a l to that i n the experiment above. 17. Nascent Peptides a) Puromycin-rel eased Nascent Peptides 15 g of n a t u r a l l y maturing trout t e s t i s (October stage) was used to prepare a c e l l suspension. Half of the suspension was incubated with 3 microcuries/ml of 35s-methionine (500 mCi/mMole, Amersham) at 18 °C f o r 10 min-utes. The other h a l f was incubated with 4 microcuries/ml - 1 2 9 -of 1 4C-formate (45.8 mOi/mMole, sodium s a l t , Amersham) at 18 °0 for 10 minutes. Puromycin was then added to both sam-ples at a concentration of 5X10""^ M and incubation was con-tinued at 18 °0 for 1 h. The c e l l s were c h i l l e d , washed and frozen. The c e l l p e l l e t s were homogenized i n TMKS and the homogenates centrifuged at 20,000 g f o r 15 minutes. The supernatants were placed i n tubes, underlayed with 6 ml of 2 M sucrose i n TMK and centrifuged at 22,000 rpm f o r 10 h i n the SW-27 rotor of the Spinco L2-65 ul t r a c e n t r i f u g e . The upper two-thirds of the supernatants were saved. Columns (1X5 cm) of Dowex-50 i n the H + form and QAE-Sephadex i n the 0H~ form were prepared. The pH of the supernatants was adjusted to 2 and they were adsorbed on the Dowex-50 columns. These columns were then washed with 0.01 N HG1 and the eluates were saved and pH adjusted to 8 with NaOH. The l a t t e r were adsorbed to the QAE-Sephadex columns and washed with water. The Dowex-50 columns were f i n a l l y eluted with 4 M; pyridine and the eluates were di l u t e d and l y o p h i l i z e d . The QAE-Sephadex columns were eluted with 2 M. acetic acid and the eluates 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 digested with por-cine t r y p s i n or t r y p s i n followed by carboxypeptidase B'. The digests were electrophoresed at pHf. 3.6 and an autoradiogram performed. Certain l a b e l l e d spots were cut out and r e - e l e c t r o -phoresed at pH 6.5 and an autoradiogram performed. Marker peptides were included i n both electropheretograms. - 1 3 0 -b) Ribosome-bound Nascent Peptides A c e l l suspension from 12 g of hormonally induced trout t e s t i s was incubated at 15 °C f o r 20 minutes with 20 microcuries/ml of 35s-methionine (690 mCi/mMole, Amer-sham). At 15 minutes cycloheximide was added to 2X10""4 M i n order to arrest nascent peptides on the ribosomes during the subsequent steps; otherwise, the chains might be com-pleted and released and i n the absence of further i n i t i a -t i o n , l i t t l e nascent protein would remain on the ribosomes. At 20 minutes, the c e l l s were washed: and then homogenized i n modified TMKS sol u t i o n CpH 6.8) containing 2X10"4" cyclo-heximide using the TRI-R tissue homogenizer at medium speed fo r 1 minute. The homogenate was centrifuged at 15,000 g f o r 10 minutes; the supernatant was adjusted to 0.5$ i n T r i t o n X-100 by addition of a 20$ s o l u t i o n of the detergent i n TMK and recentrifuged at 15,000 g f o r 20 minutes. The super-natant was layered over 2 ml of 30$ sucrose i n TMK i n 13 ml tubes and centrifuged at 60,000 rpm i n the Spinco L2-65 rotor f o r 2^ h at 4 G0. The supernatant was gently a s p i r a t -ed and the ribosome p e l l e t s washed gently with TMK. The ribosomes were resuspended i n 6 ml of TMK by use of a Pasteur pipette and any clumps were removed by c e n t r i f u -gation at 13,000 g f o r 10 minutes. The supernatant was d i -vided into three 2 ml aliquots and layered over each of three tubes containing l i n e a r gradients of sucrose from 10 to 30$ i n TMK; the gradients had been formed using a -131-Beckman density gradient former (DGF-IM-3). The tubes were centrifuged at 25,000 rpm i n the Spinco SW-27 rotor for 3-J- h and stopped without braking. The tubes were punctured at the bottom and 1 ml fractions c o l l e c t e d . The absorbance at 254 nm of each f r a c t i o n was measured and 0.2 ml aliquots were counted i n Bray's solution.-The sucrose gradient fract i o n s were pooled i n groups from the monosome and polysome regions, d i l u t e d with TMK CpHi6.8) and centrifuged at 25,500 rpm i n the SW-27 rotor f o r 6 h at 4 ° C The supernatants were decanted and the ribosome p e l l e t s treated as follows: i , Monosomes and disomes were suspended i n 1 ml of 0.2 M NH4HOO3 and digested with 0.05 mg of pancreatic RNase at 37 °C for 15 minutes and then l y o p h i l i z e d . The residue was treated with 0.2 M ethylenediamine, pH 10 at 37 °0 for 15 minutes to hydrolyze nascent peptides from adenosine; t h i s i s a modification of the method of Acs and Lipmann (297). The pH was then adjusted" to 5.Olwith-acetic-acid and some insoluble material was removed by centrifugation. The su-pernatant was dried and digested with 0.05 mg of porcine t r y p s i n i n 0.25 ml of 0.2 M NH4HC05 at 37 °G for 3 h. One-t h i r d of the digest was further treated with 0.1 mg of CpBi at 37 °0 for 4 h. The digests were pooled, dried, r e d i s s o l v -ed and electrophoresed at pH 3.6 together with marker pep-tides. An autoradiogram was performed to locate the rad i o -active marker peptides and the sample s t r i p s were cut at - 1 3 2 -0.5 inch int e r v a l s and counted i n toluene s c i n t i l l a t i o n f l u i d . As outlined i n F i g . 5 , the remainder of the sample peptide regions were cut out, sewn into a new sheet of pa-per and re-electrophoresed at pH 1 .9 with marker peptides. The paper was again cut and counted as above. i i Ribosomes from 3 regions of the sucrose gradient were incubated i n 0 .5 ml of 1 M NH^OH at 37 °0 for 30 min-utes to hydrolyze nascent peptides from tRNA. TCA was then added to 10% and,, aft e r standing at 4 °C f o r 1 h, the tubes were centrifuged at 13 ,000 g f o r 10 minutes. The supernatants were extracted 3 times with ether to remove TCA and then l y -ophilized. The residues, containing 35s-methionine l a b e l l e d nascent peptides, were submitted to Edman degradation as described e a r l i e r . Aliquots of ethyl acetate 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 for enzyme a c t i v i t i e s capable of removing methionine from protamine and the dipeptide, Met-Pro; t e s t i s extracts were also assayed f o r the a b i l i t y to remove acyl groups from methionine and the dipeptide, Met-Pro. Enzyme extracts were prepared from frozen October t e s -tes which had been stored at - 8 0 °G. The testes were par-t i a l l y thawed and then homogenized in. TMKS containing IO - 4 - M Cleland's reagent ( d i t h i o t h r e i t o l ) i n a Waring blendor at high speed f o r 2 minutes. Nuclei were