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Electrophoretic and amino acid analysis of amphibian and reptilian histones Huang, Sue-Ying 1977

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ELECTROPHORETIC AND AMINO ACID ANALYSIS OF AMPHIBIAN AND REPTILIAN HI STONES by SUE YING^HUANG B.Sc. Fu Jen C a t h o l i c U n i v e r s i t y 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE DEPARTMENT OF ZOOLOGY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1977 © Sue--Ying Huang In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for s c h o l a r l y purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or pub l i ca t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Department of 2j,oo6? "/ The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date (lc+ * , n ABSTRACT Small amounts of amphibian and r e p t i l i a n h i s t o n e s , b a s i c proteins associated w i t h DNA, can be c h a r a c t e r i z e d by a combination of e l e c t r o -phoresis and subsequent amino ac i d a n a l y s i s of the stained bands. A simple method has been devised f o r o b t a i n i n g histones f o r such a n a l y s i s from Xenopus l a e v i s , the South A f r i c a n clawed toad. Xenopus somatic histones analyzed both by sta r c h and acrylamide g e l e l e c t r o p h o r e s i s show microheterogeneity f o r the very l y s i n e - r i c h H i histones of heart and lung, but no t i s s u e - s p e c i f i c i t y f o r t h i s group of basic nuclear p r o t e i n s . Xenopus embryonic histones prepared by the method of Destree e_t al-. (1972) can also be c h a r a c t e r i z e d by starch g e l e l e c t r o -p h oresis. In t h i s procedure, the p o s s i b i l i t y of contamination of n u c l e i by yolk p l a t e l e t s and ribosomes i s reduced. No change i s observed i n the s t a r c h g e l e l e c t r o p h o r e t i c p r o f i l e s of Xenopus histones throughout e a r l y embryogenesis. This confirms the o r i g i n a l observation of Destree et al.. (1973) using polyacrylamide g e l e l e c t r o p h o r e s i s . Histones can be extracted from amphibian and r e p t i l i a n t e s t i s c e l l o suspensions and analyzed by starch and polyacrylamide g e l e l e c t r o p h o r e s i s . Since only one animal i s required i n t h i s method, the a n a l y s i s can be extended to include d i f f e r e n t species f o r which many re p r e s e n t a t i v e s are d i f f i c u l t to o b t a i n . In a d d i t i o n , using Houston's (1971) method of hy d r o l y z i n g amidoblack-stained p r o t e i n bands on polyacrylamide gels f o r amino ac i d a n a l y s i s , one can ob t a i n the chemical composition of t e s t i s -s p e c i f i c h i s t o n e s from a number of amphibians and r e p t i l e s . Amphibian t e s t i s - s p e c i f i c h istones show e n t i r e l y d i f f e r e n t patterns from each other, while the r e p t i l i a n h istones show remarkable s i m i l a r i t y toeeach other both i n t h e i r e l e c t r o p h o r e t i c p r o p e r t i e s and amino ac i d composition. - i i i -Although the present survey examines only a l i m i t e d number of species, the data do p o i n t to the r e p t i l e s as one place i n vertebrate phylogeny where the d i v e r s i t y of t e s t i s - s p e c i f i c histones i n f i s h and amphibians gives way to a r e l a t i v e constancy of such p r o t e i n s . - Iv -TABLE OF CONTENTS Page INTRODUCTION 1 PART I ELECTROPHORETIC ANALYSIS MATERIALS AND METHODS 9 A. I s o l a t i o n of histones • 9 C e l l suspension 9 Method of Byrd 9 Method of Destree et a l . 10 Modified method of Bonner et a l . 10 I s o l a t i o n of l y s i n e - r i c h histone H i 11 B. I s o l a t i o n of ribosomal b a s i c p r o t e i n s 11 C. I s o l a t i o n of yolk b a s i c p r o t e i n s 11 D. E l e c t r o p h o r e s i s Starch g e l e l e c t r o p h o r e s i s 12 Polyacrylamide g e l e l e c t r o p h o r e s i s 1M-a) Method of Bonner e_t a l . 14-b) Method of Panyim and Chalkley 15 RESULTS 16 DISCUSSION 40 PART I I AMINO ACID ANALYSIS MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES 75 76 - v -LIST OF TABLES TABLE ' N PAGE 1. P r i n c i p a l components of c a l f thymus histones and commonly used nomenclature systems 2 2. K values and t h e i r 95% confidence i n t e r v a l s f o r weighing method 53 3. Amino ac i d composition of Sigma protamine (herring) • 57 4. p-mercaptoethanol e f f e c t on a c i d h y d r o l y s i s of Xenopus t e s t i s - s p e c i f i c histone X g ' 59 5. Comparison of amino ac i d composition of Xenopus t e s t i s - s p e c i f i c histone X g prepared by d i f f e r e n t methods 61 6. Comparison of amino ac i d composition of Xenopus t e s t i s - s p e c i f i c histone X^ prepared by d i f f e r e n t methods 62 7. Comparison of amino ac i d composition of e v o l u t i o n a r i l y conservative histone HM-i n Xenopus t i s s u e and c a l f thymus 63 8. Amino ac i d composition of t e s t i s - s p e c i f i c histones from amphibian c e l l suspensions 65 9. Amino a c i d composition of t e s t i s - , .: f ductus deferens- and seman->specific histones from r e p t i l i a n c e l l suspensions 67 10. A b r i e f h i s t o r y of sperm histone c l a s s i f i c a t i o n 71 - v i - _ LIST OF FIGURES FIGURE PAGE 1. Cytochemical c l a s s i f i c a t i o n of sperm-s p e c i f i c histones i n vertebrate phylogeny 5a, b 2. E l e c t r o p h o r e t i c p r o f i l e s of Xenopus heart histones on polyacrylamide d i s c gels 17 3. E l e c t r o p h o r e t i c p r o f i l e s of Xenopus histone HI a f t e r s e l e c t i v e e x t r a c t i o n by 5% p e r c h l o r i c a c i d 18-M-. Diagram of e l e c t r o p h o r e t i c comparison of Xenopus heart histones w i t h l i t e r a t u r e data using both polyacrylamide and sta r c h gels 19 5. Starch g e l electrophoresis of Xenopus heart from f r e s h and stored organs 22 6. Two dimensional s t a r c h g e l electrophore-togram of Xenopus heart histones 24-7. Xenopus t e s t i s - s p e c i f i c histones extracted by d i f f e r e n t methods 2 6 8. E l e c t r o p h o r e t i c comparison of t e s t i s - s p e c i f i c h i stones from amphibian c e l l suspensions on polyacrylamide gels 27 9. E l e c t r o p h o r e t i c comparison of t e s t i s - s p e c i f i c h i stones from r e p t i l i a n c e l l suspensions on polyacrylamide gels . 28 10. E l e c t r o p h o r e t i c comparison of t e s t i s - s p e c i f i c histones from amphibian c e l l suspensions on the s t a r c h gels ' 29 11. Starch g e l e l e c t r o p h o r e t i c p r o f i l e s of ~.as.-- . histones from Xenopus swimming tadpoles 31 12. E l e c t r o p h o r e t i c p r o f i l e s of Xenopus embryonic histones on starch gels 33 - v i i -13. E l e c t r o p h o r e t i c p r o f i l e s of Xenopus embryonic histones on sta r c h gels during embryogenesis 35 1M-. E l e c t r o p h o r e t i c comparison of Xenopus embryonic histones and yolk basic p r o t e i n s 36 15. E l e c t r o p h o r e t i c comparison of Xenopus embryonic histones and ribosomal b a s i c p r o t e i n s • 37 16. E l e c t r o p h o r e t i c p r o f i l e s of aspermatogenic t e s t i s c e l l suspensions from l i z a r d and newt -- 39 17. Chromatogram of Beekman Amino Acid C a l i b r a t i o n Standard on the Beekman Model 118C Amino Acid Analyzer ; 51 18. E f f e c t of r e s i d u a l polyacrylamide g e l p a r t i c l e s on the amino ac i d p r o f i l e xfrom Xenopus t e s t i s -s p e c i f i c histones 5M-19. E l e c t r o p h o r e t i c p r o f i l e s of Sigma protamine 55 20. C l a s s i f i c a t i o n of sperm-specific histones i n . verteb r a t e phylogeny by amino a c i d composition 7M-V l l l DEDICATION To My Parents and My Husband - ix f -ACKNOWLEDGEMENT The author wishes to express a p p r e c i a t i o n to Dr. Harold. E. Kasinsky f o r h i s guidance, suggestions and encouragements during the course of t h i s work. A p p r e c i a t i o n i s also expressed to Drs. C.V. Finnegan, J . Gosline and J. Berger f o r t h e i r ideas and assi s t a n c e . Part I ELECTROPHORETIC ANALYSIS INTRODUCTION In order to gain a b e t t e r understanding of the molecular bio-logy of spermiogenesis and embryogenesis i n the v e r t e b r a t e s , i t i s necessary to develop micromethods to measure the important macro-molecular c o n s t i t u e n t s i n the c e l l s . At present we use biochemistry to c h a r a c t e r i z e r a t h e r l a r g e amounts o f \ m a t e r i a l and h i s t o c h e m i s t r y to analyze very small amounts of t i s s u e components. Biochemistry gives us a great deal of i n f o r m a t i o n about the chemistry of the con-s t i t u e n t s and t h e i r i n t e r a c t i o n but t e l l s l i t t l e about the s p a t i a l o r i e n t a t i o n of such components i n c e l l s or t i s s u e s . H i s t o l o g y and h i s t o c h e m i s t r y depend on a l i m i t e d number of r e a c t i o n s and o f t e n do not define the chemical components i n an adequate manner. These problems are p a r t i c u l a r l y acute i n the study of h i s t o n e s , t h e i r con-t e n t , synthesis and m o d i f i c a t i o n i n vertebrate spermiogenesis and embryogenesis, p a r t i c u l a r l y i n amphibians and r e p t i l e s . In these• organisms, the amount of t i s s u e a v a i l a b l e f o r study i s small and problems e x i s t w i t h respect to contamination w i t h b a s i c p r o t e i n s , such as those present i n ribosomes and yolk p l a t e l e t s . In t h i s t h e s i s , I w i l l examine the e l e c t r o p h o r e t i c p r o p e r t i e s and amino a c i d composition of amphibian and r e p t i l i a n h istones i n order to demonstrate that u s e f u l biochemical data can be obtained from, small amounts of m a t e r i a l when these methods are applied to somatic c e l l s , t e s t i s and embryos. Furthermore, I have chosen to look at t i s s u e s i n these lower vertebrates i n order to c o n t r a s t the d i v e r s i t y of t e s t i s - s p e c i f i c histones In these animals w i t h the greater degree:of constancy of histones TABLE 1 P r i n c i p a l Components of Calf Thymus Histones and Commonly Used Nomenclature Systems Class Nomenclature Lys/Arg Ratio T o t a l Residues Molecular Weight N terminal C terminal Very l y s i n e - H1(I, f l ) 22.0 2 1 5 21 , 500 • * Ac-Ser Lys r i c h L y s i n e - r i c h H2a(llbl, f2a2) 1 . 1 7 1 2 9 1^,001* Ac-Ser Lys H2t>(llb2, f2b) 2 . 5 0 1 2 5 13 , 7 7 ^ Pro Lys A r g i n i n e - r i c h H3(IH, f3) 0 . 7 2 1 3 5 1 5 . 3 2 U A l a A l a Hl|(lV, f2al) 0 . 7 9 102 11 , 2 8 2 Ac-Ser Gly A l l data- compiled from E l g i n et_ a l . , (1971) and DeLange and Smith, (197*0 * Ac-Ser = N-acetylated-serine - 3 -from somatic and embryonic t i s s u e s . Histones are b a s i c p r o t e i n s c l o s e l y associated w i t h DNA at some p o i n t i n t h e i r l i f e h i s t o r y . They f a l l i n t o f i v e c a t e g o r i e s : HI, H2a, H2b, H3 and HM- (Table 1). Histones H2a,. H2b, H3 and m appear to be aggregated w i t h DNA to form a nucleosome s t r u c t u r e (Kornberg, 1977; Kornberg and Thomas, 1974-; Bradbury e_t _a l . , 1972; Richards and Pardon, 1970) lo o k i n g l i k e beads on a s t r i n g (Olins and O l i n s , 197M-) . These histones show l i t t l e t i s s u e - o r species-s p e c i f i c i t y . Histones HM- i n f a c t i s the most e v o l u t i o n a r i l y conservative p r o t e i n known (De Lange and Smith, 1975). Lysine-r i c h histone H i i s thought to c r o s s l i n k the chromosome s t r u c t u r e i n a higher order of chromosome complexity (Baldwin, e_t al. , 1975) . HI g e n e r a l l y shows both tissue-and s p e c i e s - s p e c i f i c i t y (Stellwagen and Cole, 1969). The high r e s o l v i n g power of zone e l e c t r o p h o r e s i s i n st a r c h or polyacrylamide g e l s has been u t i l i z e d e x t e n s i v e l y i n studies on t i s s u e s p e c i f i c i t y of histones from various sources. Using s t a r c h g e l e l e c t r o p h o r e s i s , Vendrely e_t a_l. (1965) observed a remarkable s i m i l a r i t y i n histone patterns from c a l f thymus, l i v e r and lung as w e l l as from r a t thymus and l i v e r . Starch g e l e l e c t r o p h o r e t i c patterns of histones from domestic f o w l revealed the t i s s u e s p e c i f i c i t y of the e r y t h r o c y t e - s p e c i f i c histone (Neelin and B u t l e r , 1961), an observation which l e d to the I s o l a t i o n of chicken erythrocyte s p e c i f i c histone H5. S i m i l a r l y , polyacrylamide g e l e l e c t r o p h o r e s i s of histones from r a t spleen, thymus, l i v e r , kidney, heart and b r a i n showed only minor v a r i a t i o n s from the general p a t t e r n known f o r mammalian histones ( H n i l i c a , 1972). The most extensive and c a r e f u l study on e l e c t r o p h o r e t i c s i m i l a r -i t i e s of histones from numerous vertebrate species was undertaken by Panyim e_t al. (1970, 1971a) using a system of long polyacrylami g e l s (Panyim and Chalkley, 1969a; 1969b). The authors separated h i s t o n e s i n t o p r o t e i n bands corresponding to f r a c t i o n s HI, H2a, H2b, H3 and HM-. In a l l species analyzed, the a r g i n i n e - r i c h histones H3 and H4 had a constant e l e c t r o p h o r e t i c m o b i l i t y , i n d i c a t i n g considerable conservation of these f r a c t i o n s during the e v o l u t i o n of ve r t e b r a t e s . Only small v a r i a t i o n s were observed f o r histones H2a and H2b,while the l y s i n e ^ / r i c h histone HI v a r i e d considerably i n i t s e l e c t r o p h o r e t i c heterogeneity and m o b i l i t y , i n d i c a t i n g s u b s t a n t i a l changes i n the primary s t r u c t u r e of t h i s f r a c t i o n during e v o l u t i o n . These f i n d i n g s were i n e x c e l l e n t agreement w i t h the chemical analyses of the a r g i n i n e - r i c h and s l i g h t l y l y s i n e - r i c h histone f r a c t i o n s ( H n i l i c a , 1972; De Lange and Smith, 1974), whereas the H i Histones were found to be both t i s s u e and species s p e c i f i c , . (Bustin and Cole, 1968; De Lange and Smith, 1974). The t e s t i s i s a s p e c i a l i z e d somatic t i s s u e c o n t a i n i n g germ c e l l s : the male gametes. Both st a r c h and polyacrylamide g e l e l e c t r o p h o r e s i s have shown that amphibian and r e p t i l i a n t e s t i s c o n t a i n i n g sperm have fast-moving bands migrating f a s t e r than the somatic histone r e g i o n (Kasinsky et a l . , 1977; Bols et a l . , 1976; Bols and Kasinsky, 1973 and Bloch, 1962). These t e s t i s - s p e c i f i c h i stones may be sperm-specific as w e l l , as suggested by Kasinsky / et a l . , (1977) and Bols e_t al. (1976) . During spermiogenesis there i s g e n e r a l l y a replacement of histones by more a r g i n i n e -- 5 -r i c h p r o t e i n s i n many species. Although there i s an ample amount of cytochemical evidence about t h i s t r a n s i t i o n , the best b i o -chemically documented system i s the appearance of protamines during the te r m i n a l stages of sperm maturation i n many species of f i s h (Louie et a_l, 1973) . The components t y p i c a l f o r spermatozoa are g e n e r a l l y more b a s i c than somatic h i s t o n e s , u s u a l l y w i t h a high a r g i n i n e and serine content (5 to 18 mole%). A l s o , t y r o s i n e and c y s t e i n e are amino acids common to many h i g h l y b a s i c sperm histones (Subirana, 1975). This group of p r o t e i n s shows extreme non-conservation amongst vertebrates (Kasinsky e_t a_l. , 1977; Bloch, 1969). These diverse p r o t e i n s have been cataloged^into 5 types by Bloch (1969) (Tig. 1) based i n p a r t on the o r i g i n a l scheme of Kossel (1928). Class 1 i s Salmo type; Class 2, mammalian type; Class 3, M y t i l u s type; Class M-, Rana type and Class 5, crab type (no h i s t o n e s ) . A l l 5 types of sperm-specific histones -have been observed cytochemically and b i o c h e m i c a l l y i n d i f f e r e n t f i s h and amphibian species (Bloch, 1976). In r e p t i l e s , only two species, have been examined and both have been grouped as Type 1 (Bloch, 1976). However, a question mark i s i n d i c a t e d f o r r e p t i l i a n sperm histones i n F i g . 