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Histone synthesis during early development of Xenopus laevis (the South African clawed toad) Byrd, Earl William 1973

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ol it u ^. HISTONE SYNTHESIS DURIHG EARLY DEVELOPMENT OF XENOPUS LAEVIS (THE SOUTH AFRICAN CLAWED TOAE) by EARL WILLIAM BYHD, JR. B.A. (Biology), San Francisco State College, 1968 MA. (Cell and Molecular Biology), San Francisco State College, 1970 \ A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Zoology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1973 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Zoology The University of British Columbia Vancouver 8, Canada Date October 20, 1973 ABSTRACT Histones are the basic proteins complexed with CN A i n the chromosomes of eucaryotic organisms. The synthesis of histones and t h e i r nuclear accumulation was followed during early embryogenesis in Xenppus l a e v i s , the South African clawed toad. Histone synthesis was f i r s t examined in t o t a l c e l l homogenates of Xenop^us laevis embryos. Electrophoretic and chromatographic experiments indicate that histone synthesis takes place during the cleavage and swimming embryo stages of development i n Xenojaus l a e v i s . Furthermore, the major classes of histones (I, II and III and IV) made during these d i f f e r e n t stages appear to be q u a l i t a t i v e l y the same, as shown by electrophoresis on polyacrylamide gels and by Amberlite chromatography, and include histones similar to those found in adult somatic tissues of t h i s amphibian. Experiments u t i l i 2 i n g polyacrylamide gels show that these proteins coelectrophorese with known histone markers I, Ilb1, and III from trout testes and histone IV from peas. Individual basic proteins were pu r i f i e d from 8M urea-2M L i C l extracts of t o t a l embryc protein by carboxymethyl c e l l u l o s e chromatography and separated by Amberlite chromatography. The amino acid composition of the Amberlite peaks from swimming embryos indicates that these peaks contain histones comparable to those of other vertebrate species. Rechromatography of the f i r s t l y s i n e - r i c h peak on Amberlite with a very shallow guanidine hydrochloride gradient resolved t h i s peak into a ribosomal contaminant and at l e a s t three l y s i n e - r i c h f r a c t i o n s i n swimming embryos, sugggesting that there i s microheterogeneity in Xenojsus lag vis histone I. The synthesis and nuclear accumulation of early embryonic histones was examined next. Histones were isolated from nuclear chromatin preparations of pre-gastrula embryos and swimming tadpoles and subjected to acrylamide gel electrophoresis and amino acid analysis. Histone synthesis was demonstrated by the incorporation of »*C02 into the amino acid residues of acid hydrolyzed proteins. Gel electrophoresis indicated that each of the major groups of histones was synthesized in pre-gastrula embryos, as well as in la t e r stages, and incorporated into nuclear chromatin. These newly synthesized embryo histones were si m i l a r to those i n adult Xenojgus l i v e r , when compared by gel electrophoresis and amino acid analysis. ftctinomycin D pulse-labeling experiments u t i l i z i n g the incorporation of 3H-lysine into p a r t i a l l y dissociated blastulae showed a 20 f o l d decrease of histone synthesis i n Actinomycin D treated embryos from that of s i b l i n g controls when these basic proteins were separated by Amberlite chromatography. These data suggest that histones are synthesized almost e n t i r e l y from newly made mRNA after stages 5 to 6 in the embryo. On the whole, these observations on histone synthesis and the role of maternal template i n pre- and post-gastrula Xenons development are i n general agreement with most of the findings in sea urchin embryos. iv TABLE OF CONTENTS GENERAL INTRODUCTION ..1 INTRODUCTION PART 1 ..11 METHODS AND MATERIALS ....12 1. Mating of frogs and labeling of Xeno^us Laevis embryos .....12 A. Maintenance Of Xenojaus Laevis Stock ..............12 B. Mating Procedure ...............12 C. Labeling Of Embryos 13 2. Isolation Of Basic Proteins From Cleaving And Swimming Xeno_p_us Embryos .........14 A. Isolation Procedure ..14 B. P u r i f i c a t i o n Of Urea ,, ..17 3. Chromatography of proteins 17 A. Carboxymethyl Cellulose Chromatography ...........17 B. Amberlite Chromatography .......19 C. P u r i f i c a t i o n Of Guanidine Hydrochloride ..........19 D. Chromatography Of Proteins On Amberlite ..20 E. Acrylamide Gel Electrophoresis 21 F. Sodium Dodecyl Sulfate (SDS) Gel Electrophoresis .22 4. Amino acid analysis 24 RESULTS ..25 DISCUSSION 44 INTRODUCTION PART II ............51 METHODS AND MATERIALS 52 5. Mating Of Frogs And Labeling Of Xenppus Laevis Embryos .....52 A. **C-Labeled Embryos ..........52 B. 3 H-Lysine Labeled Embryos 52 6. Isolation Of Nuclei 53 A. Method I 53 B. Method II 54 7. Iso l a t i o n Of Chromatin ..........54 8. Extraction Of Histones 55 9. Chromatography ....55 A. Carboxymethylcellulose ...........................55 B. Amberlite Chromatography 56 10. Paper Electrophoresis Of Labeled Histones .........56 11. Fractionation Of Histones 57 A. Adult Liver 57 B. Embryonic Histones 58 12. Electrophoresis 58 13. Amino Acid Analysis .....59 RESULTS .....60 DISCUSSION 71 CONCLUDING REMARKS 77 REFERENCES .81 v i LIST OF TABLES Table Page 1 P r i n c i p a l components of c a l f thymus histone and the two commonly used systems for their nomenclature, Elgin, et a l . , 1970 3 2 Numerical data derived from histone sequences. (Johns, 1972) 4 3 Chemical components of various chromatins, ) Bonner, et a l . , 1968 5 4 Stages in the normal development of Xenppus l a e v i s , Nieuwkoop and Faber,1956. 9 5 Recoveries of t o t a l protein from stage 42 embryos. 29 6 Recoveries of t o t a l proteins from cleavage embryos. 