pelleted at 1500 g -133-for 10 minutes, mitochondria at 20,000 g f o r 30 minutes and ribosomes at 25,000 rpm i n the SW-27 rotor for 4 h. Nuclei and mitochondria were lysed i n TMK (pH 7.0) and centrifuged at 20,000 g for 20 minutes, saving the superna-tant. Ribosomes were washed with 1 M NH4CI i n TMK overnight and then centrifuged at 26,000 rpm i n the SW-27 rotor f o r 6 h, saving the supernatant. A l l extracts were preci p i t a t e d at 90% saturation with ammonium sulphate; the precipitates were co l l e c t e d by centrifugation, redissolved i n TMK (pH 7.0) and dialyzed against the same buffer at 4 °C. Material which precipitated during d i a l y s i s was removed by centrifugation. The extract, volumes were each measured and then divided i n t o several small batches; some were frozen at -80 °0 and some were stored at 4 °0 and used within a week, a) Assay f o r methionine removing enzyme i ^Snnethionine 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 with this l a b e l as described i n section #3 above. 1 4 c-arginine l a b e l l e d protamine was prepared i n the same fashion. The nuclear lysate and the postmitochondrial supernatant fractions of t e s t i s extracts were assayed as follows: 0.01 ml of l a b e l l e d protamine i n water, 0.02 ml of 2 M FaCl i n water, 0.1 ml of extract and TMK up to a t o t a l volume of 0.2 ml were mixed and incubated at 15 °0 for 40 minutes. Reaction was stopped by c h i l l i n g and addition of 0.02 ml of 3.4 N, HC1. A 0.1 ml aliquot of each reaction mix was applied to f i l t e r paper discs which -134-were plunged i n cold TCA-tungstate (10$ TCA, 0.25$ sodium tungstate, pH 2), washed with the same, boiled f o r 1 min-ute i n the same, washed again and then washed with 95$ eth-anol and f i n a l l y with ether. The dried discs were counted i n toluene s c i n t i l l a t i o n f l u i d . i i Met- 14c-Pro was prepared as described i n sec-t i o n #13 above. Assay was done by incubating 0.05 ml of enzyme extract ( i n TMK, pH 7.0) with 0.01 ml of l a b e l l e d dipeptide i n water at 16 °C f o r 45 minutes. Reaction 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 applied to paper and electrophoresed at pH 3.6 and 40 volts/cm f o r 20-40 minutes. Reaction products were located by autoradiography, cut out and counted i n toluene s c i n t i l l a t i o n f l u i d , b) Assay f o r "Deformylase" Acylated amino acids were prepared by mixing a small amount of l a b e l l e d amino acid with a large amount of the same unlabelled amino acid and then performing the acylation reaction. Thus, 20 micromoles of an unlabelled amino acid plus approximately 0.01 micromoles of unlabel-l e d amino acid were dried i n a tube and redissolved i n 0.4 ml of 0.2 M NEMO buffer (pH 8.5). Then, 0.05 ml of the appropriate acid anhydride was added and reaction allow-ed at 20 °C for 1 h. To ensure complete acylation, 0.05 ml each of r e d i s t i l l e d pyridine (concentrated) and 0.05 ml of the acid anhydride were added i n that order and the tubes -135-were shaken at 20 °0 overnight. Each tube was then extract-ed 3 times with 5 ml portions of ether to remove the excess reagents; butyric, v a l e r i c and hexanoic acids a l l formed emulsions during the reaction and had to be removed by the ether extractions since they could 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 redissolved i n 5 ml of TMK (pH 7.0). Thus, each preparation was 4X10~ 5 M i n acy-l a t e d amino acid . Assay was by incubating 0.025 ml enzyme extract, 0.025 ml TMK and 0.025 ml of 4X10"3 M substrate at 20 °0 for 30 min-utes. Reaction was stopped by c h i l l i n g and addition of 0.01 ml of 1 N KOH. The samples were then applied to paper and electrophoresed at pH 6.5 and 40 volts/cm f o r 30 minutes. Products were located by autoradiography, cut out and count-ed i n toluene s c i n t i l l a t i o n f l u i d . The reactions were stopped with KOH because the acylated substrates would be neutral i f acid was used and might co-precipitate with the protein, i n the incubation mix. -136-RESULTS AFP DISCUSSION Incorporation of Methionine A c e l l suspension was prepared from 25 g of n a t u r a l l y maturing trout t e s t i s (December stage) and was incubated with 1 microcurie/ml of 1 ^-c-methyl-methionine (11 mCi/mMole, New England Nuclear) at 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 pelleted at 3000 g f o r 10 minutes. The nuclear p e l l e t was extracted twice with 0.2 M B^SO^ and the acid-soluble proteins pre-c i p i t a t e d with addition of 4 volumes of 95$ ethanol and standing at -20 °C overnight. The p r e c i p i t a t e was r e d i s s o l v -ed and chromatographed on a column of Bio-Gel P-10 (5X 45 cm) eluted with 0.2 M acetic acid. The absorbance at 230 nm of each f r a c t i o n was measured and aliquots were counted i n Bray's s o l u t i o n . The results are shown i n Pig. 7 and i n d i -cate that methionine was incorporated into both the histones (peak 1) and protamine (peak 2). This r e s u l t was s u r p r i s i n g since routine amino acid analyses had not revealed the pre-sence of any methionine i n protamine. To ensure that the l a b e l l e d methionine was ac t u a l l y i n protamine and not i n some other protein eluting at the same pos i t i o n on the Bio-Gel P-10 column, an aliquot of peak 2 (Pig. 7) was electrophoresed with an unlabelled protamine marker on a starch-urea-aluminum l a c t a t e gel at 6 volts/cm for 12 h. The gel was s l i c e d , stained and counted as des-cribed i n section #4 of "Methods" except that the small - 1 3 7 -* i g . 7 ^ ^ C-methyl-methionine Incorporation Into Trout Testis Nuclear Basic Proteins Note: Polyacrylamide disc gel electrophoresis of peak 1 revealed histones and peak 2 had only protamine. -138-gel s l i c e s were dissolved i n 0.4 ml of tetraethylammonium hydroxide and counted i n Bray's s o l u t i o n containing 3$ Cab-O-Sil (w/v). The re s u l t s shown i n F i g . 8 indicate that a l l of the r a d i o a c t i v i t y coelectrophoresed with the unla-belled protamine or just behind i t ; newly synthesized pro-tamine i s phosphorylated (267,268) and migrates slower t o -wards the cathode at pH 3.4, the pH of the g e l , than older dephosphorylated protamine (269) which i s the predominant form i n the marker. The gel s l i c e s here were probably too wide (0.7 cm) to reveal separation of phosphorylated pro-tamine components: as observed recently by Louie and Dixon (unpublished r e s u l t s ) . However, the r e s u l t s here indicate that the l a b e l l e d methionine was incorporated into prota-mine . Since the methyl group of methionine r e a d i l y enters the 1-carbon pool, the 1^0-methyl l a b e l could have entered pro-tamine as serine or as a methyl group on a residue such as arginine (278). Accordingly, an aliquot of peak 2 ( F i g . 