1. This i s due to the f a c t that these data are based on Bloch's (1969) unpublished observation and on a s i n g l e p o s i t i v e Sakaguchi t e s t on a s i n g l e animal (Sud, 1961). By i t s e l f , the Sakaguchi t e s t i s not s p e c i f i c f o r Type 1 sperm histones (Bols and Kasinsky, 1972). In mammals these p r o t e i n s g e n e r a l l y f a l l i n t o Type 2 (Bloch, 1976). As f o r b i r d s , there has only been one species studied thus f a r , G a l l u s domesticus. The r o o s t e r sperm histone has been placed i n the Type 1 category - 5a -F i g . 1 Cytochemical c l a s s i f i c a t i o n of sperm-specific histones i n vertebrate phylogeny. Type Name Representative animal 1 Salmo type (protamine, monoprotamine) Trout 2 Mammalian type (stable protamine, b a s i c k e r a t i n ) Rat 3 M y t i l u s type (intermediate, d i - , triprotamine) Mussel 1 Rana type (somatic l i k e ) Frog 5 Crab type (no histones present ) Crab Based on data of Bloch (1969, 1976), Bols and Kasinsky (1972, 1973, 1974, 1976). Alder and Gorovsky (1975),,and P i c h e r a l (1970). Arrow i n d i c a t e s e v o l u t i o n a r y trend from r e l a t i v e d i v e r s i t y of sperm-histoneotype i n f i s h and amphibians to r e l a t i v e constancy i n r e p t i l e s b i r d s and mammals. (2) i n d i c a t e s newt s p e r m a t i d - s p e c i f i c h i s t o n e s . - 5b -Fish A m p h i -b ians Rept i les Bi rds Mammals - 6 -on the b a s i s of i t s primary s t r u c t u r e (Nakano et _al. , 1976) . I t i s not known i f t h i s observation holds true f o r other species i n the c l a s s Aves. However, there does appear to be an e v o l u t i o n a r y trend from d i v e r s i t y of sperm-specific histones i n f i s h and amphibians to a r e l a t i v e constancy of such p r o t e i n s i n r e p t i l e s and mammals (Kasinsky e_t al.. , 1977) . Although the reason f o r the replacement of histones by a r g i n i n e - r i c h protamines or s i m i l a r p r o t e i n s during spermiogenesis i s unknown, i t has been suggested that the more b a s i c a r g i n i n e -r i c h p r o t e i n s may be more e f f i c i e n t than histones i n genetic i n a c t i v a t i o n and packing of the DNA In mature sperm (Marushige and Dixon, 1969). As the DNA i s packed t i g h t e r , the p r o b a b i l i t y of i t s being damaged decreases since water, enzymes and b a c t e r i a have a smaller chance of reaching i t (Subirana and Puigjaner, 1973). The appearance of c y s t i n e bridges should r e i n f o r c e t h i s p r o t e c t i o n (Bedford and C a l v i n , 1971). The r o l e of t y r o s i n e i s not c l e a r , although i t may be i n v o l v e d i n energy t r a n s f e r processes which would p r o t e c t DNA against r a d i a t i o n damage (Subirana, 1972). I t i s l i k e l y t h a t these b a s i c p r o t e i n s also are i n v o l v e d i n other biochemical processes which take place during spermiogenesis and perhaps during f e r t i l i z a t i o n . E a r l y embryogenesis i s ( c h a r a c t e r i z e d by r a p i d cleavages, f a s t growth and d i f f e r e n t i a t i o n . Since histones are c l o s e l y associated w i t h DNA, i t i s i n t e r e s t i n g to know i f there are changes i n the content or type of histones concurrent w i t h these complex processes. Most i n v e s t i g a t o r s agree that t y p i c a l histones are present i n the n u c l e i of b l a s t u l a and g a s t r u l a embryos depending - 7 -on the p a r t i c u l a r animal species examined. A n a l y t i c a l s t udies show that i n advanced embryos, the histones are s i m i l a r to those i n a d u l t t i s s u e ( H n i l i c a , 1967). No detectable changes i n the chemical composition and e l e c t r o p h o r e t i c patterns of histones were observed during c h i c k embryogenesis (Kischer _et _al. , 1966; K i s c h e r and H n i l i c a , 1967). The e l e c t r o p h o r e t i c p r o f i l e s of t h i s group of p r o t e i n s are e s s e n t i a l l y the same during e a r l y embryogenesis i n Xenopus (Destree e_t a l . , 1973) and i n the devel-opment of f r o g tadpole (Stenroos and Reichard, 1970). Using a i ii CCv, l a b e l l i n g method, Byrd and Kasinsky (1973a, b) showed that there was extensive synthesis of each of the major classes' of h i s t o n e s p r i o r to g a s t r u l a t i o n i n Xenopus l a e v i s . These newly synthesized embryonic hist o n e s were s i m i l a r to those i n a d u l t l i v e r when compared by g e l e l e c t r o p h o r e s i s and amino a c i d a n a l y s i s . 3 Adamson and Woodland (1974), using H - l y s i n e , l a b e l l i n g , two-dimensional g e l e l e c t r o p h o r e s i s and peptide mapping of a r g i n i n e -r i c h histone H4,found that the four main histone f r a c t i o n s other than histone H i were synthesized at a l l stages of Xenopus develop-ment. HI histone (adult type) synthesis was f i r s t detected at the l a t e b l a s t u l a stage. S i m i l a r r e s u l t s were also obtained i n sea u r c h i n embryos. Changes i n the proportion, of histone c l a s s e s synthesized during development have been e s t a b l i s h e d (Easton and Chalkley, 1972; Johnson and H n i l i c a , 1971; Seale and Aronson, 1970). Aside from t h i s , synthesis of an HI c h a r a c t e r i s t i c of the morula stage gives way to synthesis of a new and e l e c t r o p h o r e t i c a l l y d i s t i n c t HI at the g a s t r u l a stage (Ruderman et a l . , 1974) A l s o , Cohen e_t a l . (197 6) observed microheterogeneity of s l i g h t l y l y s i n e -- 8 -r i c h histones H2a and H2b during e a r l y sea u r c h i n embryogenesis. The synthesis of the complete complement of ad u l t type histones f i r s t appears during b l a s t u l a t i o n i n sea u r c h i n and i n Xenopus and even l a t e r i n other animal species. The synthesis of a r g i n i n e -r i c h histones appears f i r s t , then the synthesis of l y s i n e - r i c h h i s t o n e s . Once e s t a b l i s h e d , the q u a l i t a t i v e p a t t e r n of histones i n d i f f e r e n t i a t e d t i s s u e s remains unchanged, wi t h the exception of some s p e c i a l i z e d c e l l s , such as nucleated erythrocytes or spermatozoa. What i s the s i g n i f i c a n c e of the l a t e synthesis of the e n t i r e complement of histones during embryogenesis? Is there a maternal pool of histones stored to compensate f o r t h i s l a t e s y n t h e s i s ? (Adamson and Woodland, 1977; Woodland and Adamson, 1977). The answers to these questions remain to be determined. - 9 -N . MATERIALS AND METHODS MATERIALS Eggs and embryos of Xenopus l a e v i s were obtained from a lab o r a t o r y breeding stock as described by Gurdon (1967) and were d e j e l l i e d i n 2% (w/v) cysteine hydrochloride d i s s o l v e d i n Brown's s o l u t i o n (0.035% NaCl, 0.0005% KC1, 0.001% C a C l 2 , 0.002% MgCl2« 6H 20), adjusted ..to pH 8.0 wi t h 10 N sodium hydroxide. Other amphibians and r e p t i l e s were purchased from Camosun Aquaria, Vancouver, B.C. and obtained from dealers throughout North America. They were i d e n t i f i e d by reference to standard sources (Conant, 1975; Stebbins, 1966), and di s s e c t e d a f t e r a b r i e f p e r i o d at room temperature. Organs were examined when f r e s h or stored at -70°C. METHODS A. I s o l a t i o n of Histones C e l l Suspension (Kasinsky et a l . , 1977) Somatic t i s s u e s (hearts), t e s t e s or swimming tadpoles were homogenized i n PBS s o l u t i o n (phosphate buffered s a l i n e : 1.6% NaCl, O.OM-% KC1, 0.23% Na^PO^, 0.1%MgCl 2 and 0.1% CaCl 2) , and f i l t e r e d onto the 3mm g l a s s f i b e r f i l t e r paper. Histones were extracted d i r e c t l y from the c e l l s trapped on the f i l t e r paper using 0.M- N H 2S0^ f o r 15 minutes. Method of Byrd (197MQ Somatic t i s s u e s (hearts), t e s t e s or swimming tadpoles were homogenized i n 0.14- M NaCl, 0.021 M EDTA, 1% triton-X-100 w i t h 0.05 M NaHSOa as a p r o t e o l y t i c i n h i b i t o r at pH 8.0. Nu c l e i were - 10 -p e l l e t e d by c e n t r i f u g i n g f o r 15 minutes at 1,500 g. Histones were extracted d i r e c t l y by s t i r r i n g the nuclear p e l l e t s i n 0.M- N H^SO^ f o r 1 hour. Method of Destree et al.£1972) Somatic t i s s u e s (hearts), t e s t e s and embryos were homogenized i n 2.4- M sucrose c o n t a i n i n g 3mM CaCl^,, 5 mM t r i s - H C l b u f f e r , pH 7.5, 0.05M NaHS03 and 0.5% triton-X-100. N u c l e i were p e l l e t e d by cen-t r i f u g i n g f o r 2 hours i n Beekman model L-type 50 r o t o r at 108,000,g. The nuclear p e l l e t was washed twice i n 0.02 M E1TA, 0.01 M t r i s -HCl b u f f e r , pH 8.0, 0.05 M NaHS03 and was ce n t r i f u g e d at 4-,300,;..g f o r 10 minutes. The r e s u l t i n g p e l l e t s were washed twice w i t h water and c e n t r i f u g e d f o r 10 minutes at 12,000 g (Destree et a l . 1972). Histones were ext r a c t e d from t h i s chromatin p e l l e t w i t h 0.4- N H^SQ^ f o r one hour. Modified Method of Bonner et a l . (1968) Somatic t i s s u e s (hearts), t e s t e s and embryos were homogenized i n saline-EDTA s o l u t i o n (0.075 M NaCl, 0.024 M EDTA, 0.05 M NaHS0 3, pH 8.0), and then s t r a i n e d through 4- l a y e r s of cheesecloth. The.: m o d i f i c a t i o n of the o r i g i n a l method of Bonner e_t a l . (1968) was the a d d i t i o n of sodium b i s u l f i t e to prevent p r o t e o l y s i s (Destree et a l . , 1972): The homogenate was c e n t r i f u g e d at 1,500 g f o r 15 minutes. The p e l l e t s were washed s u c c e s s i v e l y w i t h saline-EDTA s o l u t i o n and t r i s b u f f e r (0.05 M t r i s , 0.05 M NaHS0 3, pH 8.0). The p e l l e t s thus obtained were homogenized by hand i n t r i s b u f f e r and then c e n t r i -fuged at 10,000 g f o r 15 minutes. This step was repeated. Histones were extracted from the f i n a l p e l l e t w i t h 0.4- N H^SO^ f o r 1 hour. The histones were then p r e c i p i t a t e d w i t h 4- volumes of 95% ethanol. - 11 -The p r e c i p i t a t e was washed 3 times w i t h 95% ethanol and then d r i e d i n a vacuum d e s i c c a t o r . I s o l a t i o n of L y s i n e - r i c h Histone H i (John, 1976) Xenopus l a e v i s h e a r t s , lungs or embryos (5 g) was washed wi t h 0.11 M NaCl, then homogenized w i t h 20 ml of 5% HCIO^. The homoge-nate was c e n t r i f u g e d f o r 30 minutes at 1,100 g. The sediment was-extracted once more i n the same manner w i t h 10 ml of 5% HCIO^. The combined supernatant f l u i d s were c l a r i f i e d by f i l t e r i n g them through M--layered cheesecloth,and t r i c h l o r o a c e t i c a c i d was added to a f i n a l c o n c e n t r a t i o n of 18% (w/v). The p r e c i p i t a t e was recovered by low-speed c e n t r i f u g a t i o n , washed once i n a c i d i c acetone (200 ml of acetone c o n t a i n i n g 0.1 ml of concentrated HC1), then three times i n acetone and f i n a l l y d r i e d under vacuum. B. I s o l a t i o n of Ribosomal P r o t e i n s Ribosomes were obtairied by the procedure of H a l l b e r g et a l . (1973, 1975). 100 Xenopus l a e v i s eggs were homogenized i n 6-8 volumes of RS b u f f e r (0.01 M t r i s , pH 7.5, 0.0015 M MgCl 2) and the homogenate was spun a t 27,000 g (15,000 rpm) using the S o r v a l l SS-3!4 r o t o r f o r 15 minutes. The supernatant was spun under the same con-d i t i o n once again and then at 125,000 g f o r 1.25 hour (or 4-2,500 rpm f o r 1.65 hour). The ribosomal p e l l e t s were homogenized i n RS b u f f e r and were c e n t r i f u g e d again. The ribosomal b a s i c p r o t e i n s were then extracted from the ribosome p e l l e t s w i t h 0.M- N H^SO^ f o r one hour. For Xenopus embryos, the technique was the same except f o r the a d d i t i o n of 0.5% sodium deoxycholate to the i n i t i a l hoirD-g e n i z a t i o n medium. C. I s o l a t i o n of Yolk Basic P r o t e i n s - 12 -Method of Wallace and Karasaki (1963) 100 Xenopus eggs or embryos were homogenized i n 10 ml of 0.25 M sucrose s o l u t i o n c o n t a i n i n g 5% p o l y v i n y l p y r r o l i d o n e (PVP) pH 7.8. 