29 7 Amino acid composition of histones from stage 42 embryos compared with those of stage 20 newt embryos and c a l f thymus histone f r a c t i o n s . ..........40 v i i 8 Amino a c i d c o m p o s i t i o n of h i s t o n e s i n Xenojaus l i v e r and c a l f thymus. 64 9 Amino a c i d c o m p o s i t i o n of h i s t o n e s from l a t e b l a s t u l a Xeno^us embryos .............65 v i i i LIST OF FIGURES Figure Page 1 Carboxymethyl c e l l u l o s e chromatography of basic proteins synthesized by swimming embryos of Xgnopus l a e v i s . ..26 2 Tracings and photograph of acrylamide gels from CM-cellulose column. 27 3 Tracings of acrylamide gels from f r a c t i o n C and D for stage 42 embryos and cleavage embryos. ......31 4 Tracings and photograph of SDS acrylamide gels from f r a c t i o n C and D for stage 42 and swimming embryos and cleavage embryos. ........... 32 5 Amberlite chromatography of radioactive histones synthesized by cleavage embryos , stages 1-9, of Xenopus la e v i s .............34 6 Amberlite chromatography of radioactive histones synthesized by swimming embryos, i stage 42, of Xgnopus la e v i s ..35 7 Tracings from acrylamide gels of Amberlite ix peaks for stage 42 embryos, Figure 6. ...37 8 Tracings from acrylamide gels of Amberlite peaks for cleavage embryos from Figure 7. ........39 9 Chromatography of the l y s i n e - r i c h histone peak from stage 42 embryos on amberlite using a shallow gradient 43 10 Disc electrophoresis of Xenopus embryo and adult l i v e r histones on 15$ acrylamide gels according to the ' method of Panyim and Chalkley, 1969. ......61 11 Paper electrophoresis of an amino acid hydrolysate from 1*C-labeled histones of cleavage Xenogus embryos ...62 12 Amberlite chromatography of 3H-labeled histones synthesized by cleaving Xenopus embryos (stages 5-8) i n the presence of actinomycin d. ......69 13 Amberlite chromatography of 3H-lysine histones synthesized control embryos. .70 X ACKNOWLEDGMENTS The author wishes to express appreciation to Dr. Harold E. Kasinsky for his guidance, suggestions and encouragements during the course of t h i s work. Appreciation i s also expressed to Mr. B. Honda and Mr. J. Durgo for exchange of ideas and technical assistance. During the course of t h i s work E.W. Byrd, J r . , was a recip i e n t of the McLean-Fraser Memorial fellowship, 1971-1972 and a University of B r i t i s h Columbia Graduate Student Fellowship, 1972-1973. DEDICATION To Carolyn 1 GENERAL INTRODUCTION The amphibian embryo i s especially suitable for the study of c e l l differentiation because of the sequential appearance of new populations of enzymes and other molecules as development proceeds (Malacinski, 1972; Ecker and Smith, 1968). The synthesis of nucleic acids undergoes pronounced changes in the amphibian during the early period of development (Gurdon, 1968). During the mid-blastula stage the nucleus begins to exert a control over the cytoplasm with the f i r s t measurable BNft synthesis in cleaving Xenopus laevis embryos (Gurdon, 1967). L i t t l e , however, i s known about the types of proteins or patterns of synthesis during cleavage and subsequent embryonic stages (Gurdon, 1967). Several workers (Ecker and Smith, 1970; Malacinski, 1971,1972) have tried to determine the rate of protein synthesis and i t s changes during progressive developmental stages in the amphibian. Although embryonic c e l l s may be shown to synthesize a diverse range of proteins, as yet the synthesis of a protein of recognized function has not been described. We have undertaken a study of the synthesis of a class of proteins, the histones, during early development of Xenopus laevis, the South African Clawed Toad. The synthesis of histones during early development i s intresting for several reasons. Histones are a specialized group of basic proteins associated with the chromatin of almost a l l eucaryotic somatic c e l l nuclei that have been examined (Johns, 1971). Histones are acid-soluble basic proteins, rich in arginine and lysine residues. Three major classes have been 2 identified: lysine-rich, slightly lysine-rich and arginine-rich. These classes may be further broken down into five major groups using the nomenclature of Rasmussen, Murray, and Luck ,1962 (Table 1): lysine-rich histone I, slightly lysine-rich histones IIb1 and IIb2 and the arginine-rich histories III and IV. Each of these major fractions makes up about 20% of the total histone content of the c e l l (Panyim and Chalkley, 1969). Histones are the most basic proteins known, with the exception of protamine (Johns, 1971) . The amino acids lysine and arginine make up at least 23% of the total amino acid residues (table 2). A l l histones lack tryptophan, and cysteine i s present in small amounts only in histone I I I . An extensive review of the chemistry of histones can be found in Phillips (1971). Histones have been thought to function in the control of genetic activity and the maintenance of chromosome structure. Stedman and Stedman (1950) f i r s t implicated basic proteins in the nucleus as possible regulators of gene expression. This idea was based on the close correlation between the amount of histone and DNA in many species and tissues within a species (Table 3). They stated, B The basic proteins of c e l l nuclei are gene inhibitors, each histone or protamine being capable of supressing the a c t i v i t i e s of certain groups of genes. " If histones have some role in specific gene repression, then one would expect a multitude of histone types. The evidence in the histone literature (reviewed by P h i l l i p s , 1971; Hnilica, 1972) now shows that the small, but the limited heterogeneity of histones could not allow them to serve as specific repressors of 3 fable 1. Principal Components Of Calf Thymus Histones And The Two Commonly Used Systems For Their Nomenclature, Elgin, Et a l . , 1970. Subclass; nomenclature of Lys/Arg Moles/100 moles Class Ita3mussc.i cl ciV Johns and Butler ratio Molecular weight total lifatdnc N-tcrminal C-terminal Lysine-rich l a f l 22 21,000- * 0.7 Blocked Lysine lb f l 22 21,000 0.G Blocked Lysine Slightly I l b l f2a2 ~ 2 . 