7) was hydrolyzed with 6 N! HC1 at 110 °C for 22 h and the r e -s u l t i n g mixture of free amino acids was electrophoresed at pH 1.9 and 60 volts/cm for 45 minutes. Part of the sample s t r i p was stained with ninhydrin together with the region containing standard amino acids and the remainder of the sample region was cut at 0.5 inch 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-electrophoresed with the unlabelled methionine marker. This - 1 3 9 -Fig. 8 Electrophoresis of 1^c-methyl-methionine Labelled Protamine on a Starch-urea-aluminum lactate gel 1500-1000-500-DISTANCE cm 0 Note: 0, origin; P, position of unlabelled protamine marker on stained gel sli c e s . The middle gel s l i c e was cut at 0.7 cm intervals for counting. - 1 4 0 -experiment was repeated af t e r performic acid oxidation of the acid hydrolysate and the r a d i o a c t i v i t y coelectrophores-ed with an unlabelled methionine sulphone marker at pH 1 . 9 . These results showed that the 1/k<-methyl l a ^ e l w a s a l l incorporated 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 that methionine was involved i n the i n i t i a t i o n of protamine biosynthesis was obvious and sever-a l experiments were done to t r y to confirm t h i s . To determine whether the incorporated methionine was N-terminal, Edman degradation and dansylation were perform-ed on p u r i f i e d 35s-methionine l a b e l l e d protamine synthesized i n aasuspension of trout t e s t i s c e l l s . Table 4 shows that 76-89% of the 35s -methionine l a b e l can be recovered i n the ethyl acetate phase afte r one Edman degradation. To con-firm that a l l the l a b e l i n the ethyl acetate phase was PTH -35s-methionine (the phenylthiohydantoin derivative of 35s-methionine), an aliquot of the ethyl acetate phase was dried and treated as i n section #7 of "Methods". This i n -volved conversion of the thiazolinone derivative to the more stable PTH-derivative followed by thin-layer chromato-graphy on s i l i c a gel with standard PTH amino acids and -the solvent system, heptane:n-butanol:75% formic acid ( 1 0 0 : 6 0 : 1 8 , v/v). Sections of the thin-layer plate were counted and the res u l t s i n Pig. 9 indicate that a l l of the r a d i o a c t i v i t y - 1 4 1 -TABLE 4 Edman. Degradation of 35s-methionine Labelled Protamine Aqueous Ethyl Acetate % Experiment Phase Phase PTH^Met 1 1 ,660* 14,080* 89% 2 5,568 17,561 76 3 5,214 1 3 , 5 2 3 72 * CPM i n phase -142-* i g . 9 Thin-layer Chromatography of the Reaction Products of Edman Degradation of 35s-methionine Labelled Protamine t 0 2 3 4 5 6 7 DISTANCE cm 8 9 Note: Chromatography was for 2\ h i n the solvent system, heptane:n-butanol:75$ formic acid (100:60:18, v/v). Markers were located by t h e i r quenching of the fluorescent s i l i c a gel 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 . - 1 4 3 -co-chromatographed with the -unlabelled PTH-methionine marker. Thus, a l l of the counts i n the ethyl acetate phase must have come from the N-terminus of protamine. Dansylation was performed by the method of Gray (279) and aft e r acid hydrolysis and electrophoresis at pH 4 . 3 8 , the electropherogram was examined with UV l i g h t to locate the DNS amino acid markers. The sample s t r i p was cut at 0 .5 inch int e r v a l s and counted i n toluene s c i n t i l l a t i o n f l u i d with the results shown i n F i g . 10 . As can be seen, about 70$ of the 35s-methionine i n protamine was recovered as dansyl -35s-methionine. This confirmed the re s u l t s using the Edman degradation and indicated that most, i f not a l l , of the methionine incorporated i n protamine i s present at the N-terminus. Protamine i s insoluble i n the solvents employed i n these N-terminal methods and one would there-fore not expect quantitative y i e l d s of the terminal amino acid under these conditions. Structure of the Methionine Peptide When 35s-methionine-protamine synthesized i n t e s t i s c e l l s i s digested with t r y p s i n and the r e s u l t i n g peptides electrophoresed at pH 3 . 6 , a s i n g l e basic peptide, A, i s seen on the autoradiogram as i l l u s t r a t e d i n F i g . 11, A trace of a peptide that i s only s l i g h t l y basic and which was o r i g i n a l l y thought to be formylated (280) has been seen occasionally but both itsand the major peptide change -144-F i g . 10 Electrophoresis at pH 4.38 of the products of dansylation of 35s-methionine l a b e l l e d protamine UJ O I 30,000 20,000 ELECTROPHORESIS pH 4 3 8 o DNS-MET . DNS-MET SO, —T 1 1 1 I I I 1 1 1 1 1 1 1 1 I 1 € 4 2 0 2 4 6 8 10 DISTANCE ( I N C H E S ) Note: Upper frame, an aliquot of 35s-methionine protamine was dansylated, hydrolyzed i n acid and electrophoresed at pH 4.38 (Ref.279). Lower frame, free 35s-methionine. -145-F i g . 11 Autoradiogram of Electrophoretogram (pH 3.6) of Enzyme Digests of 35s-methionine Labelled Protamine 0 V t MP B 0 : Origin, A: Tryptic peptide, B: Product of action of CpB on peptide A, MP: Met- 14c-Pro marker peptide. - 1 4 6 -mobility af t e r acetylation and therefore neither can be formylated; the minor peptide i s possibly the r e s u l t of hydrolysis at the Pro-Arg bond by a contaminant of t r y p s i n under the vigorous digestion conditions used. Fig. . 1 1 shows that further digestion of peptide A with CpB yields a l a b e l -l e d peptide, B, which coelectrophoreses with the Met- 1 ^c.-Pro marker; some i n t a c t peptide A remains afte r CpB since the Pro-Arg bond i s attacked only slowly by t h i s enzyme. These results suggest a tentative sequence f o r peptide A of Met-Pro-Arg. In addition, since t r y p s i n alone yields a single l a b e l l e d peptide, a l l of the methionine enters only one sequence i n protamine and taken with the N-terminal data, the conclusion i s that t h i s must be the N-terminal t r y p t i c peptide. Chemical deformylation of the F-14-C-Met-Pro-Arg marker by treatment with anhydrous HC1 i n methanol as i n section #12 of "Methods" gave a product which streaked during subse-quent electrophoresis at pH 3 . 