20 ml of the homogenate were overlayered on 15 ml of 1.0 M sucrose-5% PVP and c e n t r i f u g e d at 1,000 g f o r 20 minutes. The p e l l e t s were washed w i t h homogenation medium and c e n t r i f u g e d as ab above f o r 3 times. The b a s i c p r o t e i n s were extracted from the f i -n a l p e l l e t w i t h 0.4 N H^SO^ f o r one hour. Method of Masui (1968) 100 Xenopus eggs or embryos were homogenized i n 10 ml of 0.25 M sucrose s o l u t i o n c o n t a i n i n g 2.5% f i c o l l and 1.2 mM CaCl^ and the homogenate was c e n t r i f u g e d at 1,000 g f o r 10 minutes to sediment the bulk of y o l k granules and n u c l e i together w i t h a small amount of pigment granules. The sediment was resuspended, layered on a 70% sucrose s o l u t i o n c o n t a i n i n g 0.5mM CaC^ and c e n t r i f u g e d at 50,000 g f o r one hour. N u c l e i and pigment granules sedimented, but y o l k granules always remained at the i n t e r f a c e and were c o l l e c t e d w i t h a p i p e t t e . The c o l l e c t e d y o l k granules were resuspended In homogenation medium and c e n t r i f u g e d at 10,000 g f o r 10 minutes. Basic p r o t e i n s were extracted from t h i s p e l l e t w i t h 0.4 N H^SO^ f o r one hour. D. E l e c t r o p h o r e s i s Starch Gel E l e c t r o p h o r e s i s The b a s i c p r o t e i n s were electrophoresed on v e r t i c a l s t a r c h g e l slabs at 250 v o l t s (4°C) i n the upwards d i r e c t i o n , using the method of Louie and Dixon (1972) (12x25x0.6 cm., pH 3.4, 4 M urea, 15 h r s ) . The gels were b i s e c t e d and stained by the s e n s i t i v e procedure of - 13 -Sung and Smithies (1969). I n t h i s procedure each h a l f of the g e l was placed f o r 30 minutes i n a t r a y c o n t a i n i n g 200 ml of 0.125% amidoblaek i n 1% a c e t i c a c i d to which 0.6 ml of 1 M c o b a l t n i t r a t e had j u s t been added. The f i n a l c oncentration of c o b a l t n i t r a t e was 0.003 M. The g e l was then destained i n 1 N s u l f u r i c a c i d . Accor-ding to Sung and Smithies (1969) the s e n s i t i v i t y of the method i s based on the d i f f e r e n t i a l . . b e h a v i o r of f r e e and protein-bound amido-blaek s t a i n . I n the presence of C o + + , f r e e dye molecules condense i n t o s m all m i c e l l e s and therefore no longer absorb' much of the i n c i d e n t l i g h t . On the other hand, protein-bound dye does not con-dense and continues to absorb the l i g h t . They i n d i c a t e that the: d e t a i l e d mechanism of t h i s p e c u l i a r behavior i s not known but can be discerned from microscopic examination of the g e l s . The presence of heavy metal i s indispensable i n t h e i r opinion. However, accor-ding to Wray and S t u b b l e f i e l d (1970), a s i m i l a r l y s e n s i t i v e s t a i n i n g of b a s i c p r o t e i n s can be accomplished i n polyacrylamide d i s c g e l using amidoblaek without c o b a l t n i t r a t e , provided that the g e l s were destained i n s u l f u r i c a c i d r a t h e r than the more conventional 7% a c e t i c a c i d . These authors therefore f e e l that the r e s u l t s do not depend on the presence of heavy metal but r a t h e r that f r e e dye i s o x i d i z e d to a c o l o r l e s s leuco form i n the presence of a strong chemical o x i d i z i n g agent l i k e s u l f u r i c acid,whereas protein-bound dye i s not. Whichever explanation i s the c o r r e c t one f o r starch g e l s , the f a c t remains that Sung and Smithies (1969) have observed a hundred-fold increase i n s e n s i t i v i t y i n the d e t e c t i o n of histones by amidoblaek using t h e i r s t a i n i n g procedure. We have therefore u t i l i z e d the c o b a l t n i t r a t e - a m i d o b l a e k - a c e t i c a c i d mixture to o b t a i n - 14 -our s t a r c h g e l electrophoretograms. Two-dimensional s t a r c h slab g e l e l e c t r o p h o r e s i s was based on the aluminum l a c t a t e - u r e a system of Sung and Smithies (1969) i n the f i r s t - d i m e n s i o n and a m o d i f i c a t i o n of the triton-X-100 polyacrylamide g e l system of Spiker et _ a l . (1976) i n the second dimension. A f t e r the f i r s t dimensional separation, the histone regions were cut and were placed on the top of the second s t a r c h g e l f o r the second-di-mensional sepa r a t i o n (7 M urea, 0.03 M aluminum l a c t a t e , 1% t r i t o n X-100). The e l e c t r o p h o r e s i s was c a r r i e d out i n the same manner as i n the f i r s t dimension run except that the t r a y b u f f e r contained 1% triton-X-100. Polyacrylamide Gel E l e c t r o p h o r e s i s a) Method of Bonner et a l . (1968)' Stock s o l u t i o n s ( s u i t a b l e f o r 3 months when stored re-f r i g e r a t e d i n amber g l a s s b o t t l e ) : 1. TEMED s o l u t i o n : 4-8 ml of IN KOH, 17.2 ml of g l a c i a l a c e t i c a c i d , 4- ml of TEMED (N, N, N T, N ' t e t r a - m e t h y l e n e d i a m i n e ) , d i s t i l l e d water up to 100 ml. 2. Acrylamide s o l u t i o n : 60 g of acrylamide, 0.4 g N, N'-methylene-bisacrylamide, d i s t i l l e d water up to 100 ml. 3. 0.2% (w/v) ammonium p e r s u l f a t e : i n f r e s h l y deionized 10 M aqueous urea s o l u t i o n . For p r e p a r a t i o n of 15% acrylamide g e l s , one p a r t of TEMED s o l u t i o n and two p a r t s of acrylamide s o l u t i o n were added to f i v e p a r t s of p e r s u l f a t e - u r e a s o l u t i o n . The mixture was then p i p e t t e d i n t o each e l e c t r o p h o r e s i s tube (0.6 cm: x 6.0 cm). This was care-f u l l y overlayered w i t h 3 M urea s o l u t i o n to allow anaerobic poly-m e r i z a t i o n of acrylamide. P o l y m e r i z a t i o n was complete a f t e r 30 - 15 -minutes. The histone samples, u s u a l l y saturated w i t h urea, were placed on the top of the g e l s . The gels were then electrophoresed i n new t r a y b u f f e r (31.2 g of ^- a l a n i n e , 8 ml of g l a c i a l a c e t i c a c i d , d i s t i l l e d water to 1 l i t e r ) , at 4-5 ma/gel f o r 90 minutes at room temperature. A f t e r e l e c t r o p h o r e s i s , the ge l s were stained w i t h 1% amidoblaek i n 20% (v/v) ethanol, 7% g l a c i a l a c e t i c a c i d , aqueous s o l u t i o n f o r 20-30 minutes, and were destained i n the same s o l u t i o n i n c h a r c o a l d i f f u s i o n d e s t a iner (Hoeffer S c i e n t i f i c Co.,). For photography, P o l a r o i d black/white f i l m (Type 107) was employed. b) Method of Panyim and Chalkley (1969b) Stock s o l u t i o n s 1. 4% TEMED (w/v) i n d i s t i l l e d water. 2. 60% acrylamide (w/v) and 0.4% N, N'- methylene-bis-acrylamide i n d i s t i l l e d water. 3. 0.2% ammonium p e r s u l f a t e (w/v) i n 10 M urea s o l u t i o n f r e s h l y prepared. 15% polyacrylamide g e l was prepared by mixing three istock s o l u t i o n s i n r a t i o s : one p a r t of s o l u t i o n 1, two p a r t s of s o l u t i o n 2 and f i v e p a r t s of s o l u t i o n 3. E l e c t r o p h o r e s i s was performed at room temperature, 2 ma/gel f o r 6 hours, using 0.6 x7.5 cm g e l tubes. The t r a y b u f f e r was 0.9 N a c e t i c a c i d . The g e l s were stained f o r 2 hours w i t h 0.1% amidoblaek i n 20% ethanol, 7% a c e t i c a c i d and water, then destained i n the same s o l u t i o n without amidoblaek. - 16 -RESULTS A. I s o l a t i o n and C h a r a c t e r i z a t i o n of Xenopus Somatic Histones Histones were extracted from Xenopus heart by d i f f e r e n t methods and electrophoresed on sta r c h g e l s and polyacrylamide g e l s . They showed a c h a r a c t e r i s t i c p a t t e r n on the gels (Fig.2, 3). The t e n t a t i v e i d e n t i f i c a t i o n of the bands on the p o l y a c r y l -s amide g e l i n F i g . 2 i s consistent w i t h the r e s u l t s of Spiker e_t a l . on pea histones (1976), Fambrough and Bonner on pea histones (1969), Panyim and Chalkley on c a l f thymus histones (1969) and Felden et a l . on c a l f thymus histones (1976) , as shown i n F i g . 4-A. For s t a r c h g e l s , the t e n t a t i v e i d e n t i f i c a t i o n of i n d i v i d u a l histones shown i n F i g . 3 i s c o n s i s t e n t w i t h the r e s u l t s of Felden e_t a l . on c a l f thymus hist o n e s (197 6), Louie on t r o u t t e s t i s h istones (1968) and Sung e_t a l . on r a t l i v e r histones (1971) , as shown i n F i g . 4-B. Histones from h i g h l y d i f f e r e n t i a t e d c e l l s such as t r o u t red blood c e l l s were used as a marker since they con t a i n r e l a t i v e l y few cytoplasmic o r g a n e l l e s which might y i e l d contaminating problems that i n t e r f e r e w i t h a n a l y s i s of hi s t o n e s . C a l f thymus HM-(a g i f t from Drs. D. Fambrough and J. Bonner), the most e v o l u t i o n -a r i l y conservative h i s t o n e , was the marker f o r the fast-moving histones i n the somatic r e g i o n . I t has been reported that very l y s i n e - r i c h H i histone could be extracted w i t h 5% p e r c h l o r i c a c i d (Johns and B u t l e r , 1962), 5% t r i c h l o r a c e t i c a c i d (De Nooij and Westerbrink, 1962), or 0.1M c i t r i c a c i d at pH 2.0 ( S e t t e r f i e l d and N e e l i n , 1972). These methods were employed to i d e n t i f y t h i s group of nuclear p r o t e i n s . A f t e r s e l e c t i v e e x t r a c t i o n w i t h 5% p e r c h l o r i c a c i d , heart H i - 17a -F i g . 2 E l e c t r o p h o r e t i c p r o f i l e s of Xenopus heart histones on polyacrylamide d i s c g e l s . Histones were prepared by e x t r a c t i n g chromatin w i t h OA N ^SO^ ( Bonner et al. , 1968). The chromatin was prepared w i t h the a d d i t i o n of sodium b i s u l f i t e to prevent p r o t e o l y s i s of the h i s t o n e s . A. The g e l system of Bonner e_t al. (15% polyacrylamide g e l contain-ing 6.25 M urea, electrophoresed f o r 90 minutes at 5 ma/gel). B. The g e l system of Panyim and Chalkley (15% polyacrylamide g e l c o n t a i n i n g 6.25 M urea, pH 3.2, electrophoresed f o r 2M-0 minutes at 2 ma/gel) . In these g e l s and a l l subsequent ones, the o r i g i n i s at the top of the g e l and p o s i t i v e l y charged b a s i c p r o t e i n s migrate towards the negative pole at the bottom. Channels from the same g e l run are grouped together. - 17b-- 18a -F i g . 3 E l e c t r o p h o r e t i c p r o f i l e s of Xenopus histone H i a f t e r s e l e c t i v e e x t r a c t i o n by 5% p e r c h l o r i c a c i d (Johns, 19761. A. Starch g e l (containing 4- M urea, 0.02 M aluminum l a c t a t e , pH 3.4; electrophoresed f o r 16 hours at 250 mamp, 250 v o l t s ) a) Calf thymus histone H4. bl Xenopus lung histone H i . c) Xenopus heart histone H i . d) Xenopus heart histones extracted by 0.4 N H^SO^ from chromatin p r e p a r a t i o n . e) Trout red blood c e l l histones extracted by 0.4 N ^SO^ using the method of N e e l i n e_t _al. (1968) i n a separate experiment. B. Polyacrylamide g e l (Bonner e r t al. , 1968) a) Heart histone HI. b) Heart whole h i s t o n e s . c) C a l f thymus histone H4. H4(-AC)= deacetylated histone H4 H3(d)= dimer of histone H3 H3(m)= monomer of histone H3 _ 18b-A B - 19a -F i g . M- Diagram of e l e c t r o p h o r e t i c comparison of Xenopus heart histones w i t h l i t e r a t u r e data using both polyacrylamide and starch g e l s . A. Polyacrylamide g e l e l e c t r o p h o r e s i s a) Pea whole histones on the g e l system of Panyim and Chalkley (1969a) . Data of Spiker and Wakim (1976) . b) Pea-bud histones on the g e l system of Bonner _et _ a l . (1968) Data of Fambrough and^ Bonner (1969). c) C a l f thymus histones on the gel- system-of vPanyim^•«cti^dCChalkley (1969a) . Data of Felden _et al. (1976). d) Same. Data of Panyim and Chalkley (1969a). e) Xenopus heart histones (extracted i n our laboratory) on the g e l system of Bonner et a l . (1968). Experimental data. B. Starch g e l e l e c t r o p h o r e s i s a) Calf thymus histon e s . Data of Felden _et al.. (1976) . b) Somatic histone r e g i o n of t r o u t t e s t i s . Data of Louie (1968) H2a (+p) = phosphorylated histone H2a. c) Rat l i v e r h i s t o n e s . Data of Sung et a l . (1971). E l e c t r o -phoresis runs a-c were performed on the g e l system of Sung and Smithies (1969) . d) Xenopus heart histones (extracted i n our laboratory) on the g e l system of Sung and Smithies (1969). Experimental data. - 19 b-PEA -- H 3 ( d ) ] H 1 - ] H 2 a , H 2 b J H 3 W - H 4 C A L F - XENOPUS • HI -H3(d)+HI —|H2a,H2b, - J H3H —H4 HI H3 H2b H23 H4 H 3 o x HI -H3 (m) - H 2 a ^ H 2 b H4 3.HI - i H 2 a H2b - I H 3 H4 B CALF mm TROUT H3(d) - | H ! . H 2 a -•H2b,H3(m) «H4 RAT ^ H 3 ( d ) — H2a(+p) H3$n) H2dT-H2b — H4(+AC) - ~ H 4 XENOPUS — H3(d) — HI — H 2 a H3(m) H2b — H 4 mm H3(d) -iHZa H2b -lm,H3(m) H 4 H4( -AC) i - 20 -his t o n e s appeared as three bands on both st a r c h g e l (Fig. 3A, channel C) and polyacrylamide g e l (Fig. 3B, channel a ) , while only one band was observed when whole histones were prepared w i t h O.M-N H^SO^. This might have been due to the p r e f e r e n t i a l e x t r a c t -i o n of HI histones by 5% TCA. I n whole histone preparations, i t appeared that H i separated more r e a d i l y from bulk histones on polyacrylamide g e l (Fig. 3B, channel b) than on starch g e l (Fig. 3A, channel d). In most organisms, histone H3 contains one or more cysteine residues and tends to form aggregates that move more slowly than H3 monomer upon e l e c t r o p h o r e s i s (Panyim et a l . , 1970; 1971a; 1971b). Xenopus and Rana histone H3 contains one cysteine (Byrd and Kasinsky, 1973; and Panyim et a l . , 1970) and r e a d i l y forms dimers. The band w i t h lowest m o b i l i t y i n the somatic histone r e g i o n on sta r c h g e l s (Fig. 3, channel d) i n our preparations i s l i k e l y to be the dimer of histone H3. This i s confirmed i n a comparison w i t h l i t e r a t u r e data ( F i g . M-B) . The m o b i l i t y of histone H3 dimer on polyacrylamide g e l s as compared to that of histone H i v a r i e s , as we can see t h a t H3 dimer of pea moves slower than HI on the g e l system of Panyim and Chalkley (1969a) (Fig. 4-A, channel a) while i t migrates f a s t e r on the g e l system of Bonner e_t al. (1968) (Fig. M-A, channel b) . Presumably histone H3 i n e i t h e r the dimer or monomer form i n my pr e p a r a t i o n should migrate between H i and H4-since the g e l system of Bonner e_t aJL. (1968) was employed (Fig. 3A, channel e). Histone H4 was i d e n t i f i e d by r a p i d m o b i l i t y , co-elec-t r o p h o r e s i s w i t h marker c a l f thymus HM- and amino a c i d a n a l y s i s (as i n d i c a t e d i n Part I I of t h i s t h e s i s ) . A band moving s l i g h t l y - 21 -f a s t e r than histone HM- was o f t e n observed both i n t r o u t blood c e l l histones ( F i g . 