5 13,000-10,000' 20.0 Blocked Lysine lycinc-rich IIb2 f2b , 2.5 13,774 2-i.C Proline Lysine Argininc-rich III f3 0.8- 13,000-15,000 IS.3 Alanine Alanine IV f2al 0.7 11,232'' 23.8 Acctylscrino Glycine 4 Table 2. Numerical Data Derived From Histone Sequences. (Johns, 1971). Histone F2B F2A2 F2A1 FS (IIb2) (Ilbl) (IV) (III) Total Number of residues per mol. 125 140* 102 101 Mol. wt. 13,774 ; 15,000* : 11,300 11,200 i Basic groups (Arg+His+Lys) 31 + 31 + 27+ 24+ Number of residues in a-helices 38. 42 31 30 Length of a-helices (A) 57A 63A 47A 45A Phosphate groups satisfied by +ve groups (excluding a-helices). B 26- 26- 23- 21-Length of DNA groove containing 116A 95A 98A these groups (see Note 1) 108A The same, + length of a-helices 165 A 179A 142A 143A Equivalent turns of the dyad DNA helix 2.4 2.6 2.0 2.0 Phosphate groups in this: C. (Total H associated with histones) 48- 52- 40- 40-Unsatisfied phosphate groups (C-B) 22- 26- 17- 19-% phosphate gToups unsatisfied 46% 50% 42% 47% Basic groups in a-helices* 5 + 5 + 4+ 3+ 468 51,300 72,300+ 113+. 177 + ' 141 212A 96--417A ,, 629A 9.0 180-84-46% (av.) 17+* The F1(I) histone has about 212 residues per molecule of 21,000 weight, including 64 positive charges due to basic amino acids. * Assumed values. ] Including histone. Note 1. These lengths include the phosphate groups by-passed because of (a) tingle non-basic acids between two basic ones; total seven groups; (b) loops containing proline residues, total 10 groups and (c) N- and C-termina! portions of the molecules, provided that they are not basic amino acids; total six gToups. The total phosphate groups by-passed in these ways is therefore about 23. 5 Table 3. Chemical Components Of Various Chromatins, Bonner, Et a l . , 1968. The chromosomal composition summarized in Table 1 indicates the mass ratios of DNA, protein and RNA found in several chromatins. Source of chromatin Content, relative to D N A , of Template-activity (% of DNA) D N A Histone Nonhistone protein R N A l'ca embryonic axis 1.00 1.03 0.29 0.26 12 l'ca vepctativo bud 1.00 1.30 .10 .11 6 Tea crowing cotyledon 1.00 0.76 .36 .13 32 Uat liver 1.00 1.00 0.67 .043 20 Rat ascites tumor 1.00 1.16 1.00 .13. 10 Human HeLa cells 1.00 1.02 . 0.71 .09 10 Cow thymus 1.00 l . H .33 .007 15 Sea urchin blastula 1.00 1.04 0.48 .039 10 Sea urchin plutcus 1.00 0.86 1.04 .078 20 6 genes the biochemical means by which DNA , presumably containing a l l the genetic information of the parents, i s specifically restricted during cellular differentiation s t i l l remains a challenging problem. The function of histones in chromatin, however, i s s t i l l unclear. Their role may be a structural one. The reconstitution experiments of Zubay and Wilkins (1964) showed that DNdA and histones prepared separately can recombine to give patterns characteristic of the native nucleohistone. But, l i t t l e i s known of the precise factors involved in the histone-DNA interactions that lead to the formation of a " supercoiled " structure (Bradbury and Crane-Bobinson, 1971). Thus, i t can be suggested that histones possibly serve a structural function. There i s a very close interrelationship between DNA synthesis and histone synthesis. Both syntheses are closely linked in rapidly dividing c e l l s (Butler and Mueller, 1973). The early embryonic stages in the frog are characterized by a switch from a complete absence of c e l l division and DNA synthesis in the ooctye to an extremely rapid rate of division (15 minutes or less in the embryo (Gurdon, 1968). At the end of this period of rapid c e l l division or cleavage the egg has undergone 12-15 divisions and consists of some 15,000 c e l l s . Therefore, one would expect the cleavage period in the amphibian to be characterized by histone biosynthesis. However, Asao (1969, 1970) has reported the complete absence of histone synthesis during cleavage in the Japanese newt. It i s only at gastrulation that, according to Asao, arginine-rich histones 7 appear followed by the other classes of histones as development proceeds. He have examined histone synthesis in Xgnopus laevis to see i f the synthesis of histones takes place during a period of rapid DNA synthesis and to determine i f a l l classes of histones are synthesized during embryonic development. Finally, an understanding of histone synthesis may be of importance in understanding the way in which proteins synthesis in general is regulated in the embryo. There is a growing amount of evidence to show that protein synthesis is regulated at the translational level in the i n i t i a l stages of development in the amphibian. The pattern of early morphogenesis appears to be largely programmed by the egg cytoplasm. Gene products are synthesized during oogenesis and distributed to the oocyte cytoplasm prior to ovulation and stored for later use (Gurdon, 1967). With the first measurable RNA synthesis appearing at the mid-blastula stage of the Xenopus embryos (Davidson, 1968), i t is apparent that the nucleus does not exert a measurable control over the cytoplasm until this stage. There is l i t t l e information avialable which refers to either the temporal appearance of proteins during pregastruala stages of development or the genetic control that underlies the maternal influence over early embryonic protein synthesis (Ecker and Smith, 1970). Analysis of histone synthesis during development may shed some light on how protein synthesis is regulated during early embryonic development. This study characterizes histone synthesis during the early development and differentiation of the embryos of Xenopus 8 laevis, the South African Clawed Toad. In part one of this thesis histone synthesis i s examined in total c e l l homogenates of Xeno£US embryos to determine 1) when histones are synthesized, 2) whether a l l classes of histones are synthesized throughtout develeopment, and 3) i f there are any qualitative changes in the types of histones present throughout development. Part two of this thesis examines histone synthesis and accumulation in nuclei of Xenopus laevis embryos. The results show that a l l classes of histones are synthesized, and that they accumulate in nuclei at a l l stages of development. The data also suggests that these histones are probably made almost entirely from newly synthesized histone mRNA after stages 5 to 6 (table U) in this embryo. 