6 . Therefore, an alternative approach was taken whereby 35s-methionine protamine bi 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 chemically f o r -mylated (276) i n order to compare the l a b e l l e d t r y p t i c pep-tide from i t with synthetic formylated marker&peptides. Thus, the chemically formylated, b i o s y n t h e t i c a l l y l a b e l l e d 3 5 s -methionine protamine was digested with t r y p s i n and a portion further digested with CpB. The digests were then electrophoresed at pH 3 . 6 and an autoradiogram i s shown i n - 1 4 7 -Pig. 12 . Trypsin alone yielded a weakly basic peptide, Af, 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 chemically formylated protamine which coelectrophpresed with the P - ^o-Met-Pro-Arg marker. This was confirmed by re-electrophoresis at pH 1.9 as shown i n Pig. 13 . The P- 1 4C-Met-Pro-Arg marker had been through several procedures as outlined e a r l i e r and existed mainly i n the sulphoxide form which migrates s l i g h t l y slower at pH 1.9 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 portion of the marker have i d e n t i c a l m o b i l i t i e s . Trypsin followed by CpB gave a l a b e l l e d peptide, Bf, which coelectrophoreses with the P-Met-1 4C-Pro marker at pH. 3 .6 (Pig. 12) and upon re-electrophore-s i s at pH 6 .5 (Pig. H a ) and upon descending chromatography (Pig. H h ) . I t i s clear, therefore, that methionine i s i n -corporated into protamine i n the N-terminal sequence Met-Pro-Arg... during the biosynthesis of protamine i n trout spermatid c e l l s . Generality of Methionine Incorporation The p o s s i b i l i t y remained that methionine was only i n -corporated into a minor component of protamine rather than into a l l components as would be required i f i t played a general role i n the i n i t i a t i o n of synthesis of protamine. A c e l l suspension was therefore incubated with 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 - 1 4 8 -Pig. 12 Autoradiogram of Electrophoretogram (pH 3 . 6 ) of Enzyme Digests of Chemically Pormylated 35s-methionine Protamine A f: Tryptic peptide, B f: Product of action of CpB on peptide A f, Markers: 2, F-Met-HC-Ero; '» Met-HC-Pro 4, F- 1 4C-Met-Pro-Arg; 5 , 1 *C-Met-Pro-Arg. -149-F i g . 13 Re-electrophoresis at pH 1.9 of peptide A f and the marker peptide, P-Hc-Met-Pro-Arg, from Pig. 12. : e I A , © 0 Note: Af, t r y p t i c peptide of b i o s y n t h e t i c a l l y l a b e l l e d , chemically formylated 35s_me+,hionine protaminei 4, F-Hc-Met-Pro-Arg marker peptide. 0, o r i g i n . - 1 5 0 -Pig. 14 a) Autoradiogram of Electrophoretogram (pH 6.5) of Peptide B f from Pig. 12. b) Autoradiogram of Descending Chromatogram of Peptide B^ from above (butanol/acetic acid/water/pyridine, 15:3: 12:10, v/v, f o r 16 h). • 4 #-§ < - 1 5 1 -chromatographed on a CM-cellulose column eluted with a l i n e a r gradient of L i C l i n order to achieve p a r t i a l separa-t i o n of the protamine components as previously described (265) . The e l u t i o n diagram i n Pig. 15 shows incorporation of the l a b e l across the o p t i c a l density p r o f i l e with 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 previously, newly synthesized protamine i s phosphorylated on i t s s e r y l residues and t h i s modification affects the e l u t i o n behavi-our on CM-cellulose (268) . Therefore, another preparation of 35s_methionine protamine was digested with E. c o l i a l k a -l i n e phosphatase (Worthington) and chromatographed on a column of CM-cellulose under s l i g h t l y d i f f e r e n t conditions from those used i n Pig. 1 5 . As can be seen i n Pig. 16 , the l a b e l was s t i l l incorporated across the o p t i c a l density pro-f i l e with 3 major peaks and 3 or 4 minor peaks. C l e a r l y then, methionine i s incorporated into several protamine components and must be of general importance i n t h e i r biosynthesis. Transient Nature of Methionine Incorporation In b a c t e r i a l systems where the r o l e of N-formyl-methio-nine i s well established i n protein chain i n i t i a t i o n (185) , the N-terminal methionine i s usually removed after chain completion. To t e s t for methionine removal i n the t e s t i s system, c e l l suspensions were pulsed with l a b e l l e d arginine and methionine and then chased i n f r e s h medium with unlabel-l e d amino acids as described i n section #15 of "Methods". -152-F i g . 15 Ionr-Exchange Chromatography of 35s-methionine Labelled Protamine on CM-cellulose C M C 2 - 9 x ae C M - L I N E A R G R A D I E N T L i C I F R A C T I O N NO-Note: CM-cellulose column (2.5X26 cm) eluted with a l i n e a r gradient of LiCI generated from 600 ml each of 0.6 and 1.3 M LiCI i n 0.01 M li t h i u m acetate (pH 5.0). - 1 5 3 -F i g . 16 lion-exchange Chromatography of Alkaline Phosphatase Treated -methionine Labelled Protamine on CM-cellulose. Note: CM-cellulose column (2X30 cm) eluted with l i n e a r gradient of LiCI generated from 500 ml each of 0.6 and 1.4 M LiCI i n 0.01 M l i t h i u m acetate (pH 5.0). Fraction volumes about 10 ml each. 25 mg of l a b e l l e d prota-mine digested with 0.25 mg E. c o l i a l k a line phosphatase f o r 2 h at 37 °C in. 0.2 M NH4HCO3. - 1 5 4 -F i g . 17 shows the re 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 both labels i n protamine were follow-ed f o r 2 h aft e r a 1G minUte pulse. The incorporated a r g i n -ine i s stable during the chase i n d i c a t i n g that protamine i s not degraded a f t e r i t s synthesis; however, the s p e c i f i c ac-t i v i t y of methionine (35s) f a l l s during the chase by about 50% at 2 h. Even aft e r a 10 minute pulse one may note that the s p e c i f i c a c t i v i t y of methionine i n protamine continues to r i s e f o r the f i r s t 30 minutes of the chase. Fi g . 18 and Table 5 show the re s u l t s of Experiment B i n which a 6 minute pulse was followed by a 5% h chase. Bas-i c proteins were fractionated on a starch-urea-aluminum l a c t a t e gel and the gel s l i c e s were counted for 35s-methio-nine and -arginine by counting i n two channels i n a Unilux l i q u i d s c i n t i l l a t i o n counter (counting at A-JQ and H^Q, A standard 35s-methionine sample overlapped into the lower channel to the extent of 27 .3$ of the upper channel counts whereas no 3 H -arginine counts entered the upper channel). On the stained gel s l i c e s , mature dephospho-protamine mi-grated 1 3 . 0 - 1 5 . 0 cm from the o r i g i n and "nascent" prota-mine, 1 0 . 0 - 1 3 . 0 cm. F i g . 18 shows a plot of the r a t i o of 35s-methionine to 3H^arginine i n gel s l i c e s at various times during the experiment. In. frames A - 0 , one may see several peaks mainly in. the region behind protamine where the newl'y synthesized, phospho-protamines migrate. The peaks i n the region from 7 . 