3A, channel e) and i n Xenopus heart histones (Fig. 3A, 5A). Since red blood c e l l histones are l e s s l i k e l y to be contaminated by b a s i c ribosomal p r o t e i n s t h i s e xtra band could be the deacetylated form of histone HM- (Adamson and,-Woodland, 197M-; Panyim and C h a l k l e y , 1969a; Pogo et a l . , 1968). D i f f e r e n t histone e x t r a c t i o n methods were employed i n the hope of d i s c o v e r i n g a r e l a t i v e l y simple one f o r i s o l a t i n g histones from a small amount of somatic t i s s u e s . A method was devised by Dr. E. W. Byrd, J r . (197M-) that i n v o l v e s preparing n u c l e i and a c i d - e x t r a c t i n g them to o b t a i n h i s t o n e s . This d i f f e r s from the methods of Bonner et . a l . (1968) and Destree et al.. (1972), which r e l y more h e a v i l y on chromatin preparations from which the histones are a c i d - e x t r a c t e d . . As shown i n F i g . 5A, heart histones i s o l a t e d from f r e s h organs e i t h e r by n u c l e i p r e p a r a t i o n or chromatin ex-t r a c t i o n appear to y i e l d s i m i l a r r e s u l t s . This f i n d i n g was f u r t h e r confirmed by using stored organs. I t was found, however, that h i s t o n e s extracted from organs stored at -70°C showed p r o t e o l y t i c degradation ( F i g . 5C). Of the three d i f f e r e n t p r e parations employed, i t appears that Byrd's method y i e l d e d the l e a s t degraded histone p r e p a r a t i o n from stored organs (F i g . 5B, channels c and d). This was probably due to the f a c t that a f t e r thawing, p r o t e o l y t i c enzymes were released. Less p r o t e o l y s i s should have taken place I f the e x t r a c t i o n procedure had been per-formed more r a p i d l y . In Byrd's method the e n t i r e e x t r a c t i o n of histones took place i n about two hours. This i s l e s s than h a l f the time re q u i r e d f o r the other procedures. - 22a.-F i g . 5 Starch g e l e l e c t r o p h o r e s i s of Xenopus heart histones from f r e s h and stored organs. A. Fresh organs a) Modified method of Bonner et _ a l . (1968) . b) Byrd's method (1974'). B. Stored organs (-70°C, 1 month) a) Method of Destree e_t a l . (1972) b) Modified method of Bonner e_t _ a l . (1968) c) Byrd's method . (197 M-) . d) Repeat. C. Comparison of heart histones prepared from f r e s h and stored organs by modified method of Bonner et al. (1968") a") Fresh organs, b) Stored organs. SH= somatic histones The dotted arrows correspond to products of p r o t e o l y t i c u u u L i , ) " degradation. - 22b-- 23 -Two-dimensional g e l e l e c t r o p h o r e s i s has been attempted on polyacrylamide g e l s w i t h the a d d i t i o n of triton-X-100 (Alfageme et a l . , 1974- and Spiker e_t a_l. , 1976) i n the hope of o b t a i n i n g b e t t e r r e s o l u t i o n of histon e s . We applied t h i s method to histone separation on s t a r c h g e l s . In F i g . 6 we see the e f f e c t of 1% triton-X-100 treatment on the separation. The bulky doublet bands (HI + H2a + H2b + H3 reduced) s p l i t i n t o three spots i n the second dimension i n the presence of triton-X-100 (Fig. 6B), while the doublet bands s t i l l remained as two spots without detergent treatment (Fig. 6A). Separation on triton-X-100 -c o n t a i n i n g g e l s i s based on the hydrophobic character of the histones r a t h e r than charge p r o p e r t i e s . According to Spiker e_t a l . (197 6) the order of c a l f thymus histone m o b i l i t y i n t r i t o n -c o n t a i n i n g polyacrylamide gels was H i ~> H2b > HM- > H3 >H2a. . This does not seem to be the case f o r s t a r c h g e l where the m o b i l i t y of histone H4- i s s t i l l greater than histone H i i n the presence of detergent. More work has to be done on the ident-i f i c a t i o n of the three spots i n F i g . 6B. I n a d d i t i o n , the experimental c o n d i t i o n s have to be tested i n order to o b t a i n as s a t i s f a c t o r y r e s o l u t i o n on two dimensional s t a r c h g e l as on polyacrylamide g e l s . B. I s o l a t i o n and C h a r a c t e r i z a t i o n of T e s t i s - S p e c i f i c Histones  i n Amphibians and R e p t i l e s D i f f e r e n t methods were employed to i s o l a t e t e s t i c u l a r histones from Xenopus l a e v i s . I n F i g . 7, the electrophoretograms showed that Xenopus t e s t i s contained two fast-moving bands, X g and X f, on both s t a r c h g e l (Fig. 5A) and polyacrylamide g e l (Fig. - 24a -F i g . 6 Two-dimensional st a r c h g e l electrophoretogram of Xenopus heart h i s t o n e s . A. The f i r s t dimension ( l e f t to r i g h t ) i s an aluminum lac t a t e - u r e a g e l system (4 M urea, 0.02 M aluminum l a c t a t e , pH 3.4). The second dimension (top to bottom) i s a s i m i l a r g e l system (7 M urea, 0.03 M aluminum l a c t a t e , pH 2.7). B. The f i r s t dimension ( r i g h t to l e f t ) and the second dimension are the same as those i n A except that i n the second dimension run 1% triton-X-100 i s added. For reference, a diagram of each of the two g e l channels of the f i r s t dimension i s p i c t u r e d above the photograph and a diagram of the. two-dimensional g e l i s shown below. - 24b-j H1,H2<U lH2b,H3J H3(ox.) I H4 I e e v CalfH4 CalfH4 1 HI, I-1 H2b, H2<3, H3 H4 H3(ox) B H3(ox) J )Hl,H2a, 0 H2b,H3 I H4 > CalfH4 J CalfH4 H3 (ox) \H2b,H3 o / H4 + Triton-X-100 - 25 -7B). Comparing the d i f f e r e n t e x t r a c t i o n procedures used, i t appears that the t e s t i c u l a r c e l l suspension i s as good as more c a r e f u l p u r i f i c a t i o n procedures i n c h a r a c t e r i z i n g the t e s t i s - s p e c i f i c h i stones of Xenopus. Because i t i s a much simpler procedure re-q u i r i n g only small amounts of m a t e r i a l , a s i n g l e t e s t i s from one small animal being s u f f i c i e n t , we have been able to examine the t e s t i s - s p e c i f i c h istones from s e v e r a l species amongst amphibians and r e p t i l e s . The comparison of amphibian t e s t i c u l a r histones i s shown i n F i g . 8'. Sigma protamine prepared from h e r r i n g was used as a marker (channel g). T e s t i s - s p e c i f i c histones of three anuran species (channels b, d and f ) and urodeles species (channel c) sjtdwed marked d i v e r s i t y upon polyacrylamide g e l electrophoretogram. Rana had only somatic histones (channel b); Xenopus had two bands moving more r a p i d l y than HM- (channels d and e) ; Buf o showed a s i n g l e band that moved somewhat f a s t e r than Sigma protamine (channel f ) ; Cynops, the Japanese f i r e - b e l l y newt, also showed a s i n g l e band that moved to the r e g i o n between Xenopus X^'.and Sigma protamine (channel c ) . The t e s t i s - s p e c i f i c histones of r e p t i l e s also have been examined. As i n d i c a t e d i n F i g . 9, A n o l i s (channel c) ; Terrapene, the F l o r i d a box t u r t l e (channel f) and Sceloporous, the desert spiny l i z a r d (channel h ) , each had a s i n g l e band migrating i n the r e g i o n of the g e l c l o s e to Sigma protamine (channels a, e and g ) , as d i d the d u c t u s - d e r e r e n s - s p e c i f i c histone of Thamnophis (channel b) and the semen-specific histone of Elaphe (channel d). Amphibian t e s t i s - s p e c i f i c histones were also c h a r a c t e r i z e d by s t a r c h g e l e l e c t r o p h o r e s i s ( F i g . 10). Rana showed no bands - 26a -F i g . 7 Xenopus t e s t i s - s p e c i f i c histones extracted by d i f f e r e n t methods. A. Starch g e l electrophoretogram a,b) C e l l suspension. c) Byrd's method (1974-). d) Modified method of Bonner et a l . (1968). e) Method of Destree et al. (1972). f) Xenopus heart histones from chromatin p r e p a r a t i o n (modified method of Bonner et a l . (1968). B. Polyacrylamide g e l s (Bonner et a_l. , 1968) a) Modified method of Bonner et a l . (1968). b) Byrd's method (1974). c) C e l l suspension. d) Repeat. e) Calf thymus histone HM-. f) Xenopus heart histones extracted from c e l l suspension. X and X I = slow and f a s t t e s t i s - s p e c i f i c Xenopus h i s t o n e s . - 2 6 b -a b c d e f - 2 7 a -F i g . 8 E l e c t r o p h o r e t i c comparison of t e s t i s - s p e c i f i c histones from amphibian c e l l suspension on polyacrylamide g e l s . Gel system of Bonner e_t _ a l . (1968) was employed. a) C a l f thymus HM-. b) Rana p i p i e n s (frog) t e s t i s . c) Cynops pyrrhogaster (newt) t e s t i s . d) Xenopus l a e v i s (toad) t e s t i s . e) Xenopus l a e v i s (toad) t e s t i s . f ) Bufo marinus (toad) t e s t i s . g) Sigma protamine ( h e r r i n g ) . P= Sigma protamine prepared from h e r r i n g . - 27b-- 28a -F i g . 9'. E l e c t r o p h o r e t i c comparison of t e s t i s - s p e c i f i c histones from r e p t i l i a n c e l l suspension on polyacrylamide g e l s . The g e l system of Bonner e_t _al. (1968) was employed. Sigma protamine (h e r r i n g ) . Thamhophis S i r t a l i s (snake ductus deferens. A n o l i s c a r o l i n e n s i s ( l i z a r d ) t e s t i s . Elaphe guttata guttata (snake) semen. Sigma protamine (herrin g ) . Terrapene c o r o l i n a b a u r i ( t u r t l e ) t e s t i s . Sigma protamine ( h e r r i n g ) . Sceloporous magister ( l i z a r d ) t e s t i s . A n o l i s c a r o l i n e n s i s ( l i z a r d ) t e s t i s . - 28b-d b c d e f q h i - 29a -F i g . 10 E l e c t r o p h o r e t i c comparison of t e s t i s - s p e c i f i c histones from amphibian c e l l suspensions on..starch g e l s . a) Cynops pyrrhogaster (newt). b) Xenopus l a e v i s (toad). c) C a l f thymus HM-. d) Rana p i p i e n s (frog) . Cp = newt t e s t i s - s p e c i f i c h i s t o n e s . 29o CP-CO-SH calf H4" l - X c c d moving f a s t e r than HM- (channel d) ; Xenopus had two faster-moving bands (channel b ) . Those were the same as bands on polyacrylamide g e l s . Instead of showing only one band as on polyacrylamide g e l (Fi g . 8,channel c ) , a t e s t i s c e l l suspension from Cynops showed a doublet moving more r a p i d l y than X^ and a t h i r d band migrating i n the r e g i o n of X . (Fig. 10, channel a). A v a r i e t y of banding patterns due to d i f f e r e n t g e l systems employed was also observed by Bols et _ a l . (1976) . They found that the t e s t i s - s p e c i f i c h i stones from Notophthalmus, the eastern red spotted newt, showed one band on the polyacrylamide g e l system of Panyim and Chalkley (1969) , but 3 bands on the polyacrylamide g e l system of Bonner e_t a l . (1968), and the s t a r c h g e l system of Sung and Smithies (1969). I t seems that s t a r c h g e l y i e l d e d greater separation than d i d polyacrylamide g e l f o r t e s t i s - s p e c i f i c histones when the sample was r e l a t i v e l y u n p u r i f i e d . This phenomenon could also be observed f o r Sigma protamine extracted from h e r r i n g (Fig. 19) . However, the urodele bands u s u a l l y were more d i f f u s e on s t a r c h .thai* on polyacrylamide gels (Kasinsky, Byrd, Kwauk and Yee, unpublished data). C. I s o l a t i o n and C h a r a c t e r i z a t i o n of Xenopus Embryonic Histones Histones i s o l a t e d from Xenopus swimming tadpoles (stage 50) showed the same e l e c t r o p h o r e t i c p a t t e r n as d i d those from a d u l t heart upon s t a r c h g e l e l e c t r o p h o r e s i s (Fig. 11). Whether the h i s -tones were obtained by Byrd's (197M-) nuclear p r e p a r a t i o n (channel b) or by the chromatin p r e p a r a t i o n of Destree et al. (1972)„ (channel a ) , they showed adequate r e s o l u t i o n . However, f o r e a r l y embryos before stage M-0, Byrd's method was not a p p l i c a b l e because of the presence of a large amount of y o l k p r o t e i n s as w e l l as - 31a -F i g . 11 Starch g e l e l e c t r o p h o r e t i c p r o f i l e s of histones from Xenopus swimming tadpoles (stage 50) (Nieuwkoop and Faber,1967). a) Method of Destree _et al. (1972). b) Byrd's method (10 tadpoles'). c) Calf thymus H4-. - 31b-H3(ox) — [ H2b,H3 J L_ W H4 — H4(-Ac) — — Calf HA a b c - 32 -newly-made ribosomal p r o t e i n s (Hallberg and Brown, 1973). Therefore, the method of Destree e_t a_l. (1972) was employed to e x t r a c t histones from Xenopus g a s t r u l a and neurula. In the method of Destree e_t a l . (1972), 2.4- M sucrose c e n t r i f ugation was capable of separating the bulk of the yolk granules and cytoplasmic organelles from the n u c l e i , which were p e l l e t e d by c e n t r i f u g a t i o n . Yet yolk granules i n the e a r l y embryos were not completely removed. This p o s s i b l e contamination makes the histone banding p a t t e r n of the e a r l y embryos d i f f i c u l t to c h a r a c t e r i z e . However, the method of Destree et a l . (1972) appears to be the best method known so f a r . The other advantage of using t h i s method, combined w i t h starch g e l e l e c t r o p h o r e s i s , i s that only a small number of embryos (ca. 150) are required to o b t a i n c l e a r r e s o l u t i o n . The banding patterns were e s s e n t i a l l y s i m i l a r f o r Xenopus gas-t r u l a , neurula and swimming tadpoles (Fig. 12). In order to com-p l e t e l y c h a r a c t e r i z e histones from e a r l y embryos, amino a c i d ana-l y s i s of the bands w i l l be re q u i r e d . The l y s i n e - r i c h histone H i from tadpoles also showed three bands ( F i g . 12, channel g) that were 3 s i m i l a r to those of somatic histone H i (Fig. 3A, channels b and c ) . Histones extracted w i t h 0.1 M c i t r i c a c i d at pH 2.0 showed two bands on sta r c h electrophoretogram (channel i ) , p o s s i b l y l y s i n e - r i c h h i s -tones H i , H2a+H2b. Since the order of release of histone f r a c t i o n s from DNA i s a f u n c t i o n of pH and in v o l v e s the breakdown of e l e c t r o -s t a t i c linkages between histone molecules and DNA, histone H i and H2a+H2b were more r e a d i l y extracted than H3 and H4 at pH 2.0 (Murray, 1966) . I t i s assumed that the f i r s t band was H i and the second band was H2a+H2b when channels e,g and i are compared. Because histones from e a r l y embryos could be assayed by sta r c h - 33a -F i g . 