9 Table H. Stages In The Normal Development Of Xenopus Laevis Nieuwkoop And Faber,1956. St. 5 — Dor«. St. 5 — A n . — Dori.-!at. St. 6—An. St. V/i — An. St. 8 — Vehtt. St. 9 —Vegetative view Dorsal side above ( V e g . l St. 15 — Post^dort. St. H —Lateral view (leftside) (Lit) St. 15 — Anterior view (Aat.) St. 42 Lat. St. 11— Ut. St. il — Vcnir. St, HO — Lat. St. 39 — Lat. St. 37.38 — U t . f j 1 (Small Ind.) (Small Ind.) 10 Part 1 . Histone Synthesis In Total C e l l Homogenates Of Xeno^us Laevis, The South African Clawed Toad O ) 11 INTRODUCTION PART 1 We have investigated the pattern of histone synthesis in t o t a l c e l l homogenates of Xenopus laevis both pre- and post-gastrula, using a combination of chromatographic and electrophoretic techniques. In p a r t i c u l a r , we have focused on the synthesis of basic proteins during the cleavage and swimming embryo stages when the patterns of RNA synthesis are known to be d i f f e r e n t . In the course of a study on ribosomal protein synthesis in X§£2£J2J> 2§S2iS embryos, Hallberg and Brown (1969) noted that about 8% of the t o t a l protein synthesized by cleaving embryos pulsed with 1 4C02 eluted as a single peak from a carboxymethylcellulose column. Kasihsky (1969) observed that electrophoresis of t h i s radioactive peak on acrylamide gels showed the presence of two radioactive bands which were d i s t i n c t from known ribosomal proteins and migrated s i m i l a r l y to histone f r a c t i o n s i s o l a t e d from adult somatic tissues. The experiments discussed here demonstrate that these two radioactive bands present i n extracts both of cleaving and swimming Xenopus laevis embryos represent the synthesis of the major classes of histones. 12 METHODS AND MATERIALS 1. MATING OF FROGS AND LABELING OF XJNOPUS LAEVIS EMBRYOS A. Maintenance Of Xenopus Laevis Stock ^S2£iil l a e v i s adult toads were obtained from the Snake Farm, South A f r i c a . Adults were kept for laboratory use over a period of months to years u n t i l death or disease eliminated them from the breeding stock. They were maintained i n a constant environment room at 19° CC in dechlorinated tap water. The l i g h t cycle was set at 16 hours of l i g h t and 8 hours of darkness. Toads were allowed a refractory period of 4 months between matings. B. Mating Procedure l a e v i s males were injected i n the dorsal lymph sac with 0.2 ml of chorionic gonadotropin hormone (Sigma Chemical Corp. 1000 I.U./m'l, diluted with Holtfreters solution) and females with 0.25 ml one week prior to mating. On the day of mating, males were injected with 0.25 ml of hormone and females with 0.75 ml. The mating pairs were placed in dechlorinated water i n breeding tanks overnight at 19° C. Eggs were shed and f e r t i l i z e d overnight. The embryos were collected the next day and deje l l e d for four minutes i n a 2% cysteine hydrochloride solution, pH 7.8 (Gusseck and Hedrick,1971). 13 C. Labeling Of Embryos Batches of 500 cleaving embryos were labeled for 6 hours after f e r t i l i z a t i o n (stages 1-9) in small disposable tissue culture flasks (50ml) containing ^ 100 C i (0.5 ml) of NaH14C03 (spe c i f i c a c t i v i t y ca. 50 mCi/mmol, Schwarz/Mann, No. 0023-92) in 10 ml of 1/10 Holfreter's solution modified to remove excess C02 by deletion of NaHC03. Modified Holtfreters consists of 3.5 gm of NaCl, 0.05 gm of KCl, 0.1 gm of CaCl2. Incubation medium i s made up of 75 ml of 1/10 modified Holtfreters and 25 ml of sodium phosphate buffer, pH 6.4. Both solutions are boiled before mixing to remove excess C02. X2J32£Iis la e v i s embryos were labeled with the **C02 precursor as they are impermeable to radioactive amino acids under normal conditions (Kutsky, 1950). Bromocresol purple (0.01%) was added as a pH indicator and 0.1N HCl was used to adjust pH to 6.4. The f l a s k s were then gently shaken at low speed on an Eberbach shaker in the hood. Labeling was stopped by cooling the embryos to 4° C when they reached late blastula (stage 9, Nieuwkoop and Faber, 1956). Embryos were then frozen and stored at -20° C. Swimming tadpoles were f i r s t labeled at stages 37-38 with 50 Ci of NaH**C03 for 8 hours as described for cleaving embryos. The embryos were then washed in dechlorinated water to remove excess l a b e l and were allowed to develop overnight to stage 40-41. They were then pulsed for 6 hours with. SO^Ci of NaH 1 4C03, 14 the embryos again were washed i n dechlorinated water to remove excess label and then the embryos were allowed to develope to stage 42 at room temperature, When the embryos reached the proper stage they were collected and stored at -20° CC. 2. ISOLATION OF BASIC PROTEINS FROM CLEAVING AND SWIMMING XENOPUS EMBRYOS A. Isolation Procedure Basic proteins were is o l a t e d by following a modified procedure of Hallberg and Brown (1969). A l l i s o l a t i o n procedures were done at room temperature C and urea used in t h i s i s o l a t i o n was always r e p u r i f i e d from the reagent grade. Approximately six thousand embryos were homogenized in 150 ml of 2M LiCl-8M urea, using a Bounce glass homogenizer, to so l u b l i z e the bulk of the proteins and precipitate RNA. The homogenate was then centrifuged at 27,000 g for 15 minutes. After c e n t r i f ugation, the supernantant was incubated with 105? t r i c h l o r o a c e t i c acid (solid TCA was added to a f i n a l concentration of 103? w/v) for 24 hours at 37° C, a method which s o l u b