0 - 1 0 . 0 cm are probably highly phosphorylated -155-17 Pulse-Chase Experiment A r 80 60 TIME (min) "600 -400 I C D < i X to < - 2 0 0 CL 1 CO Rote: The pulse was from minus 10 minutes to 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. -156-F i g . 18 Pulse-Chase Experiment B P DISTANCE FROM ORIGIN (cm) Frames represent: A, end of 6 minute pulse; 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 of chase. P, p o s i t i o n of unlabelled protamine marker on stained gel s l i c e s . -157-TABLE 5 Pulse-Chase Experiment B: Total Total GPM 35s-Met per Time 3H-Arg CPM 35s,Met 0PM 1000 0PM 3H-Arg End of pulse 2931 5883 2010 Minutes a f t e r resuspension: 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 Note: Total CPM refers to the t o t a l r a d i o a c t i v i t y i n the gel 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 of 3R^arginine i s prob-ably due to the d i f f i c u l t y i n wetting the glass f i b r e discs i n the gel s l o t s and thus a variable amount of basic proteins i s extracted by the acid i n the s l o t s . -158-protamine components which have been observed i n th i s r e -gion i n recent experiments of Louie and Dixon (unpublished r e s u l t s ) . By 30 minutes of the chase, both the Met:Arg r a t i o ( F i g . 18, frame D) and the absolute amount of methionine i n the protamine region (Table 5) had f a l l e n markedly. This rapid removal of methionine from newly synthesized prota-mine must mean that the c e l l s used i n this experiment had very active methionine aminopeptidase a c t i v i t y . The c e l l s were from hormonally induced trout t e s t i s i n which matura-t i o n i s accelerated and th i s may explain the rapid removal,, of methionine. The fa s t e r apparent r a t e o f removal .;> during:;^ the period 10-30 minutes as compared with the period 0-10 minutes may be due to continued incorporation of 35 S -methio-nine from c e l l pools during the early chase period. This could be prevented by early addition of cycloheximide at the beginning bf the chase. Experiment A ( F i g . 17) measured the rate of methionine removal i n protamine present i n chromatin whereas experiment B measured the removal from whole-cell protamine; the l a t t e r would include newly synthesized protamine not yet trans-ported into the chromatin. Thus, experiment A shows that methionine can be removed from completed protamine even i n chromatin! since t h i s experiment measured methionine removal' only i n chromatin, t h i s may explain the slower rate of r e -moval than i n experiment Bi Cytoplasmic protamine.may have -159-i t s N-terminal methionine removed faster 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 cleaving the Met-Pro bond i s predominantly i n the cyto-plasm although 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 nucleus. Nascent Protamine a) Puromycin Released Nascent Peptides As described i n section #17 of "Methods'1, t e s t i s c e l l suspensions were incubated with 14C-formate or 35s-methio-nine f o r 1 0 minutes, puromycin was added to 5X1 0 ~ 4 M and incubation was continued f o r 1 h. Since puromycin causes premature release of nascent protein chains by acting as an analogue of the 3 ' terminus of aminoacyl-tRNA (298) , i t was f e l t that this would be a means to determine i f the methionine incorporated at the N-terminus of protamine i s formylated. Thus, l a b e l l e d products i n the soluble cytoplasm from the above incubations were fractionated on ion-exchange columns, digested with tr y p s i n or t r y p s i n and GpB,, e l e c t r o -phoresed with marker peptides and autoradiograms performed. F i g . 19 shows the re s u l t s of electrophoresis at pHi 3 .6 of the 35s-methionine l a b e l l e d products of the above pro-cedures. 'Peptide'A has the mo b i l i t y of Met-Pro but was not further investigated. 'Peptide' B. migrated close to the F^Met-14(j-Pro marker at th i s pH but when eluted from the paper and re-electrophoresed at pH 6 . 5 , the r e s u l t i n F i g . 20 indicates that 'peptide' B i s not i d e n t i c a l with F-Met- 1 4 0 --160-Fi g . 19 Autoradiogram of Electrophoretogram (pH 3.6) of 35s_ methionine Labelled Products i n the Cytoplasm of Testis Cells Incubated with Puromycin. Note: 0, o r i g i n ; 1, F - 1 4 c-Met marker; 2, P-Met-14-C-Pro marker; 3, Met-14-C-Pro marker. The 6 samples are, l e f t to r i g h t : Dowex-50 retained material - control, t r y p s i n and trypsin-CpB digests; and QAE-Sephadex retained material - c o n t r o l , trypsin and t r y p s i n -CpB digests. The 3 'peptides' to the l e f t of B were eluted and re-electrophoresed at pH 6.5 (Pig. 20). - 1 6 1 -Pig. 20 Re-electrophoresis at pH 6 . 5 of 'peptides 1 from Pigs. 19 & 21 f f • I I I - 2 Note: 0 , o r i g i n ; 1 , P-Met- 1 4C-Pro marker; 2 , P-14-C-Met marker. Samples are: B, 5 5 S -methionine l a b e l l e d material from Pig. 1 9 ; C-E, l 4C-formate l a b e l l e d material from Pig. 2 1 . -162-Pro. The trace of l a b e l l e d material from the 'peptide' B1 sample and migrating close to the P-Met-14-0-Pro marker i n Pig. 20 separated from the l a t t e r upon descending chromato-graphy. Pig. 21 shows the H o-formate l a b e l l e d products of the experiment above a f t e r high voltage electrophoresis at pH 3.6. 'Peptides' 0 and D have the mobility of the P-Met-H©-Pro marker and 'peptide' E has the m o b i l i t y of P-14-0-Met marker; however, when re-electrophoresed at pH 6.5 as shown i n Pig. 20, none of the sample 'peptides' coelectrophorese with the marker peptide or amino acid. The results above strongly suggest that the methionine at the Ni-terminus of nascent protamine i s never formylated as i t i s i n b a c t e r i a l systems as discussed;earlier;- The only other p o s s i b i l i t y i s that the formyl group i s removed very soon after chain i n i t i a t i o n and very r a p i d l y . b) Ribosome Bound Nascent Peptides Testis c e l l s from hormonally induced trout at the protamine stage of maturation were incubated with 35s_ methionine (20 microcuries/ml) f o r 20 minutes at 15 °C with addition of cycloheximide to 2X10" 4 M a f t e r 15 minutes of the incubation i n order to arrest nascent protein chains on the ribosomes. Ribosomes were then i s o l a t e d and f r a c t i o n -ated on 10-30$ sucrose gradients as described i n section #17 of "Methods". One of the gradients i s shown i n Pig. 22. One may see that methionine i s incorporated generally across -163-Pig. 21 Autoradiogram of Electrophoretogram (pH 3.6) of 1 4 C -formate Labelled Products i n the Cytoplasm of Testis Cells Incubated with Puromycin. I Note: 0, o r i g i n ; 1, P-Met-14-C-Pro marker; 2, P-Ho-Met marker; 3, Met-14-C-Pro marker. The 6 samples are, l e f t to r i g h t : Dowex-50 retained material - control, t r y p s i n and trypsin-CpB digests; QAE-Sephadex r e t a i n -ed material - control, t r y p s i n and trypsin-CpB digests The 2 'peptides' to the r i g h t of C and the 3 to the l e f t of I) and E were eluted and rerun (Pig. 20). - 1 6 4 -F i g . 