12. E l e c t r o p h o r e t i c p r o f i l e s of Xenopus embryonic histones on st a r c h g e l s . a) G a s t r u l a (stage 9) , method of Destree e_t _ a l . (1972) . (100-150 embryos) . b) Neurula (stage 13), method of Destree et a l . (1972). (100-150 embryos) . c) Swimming tadpoles (stage 50), Byrd's (1974) method (10 embryos). d) Xenopus heart histones from chromatin, method of modified method of Bonner e_t j a l . (1968) . e) Swimming tadpoles (stage 50), Byrd's (1974) method. f) Gastrula (stage 9) , method of Destree e_t a l . (1972) . g) Swimming tadpoles (stage 50), 5% p e r c h l o r i c a c i d e x t r a c t i o n . h) Swimming tadpoles (stage 50) . i ) L y s i n e - r i c h h i s t o n e s s e l e c t i v e l y e x tracted w i t h 0.1 M c i t r i c a c i d at pH 2.0. ( S e t t e r f i e l d and N e e l i n , 1972) j) Calf thymus H4. - 33b_ d b c d e f g h ' J - 34 -g e l e l e c t r o p h o r e s i s using small numbers of animals using the method of Destree e_t _ a l . (1972) , a study of histone changes during Xenopus embryogenesis was undertaken.. The r e s u l t s (Tig. 13') i n d i c a t e d the e l e c t r o p h o r e t i c p r o f i l e s of histones remained the same during e a r l y embryogenesis, as p r e v i o u s l y noted by Destree et_ a l . (1973) using polyacrylamide g e l e l e c t r o p h o r e s i s . The bands slower than histone H3 (oxidized form) and the band moving between H3 and the r e s t of the ,.* • somatic his t o n e s were probably y o l k p r o t e i n s (Fig. 13C). These bands disappeared a f t e r the embryos s t a r t e d to feed (stage 41) when the yolk p o o l waS-dfip&e'ted. I n order to check the p o s s i b l e contamination of embryonic histones by ribosomal and yolk b a s i c p r o t e i n s , the l a t t e r two cl a s s e s of p r o t e i n s were i s o l a t e d and c h a r a c t e r i z e d e l e c t r o p h o r e t i c a l l y on sta r c h and polyacrylamide g e l s . Yolk b a s i c p r o t e i n s were extracted from yol k granules by two methods. Masui's method (1968) could not be repeated because of the s u b s t a n t i a l contamination of yolk ++ + granules by n u c l e i . I t has been reported that Ca , Na , e t c , would cause the breakdown and the extensive aggregation of yolk granules (Greenhouse and Morrisey, 1974; Essner, 1954). There-f o r e , a simple sucrose s o l u t i o n c o n t a i n i n g 5% p o l y v i n y l - p y r r o l i d o n e (PVP) without c a t i o n s was used. From F i g . 14, i t appears that the major yolk b a s i c p r o t e i n s ran slower than somatic histones on starch g e l w i t h one band running between H3 (oxidized form) and H1+H2+H3(TI) ( F i g . 14B). A s i m i l a r r e s u l t has been obtained upon polyacrylamide g e l e l e c t r o p h o r e s i s (Fig. 14A). In F i g . 15, we see that ribosomal p r o t e i n s overlapped the histones i n some regions of the g e l . Because the ribosomes were - 35a -F i g . 13 E l e c t r o p h o r e t i c p r o f i l e s of Xenopus embryonic histones on s t a r c h gels during embryogenesis. Histones were extracted accor-ding to the method of Destree et a l . (1972). A. a) C a l f thymus histone H i . b-d) Stages 33-34. 1 e-h) Stages 8-11. i ) Stages 1-4. B. a) Ca l f thymus H4. b) Swimming tadpoles a f t e r stage 45. c-d) Stage 40 e-f) Stages 22-26. g-h) Stage 13. i - j ) Stages 10%-11. C. Diagram of Xenopus histones during embryogenesis. a) C a l f thymus H4. b) Swimming tadpoles a f t e r stage 45. c) Stages 33-34. d) Stages 22-26. e) Stages 13-15. f) Stages 9-10%. g) Stages 1-4. About 100 embryos were used i n each channel. The l o c a t i o n of y o l k b a s i c p r o t e i n s i s i n d i c a t e d i n C. - 3 5 b -- 36a -F i g . 14- E l e c t r o p h o r e t i c comparison of Xenopus embryonic histones and yolk b a s i c p r o t e i n s . A. Polyacrylamide g e l (Bonner e_t al. , 1968) a) Swimming tadpole histones (stage 50). b) E a r l y embryo ( f e r t i l i z a t i o n to stage 12) yolk b a s i c p r o t e i n s ( s o l i d l i n e s ) B. Starch g e l ""(Sung and Smithies, 1969) a) Tadpole h i s t o n e s . b) E a r l y embryo yolk basic p r o t e i n s , ( s o l i d l i n e s ) . - 36b-- 37a -F i g . 15 •-. E l e c t r o p h o r e t i c comparison of Xenopus embryonic histones and ribosomal b a s i c p r o t e i n s . A. Starch g e l (Sung and Smithies, 1969) a) Swimming tadpole! h i s t o n e s . b) Gastrula ribosomal b a s i c p r o t e i n s extracted without 0.5% sodium deoxycholate. c) Gastrula ribosomal b a s i c p r o t e i n s extracted i n the presence of 0.5% sodium deoxycholate. B. Polyacrylamide g e l (Panyim and Chalkley, 1969) a) Gastrula ribosomal b a s i c p r o t e i n s . b) Swimming tadpole h i s t o n e s . c) Calf thymus histone HM-. The dotted arrow, i n d i c a t e s an a r t i f a c t due to a loose piece of g e l . -37b-A B - 38 -spun down from a post-nuclear supernatant which was c e n t r i f u g e d at high speed twice, i t was u n l i k e l y that they were contaminated by n u c l e i . I n a d d i t i o n , ribosomal b a s i c p r o t e i n s showed bands moving f a s t e r than those of the somatic h i s t o n e s . Whether these f a s t e r bands represent-.: degraded p r o t e i n s i s not known at t h i s time. However, t h i s r e s u l t seems to be c o n s i s t e n t w i t h the r e s u l t s of N e e l i n and V i d a l i (1968) on st a r c h g e l and of Felden _et _ a l . (1976) on polyacrylamide g e l i n that both show overlapping and f a s t moving bands f o r ribosomal p r o t e i n s i n b i r d s and f u n g i . Histones extracted from aspermatogenic t e s t i s c e l l suspensions showed no bands moving f a s t e r than histone HM- upon elec-t r o p h o r e s i s (Fig. 16) while those from spermatogenic t e s t i s showed fast-moving bands (Fi g . 8c, F i g , 9c). This observation supports the view that the f a s t moving t e s t i s - s p e c i f i c bands appearing upon e l e c t r o p h o r e s i s are not due to p o s s i b l e contamination of t e s t i c u l a r h i s t o n e s by ribosomal b a s i c p r o t e i n s . Otherwise, one would expect the same p a t t e r n i n both aspermatogenic and spermatogenic t e s t e s . - 39a -F i g . 16 E l e c t r o p h o r e t i c p r o f i l e s of aspermatogenic t e s t i s c e l l suspensions from l i z a r d and newt. a) Sigma protamine (herring) b) A n o l i s c a r o l i n e n s i s ( l i z a r d ) t e s t i s , no sperm observed by l i g h t microscopy. c) Cynops pyrrhogaster (newt) t e s t i s , no sperm observed by l i g h t microscopy. d) Sigma protamine (herring) - 39b-a b c d - 4-0 -DISCUSSION Xenopus heart h i s t o n e s have been extracted and c h a r a c t e r i z e d on both s t a r c h gels and polyacrylamide g e l s . The order of m o b i l i t y of the f i v e histone ' f r a c t i o n s are H3 (dimer) ^ > H i + H2a + H2b + H3 (monomer) > m on s t a r c h g e l and H i >H2a + :H2b + H3 (d,m) > m on polyacrylamide g e l . The t e n t a t i v e i d e n t i f i c a t i o n of the banding pat t e r n s on g e l s needs f u r t h e r c o n f i r m a t i o n by amino a c i d a n a l y s i s . D i f f e r e n t methods of histone i s o l a t i o n have been t r i e d i n order to f i n d a simple method to ensure the minimal l o s s and degradation of m a t e r i a l during the pr e p a r a t i o n procedures from a small amount of m a t e r i a l . The method of Bonner e t a l . (1968) i n v o l v e s a " d i r e -c t " chromatin p r e p a r a t i o n and that of Destree et a_l. (1972) invo-l v e s an " i n d i r e c t " chromatin p r e p a r a t i o n from i s o l a t e d n u c l e i . I t has been reported (Furlan et a l . , G a r r e l s , et jal.,1972) that there are nuclear histone proteases i n c a l f thymus n u c l e i , chromatin and r a t l i v e r chromatin. Destree et a l . (1975) and H e i n r i c h e_t a l . (197 6) observed the s u b c e l l u l a r d i s t r i b u t i o n of histone-degrading enzyme a c t i v i t i e s from Xenopus l i v e r and r a t l i v e r . These f a c t s suggest that the time-consuming chromatin p r e p a r a t i o n s , e i t h e r prepared d i r e c t l y or i n d i r e c t l y from i s o l a t e d n u c l e i , are l i k e l y to be a c c e s s i b l e to p r o t e o l y t i c degradation. Histones extracted from a nuclear p r e p a r a t i o n ( s u i t a b l e f o r heart) and from a c e l l suspension ( s u i t a b l e f o r tes t i s ) s e e m to undergo l i t t l e proteo-l y s i s , i f any, as revealed by our experimental r e s u l t s . For em-bryonic h i s t o n e s , because of the serious cytoplasmic contamination (Destree et a l . , 1972; Asao et a l . , 1969), the chromatin prepara-*-.-- V i -r i o n from i s o l a t e d n u c l e i using the method of Destree e_t a l . (1972) i s r e q u i r e d . E l e c t r o p h o r e t i c c h a r a c t e r i z a t i o n i n d i c a t e s the existence of microheterogeneity i n histone H i . This i s c o n s i s t e n t w i t h the general agreement that there are s u b f r a c t i o n s separable by e l e c -t r o p h o r e t i c and chromatographic means w i t h i n t h i s histone c l a s s (Stellwagen and Cole, 1969; Sherod et a l . , 197•+) . D i f f e r e n t Xe-nopus t i s s u e s , i n c l u d i n g lung and heart have been examined and each one shows a l l the H i histone bands. This r e v e a l s no t i s s u e s p e c i f i t y , although Panyim et a l . (1971) have suggested that the r e l a t i v e i n t e n s i t y of d i f f e r e n t H i bands might be tissue-dependent. I t i s not known whether the microheterogeneity i s caused by such f a c t o r s as phosphorylation or a c e t y l a t i o n of the same molecule or by-molecules w i t h s l i g h t l y d i f f e r e n t sequences. A c o n t r i b u t i o n from a l l three p o s s i b i l i t i e s i s l i k e l y . Swimming tadpole histone H i a l s o shows three bands upon s t a r c h g e l e l e c t r o p h o r e s i s . This i s c o n s i s t e n t w i t h the previous observation of synthesis of three H i h i s t o n e s i n Xenopus swimming tadpole as revealed by chromato-graphy on amberllte (Byrd and Kasinsky, 1973). I t appears that the microheterogeneity of histone H i i n a d u l t tissues seems to have i t s o r i g i n i n e a r l y development. S i m i l a r r e s u l t s ( s u b f r a c t i o n a t i o n of histone Hi) have also been reported by Imoh and Minamidani (1973) i n newt embryos and S e v a l j e v i c (1973) i n sea u r c h i n embryos. I t can be seen from our e l e c t r o p h o r e t i c data that during the development of Xenopus the histone complement remains q u a l i t a t i v e l y u n altered. I t i s d i f f i c u l t to d i s c e r n changes i n r e l a t i v e quan-t i t i e s of d i f f e r e n t histone f r a c t i o n s because of the d i f f e r e n c e s - 42 -In histone e x t r a e t a b i l i t y from chromatin during successive deve-lopment stages. For i n s t a n c e , s t r u c t u r a l changes i n the n u c l e i of i n t a c t embryos have been observed with/the l i g h t microscope (Immers, 1972) and w i t h the e l e c t r o n microscope (Runnstrom, 1967) and these changes might be c o r r e l a t e d w i t h d i f f e r e n c e s i n the ex-t r a e t a b i l i t y of histones during development. Adamson and Woodland (1974) have found f o u r of the f i v e main histone f r a c t i o n s i n Xenopus being synthesized a t a l l stages of e a r l y development, except f o r histone HI,which was f i r s t detected at the l a t e b l a s t u l a stage. Byrd and Kasinsky (1973a) have found t h a t major c l a s s e s of histones are synthesized both i n cleavage and swimming Xenopus embryos and appear to be q u a l i t a t i v e l y the same. However, Adamson and Woodland (1974) were able to examine s e v e r a l embryos from each stage using m i c r o i n j e c t i o n techniques to l a b e l the histones w i t h H - l y s i n e , while Byrd and Kasinsky (1973a) had to average t h e i r data from the t w o - c e l l stage to g a s t r u l a em-14 bryos as t h e i r CO2 l a b e l was much l e s s s e n s i t i v e i n l a b e l i n g the h i s t o n e s . I n sea u r c h i n embryos, an increase i n HI or i t s subfra-c t i o n s and a decrease i n s l i g h t l y l y s i n e - r i c h histones during e a r l y development have been observed by Benttinen et a l . (1971) i n Ly- techinus p i c t u s and by Easton et _ a l . (1972) i n Arbacia punctulata. r e s p e c t i v e l y . S e v a l j e v i c (1973) has found an Increase i n the sy-n t h e s i s of r e l a t i v e amounts of HI and H3 histones compared to that of the H2a + H2b, and a q u a l i t a t i v e change of HI histone r e l a t e d to the t r a n s i t i o n of embryos from b l a s t u l a to g a s t r u l a stage i n Paracentrntus l i v i d u s . Ruderman et a l . (1974) a l s o have observed that i n Lytechinus and A r b a c i a , the synthesis of d i f f e r e n t kinds - 143 -and amounts of hiatones do i n f a c t d i f f e r c h a r a c t e r i s t i c a l l y from one stage of development to the next. Such a p a t t e r n of histone changes during development of the surf clam, S p i s u l a s o l i d i s s i m a , has also been reported by G a b r i e l l i and B a g l i o n i (1975). The discrepancy between ob-servations reported here and those c i t e d l i t e r a t u r e studies might be due to the f a c t that the c i t e d works emphasize- " s y n t h e s i s " of histones while the present work concerns mainly the "content" of h i s t o n e s . D i f f e r e n c e s i n e x t r a c t a b i l i t y and/or degradation of histones could i n f l u e n c e the r e s u l t s c i t e d i n the above l i t e r a t u r e . I n a d d i t i o n , the maternal p o o l of histones accumulated during oogenesis to provide enough histones f o r subsequent development (Adamson and Woodland, 1974-, 1977; Woodland and Adamson, 1977; Gagnetti, 1974-) might play a r o l e i n rendering the content of each histone f r a c t i o n q u a l i t a t i v e l y constant. The present r e s u l t s i n d i c a t e that there i s no dramatic change i n the e l e c t r o p h o r e t i c p r o f i l e s of h i s t o n e s during e a r l y embryogenesis In Xenopus. The r e s u l t s are en-t i r e l y c o n s i s t e n t w i t h the e a r l i e r observations of Destree et _al. (1973) on the histone content of Xenopus embryos. However, employing a deter-gent-polyacrylamide g e l system might r e v e a l d i f f e r e n c e s i n H2a and H2b s u b t r a c t i o n s as Cohen ejt a_l. (1976) observed i n sea u r c h i n embryogenesis. Before going i n t o the d i s c u s s i o n of e l e c t r o p h o r e t i c p r o p e r t i e s of t e s t i s - s p e c i f i c h i s t o n e s , I would l i k e to p o i n t out that " t e s t i s - s p e c i f i c " are p o s s i b l y "sperm-specific" as w e l l because they only show up e l e c t r o -p h o r e t i c a l l y when the t e s t i s contains sperm. However, c e l l separation techniques, such as those of Eckhardt and R i s l e y (1976) w i l l be needed to d i s t i n q u i s h histones i n spermatids from those i n _ Ui4 _ sperm. I n i t i a l experiments (Kasinsky e_t a l . 