i l i z e s nucleic acids while p r e c i p i t a t i n g the protein. The solution was then centrifuged at 27,000 g for 20 minu/tes. The prec i p i t a t e was s t i r r e d into 150 ml of 10% TCA and centrifuged at 27,000 g for 15 minutes. The precipitate was collected and s t i r r e d into 180 ml of cold ether to remove l i p i d s and 15 centrifuged at 12,000 g for 5 minutes. This was repeated twice with the p e l l e t using ether at room temperature. The p r e c i p i t a t e was then s t i r r e d into 180 ml of 1/1 100% ethanol/anhydrous ether and centrifuged for 15 minutes at 27,000 g. The preciptate was then incubated i n 90 ml of 1/1 ethanol-ether for 60 minutes at 37° C i n a t i g h t l y stoppered f l a s k . After incubation the solution was centrifuged at 27,000 g for 15 minutes and then washed twice more. The precipatate was washed three more times i n 180 ml of anhdrous ether. After f i n a l centrifugation the residue was a i r dried in a fume hood at room temperature. The protein p r e c i p i t a t e was dissolved i n 0.1N HCl and then dialyzed against several changes of of 0.1N HCl for 24 hours to s o l u b i l i z e the basic proteins. This solution was then centrifuged for 90 minutes at 63,000 g i n a Spinco SW 27 rotor. The supernantant was dialyzed against 1,000 ml of 9M urea, 0.1N HCl, pH 3.0 at room temperature for 12 hours. The pH of the solution was then adjusted to pH 8.7 after the addition of d i t h i o t h r e i t o l (DTT) to a f i n a l concentration of 0.05M. DTT i s a sulfhydryl compound that reduces d i s u l f i d e bonds i n proteins to sulfhydryls i n order to minimize protein aggregation. After 60 minutes the solution was adjusted to pH 5.5 with concentrated g l a c i a l acetic acid and dialyzed against s t a r t i n g buffer (8M urea, 0.05 H sodium acetate, 0.001M DTT, pH 5.5). This procedure has been represented on the following page i n the form of a flow sheet. 16 Flow sheet: I s o l a t i o n procedure for basic proteins from embryos of Xenopus l a e v i s . Embryos (6,000) homogenize i n 150 ml of 2M L i C l , •'8M urea, centrifuge at27,000g for 15 rrtin. Ppt. discard : j Add s o l i d TCA to 10% (w/v), incubate 24 hours at 37°C, centrifuge 27,000g f o r 15 min. Ppt. Add 150 ml of 10% TCA, vortex, centrifuge 27,000 g for 15 min. 1 supernantant-discard II S t i r Ppt. into 180 ml cold ether, centrifuge 1,200 g f o r 5 min. 1 super nan tan t- d i s c a r d II Repeat above step twice with room temperature ether | 1 Super nan tan t-d i s c a r d Incubate in'90 ml of 1/1 (ethanol/ether) at 3 7° for 60 min., centrifuge 15 min. at 27,000 g I 1 super nan tan t-•discard Wash ppt. twice with 1/1 ethanol:ether, centrifuge at 27,000 g for 15 min. 1 supernantant- -discard Wash ppt. tfwice with 180 ml of ether, centrifuge supernantant- -discard at 27,000 g f o r 15 min. Dry ppt. i n fume hood, resuspend i n 180 ml of O.lN HCl, d i a l y z e against 4 l i t e r s o£ O.lN HCl, two changes of d i a l y s i s s o l n . , at 4 c . centrifuge 90 m i n at 63,000a. n — protein and DNA (acidic) Supernantant, d i a l y z e against 2000 ml of 9M urea, 0.01N HCl, pH.3, at 15 C for 7-12 hours, j Add IN NaOH, adjust pH to 8.7 to 8.9 add d i t h i o t h r e i t o l to 0.05M, s t i r for 60 min. at room temperature. Adjust pH to 5.J with g l a c i a l a c e t i c a c i d , d i a l y z e against s t a r t i n g b u f f e r The b a s i c protein extract i s ready to apply to a CM-cellulose column buffered witli s t a r t i n g I. b u f f e r . 17 B. P u r i f i c a t i o n Of Orea To insure r e p r o d u c i b i l i t y and good column recoveries, urea solutions were deionized according to Hallberg and Brown, 1968. Four l i t e r s of freshly made 9M urea (reagent grade) solutions were s t i r r e d at room temperature f o r 18 hours with 125 grams of Dowex AG 501-X8 re s i n (Biorad Co., AG50 i s a strongly a c i d i c cation exchange resin composed of nuclear s u l f o n i c acid exchange groups attached to a styrenedivinylbenzene polymer l a t t i c e . ) and then f i l t e r e d through a large scintered glass f i l t e r . Urea solutions were stored for up to 1 week at 4° C. 3. CHROMATOGRAPHY OF PROTEINS A. Carboxymethyl Cellulose Chromatography Proteins extracted by the preceding procedure were fractionated on microgranular carboxymethyl c e l l u l o s e (Whatman microgranular CM-32). CM-cellulose was precycled according to the Whatman information booklet. The CM-32 ce l l u l o s e was s t i r r e d into 0.5N NaOH (15:1; volume liquid/dry weight) and equilibrated for 30 minutes, washed with water to an intermediate pH of 8, s t i r r e d into 0.5N HCl (15:1; liquid/dry weight) and equilibrated again for 30 minutes before washing. After washing the CM-cellulose was stored i n water or equilibrated with s t a r t i n g buffer. 18 The basic protein f r a c t i o n from 3,000 embryos (st. 42 embryos, 500 mg average; cleavage embryos, 210 mg average) was washed into a 2X25 cm column with 100 ml of sta r t i n g buffer and eluted with a 400ml l i n e a r gradient of NaCI (0 to 0.25M) in st a r t i n g buffer. Any remaining protein adhering to the CM-c e l l u l o s e column was eluted with 200 ml of 815 urea, pH 2. Fractions of 3.3 ml were collected at a flow rate of 0.5 ml/inin. The proteins eluted were measured by absorbance at 278nm in a G i l f o r d spectrophotometer. Radioactive assays were performed by the f i l t e r paper disk technique of Bollum (1968) using 50 1 samples taken from alternate tubes. Samples were applied to 2 cm square pieces of Whatman No. 3MM f i l t e r paper. The samples were dried at room temperature, then incubated for f i v e minutes in room temperature TCA (10%) and f i v e minutes i n 5% TCA at room temperature (repeated twice). The samples were then incubated for 15 minutes at 90° CC i n 5% TCA to s o l u b i l i z e the nucleic acids and then incubated again in 5% TCA at room temperature. Samples were incubated for 5 minutes in 1/1 100% ethanol/anhydrous ether, t h i s step was repeated twice and then followed by a f i n a l incubation for 1 minute i n anhydrous ether. The samples were then a i r dried at room temperature. Samples were counted i n a Nuclear Chicago l i q u i d s c i n t i l l a t i o n counter (Unilux II-A). The c o c k t a i l used for s c i n t i l l a t i o n was a toluene based s c i n t i l l a t i o n f l u i d (4 l i t e r s s c i n t i l l a t i o n grade toluene, 16 gm 2,5-Diphenyloxazole (PPO, Eastman Kodak co.), 0.2 gm 2,2-p-phenylenebis (5-phenyloxazole, POPOP, Eastman Kodak Co.) . 