22 35s-methionine Labelled Ribosomes Fractionated on Sucrose Density Gradients BOTTOM TOP FRACTION NO. Fractions 15 -27 were pooled for release of nascent protamine. Fractions 1 - 7 , 8-13 and 14 -20 from two other gradients were pooled f o r release of nascent peptides and Edman degradation. - 1 6 5 -the polysome region with the s p e c i f i c a c t i v i t y increasing with polysome s i z e ; that i s , CPM.of 35s-methionine per A254 increases towards the bottom of the gradient. The pattern i s sim i l a r to that observed for arginine incorporation at the November stage of natural maturation (266). Some of the increase i n s p e c i f i c a c t i v i t y i n higher polysomes could be due to the presence of nascent proteins with i n t e r n a l meth-ionine residues; data supporting t h i s hypothesis w i l l be presented below. Another reason f o r the higher s p e c i f i c a c t i v i t y of higher polysomes could be the nonspecific bind-ing of newly synthesized proteins including protamine to polysomes a f t e r completion of synthesis and release; i f this were the case, one would have to postulate .that the higher polysomes are capable of binding more protein per ribosome than lower polysomes. Unpublished work of G-ilmour, Louie and Dixon has revealed the presence of newly synthe-sized protamine r i g h t across the ribosome p r o f i l e as deter-mined by extraction of various regions and electrophoresis on starch-urea-aluminum lactate gels. The presence of pro-tamine across the ribosome p r o f i l e and extractable with acid implies that newly synthesized protamine i s binding non-s p e c i f i c a l l y ; p r i o r to chain completion and release, pro-tamine would not extract i n acid as i t would s t i l l be attach-ed to tRNA. I f protamine were synthesized on polysomes high-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 that on monosomes since one expects only one - 1 6 6 -nascent protein per ribosome. The monosome and disome peaks from one gradient were pooled, d i l u t e d and pelleted by centrifugation; nascent peptides were released by digestion with pancreatic RFase followed by mild alkaline hydrolysis. The released peptides were digested with t r y p s i n and a portion was further digest-ed with OpB. The pooled digests were electrophoresed at pH 3.6 with marker peptides; the markers were located by auto-radiography and the sample peptides by cutting out part of the sample s t r i p and counting as described i n "Methods". The r e s u l t s shown i n Pig. 23(a) suggested the presence of free methionine, Met-Pro and Met-Pro-Arg. This was confirm-ed by cutting out the remainder of the sample peptides above and re-electrophoresis at pH 1 . 9 with the r e s u l t shown i n Pig. 23(b). V i r t u a l l y a l l of the methionine l a b e l on the monosomes and disomes was therefore present as free methio-nine, Met-Pro or Met-Pro-Arg; there was only a trace of l a b e l i n the a c i d i c region i n Pig. 23(a) where the formyla-ted marker peptides migrate. Since the ribosomes were washed to remove any free methionine p r i o r to release of nascent peptides, the pre-sence of free methionine i n the material released from mono-somes 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 that i t might have been present as 35s-.Met-tRNAf * on the mono-somes as part of an i n i t i a t i o n complex. In b a c t e r i a l systems, the formyl group on P-Met-tRNAf i s not attacked by the de-- 1 6 7 -F i g . 23 a) Electrophoresis at pH 3 .6 of trypsin-CpB digests of 35s-methionine l a b e l l e d nascent peptides from disome region. 3 0 0 • UJ 2 0 0 in Q . I 0 0 O F M FMP a N MP o a CD MPA j-n-\ n 0 4 3 2 1 0 1 2 3 4 0 D I S T A N C E FROM ORIGIN ( i nches ) b) Re-electrophoresis (pH 1 .9 ) of the peptides above. I20-L U 8 0 CO 4 0 -® ® LL IO 2 0 D I S T A N C E (cm) 3 0 0 Note: FM, F-HG>Met; FMP, F-Met-14-G-Pro; Ni, neutral marker (epsilon-DNP-lysine); MP and 1 , M e t - H 0 - P r o ; 2 , 5 5 S -Met; MPA and 3 , l 4G-Met-Pro-Arg. -168-formylase and the above treatment would lead to the release of F-Met; thus the presence of Met and the absence of F-Met i n the material released from t e s t i s ribosomes suggests that the methionine i s never formylated i n the t e s t i s sy-stem. The other two sucrose gradients of 35s-methionine l a b e l -led ribosomes were processed as follows. Fractions 1-7, 8-13 and 14-20 were pooled, d i l u t e d with TMK and pell e t e d by centrifugation. Nascent peptides were released by hydro-l y s i s with 1 M NH^OH at 37 °0 f o r 30 minutes and extracted as described i n section #17 of "Methods". The released ma-t e r i a l was subjected to one Edman degradation with the r e -sults shown i n Table 6. V i r t u a l l y a l l of the methionine in. the disome region reacted and therefore must be N-terminal or f r e e . A lower proportion of the methionine i n the higher polysomes reacted; this suggests that some of the l a b e l l e d methionine i s i n t e r n a l i n nascent proteins on the ribosomes i n t h i s region. However, the large proportion of N-terminal methionine i n the higher polysomes may mean that proteins other than protamine are also i n i t i a t e d with methionine i n the trout t e s t i s system. Inhibitors F i g . 24 shows the s e n s i t i v i t y of methionine incorpora-t i o n into protamine to i n h i b i t i o n . b y cycloheximide and chloramphenicol. The extreme s e n s i t i v i t y to cycloheximide - 1 6 9 -; TABLE 6 Edman Degradation of Ribosome-Bound -methionine Labelled Nascent Peptides Region on CPM: i n gradient aqueous CPM' i n ethyl %- of 35s-Met that (fra c t i o n s ) phase acetate phase i s N.-terminal , 1 -7* 1848 6278 77% 8-13 1065 3256 75 14-20 1091 9617 90 * Assuming that a l l the r a d i o a c t i v i t y i n the ethyl acetate phase i s from N-termini.; a c t u a l l y , as discussed i n the text, some of the r a d i o a c t i v i t y must come from free methionine. ** Fraction 1 i s at the bottom of the gradient. -170-* i g . 