197M-) using STAPUT c e l l s eparation i n bovine serum albumin gradients (Lam e t a l . 1970) i n d i c a t e t h a t sperm c o n t a i n the two main Xenopus bands and the m u l t i p l e Bufo bands upon sta r c h g e l e l e c t r o p h o r e s i s of acid-ex-t r a c t e d sperm h i s t o n e s . The s t a r c h and polyacrylamide e l e c t r o p h o r e t i c comparisons of nuclear basic p r o t e i n s present i n Rana, Xenopus, Bufo, and Cynops te s t e s r e v e a l a broad spectrum of histones i n these amphibians. Rana t e s t i s contains only the somatic complement of histone s . This i s i n agreement w i t h s e v e r a l e a r l i e r s t u d i e s . Vendrely (1957) found that the amino ac i d composition of histones extracted from Rana sperm and from somatic t i s s u e s were s i m i l a r , while Bloch (1962), and Bols and Kasinsky (1972) found no cytochemical d i f f e r e n c e s between histones from t e s t e s and somatic t i s s u e s . Bols and Kasinsky (1973) saw s i m i l a r e l e c t r o p h o r e t i c patterns i n t e s t i s and somatic t i s s u e but Alder and Gorovsky (1975) d i d f i n d a new l y s i n e - r i c h histone H i i n Rana sperm by e l e c t r o p h o r e s i s on long polyacrylamide g e l s . Unlike Rana p i p i e n s , e l e c t r o p h o r e s i s of histones from Xenopus l a e v i s t e s t i s r e v e a l s two fast-moving bands, X s and Xf , which are probably sperm-specif i c (Kasinsky e_t a l ., 197M-; Eckhardt and R I s l e y , 1976). Bands X s and Xf demonstrate m o b i l i t i e s intermediate between somatic histones and the very a r g i n i n e - r i c h protamines. Thus the M y t i l u s (mussel) c l a s s of sperm histone to which Xenopus sperm histone belongs i s not only an "intermediate" type c y t o l o g i c a l l y (Bloch, 1969) but a l s o appears to be "intermediate" w i t h respect to b a s i c i t y and molecular weight upon e l e c t r o p h o r e s i s . - M-S -One fast-moving band on polyacrylamide g e l i s s p e c i f i c to the t e s t i s of Bufo and migrates i n the v i c i n i t y of Sigma protamine. I t i s probably also sperm-specif i c (Kasinsky et a l . 197'+) . Bols and Kasinsky (1972) t e n t a t i v e l y suggested that Bufo sperm might be c l a s s i f i e d as a Rana type r a t h e r than a Salmo protamine based on the unusual cytochemical data. Bloch (1969), on:, the other hand, c l a s s i -f i e d the spErm histones of Bufo v u l g a r i s as the M y t i l u s type on the b a s i s of h i s unpublished cytochemical data. The present e l e c -t r o p h o r e t i c experiments favor the Bufo sperm histone being s i m i l a r to the salmon type. However, since the e l e c t r o p h o r e t i c c h a r a c t e r i -z a t i o n i s based on charge, molecular s i z e and shape, i t i s d i f f i c u l t to r e c o n c i l e the cytochemical c l a s s i f i c a t i o n w i t h the e l e c t r o p h o r e t i c p r o p e r t i e s . E l e c t r o p h o r e s i s of Cynops pyrrhogaster histones extracted from c e l l suspensions r e v e a l s that the p a t t e r n of t e s t i s - s p e c i f i c b a s i c p r o t e i n s i n t h i s urodele i s d i s t i n c t from that i n the anurans, Rana, Bufo and Xenopus. Cynops t e s t i s - s p e c i f I c histones show a s i n g l e band migrating a b i t slower than h e r r i n g protamine on the polyacrylamide g e l system of Bonner et a l . (1968). On s t a r c h gels i t shows three bands i n the r e g i o n between histone HM- and Sigma protamine. This i s c o n s i s t e n t w i t h the e l e c t r o p h o r e t i c r e s u l t s obtained w i t h t e s t e s from newt Notophthalmus (Bols and Kasinsky, 1976). I t has been reported ( P i c h e r a l , 1970; Bols and Kasinsky, 197 6) that during spermiogenesis i n the newts Pleurodeles w a l t l i i and Notophthalmus v i r i d e s c e n s c e l l s undergo a progressive s h i f t from somatic h i s t o n e s - ^ " s t a b l e protamines'Vprofamines •'in the mature sperm. Thusj the three-banded patern of sperm - s p e c i f i c histones i n - 46 -the s t a r c h g e l might r e f l e c t molecular species i n d i f f e r e n t t r a n -s i t i o n stages or might represent heterogeneity i n the;:s;ame sperm nucleus, as p r e d i c t e d by Bedford and C a l v i n (1974). Cytochemically, the newt's t e s t i s - s p e c i f i c histones are c l a s s i f i e d as the "salmon*-, type" (Bols and Kasinsky, 1976; P i c h e r a l , 1970). However, the e l e c t r o p h o r e t i c r e s u l t s i n d i c a t e that they are i n f a c t d i s t i n c t from those i n salmon. Thus, the cytochemical c l a s s i f i c a t i o n of newt sperm'as the salmon type i n Bloch's scheme (1969) probably i n d i c a t e s that a d i s t i n c t range e x i s t s f o r p o s s i b l e b a s i c p r o t e i n s f a l l i n g i n t o t h i s cytochemical category. In c o n t r a s t to the amphibian s t o r y , the polyacrylamide g e l e l e c t r o p h o r e t i c comparison of t e s t i s - , ductus deferens- and semen-s p e c i f i c h istones present i n Thamnophis, A n o l i s , Elaphe, Terrapehe and Sceloporous r e v e a l s a marked s i m i l a r i t y i n the m o b i l i t y of these b a s i c p r o t e i n s . They a l l show a s i n g l e band that migrates i n the v v i c i n i t y of Sigma protamine. Our e a r l i e r observations by s t a r c h g e l e l e c t r o p h o r e s i s (Kasinsky et a l . , 1977) i n d i c a t e d that the t e s t i s s p e c i f i c h istones of l i z a r d s ( L a c e r t i l i a ) and snakes (Sauria) also showed a remarkably s i m i l a r two-banded p a t t e r n i n t h i s medium, one band moving cl o s e to the fast-moving band of Xenopus and the other close ;_to that of t r o u t protamine. The r e s u l t s from both g e l systems showcfthe s i m i l a r i t y i n the t e s t i s - and sperm - s p e c i f i c histones of the r e p t i l e s thus f a r examined. In general, then, both e l e c t r o p h o r e t i c and cytochemical pro-p e r t i e s show the s i m i l a r i t y among r e p t i l i a n t e s t i s - s p e c i f i c histones and the d i v e r s i t y among amphibian ones. This i s also true of the chemical data thus f a r obtained, as w e ^ w i l l see when we discuss - 47 -the amino a c i d a n a l y s i s of these b a s i c p r o t e i n s i n Part I I of the Discussion. 1 i - 47a -Part I I AMINO ACID ANALYSIS - 48 -MATERIALS AND METHODS The amino a c i d analyses were performed on the amidoblack-stained bands from polyacrylamide g e l s i n tubes or from s t a r c h g e l s l a b s . Methods f o r the e l e c t r o p h o r e s i s of these gels have been described i n Part I . The a n a l y s i s of bands on polyacrylamide g e l was based mainly on the methods of Houston (1971), and P a l l o t t a and Te s s i e r (197 6). The i n d i v i d u a l s t a i n e d histone bands were c a r e f u l l y cut out using a new s i n g l e edge razor blade and t r a n s f e r r e d i n t o a clea n t e s t tube(no. 9800, Pyrex). H y d r o l y s i s was c a r r i e d out i n 0.5 ml of 6 N HC1 at 110°C f o r 24 hours or at 145°C f o r M- hours (Roach and Gehrke, 1970). To each tube 10 /al of 0.8 M B- mercaptoethanol was added. Following the h y d r o l y s i s , the tubes were allowed to c o o l before they were placed i n an i c e bath f o r 1 hour. To e l i -minate the acrylamide r e s i d u e , the tubes were c e n t r i f u g e d at 3,000 g f o r 10 minutes i n a S o r v a l c e n t r i f u g e equipped wi t h a SS-34 r o t o r . ;V C The r e s u l t i n g supernatants were d r i e d i n a vacuum desiccator and the m a t e r i a l was d i s s o l v e d i n 0.4- ml of 0.2 N sodium acetate d i l u t i o n b u f f e r , PH 2.2. A f t e r f i l t r a t i o n , the sample was ready f o r amino a c i d a n a l y s i s . For s t a r c h g e l s , the i n d i v i d u a l stained histone bands were c a r e f u l l y cut out and the p r o t e i n i n the bands was eluted w i t h 0.5 ml of 6 N HC1 overnight at 4°C. The eluate was c o l l e c t e d by f i l t r a t i o n through a m i l l i p o r e f i l t e r and hydrolyzed at 110°C f o r 24- hours (Durgo, 1977). A f t e r h y d r o l y s i s the m a t e r i a l was d r i e d and then d i s s o l v e d i n the same d i l u t i o n b u f f e r . The samples were - 49 -f i l t e r e d before being a p p l i e d to the column i f charred residues we were present. The amino a c i d analyses were performed on a Beekman Model 118C Amino Acid Analyzer, using a s i n g l e 6 x 510 mm column, 3 hours run. The t o t a l flow r a t e was SiSv-S-^ml/hr. The s e n s i t i v i t y of recording was maximal on the 0.1 O.D. sc a l e . The time s e t t i n g s were as f o l -lows: b u f f e r 1, 51 minutes; b u f f e r 2, 16 minutes; b u f f e r 3, 88 minu-tes and NaOH wash, 12 minutes. To qu a n t i t a t e amino acids appearing i n the chromatogram of a p a r t i c u l a r sample, the area under the peak was i n t e g r a t e d by weighing IBM Copier traces of the chromatogram on a M e t t l e r semimicro-balance. Nanomoles of each amino a c i d were c a l c u l a t e d by comparison w i t h the standard curve obtained using the Beekman Amino A c i d C a l i b r a t i o n Standard. weight of peak area of standard amino a c i d K = — * known nanomoles of standard amino a c i d n „ „ . weight of peak area of unknown nanomoles of amino _ aci d i n unknown ^ K i s a constant s p e c i f i c f o r each amino a c i d . To ob t a i n a confi d e n t K value, 10 runs of 6.25^nmoles of Beekman Amino Acid C a l i b r a t i o n Standard were done and the mean of 10 runs was taken f o r the above c a l c u l a t i o n . Two runs were performed on each stained g e l band examined. The average of two r e s u l t s i s shown i n the table i n the R e s u l t s . - 50 -RESULTS Houston (1971) as w e l l as P a l l o t t a and T e s s i e r (1976) have p r e v i o u s l y described a method f o r analyzing the amino a c i d compo-s i t i o n of p r o t e i n s d i r e c t l y from stained polyacrylamide g e l s l i c e s . The technique proved s u c c e s s f u l w i t h serum albumin and ovalbumin using the g e l system of Ornstein and Davis (Houston, 1971), as w e l l as w i t h somatic histones using the acrylamide-urea g e l system of Panyim and Chalkley ( P a l l o t t a and T e s s i e r , 1976). However, amino a c i d a n a l y s i s of histones which are separated on the acrylamide g e l system of Bonner et a l . (1968) has never been attempted. For t h i s reason we d i d s e v e r a l c o n t r o l experiments using protamine (herring) prepared by the Sigma Chemical Co. and c a l f thymus HM-( a g i f t from Drs. D. Fambrough and J . Bonner) before analyzing the t e s t i s - s p e c i f i c histones of amphibians and r e p t i l e s . Standard Amino A c i d Chromatography A mixture of standard amino acids (6.25 nmoles of each amino a c i d , 3.125 nmoles c y s t i n e , Beekman Co.,) were run on a Beekman model 118C Amino Acid analyzer using a 6 x 510 mm column f o r 3 hours.. The chromatogram i s shown i n F i g . 17. The o r i g i n a l r e cording i s a s e r i e s of dots which we have redrawn f o r c l e a r e r r e p r e s e n t a t i o n . A s h i f t upwards i n the b a s e l i n e a f t e r the second b u f f e r i s sometimes observed. This i s probably due to the storage of n i n h y d r i n . I t does not a f f e c t the a c t u a l area under each peak. The i n t e g r a t i o n of each amino a c i d i s obtained by weighing an IBM photocopy of the peak on a M e t t l e r semi-micro a n a l y t i c a l balance. This method has the shortcoming of i n v o l v i n g many v a r i a b l e s , such - 51a -F i g . 17 Chromatogram of Beekman amino a c i d c a l i b r a t i o n standard on the Beekman Model 118C Amino Acid Analyzer using a 6 x 510 mm column, 3 hours run. 6.25 nmole per amino a c i d except f o r c y s t i n e (3.125 nmole). T o t a l flow r a t e : 52.5 ml/hr. S e n s i t i v i t y : 0.1 O.D. s c a l e . The readout i s presented as dots by the recorder; we have drawn a continuous trace by connecting the dots. - 52 -as i n c o n s i s t e n c y i n c u t t i n g the paper by hand and i n c o n s i s t e n c y in-' the s e n s i t i v i t y of the balance. However, according to Table 2, which shows the K value and i t s 95% confidence i n t e r v a l , o f each amino a c i d , K values c a l c u l a t e d f o r weighing method are r e l i a b l e since t h e i r ranges of the 95% confidence i n t e r v a l are very s m a l l . Therefore, to deal w i t h a considerably l a r g e set of data, the weighing method has proved to be the most convenient and time-saving. R e s i d u a l Polyacrylamide Gel P a r t i c l e s E f f e c t A f t e r a c i d h y d r o l y s i s , the polyacrylamide g e l pieces i n the sample have to be removed completely. I f the sample contains re-s i d u a l m a t e r i a l from hydrelyzed g e l , the 'peaks on the chromatogram w i l l show shoulder and t a i l i n g e f f e c t s . I n F i g . 18, we see a p o r t i o n of the chromatogram of Xenopus t e s t i s - s p e c i f i c histone Xs i n a c l a r i f i e d s o l u t i o n (Fig.l8A) and i n the s o l u t i o n w i t h r e s i d u a l m a t e r i a l from hydrolyzed gels s t i l l present due to incomplete cen-t r i f u g a t i o n ( F i g . 