19 B. Amberlite Chromatography Amberlite (CG 50) , a mildly a c i d i c r e s i n , was prepared by the method of Luck, et a l . , 1958. A thin s l u r r y of Amberlite was prepared by s t i r r i n g 100 gm of Amberlite (Mallinckrcdt, l o t #3341, 200-400 mesh, chromatographic grade) into 200 ml of d i s t i l l e d water. After the heavier material had s e t t l e d , the fines were decanted and the residue was washed 4 more times. The Amberlite was then suspended successively in the following solutions; one l i t e r of 2N HCl for 24 hours; washed with water u n t i l pH was 5.5; suspended in 2N NaOH for 15 minutes; washed with water u n t i l pH 8.0; suspended in 2N HCl for 15 minutes; washed with water ; f i n a l l y supended in two l i t e r s of 2N NaCI and t i t r a t e d to pH 7 with 2N NaOH. The resin was allowed to egui l i b r a t e for several days and the pH was adjusted with 2N NaOH when i t changed from pH 7. After f i n a l e q u i l i b r a t i o n , i t was f i l t e r e d through a Buchner funnel and resuspended in 8% guanidine hydrochloride in 0.1M phosphate buffer, pH 6.8. C. P u r i f i c a t i o n Of Guanidine Hydrochloride Guanidine hydrochloride (reagent grade, Eastman Kodak) was pu r i f i e d on a Celite-charccal column. The Celite-activated charcoal column was prepared by mixing 200 gm of C e l i t e (#545 Johns-Manville) with 100 gm of activated charcoal (#655,Matheson Co.) in d i s t i l l e d water to make a thin mixture of charcoal and C e l i t e . This slurr y of Celite-charcoal was then poured into a 4X60 cm column to a height of 50 cm and allowed to drain. One 20 l i t e r of 60% guanidine hydrochloride i n d i s t i l l e d water was poured onto the column and c o l l e c t e d . The column was then washed with one-half holdup volume of water. The guanidine hydrochloride fractions were collected and f i l t e r e d through a Buchner funnel. The concentration of guanidine hydrochloride was determined by i t s r e f r a c t i v e index . The Celite-charcoal column was discarded af t e r one p u r i f i c a t i o n of guanidine hydrochloride. D. Chromatography Of Proteins On amberlite After e q u i l i b r a t i o n , the resin was suspended in 8% (w/v) guanidine hydrochloride in 0.1M sodium phosphate buffer, pH 6.8. A 0.6X55 cm column was packed with resin under a i r pressure. Lyophilized samples (8-10 mg) of f r a c t i o n C S D proteins (proteins eluted i n 0.175 to 0.25M s a l t gradient i n urea buffer. Figure 1) were brought up in a few mis of 8% guanidine hydrochloride and applied to the column. A l i n e a r gradient of 8-14% guanidine hydrochloride was used to separate histones from each other according to the procedure of Luck, et a l . , 1958. After the gradient was completed, 25 ml of 40% guanidine hydrochloride was added to elute any adhering protein. The eluted protein was collected in 0.3 ml f r a c t i o n s during a 3-5 day run at room temperature (22-25° CC) and 1 4 C - l a b e l e d proteins were assayed as described for CM-cellulose chromatography. Protein concentration was measured by TCA p r e c i p i t a t i o n . Fractions of 0.3 ml from the column were diluted with 0.9 ml of water and 0.6 ml of 3.3M TCA. The tubes were shaken and 13 21 minutes l a t e r the o p t i c a l density of the tubes was read at 400nm, according to the procedure of Bonner, et a l . , 1968. E. acrylamide Gel Electrophoresis Disc electrophoresis was performed using 15% polyacrylamide gels made 6M with urea, pH 4.5, according to the method of Bonner, et a l . , 1968. Stock solutions (stored for up to 2 months and discarded): N,N,N,N, tetramethylenediamine (Temed, Eastman Kodak Co.) solution(48 ml KOH, 17.2 ml g l a c i a l acetic acid, 4 ml TIMED, d i s t i l l e d water up to 100 ml). Acrylamide (Eastman Kodak Co.) solution (60 gm acrylamide, 0.4 gm methylenebisacrylamide, d i s t i l l e d water to 100 ml). 0.295 {v/v) ammonium persulfate in freshly deionized 10M urea. For the preparation of 8 (0.5X7 cm) electrophoresis gels, 1,4 ml of TEMED solution and 2.8 ml of acrylamide solution were added to 7 ml of persulfate-urea, the mixture was mixed and pipetted into the 8 capped tubes. The gels were then overlaid with 3M urea solution to allow for anaerobic polymerization of the acrylamide. After polymerization the gels were f i r s t pre-electrophoresed at room temperature to remove persulfate at 4 mA/gel for 90 minutes at room temperature i n tray buffer (31.2 gm beta-alanine, 8 ml acetic acid and water to one l i t e r ) . The 22 gels were then electrophoresed in new tray buffer at 4 ma/gel for 90 minutes with the protein sample. Protein samples were reduced (1mg/ml) i n 8M urea, 0.05M DTT for 30 minutes at 37° C to prevent histone aggregation. The samples were run and the gels were stained with 155 amido black in 1% g l a c i a l acetic acid and destained i n 1% acetic acid i n a charcoal d i f f u s i o n destainer (Hoefer S c i e n t i f i c Co.). Densitometric tracings were obtained on the G i l f o r d spectrophotometer, l i n e a r scanner model 2400, at 660nm. For autoradiography, gels were s l i c e d l o n g i t u d i n a l l y , vacuum dried onto f i l t e r paper , and subjected to autoradiography (30 days) using Kodak No-Screen Medical X-ray f i l m according to the method of Fairbanks, et a l . , 1965. Radioactive assays were performed by making 0.5 mm s l i c e s of gels frozen on dry i c e on a gel s l i c e r (Mickle Laboratory Engineering Co.). The gel s l i c e s were dissolved by incubating then in i n 0. 