24 Ef f e c t of Inhibitors on 35s-methionine Incorporation i n t o Protamine Log Molarity Inhibitor Fote: S p e c i f i c a c t i v i t y refers to CPM 3 5s-Met per A230 °^ protamine. Procedures described i n section #16 of "Methods". -171-i s c h a r a c t e r i s t i c of eukaryotic cytoplasmic protein synthe-s i s and has been described previously f o r arginine incorp-oration into protamine (264). Cycloheximide has been found to i n h i b i t both chain i n i t i a t i o n and elongation (281 ) and i s known to 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 great s e n s i t i v i t y of methionine incorpora-t i o n to this i n h i b i t o r rules out a mechanism involving N-terminal addition of amino acids as described i n E. c o l i (282,283) and i n rat l i v e r (284). The i n s e n s i t i v i t y of pro-tamine synthesis to a l l but high l e v e l s of chloramphenicol together with work on the i n t r a c e l l u l a r s i t e of protamine synthesis (265,266) eliminates mitochondria as the l o c a t i o n of synthesis. As described i n section #16 of "Methods", aminopterin was tested f o r i t s e f f e c t on methionine incorporation into protamine. The results given i n Table 7 indicate very l i t t l e i n h i b i t i o n by this agent. Since aminopterin i s known to i n h i b i t eukaryotic dihydrofolate reductase (299), formyla-t i o n of Met-tRNAf*, i f i t occurred, would be i n h i b i t e d and one would then expect i n h i b i t i o n of protein i n i t i a t i o n . The re s u l t s with methionine incorporation into protamine there-fore imply that formylation of the methionine does not oc-cur or at l e a s t i s not necessary. -formate Incorporation, into Protamine C e l l suspensions incubated with Hc-formate were found - 1 7 2 -TABLE 7 E f f e c t of Aminopterin on 35s-methionine Incorporation, into Protamine S p e c i f i c a c t i v i t y * Sample (OPM per A 2 3 0 ) % of Control Control 1 1910 2 2160 Aminopterin 1 0 " 6 M 1730 85% IO" 5 M 1660 82 IO - 4" M 1620 80 10-3 M 1800 88 * CPM 35s-Met per A23O of protamine measured as outlined,in section #16 of "Methods". - 1 7 3 -to incorporate the l a b e l into protamine but none of the incorporated l a b e l was v o l a t i l e a f t e r acid hydrolysis. In f a c t , acid hydrolysis and electrophoresis at pH 1.9 re v e a l -ed that a l l of the formate l a b e l i n protamine had entered as serine. The incorporation of formate into serine i s pre-sumably due to the presence i n trout t e s t i s of a hydroxy-methyl transferase which can synthesize serine from glycine and -methylene tetrahydrofolate, the methylene moiety being derived from formate. This pathway i s outlined below: Formate + THFA + ATP * FfO-Formyl THFA 1 N 1 0-Formyl THFA » N5,1O-Methenyl THFA 2 E5» 1 °-Methenyl THFA > H5-, 10-Methylene THFA + H + + NADPH. 3 + NADP+ N5,10_Methylene THFA + glycine > Serine + THFA Note: 1, formyltetrahydrofolate synthetase; 2, methenyltetrahydrofolate cyclohydrolase; 3 , hydroxymethyltetrahydrofolate dehydrogenase; 4, 1-serine hydroxymethyl transferase. Enzyme Assays. Preliminary studies were done to assay for enzymes i n trout testes capable of removing acyl groups from methio-nine or Met-Pro and f o r enzymes capable of removing the - 1 7 4 -methionine residue from protamine or the dipeptide, 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 high speed supernatant using the assay described i n section #18 of "Methods". Pig. 25 shows an autoradiogram of an e l e c t r o -phoretogram at pH ' 6 . 5 of the reaction products a f t e r i n c u -bating t e s t i s extracts with either F -35s-Met or Ac -35s-Met. The l a b e l l e d product of deacylation of either substrate i s free 35s-methionine which remains near the o r i g i n during electrophoresis at pH 6 . 5 . The preference of the t e s t i s de-acylase f o r P-Met rather than Ac-Met i s obvious just by "inspecting these autoradiograms. Table 8 summarizes the properties of the t e s t i s deacy-lase. The a c t i v i t y i s mainly i n the high speed supernatant and prefers the formyl group over the other acyl groups t r i e d . There i s s p e c i f i c i t y for the amino acid as well since P-Ileu and even R-formyl-methionine sulphone are i n -active as substrates. There i s very l i t t l e a c t i v i t y with P-Met-Pro as substrate. A s i m i l a r a c t i v i t y i s present i n E. c o l i ( 3 0 0 ) and has been attributed to the enzyme N-acetyl-ornithinase (N-acetyl-1-ornithine- amidohydrolase) which i s an enzyme involved i n ornithine biosynthesis i n bacteria (301). This enzyme has a marked s p e c i f i c i t y f o r P-Met as substrate as shown i n Table 9 which i s taken from a paper by Pry and Lamborg ( 3 0 0 ) . -17 5 -F i g . 25 Assay f o r Testis Deacylase a) F - 3 5 s - M e t Substrat< © M f f M b) Ac -35s-Met as substrate © Note: F, F -35s-Met; Ac, Ac -35s-Met; 1 , control; 2 , nuclear ly s a t e ; 3 , high speed sup.; 4 , mitochondrial ly s a t e ; 5 , ribosome wash. 0 , o r i g i n . Incubation was with 0.05 ml extract, 0.01 ml substrate, and TMK up to 0.11 ml at 18 °0 for 1 h. I 2 3 4 5 o • • • , • ^ -176-TABLE. 8 Deacylase A c t i v i t y 1 ) C e l l Fraction. , Rate* $ Total Nucleoplasm 580 13$ High speed sup. 3540 82 Ribosome wash 40 1 Mitochondrial lysate 167 4 2) Substrate Rate** Formyl-Met 28.0 Acetyl-Met 12.2 Propionyl-Met 0 Butyryl-Met 0.9 Valeryl-^Iet 1.3 Hexanoyl-Met 0.8 Formyl-Met sulphone 0.7 Formyl-lieu 0 F-Met-Pro Rate*** High speed sup. 1.5 Nuclear lysate 0 Ribosome wash 0 * nMoles Formyl-Met deacylated per 30 min per g t e s t i s at 20 °0 ** nMoles deacylated per 0.025 ml HSS per 30 min at 20 °0 *** $ deacylated per 0.05 ml extract per 45 min at 16 °0 -177-TABLE 9 Properties of E. c o l i N-acetyl-ornithinase (Ref.3Q0) Rel a t i v e * (formy- R e l a t i v e * (acety-Acylated Amino Acid A c t i v i t y -lated) A c t i v i t y lated) 1-methionine 100 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 rates are r e l a t i v e to the rate of hydrolysis of F-Met set at 100. Incubation at 25 °C for 13 minutes. -1 7 8 -The enzyme cannot be important in. b a c t e r i a l protein syn-thesis since mutants d e f i c i e n t i n i t show normal growth and do not have enhanced leve l s of F-Met at the, N^termini of t h e i r proteins (300). With i t s rapid hydrolysis of F-Met and very slow hydro-l y s i s of F-Met-Pro, the testis deacylase i s quite the oppos-i t e to the b a c t e r i a l deformylase (195-197) responsible f o r removal of formyl groups from nascent proteins. It thus seems that the t e s t i s a c t i v i t y must be due to a deacylase not involved d i r e c t l y i n protein synthesis. Previously described deacylases i n animal tissues i n -clude Aryl-formylamine amidohydrolase i n l i v e r (302) which hydrolyzes F-formyl-l-kynurenine, Aryl-acylamine amidohydro-lase i n chick kidney (303) which hydrolyzes F - a c y l - a n i l i d e s , 10-formyltetrahydrofolate amidohydrolase i n l i v e r (304) which removes formate from F 1°-formyl-tetrahydrofolate, F-acylaminoacid amidohydrolase (305,306) which removes a wide v a r i e t y of acyl groups from amino acids, and F - a c y l -aspartate amidohydrolase from kidney (305,306). A meaning-f u l comparison of the t e s t i s deacylase with any of the above a c t i v i t i e s i s not possible with the data available. Hog kidney acylase T i s quite active with Ac-Met as substrate (306) but F-Met was not compared; acylase TT from the same source i s about one-third as active with Ac-Met as with i t s natural substrate, Ac-Asp, but F-Met was not compared. At present, then, i t i s not possible to say i f F-Met i s the -179-natural substrate of the t e s t i s deacylase. b) Methionine Aminopeptidase A c t i v i t y Testis extracts were assayed with 35 s -methionine and -arginine 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 incubated with the labels separately. The r e s u l t s given i n Table 10(a) show that t e s t i s extracts cause a de-crease i n TCA-tungstate p r e c i p i t a b l e 35s-methionine prota-mine counts. However, the extracts also giveL-a considerable decrease i n acid precipitable 1 4 C - a r g i n i n e protamine counts, in d i c a t i n g that there must be extensive proteolysis of pro-tamine. I t was decided to discontinue t h i s type of assay but i t could be a useful assay f o r an attempt to i s o l a t e ahspecific methionine aminopeptidase. The l a t t e r would give a decrease i n ^5s-methionine but not 140-arginine acid p r e c i p i t a b l e counts i n protamine. Met- l 40-Pro, prepared as i n section #13 of "Methods", was also t r i e d as a substrate i n these studies. The r e s u l t s given i n Table 10(b) reveal the presence of a c t i v i t y capable of hydrolyzing t h i s dipeptide mainly i n the high speed su-pernatant but s i g n i f i c a n t a c t i v i t y was also present i n the nuclear lysate and ribosome wash. As mentioned e a r l i e r , the nuclear a c t i v i t y detected here may be responsible for remov-a l of N-terminal methionine from any protamine that escapes the cytoplasmic enzyme. The t e s t i s enzyme;.has not been as-sayed with other substrates>;such as longer peptides with - 1 8 0 -TABLE 1,0 Methionine-removing A c t i v i t y a) Protamine as substrate 35s-Met Protamine 14c?-Arg Protamine Enzyme as substrate as substrate High speed sup. 41.3$ * 73.2$ Nuclear lysate 14.2 40.9 * $ decrease i n TCA-tungstate pre c i p i t a b l e counts. Incubations were with 0.1 ml extract, 0.02 ml 2 M NaCl, 0.01 ml substrate i n water, and TMK up to 0.2 ml for 40 min at 15 °0. b) Met - H c-Pro as substrate R e l a t i v e * * $ Enzyme Rate* a c t i v i t y Total High speed sup. 73.9 100 57 Nuclear lysate 65.8 50 28 Ribosome wash 53.4 27 15 Fote: * $ hydrolyzed i n 45 min at 16 °0 ** Corrected f o r volume of extract -181-methionine i n the Nr-terminal p o s i t i o n ; this 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 that X-Pro bonds are r e s i s t a n t to many proteases with the possible exception of imidodipeptidase (289), the t e s t i s a c t i v i t y i s unusual and could well be involved i n vivo i n removing methionine from the N-terminus of newly synthesized proteins. Matheson and Dick (198) have recently described some of the properties of an aminopepti-dase present on ribosomes i n E. c o l i and Table 11 i s taken from t h e i r paper. With Met-X dipeptides, the b a c t e r i a l en-zyme strongly prefers leucine or methionine i n the 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 against Ala-X or Ser-X dipeptides ; the authors state that the enzyme i s also i n -active against tripeptides with serine or alanine i n the N-terminal position. These properties seem well suited to account for the incidence of N-terminal residues i n bulk protein from E. c o l i (193) where serine, alanine, methio-nine and threonine are the only N-termini found i n most proteins. Unfortunately, Met-Pro was not t r i e d as a sub-strate . Conclusion Protamine synthesis i n trout t e s t i s has been shown to involve transient incorporation of methionine at the N-terminus of the newly synthesized chains. At no time, even on the ribosomes, i s there any evidence that the amino -182-TABLE 11 Properties of an Aminopeptidase on Ribosomes of E. c o l i * C-terminal residue N-t erminal r es i due Methionine Alanine Serine leucine methionine phenylalanine glycine valine serine alanine gl y c y l - g l y c i n e alanyl-serine glycyl-methionine Gly-Met-Met 9 6 * * 80 25 25 1.3 13 11 87 88 155 165 30 18 7 0 0 0 0 0 14 10 5 1 3 0 0 * From Matheson and Dick (198) ** Rates are given as a percentage of the rate of hydrolysis of leucyl-leucine (0.026 micro-moles per min per mg protein) at 30 °C. -183-group of the methionine i s formylated. The incorporation of methionine into the several com-ponents of protamine at t h e i r N-termini and the subsequent removal of methionine from t h i s p o sition i s strong presump-t 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 protein synthesis i h the trout t e s t i s system. There are several features of this system which have probably aided a demonstration of methionine incorporation. Pish t e s t i s matures r e l a t i v e l y synchronously and testes may be obtained which contain mainly spermatids which are i n the process of completely replacing the histones i n chromosomes by prota-mine. Thus, protamine i s being synthesized i n large amounts and since methionine i s absent from i n t e r n a l positions, at l e a s t i n the major components (261 ), the incorporation of methionine at the N-terminus was e a s i l y observed. The small s i z e of protamine, 32-33 residues long, means that the entire molecule can be protected or "shielded" by the ribosome during i t s synthesis; Rich, Eikenberry and Malkin (287,288) established that ribosomes protect nas-cent proteins up to 35 residues long from external attack by p r o t e o l y t i c enzymes. 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