18B). The reason f o r the t a i l i n g i s not known; i t i s p o s s i b l y due to the i n t e r a c t i o n of hydrolyzed polyacrylamide w i t h amino a c i d s . Amino A c i d A n a l y s i s Before And A f t e r Gel E l e c t r o p h o r e s i s Sigma protamine was run on polyacrylamide g e l s c o n t a i n i n g 6.25 M urea (Fig. 19) . The stained band was cut out and hydrolyzed i n 6 N HC1 i n the presence of B-mercaptoethanol. The r e s u l t s are presented i n Table 3 (column D) and can be compared w i t h those of -Sigma protamine powder hydrolyzed d i r e c t l y (Column B). There i s no s i g n i f i c a n t d i f f e r e n c e between these two amino a c i d analyses - 53 -Table 2 R v a l u e s And Their 95% Confidence I n t e r v a l s f o r Weighing Method. Amino Acid K (xlO 3) C . I . ( x l 0 3 ) Lys 23.4-2 - 3.14 His 18.28 - 2.97 Arg 25.51 - 1.24 Asp 24-.61 - 0.90 Thr 15.53 - 0.71 Ser 16.21 - 1.34 Glu 29.54 - 1.27 Pro 7.45 i 0.30 Gly 25.55 - 1.05 Ala 22.83 - 0.93 Cys/2 14.07 t 0.48 V a l 22.53 - 0.87 Met 30.58 - 1.00 H e 22.79 - 0.67 Leu 26.01 - 1.35 Tyr 23.92 - 'l.;04 Phe 20.33 - 1.12 K i s a constant, s p e c i f i c f o r each amino a c i d , (see M a t e r i a l s And Methods, Part I I ) . The K value i n d i c a t e d above i s the mean of 10 runs. C.I. i s the 95% confidence i n t e r v a l of K. - 54-a -F i g . 18- E f f e c t of r e s i d u a l polyacrylamide g e l p a r t i c l e s on the amino a c i d p r o f i l e from Xenopus t e s t i s - s p e c i f i c h i s t o n e s . A. C l a r i f i e d s o l u t i o n . B. Res i d u a l polyacrylamide g e l p a r t i c l e s present. A and B are from the same preparation. i - 54b-Ser A-Gly A Elution Time - 55a -F i g . 19 E l e c t r o p h o r e t i c p r o f i l e s of Sigma protamine (herring) . A. Starch g e l , (Sung and Smithies, 1969). Dotted arrows i n d i c a t e p o s s i b l e i m p u r i t i e s . B. Polyacrylamide g e l (Bonner et a l . , 1968). - 55 b-- 56 -except t h a t Sigma protamine on the g e l has a b i t higher percentage of s e r i n e and g l y c i n e . This might be due to b a c t e r i a l contamination i n the d i s t i l l e d water. When d i s t i l l e d water i t s e l f was analyzed, i t showed very small peaks i n the h i s t i d i n e , Serine and g l y c i n e regions of the chromatogram. The amino a c i d composition of Sigma protamine (Table 3, Column A and B) was compared to the r e s u l t s of Ando and Suzuki (1967) (column 1F) based on the primary s t r u c t u r e of clupeine (herring protamine) pr e p a r a t i o n . The d i f f e r e n c e between these two sets of data might due to i m p u r i t i e s i n the Sigma protamine. This can be seen when the Sigma protamine i s electrophoresed on s t a r c h g e l s (Fig. 19B) . Although i t only shows a s i n g l e band (Fig. 19A) on the polyacrylamide g e l system of Bonner et a l . (1968), i m p u r i t i e s are c l e a r l y evident on the s t a r c h g e l . The bands moving f a s t e r than P on the s t a r c h g e l might represent modified forms of the protamine, as seen i n t r o u t protamine (Louie and Dixon, 1972). H y d r o l y s i s Time and Temperature E f f e c t D i f f e r e n t h y d r o l y s i s c o n d i t i o n s were examined i n order to f i n d s u i t a b l e c o n d i t i o n f o r the amino a c i d a n a l y s i s . H y d r o l y s i s at 1M-5°C f o r M- hours (Roach and Gehrke, 1970) and h y d r o l y s i s at 110°C f o r 2M-hours (Moore and S t e i n , 1963) were studied since these two c o n d i t i o n s were reported to be the best. The r e s u l t s , shown i n Table 3 (column A. and B), show no s i g n i f i c a n t d i f f e r e n c e s i n the data obtained by these two methods. However, h y d r o l y s i s at 110°C f o r 2M- hours i s p r e f e r a b l e i f time permits since f l u c t u a t i o n s i n the t o t a l time of h y d r o l y s i s w i l l not be so c r i t i c a l f o r the longer 24-hour period (1M-5°C) . - h i -TABLE 3 Amino A c i d Composition o f Sigma Protamine ( H e r r i n g ) Amino a c i d A (mole %) B (mole'5?) C (mole %) D '(mole %) E (mole %) F (mole %) Lys Tr Tr 1.7 Tr 2.3 _ H i s 2.2 1.2 Tr 1.7 Tr -Arg 56.1 57.1* 70.6 57.3 58.8 61*.5 Asp 1.2 1.1 Tr Tr Tr -Thr 2.6 2.5 3.6 2.5 1*.7 6.5 Ser 9.3 • 9.1* 7.6 11.6 10.1 9.7 G l u Tr 1.3 Tr l . l 1.2 -Pro 6.6 6.3 IE 5.1* Tr 6.5 G l y 3.0 2.6 2.2 1*.2 l * . l 3.2 A l a 8.5 8.3 8.3 8.3 . 11.3 6.5 Cys/2 - - - - - -V a l 7..1* 7.3 5.0 5.9 6.5 6.5* Met - - - - - - -l i e 1.6 - 1.1 - (3.2)** Leu Tr Tr - Tr - -Tyr - - - - - -Phe - - - - - - • Lys/Arg - — 0.02 — o.oi* -B a s i c / A c i d i c 29.91* '2k.9h 8U.28 31*. 07 31.78 -A. Sigma protamine powder h y d r o l y z e d a t ll*5*C f o r k hours (no-NaOH tr e a t m e n t ) and chromatographed on a Beekman 118C Amino A c i d A n a l y z e r , s i n g l e 51 cm column, 3 hour r u n , 0.1 0.D. s c a l e . No c o r r e c t i o n i s made f o r h y d r o l y t i c l o s s e s . B. Sigma protamine powder h y d r o l y z e d a t 110*C f o r 2h hours (no-NaOH t r e a t m e n t ) . l l 8 C A n a l y z e r . C. Sigma protamine powder h y d r o l y z e d at lU5*C f o r k hours (NaOH t r e a t e d ) a n a l y z e d on a Beekman 120B Amino A c i d A n a l y z e r m o d i f i e d by Dr. E. A. Boeker f o r a s i n g l e 60 cm column, 5 hour r u n . P r o l i n e i s p r e s e n t o n l y i n t r a c e amounts. D. Sigma protamine on p o l y a c r y l a m i d e g e l h y d r o l y z e d at 110*C f o r 2h hours (no-NaOH treatment) a c c o r d i n g t o Houston (1971). 118C A n a l y z e r . E. Sigma protamine on p o l y a c r y l a m i d e g e l h y d r o l y z e d a t 1^ 5*C f o r k hours (NaOH t r e a t e d ) a c c o r d i n g t o P a l l o t a and T e s s i e r (1976). 120B m o d i f i e d A n a l y z e r . Only a t r a c e o f p r o l i n e i s seen. F. Clupeus harengus Y (Ando and S u z u k i , I967). Data based on p r i m a r y s t r u c t u r e o f p r o t e i n . • o n l y i n YI ** ( o n l y i n YII) * B a s i c / A c i d i c = (Lys + H i s + Arg) / (Asp + Glu ) . No c o r r e c t i o n i s made f o r h y d r o l y t i c l o s s e s . - 58 -E f f e c t of Sodium Hydroxide on Amino A c i d A n a l y s i s A large amount of ammonia i s released during the h y d r o l y s i s of the stained g e l pieces as the polyacrylamide i s converted to p o l y a c r y l i c a c i d (Houston, 1971). I t has been suggested that the a d d i t i o n of 10 N NaOH e l i m i n a t e s much of the ammonia when bas i c amino acids are analyzed on the short column of a two-column Beckman model 120C analyzer ( p o l l o t t a and T e s s i e r , 1976). However, the analyzer used i n t h i s study i s a s i n g l e column type (Beckman 118C) that r e a d i l y separates ammonia from ba s i c amino ac i d s . There-f o r e , s tudies of the e f f e c t of sodium hydroxide treatment were • undertaken. Comparing the r e s u l t s i n Table 3, column A and column B w i t h those of column C, or comparing column D w i t h column E, one can see that sodium hydroxide treatment i n t e r f e r e s w i t h the a n a l y s i s of p r o l i n e and i s o l e u c i n e . P a l l o t t a and Tes s i e r (1976) also observed the d e s t r u c t i o n of p r o l i n e , phenylalanine and t y r o s i n e by t h i s treatment. Therefore, sodium hydroxide treatment was not employed i n subsequent amino a c i d a n a l y s i s on the 118C analyzer i n order to o b t a i n the true composition of the p r o t e i n s . B-Mercaptoethanol E f f e c t on Amino Acid A n a l y s i s I t has been reported that t y r o s i n e and h i s t i d i n e are destroyed during p r o t e i n h y d r o l y s i s without the p r o t e c t i o n of B-mercaptoethanol (Houston, 1971). When Xenopus t e s t i s - s p e c i f i c histone X s was examined without B-mercaptoethanol, i t was found (Table 4) that methionine and t y r o s i n e were degraded, but not h i s t i d i n e . This d e s t r u c t i o n may r e s u l t from o x i d a t i o n since i t i s d i f f i c u l t to remove a l l trapped oxygen w i t h i n the g e l piece. - 5 9 -TABLE k P-mercaptoethanol E f f e c t on Acid Hydrolysis of Xenopus T e s t i s - S p e c i f i c Histone. 'X s Amino acid A (mole %) B (mole %) Lys 1.3 1.8 His 2.5 3.U Arg 30.1 30.1+ Asp h.9 5.0 Thr 10.2 9.8 Ser 15.6 16.6 Glu h.3 U.ai Pro 1.1 1.1 Gly 9.3 9.3 A l a 12.2 11.6 Cys/2 - -Val 2.8 2.6 Met - 2.2 Tr H e Tr Tr Leu l.h 1.9 Tyr 1.8 Tr Phe Tr Tr A. X g from polyacrylamide g e l hydrolyzed with 0.8 M p-mercaptoethanol. X^ was prepared from a t e s t i s chromatin preparation using the modified method of Bonner et a l . (1968). Methionine and tyrosine ar.eepresehtvt 0„8' H- —mercaptcethe-aul-. . prepared i. ; . t . . B. Samejiiwithout 0.8 M ^-mercaptoethanol, prepared from a t e s t i s c e l l suspension. - 60 -T e s t i c u l a r Histone Preparation E f f e c t Table 5 and 6 show the amino a c i d composition of Xenopus t e s t i s -s p e c i f i c h i s t o n e s X^ and X^ using d i f f e r e n t p r e p a r a t i o n methods. The o v e r a l l compositions are b a s i c a l l y s i m i l a r amongst the histones obtained from a c e l l suspension p r e p a r a t i o n (Fig.,> 7B, channel c) , a nuclear p r e p a r a t i o n (Fig. 7B, channel.b) and from a chromatin p r e p a r a t i o n (Fig. 7B channel a ) , wit h an exception f o r X ^ i n the l a t t e r instance. The X^ band from a. chromatin p r e p a r a t i o n (Table 6, column A) shows a high content of a r g i n i n e and a lower content of h i s t i d i n e than X^ obtained i n the two other methods (Table 6. co-lumns B and C). Xenopus t e s t i s - s p e c i f i c histones have been reported to show microheterogeneity on sta r c h gels (Bols.St al.,,1976) and on polyacrylamide g e l s (RisLey -and Eckhardt, 1975). I t i s p o s s i b l e that the chromatin p r e p a r a t i o n s e l e c t i v e l y e x t r a c t s a p a r t i c u l a r species of X^. This could e x p l a i n the d i f f e r e n c e s between columns B and C i n Table 6. Amino a c i d Composition of Histone HM- from C a l f Thymus And Xenopus Table 7 shows the amino a c i d composition of the e v o l u t i o n a r i l y conservative histone H4- from Xenopus and from c a l f thymus. Calf thymus histone H4, a r e l a t i v e l y p u r i f i e d powder, was electrophoresed on polyacrylamide g e l (Fi g . 7, channel e) . Xenopus histone H4-from a c e l l suspension p r e p a r a t i o n was also electrophoresed i n the same manner (Fig. 7, channels c and d). The r e s u l t s shown i n Table 7, columns A and B, are compared to those of John's (1971) f o r c a l f thymus HM- histone and Byrd and Kasinsky (1973) f o r Xenopus l i v e r h i stone H4- p u r i f i e d from chromatin by s e l e c t i v e e x t r a c t i o n and chro-- 61 -TABLE 5 Comparison of Amino Acid Composition of Xenopus T e s t i s - S p e c i f i c Histone X Prepared by D i f f e r e n t Methods Amino a c i d A (mole %) B (mole %) C (mole ! Lys 1 j 1.3 2.0 1.9 His 2.5 3.h 3.8 Arg 30.1 25.9 25.8 Asp U.9 5.T 5.5 Thr 10.2 8.7 9.h Ser 15.6 17.1 16.5 Glu •k.3 5.2 h.l Pro 1.1 1.1 l.h Gly 9.3 11.2 9.6 A l a 12.2 11.0 11.9 Cys/2 - - -Val 2.8 2.8 2.9 Met 2.2 l.U 2.1 H e Tr Tr Tr Leu l.h 1.9 1.9 Tyr 1.8 1.8 2.6 Phe Tr Tr Tr Lys/Arg 0.0k 0.08 0.07 Basic/Acidic 3.68 2.87 3.29 A. X^ extracted from a t e s t i c u l a r chromatin preparation by the modified met;ho,d" efS c,B.o-hneis;ee.-fe^al. (1968) ( g e l ) . B. X s extracted from a t e s t i s nuclear preparation by Byrd's method , ( g e l ) . C. X__ extracted from a t e s t i s c e l l suspension ( g e l ) . - 62 -TABLE 6 Comparison of Amino Acid Composition of Xenopus T e s t i s - S p e c i f i c Histone X Prepared by D i f f e r e n t Methods Amino a c i d A (mole %) B (mole %) C (mole ! Lys 2.h 3.0 2.1 His 2.6 l.h 10.7 Arg 28.1 19. h 16.6 Asp 3.5 h.5 3.9 Thr 10. U 5.3 .8.3 Ser IT.5 . 23.h 22.5 Glu k.l 3.6 Pro 2.3 Tr 1.3 Gly 9.5 16.9 11.7 A l a 13.7 10.0 10.2 Cys/2 - - -Val 5.1 h.2 Met Tr Tr Tr l i e Tr Tr 1.3 Leu Tr Tr 1.8 Tyr Tr Tr Tr Phe Tr Tr 1.3 Lys/Arg 0.09 0.16 0.10 Basic/Acidic i+.05 3.32 h.91 %) A. X extracted from a t e s t i c u l a r chromatin preparation by the;'modified method Q-ftBonneBfeet - a l . (1968) ( g e l ) . B. X^ extracted from a t e s t i s nuclear preparation by Byrd's method (g e l ) . C. X_p extracted from a t e s t i s c e l l suspension ( g e l ) . - 63 -TABLE 7 Comparison of the Amino Acid Composition of E v o l u t i o n a r i l y Conservative Histone1.' H4 i n Xenopus Tissues and C a l f Thymus Amino .acid A (mole %) B (mole %) C (mole %) D (mole ' Lys 9.k 11.9 10.2 13. h His 5.0 2.3 2.2 2.2 Arg 11.2 12.8 12.8 13.2 Asp 6.0 5.3 5.2 6.2 Thr 7.1 6.9 6.3 h.l Ser l.h 2.2 2.2 2.7 Glu 6.6 7.3 6.9 6 .U Pro 1.5 Tr 1.5 2.6 Gly 16.U 16.6 _ 12.0 A l a 8.0 8.1 7.7 7.1 Cys/2 - - - -Val 7.7 10.0 8.2 6.5 Met Tr Tr 1.0 1.2 l i e 3.9 1+.3 5.7 5.9 Leu 7.0 7.3 8.2 8.6 Tyr Tr 3.0 3.8 3.9 Phe 2.2 1.1+ 2.1 3.6 Lys/Arg 0.81+ 0.93 0.80 1.02 Basic/Acidic 2.01+ 2.11+ 2.08 2.28 A. Histone Hl+ from Xenopus t e s t i s c e l l suspension ( g e l ) . B. Histone Hl+ from c a l f thymus, a g i f t from Drs. D. Fambrough and J . Bonner ( g e l ) . C. Histone Eh from c a l f thymus (Johns, 1971). D. Histone Eh from Xenopus l i v e r chromatin (Byrd and Kasinsky, 1973a). - 64 -matography. They show considerable s i m i l a r i t y . The higher h i s t i d i n e , s e r i n e and g l y c i n e content i n the Xenopus t e s t i s histone H4 might be due to slight': contamination of d i s t i l l e d water wi t h b a c t e r i a , as mentioned e a r l i e r . Amino Acid A n a l y s i s of T e s t i s - S p e c i f i c Histones from Amphibians And  R e p t i l e s T e s t i s - s p e c i f i c histones extracted from c e l l suspensions of the South A f r i c a n clawed-toad Xenopus l a e v i s , the toad Bufo marinus, the Japanese f i r e - b e l l y newt Cynops pyrrhogaster, the snakes Thamno-phis s i r t a l i s and Elaphe guttata guttata and the l i z a r d A n d l i s  c a r o l i n e n s i s . were electrophoresed on polyacrylamide gels (Fig.8 and 9) and bands were cut out f o r amino ac i d a n a l y s i s . Table 8 i n d i c a t e s the amino a c i d composition of fast-moving t e s t i s - s p e c i f i c histones from three amphibian.species (Xenopus, Cynops and Bufo). They show considerable d i v e r s i t y i n t h e i r amino a c i d composition. The b a s i c / a c i d i c r a t i o ranges from 1.58-10.3. Bufo shows a very high content of a r g i n i n e (M-5 nifole %) and a very low content of serine and g l y c i n e ; Xenopus shows an intermediate content of ar g i n i n e (20 mole%) and a high content of serine and g l y c i n e ; Cynops has only about lQmole% a r g i n i n e but shows a high content of serine and g l y c i n e . Rana does not have any f a s t - m i g r a t i n g band and the amino a c i d composition of i t s t e s t i c u l a r histones has been shown to be s i m i l a r to that of somatic histones (Vendrely, 1957). This d i v e r s i t y i n amino a c i d composition i s c o n s i s t e n t w i t h the d i v e r s i t y p r e v i o u s l y observed i n the e l e c t r o p h o r e t i c p r o p e r t i e s of these t e s t i s - s p e c i f i c histones from amphibians. (see Part I ) . - 65 -TABLE 8 Amino. Acid Composition of Testis-Specific Histones from Amphibian Ce l l Suspensions Amino acid A (mole %) B (mole %) C (mole %) D (mole %) Lys 1.9 2.1 h.8 1.8 His 3.8 . 