1 ml of 305? hydrogen peroxide i n s c i n t i l l a t i o n v i a l s for 60 minutes at 50° C. Five mis of Aguasol (New England Nuclear) were added to the s c i n t i l l a t i o n v i a l s , the v i a l s were shaken and then counted on the l i q u i d s c i n t i l l a t i o n counter. F. Sodium Dodecyl Sulfate (SDS) Gel Electrophoresis SDS gel electrophoresis was performed using 105? acrylamide gels with 13? SDS according to the method of Weber and Osborne (1969) . Stock Solutions (stored for up to two months and discarded): 23 Gel buffer. Gel buffer contained 7.8 gtn flaH2P04«H20, 38.6 gm Na2 H2P04»H20, 2 gm SDS/liter. Acrylamide solution. 22.2 gm acrylamide, 0.6 gm methylenebisacrylamide were dissolved i n d i s t i l l e d water to give 100 ml of s o l u t i o n . To make a 10% acrylamide solution, 15 ml of gel buffer and 13.5 ml of acrylamide solution were mixed and aspirated under vacuum. Then 0.75 ml of a freshly made solution of ammonium persulfate (15mg/ml) mixed with N,N,N,N, tetramethylenediamine (45 1/ml) was added and mixed. This solution was then pipetted into 0.6X10 cm glass tubes capped with parafilm. The solutions were topped with water. After polymerization gels were pre-electrophoresed for 3 hours at 8 mA/tube to remove persulfate. The electrophoresis compartments were f i l l e d with gel buffer diluted 1:1 with water. Tray buffer containing persulfate was replaced after each run. Protein samples (1 mg/ml i n 0.1 M sodium phosphate, 0.1% SDS and 0.1% mercaptoethanol, reduced for two hours at 37° C) were then applied to the gels and run for 2-3 hours at 8 mA/gel. After electrophoresis gels were incubated i n 12% TCA at 65° CC f o r 30 minutes. Gels were then stained i n Coomassie blue solution (0.2% Coomassie blue v/v ; 45 ml absolute ethanol; 10 ml g l a c i a l acetic acid and d i s t i l l e d water to 100 ml) for 30 minutes at 65° C. Gels were destained i n a Hoeffer destaining apparatus containing 7% [v/v) acetic acid, 20% (v/v) ethanol, 73% d i s t i l l e d water. 24 4. AMINO ACID ANALYSIS Samples of the various histones were hydrolyzed i n vacuo at 110° C fo r 24 hours in 6N NCl . After evaporation to dryness, the hydrolysates were dissolved in d i s t i l l e d water and their amino acid composition was determined on a Technicon amino acid analyzer. The presence of tryptophan was measured by the colormetric method of Opienska-Blauth, et a l . , 1963. Reagent A (0.27 gm of FeCl2«6H20 i s dissolved in 0.5 ml of water and g l a c i a l acetic acid i s added to one l i t e r ) i s mixed with one ml of sample that contains 2 to 40 g of tryptophan and 2 ml of concentrated s u l f u r i c acid i s added. The solution i s shaken and allowed to stand 15 minutes (10 hours maximum) before the' absorbance i s measured at 545 nm. This assay was run against a known standard for c a l i b r a t i o n . 25 RESULTS When subjected to chromatography on carboxymethyl c e l l u l o s e , the basic proteins synthesized either in cleaving or swimming embryos of Xenojus laevis were fractionated as shown in figure 1. Fractions A through D from the CM-cellulose column were pooled, concentrated and dialyzed to remove s a l t . Each f r a c t i o n was then electrophoresed on 15% acrylamide gels. Figure 2 shows the tracings of fractio n s A through D from stage 42 embryos, while s i m i l i a r tracings were also obtained from cleavage embryos. Hallberg and Brown (1968) found that most of the materials present i n fra c t i o n s A and B were ribosomal proteins, as shown by co-electrophoresis with adult ribosomal proteins i s o l a t e d as markers. The region of par t i c u l a r i n t e r e s t consisted of fr a c t i o n C and D i n which Kasinsky (1969) observed proteins s i m i l a r to adult somatic histones of Xenopus laevis i n t h e i r electrophoretic properties. This f r a c t i o n was found to elute between 0.175 and 0.25M NaCI i n the gradient on the 2x25cm column. This presumptive histone f r a c t i o n made up about 6% of the t o t a l radioactive proteins in stage 42 embryos and about 9% in cleavage , can be seen in Tables 5 and 6. Of the t o t a l proteins i n the o r i g i n a l homogenate, th i s f r a c t i o n represents about 9% i n cleavage and about 12% in swimming tadpoles. In order to determine i f these proteins were histones, f r a c t i o n s C and D were reduced with d i t h i o t h r e i t o l and examined by electrophoresis i n 15% ,acrylamide gels by the method of 26 Figure 1. Carboxymethyl Cellulose Chromatography Of Basic Proteins Synthesized By Swimming Embryos Of Xenopus Laevis. The basic protein fraction from 3,000 embryos (average sample, 250 mg) was washed into a 2 X 25 cm CH-cellulose column with 100 ml of starting buffer and eluted with a 400 ml linear gradient NaCl (0 to 0.25H) in starting buffer. Any adhering protein was eluted with 200 ml of 8H urea, pH 2. Fractions of 3.3 ml were collected at a flow rate of 0.5 ml/min. Radioactive assays were performed by the f i l t e r paper disc technique of Bollum (1968) using 50^ .1 samples from alternate tubes. 