10.7 7.8 9-5 Arg 25.8 16.6 ^5.5 10.7 Asp 5.5 3.9 Tr 6.5 Thr 9.h 8.3 6.h 2.6 Ser 16.5 22.5 8.5 23.0 Glu U.i 3.6 5.h 7.3 Pro l.h 1.3 •6.1 2.6 Gly 9.6 11.7 1.5 16.6 Ala H.9 10.2 6.1 7.9 Cys/2 - - - -Val 2.9 h.2 5.U 5.3 Met 2.1 Tr Tr Tr l i e Tr 1.3 Tr Tr Leu 1.9 1.8 Tr 2.7 Tyr 2.6 Tr 1.2 Tr Phe Tr 1.3 Tr 1.7 Lys /Arg 0.07 0.15 0. H) 0.16 Basic/Acidic 3.29 U.91 10.30 1.58 A. F i r s t fast-moving band (X ) of test is-specif ic histones of Xenopus (gel). B. Second fast-moving band (X f) of test is-specif ic histones of Xenopus (gel). C. Testis-specific histone of the toad, Bufo.marinus,(gel). D. Testis-specific histone of the Japanese f i re-bel ly newt, Cynops pyrrhogaster (gel). - 66 -Table 9 shows the r e s u l t s of amino a c i d a n a l y s i s of fast-moving histones from t e s t i s , ductus deferens and semen of three r e p t i l i a n species (Thamnophis, Elaphe and Ano lis*) . Unlike the amphibian species the r e p t i l e s show a remarkable s i m i l a r i t y i n t h e i r t e s t i s -or semen-specif i c h i s t o n e s . The a r g i n i n e contents are i n the range of 23 mole% to 29 mole%; serine/fcontents range from 12 to 16 mole% and g l y c i n e contents are i n the range of 18-24- mole%. This confirms the s i m i l a r i t y p r e v i o u s l y observed i n the e l e c t r o p h o r e t i c p r o p e r t i e s of the t e s t i s - s p e c i f i c histones i n these snakes and l i z a r d s (see Part I ) . However, i t should be noted that Bufo,.Anolis, and snake bands show microheterogeneity on s t a r c h gels (Kasinsky e_t ' a l . , 1977) so that these analyses might represent e i t h e r an averaging of micro-heterogeneous p r o t e i n s w i t h s i m i l a r amino a c i d compositions, as has been observed i n t r o u t protamine (Ling, e_t a_l. , 1971), or one p r o t e i n w i t h d i f f e r e n t degrees of phosphorylation of serine residues or a c e t y l a t i o n of l y s i n e residues ( Louie and Dixon, 1972; Sung _et a l . , 1977) . Amino Acid A n a l y s i s of Stained Bands on Starch Gels For amino a c i d a n a l y s i s of s t a r c h g e l bands, Durgo's method (1977) was employed. Instead of h y d r o l y z i n g the whole band as i n the case of polyacrylamide g e l s , the p r o t e i n s were f i r s t e luted from the stained band before h y d r o l y s i s , since i t has been reported by T r i s t a n (1939), B a i l e y (1937) that i n the presence of carbohy-drates a r g i n i n e and methionine were destroyed e x t e n s i v e l y . The amount of degradation was p r o p o r t i o n a l to the concentration of carbohydrates. Johns et a l . (1961) extracted histones from stained - 67 -TABLE 9 ti" Amino Acid ComposLpn of Testis-Ductus Deferens-and Semen-specific Histones from R e p t i l i a n C e l l Suspensions Amino acid A (mole %) B (mole %) C (mole %) Lys h.k 7.0 His .7.0 .7.7 .9.3 Arg 23 .0 29.5 26.3 Asp 5.h h.3 h.2 Thr 1.9 1.9 IT Ser 16.2 lh.6 . 12.3 Glu U.l h.2 3.6 Pro 2.1 1.0 Tr Gly 18. U 18.8 2U.5 A l a 5.9 3.8 h.2 Cys/2 - - -Val 5.2 3.5 2.1 Met - -l i e 1.3 1.7 Tr . Leu 2.7 1.6 1.2 Tyr Tr Tr Tr Phe 1.6 Tr Tr Lys/Arg 0.19 0.25 0.38 Basic/Acidic 3 . 6 l •U.8U 5.77 A. Ductus deferens-specific histone of the snake Thamnophis s i r t a l i s ( g e l ) . B. Semen-specific histone of the snake Elaphe guttata guttata ( g e l ) . C. T e s t i s - s p e c i f i c histone of the l i z a r d Anolis c-arolinensis ( g e l ) . The histones i n A and B are probably sperm-specific as t h i s i s the predominant c e l l type. - 68 -s t a r c h g e l s w i t h 0.1 N HCl by f r e e z i n g and thawing methods. In our experiments, water, 0.M- N HCl and 6N'HCl were used to e x t r a c t histones from stained bands. The r e s u l t s (not shown) i n d i c a t e d that 6 N HCl was much b e t t e r than the other two e l u t i n g agents: How-ever, unknown peaks a l s o appeared i n the chromatogram, and accurate r e s u l t s could not be obtained. This was probably due to the f a c t that s t a r c h g e l i t s e l f was a good medium f o r b a c t e r i a l growth, even at low pH. The unknown peaks picked i n the a n a l y s i s were probably s p e c i f i c to contaminating b a c t e r i a . Further experiments are r e -quired to b r i n g the amino ac i d a n a l y s i s of s t a r c h g e l bands up to the accuracy of the polyacrylamide g e l band a n a l y s i s . - 69 -DISCUSSION S t a r t i n g from the methods of Houston (1971) and P a l l o t t a and T e s s i e r (1976), I was able to analyze the amino ac i d composition of the p r o t e i n i n a s i n g l e band on the polyacrylamide g e l system of Bonner et a l . (1968). Both c o n t r o l experiments on Sigma pro-tamine and c a l f thymus histone HM- show the same amino a c i d a n a l y s i s before and a f t e r g e l e l e c t r o p h o r e s i s . That i s to say, the g e l and the dye used, amidoblaek, do not a f f e c t the amino a c i d a n a l y s i s i f the g e l residue a f t e r h y d r o l y s i s i s completely removed. Therefore, one does not have to r e l y on matching an unstained g e l w i t h a stained g e l , and then t r y i n g to excise the corresponding band and e l u t i n g the p r o t e i n out of i t . f o r f u r t h e r a n a l y s i s . One can hydro-l y z e the amidoblaek-stained p r o t e i n d i r e c t l y i n the g e l f o r amino ac i d a n a l y s i s . T e s t i s - s p e c i f i c histones from d i f f e r e n t p r e p a r a t i o n s , i . e . , chromatin, n u c l e i preparations and c e l l suspensions show a s i m i l a r composotion. Combining the ease of preparing h i s t o n e s from c e l l suspensions and analyzing a s i n g l e band on polyacrylamide g e l , I was able to examine the t e s t i s - s p e c i f i c histones from a s i n g l e animal of d i f f e r e n t amphibian and r e p t i l i a n species. Using Bloch's c a t e g o r i e s (1969), we f i n d t h a t the t e s t i s - , or sperm-specific histones of the three amphibians examined, Xenopus, Bufo and Cynops and the three r e p t i l e s , Thamnophis, Elaphe and A n o l i s , a l l f a l l i n t o the M y t i l u s or intermediate type (Type 3) since they c o n t a i n more than one d i b a s i c amino a c i d and no c y s t e i n e . However, when one examines the amino a c i d composition - 70 -of each one, one would see that the r e l a t i v e q u a n t i t y of the three b a s i c amino acids v a r i e s markedly from an a r g i n i n e content of M-5 mole% to one of only 10 mole% and a l y s i n e content of 1.3 mole% to 9.8 mole%. L i t e r a t u r e data show that L o l i g o p e a l e i i sperm h i s t o n e , which i s i n the M y t i l u s category cytochemically, has an a r g i n i n e content as high as 75 mole% while i t s l y s i n e and h i s t i d i n e contents are as low as 0.8 mole% and 0.1 mole% respect-i v e l y (Subirana et_ al., 1973). F e l i x (1952) c l a s s i f i e d Salmo / f o n t i n a l i s sperm histones as the Salmo type cytochemically. However he found 2 mole% l y s i n e content i n the amino a c i d composi-t i o n of the p r o t e i n . Bols and Kasinsky (1973) c l a s s i f i e d Bufo  americanus sperm histone as e i t h e r the Salmo or the M y t i l u s type. Bufo boreus sperm h i s t o n e , which was thought to be the Rana type cytoc h e m i c a l l y (Bols and Kasinsky, 1972), a c t u a l l y showed a f a s t -moving band migrating f a s t e r than somatic histones (Bols and Kasinsky, 1973; Kasinsky et al.,1977). A l l these c i t e d data p o i n t out t h a t the cytochemical c l a s s i f i c a t i o n of sperm-specific histones i s a very loose one, e s p e c i a l l y w i t h regard to the M y t i l u s type of sperm h i s t o n e s . This i s probably due to 'the l i m i t a t i o n s and the r e l a t i v e i n s e n s i t i v i t y of the cytochemical r e a c t i o n s i n s i t u . The cytochemical approach, although promising, i s f r u s t r a t e d by the l a c k of i n f o r m a t i o n on b a s i c p r o t e i n composi-t i o n of most sperm, by the problems of comparing conclusions from biochemical work w i t h those drawn from a cytochemical approach. A more exacting c l a s s i f i c a t i o n based on the amino a c i d composition i s recommended, such as t h a t i n d i c a t e d i n Table 10. This scheme continues to be a m o d i f i c a t i o n of Kossel's (1928) - 71 -Table 10 A B r i e f H i s t o r y of Sperm Histone C l a s s i f i c a t i o n . Date 1928 1950's 1960 Ts 1977 I n v e s t i g a t o r Kossel F e l i x Bloch and A l f e r t Huang and Kasinsky Methods used chemical chemical chemical and cytochemical C l a s s i f i c a t i o n  scheme devised A monoprotamine (1) diprotamine (2) ± t r i p r o famine (3) same Class 1-Salmo (mono-) type Class 2-mammalian type Class 3-Mytilus ( d i - , t r i - ) type Class 4-Rana (somatic) type Class 5-crab type (non-hi stone) Proposed scheme Type i-monoprotamine Type 2-basic k e r a t i n (cysteine containing) cytochemical, i m o x . l i r i n / , e l e c t r o p h o r e t i c Type 3A - < 40% Arg , . . , J i f and amino a c i d ; Type 3B - > 4-0% Arg a n a l y s i s Type 4-Rana type Type 5-Crab type * (!) » (2) , (3) = the number of d i f f e r e n t kinds of ba s i c amino a c i d residues (arg, l y s or h i s ) i n p r o t e i n s . -7,2 -o r i g i n a l c l a s s i f i c a t i o n of sperm histones i n t o mono-, d i - , or t r i p r o t a m i n e s depending on the presence of a r g i n i n e (mono-), a r i n i n e + l y s i n e ( d i - ) , a r g i n i n e + h i s t i d i n e ( d i - ) , or arginine+lysine+ h i s t i d i n e (triprotamine) i n the p r o t e i n . In t h i s scheme, categories 1, 2 and M- of sperm histones remain unaltered, as does category 5 of sperm l a c k i n g h i s t o n e s . Type 3, by Bloch's d e f i n i t i o n , i n c l u des the d i - and t r i p r o t a m i n e s of Kossel's c l a s s i f i c a t i o n scheme. Examining the amino a c i d compositions of amphibian and r e p t i l i a n sperm histones a c t u a l l y obtained i n t h i s t h e s i s , one f i n d s that they encompass a wide range of a r g i n i n e c o n t e n t s w i t h i n two broad c a t e g o r i e s . There-f o r e , a content of M-0 mole% ar g i n i n e has been set as an a r b i t a r y but r e a l i s t i c l i n e to separate Type 3 i n t o low-arginine Type 3A (arginine content l e s s than 4-0V;mole%) and h i g h - a r g i n i n e Type 3B (arginine content greater than M-0 mole%) subclasses. I n t h i s sense, amongst the amphibians, Bufo should be categoried as Type 3B and Xenopus and Cynops should be placed i n Type 3A. Amongst the r e p t i l e s , Thamnophis, Elaphe and A n o l i s a l l f a l l i n t o the Type 3A category. Comparing the amino a c i d composition of these t e s t i s - (sperm-) s p e c i f i c h i s t o n e s , one can see the marked d i v e r s i t y amongst amphibian and the s i m i l a r i t y amongst r e p t i l i a n p r o t e i n s . This i s a l s o r e f l e c t e d i n t h e i r e l e c t r o p h o r e t i c p r o p e r t i e s . Both cytochemical and biochemical studies show t h a t there i s marked d i v e r s i t y i n f i s h sperm histones (Bloch, 1976); that mammalian sperm histones show conservation and f a l l i n t o e i t h e r Type 1 or Type 2 categories (Bloch, 1976). I f one can g e n e r a l i z e from the l i m i t e d number of r e p r e s e n t a t i v e s examined so f a r , I t would appear that the r e l a t i v e conservation of - 73 -t e s t i s - o r sperm-specific histones i n r e p t i l e s represents a crossover p o i n t from the d i v e r s i t y of such p r o t e i n s i n f i s h and amphibians to r e l a t i v e constancy of such p r o t e i n type i n mammals (Fig. 20). As the only b i r d sperm histone that has been examined thus f a r i s the r o o s t e r protamine (Nakano, et a l . , 1976), we cannot say whether t h i s r e l a t i v e constancy w i l l a l s o include the sperm histones of b i r d s . - 74a F i g . 20 C l a s s i f i c a t i o n of Sperm-SpecifIc Histones i n Vertebrate Phylogeny by Amino A c i d Composition Based on data of Bloch (1976), Nakano ,et _ a l . (1976), Vendreley (1957) and present chemical analyses. Parentheses denote sperm ' i t . : : histone type determined only by cytochemical means. As i n Figure 1 arrow denotes e v o l u t i o n a r y trend towards r e l a t i v e constancy of sperm histone type i n vertebrate phylogeny. - 74b -CONCLUSION Combining the use of the polyacrylamide e l e c t r o p h o r e s i s w i t h amino a c i d a n a l y s i s of the stained bands has proved to be an appro-p r i a t e method to analyze small amounts of hi s t o n e s . I n t h i s study, I have demonstrated b i o c h e m i c a l l y the c o n t r a s t between the d i v e r s i t y of t e s t i s - s p e c i f i c histones and the constancy of somatic and embryoni histones by these methods of g e l e l e c t r o p h o r e s i s and amino a c i d a n a l y s i s . Whether r e d e f i n i t i o n of the intermediate c l a s s of sperm histones i n t o h i g h - a r g i n i n e and low-arginine subgroups w i l l prove to be v a l i d f o r other vertebrates awaits a great deal more e x p e r i -mental work on these p r o t e i n s . 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