26a 27 Figure 2. Tracings Of Acrylamide Gels From CM-Cellulose Column. Fractions A through D and the pH2 wash for stage 42 swimming embryos were electrophoresed on 1595 polyacrylamide gels made 6H with urea, pH 4.5. Proteins (75/Wwg samples) were electrophoresed for 90 minutes at room temperature according to the procedure of Bonner, et a l . , (1968). A l l samples were reduced with 0.05M dithiothreitol to prevent histone aggregation. Gels were pre-electrophoresed for 90 minutes to remove persulfate. After the f i n a l run, gels were stained with 1% amido black in 7% acetic acid and destained in 7% acetic acid in a diffusion destainer. Tracings were taken using the Gilford linear scanner (model 2400) at 660nm. (A) . Fraction A (B) . Fraction B (C) . Fraction C (D) . Fraction D (£). PH 2 wash 28 Table 5. Recoveries Of Total Protein From Stage 42 Embryos. Approximately 6500 swimming embryos were homogenized in 8M urea-2B L i C l . They were processed as described in the text. Protein was assayed according to Lowry, et a l . , 1953. Radioactive assays were performed by the f i l t e r paper disc technique of Bollum (1968) using SO^J. samples. Flow sheet i s presented on page 16. * 28a Processing Of Stage 12 Embryos protein Radioactivity total * of total 5 of (ng) original (cpm) origii Stage of processing 8 H Urea-2 K L i C l Homogenate 1,095 1005 23.2x10 « 1005 Fi r s t homogenization before TCA 1.017 935 19.7x10 6 855 Fi r s t TCA Supernantant 165 155 2.2x10 ' 9.55 Second TCA supernantant 30 35 5.8x10 s 2.55 0.1 N HC1 Dialysate 797 735 13.4x10 ' 585 Supernantant after centrifugation 667 615 12.3x10 • 535 DTT Reduced Sample Before CMC 576 535 9.6x10 * 41.55 Fractions Recovered From Column fraction A 192 17.55 4.9x10 « 20. 65 Fraction B 88 8.85 1.2x10 « 4.75 fraction C + D 100 9. 15 1.4x10 * 6. 4 5 PH 2 Wash U3 4.45 8.Hx10 5 3.65 I n i t i a l wash 97 8.85 1.3x10 « 6.05 Total Protein Recovered From Column 517 48.75 9.5x10 « 41. 35 29 Table 6. Recoveries Of Total Proteins From Cleavage Embryos. approximately 6900 cleavage embryos were homogenized in 8M urea-20 L i C l . They were processed as described in the text. Conditions are the same as in table 5. 29a P r o c e s s i n g O f C l e a v a g e E m b r y o s S t a g e o f p r o c e s s i n g 8 M O r e a - 2 H L i C l H o m o g e n a t e F i r s t h o m o g e n i z a t i o n b e f o r e T C A F i r s t T C A S u p e r n a n t a n t S e c o n d T C A s u p e m a n t n a . t 0.1 H H c L D i a l y s a t e s u p e r n a n t a n t a f t e r c e n t r i f n g a t i o n D T T S a m p l e B e f o r e C H - C e l l u l o s e F r a c t i o n s R e c o v e r e d F r o m C o l u m n f r a c t i o n A F r a c t i o n B f r a c t i o n C + D P H 2 W a s h i n i t i a l w a s h T o t a l R e c o v e r e d F r o m C o l u m n p r o t e i n R a d i o a c t i v i t y t o t a l 55 o f t o t a l X o f ( m g ) o r i g i n a l ( c p o ) o r i g i i n o 1 0 0 * 3 5 . 2 x 1 0 s 1 0 0 5 5 3 8 5 9 1 T . 3 3 . 5 x 1 0 s 9 5 . 3 5 ! 5 3 1 2 . 835 2 . 7 x 1 0 s 7 . 7 5 5 1 1 . 2 . 7 5 5 9 . 2 x 1 0 * 2 . 6 S 2 9 1 7 2 ? 2 5 6 6 2 . 556 2 1 . 2 x 1 0 s 6 0 . 3 5 5 2 2 5 5 5 * 1 8 . 7 x 1 0 s 5 3 5 5 9 1 2 2 . 9 5 5 7 . 3 x 1 0 s 2 0 . 755 2 7 . 2 6 . 7 5 5 2 . 6 x 1 0 s 7 . M S 5 0 . 2 1 2 . 2 5 5 3 . 3 x 1 0 s 9 . 1 5 5 1 2 . 7 3 . 1 5 5 1 . 7 x 1 0 s 1 . 8 5 5 1 8 . 7 1 . 5 5 5 2 . 8 x 1 0 5 1 . 8 5 5 2 0 2 • 1 9 . 5 3 5 1 7 . 7 x 1 0 s 30 Bonner, et a l . , 1968 (figure 3). Tracings of these gels stained with amido black showed the presence of a fast moving doublet i n both swimming tadpole and cleaving embryos that coincided with a radioactive doublet as determined by counting gel s l i c e s in a l i q u i d s c i n t i l l a t i o n counter. Electrophoresis on the same gel with a known histone marker, the arginine-rich histone IV from pea seedlings, indicated that the fastest migrating band in both cleaving and swimming embryos histone migrated together with pea histone IV. These data indicated that we were looking at a histone f r a c t i o n . Duplicate runs of f r a c t i o n C and D from swimming tadpole and cleavage embryos and pea histone IV marker (a g i f t of Drs. Fambrough and Bonner) were also performed on SDS gels by the method of Weber and Osborne (1969), for comparative purposes on a d i f f e r e n t electrophoretic system (Figure 4). Tracings of these gels also showed the presence of several proteins, the fastest migrating protein in fractions from either swimming tadpole or cleavage embryos co-electrophoresing with histone IV as a marker. This was a further i n d i c a t i o n that at least one protein present was histone IV and that the rest of the histones probably were also present i n t h i s f r a c t i o n . As we s h a l l see from the subsequent data on Amberlite chromatography and gel electrophoresis, the large peak of the radioactive doublet i n Figure 3, f r a c t i o n C and D, represented the synthesis of the other major classes of histones i n the cleavage and swimming embryos. Further characterization of the newly synthesized basic proteins present in fractions C and D as presumed histones was 31 Figure 3. Tracings Of Acrylamide Gels Of Fraction C And D From Stage 42 And Cleavage Embryos. Solid l i n e s , optical density at 660 nm. Dotted profiles, radioactivity. Conditions were the same as described in figure 2 for electrophresis on 15% acrylamide gels. Radioactive assays were performed on 0.5 mm gel slices after peroxidation and solubilization with Aguasol for s c i n t i l l a t i o n counting. (A) radioactive fraction C • D, swimming embryos (B) fraction C • D swimming embryos electrophoresed on the same gel with unlabeled pea histone IV (C) radioactive fraction C +D, cfleavage embryos (D) fraction C + D cleavage embryos electrophoresed on the same gel with unlabeled pea histone IV 31a mm 32 Figure 4. Tracings Of SDS Acrylamide Gels Of Fraction C And D From Stage 42 And Cleavage Embryos. Solid l i n e s , optical density at 660 nm. Gel electrophoresis was performed using 10% acrylamide gels with 1% SDS. Proteins (75 g sample) were run for 3 hours at room temperature according to the procedure of Weber and Osborne (1969). A l l samples were reduced in 0.1% mercaptoethanol for 2 hours at 37° C. Gels were pre-electrophoresed 3 hours to remove persulfate After the run gels were stained with 0.2% Coomassie Blue and destained in a diffusion destainer. The arrow indicates known histone IV marker protein. (A) fraction D and C, swimming embryo. (B) fraction C +D electrophoresed on the same gel with pea histone IV. (C) fraction C • D , cleavage embryos. (D) fraction C and D electrophoresed on the same gel with pea histone IV. 32a A 33 obtained from Amberlite chromatography (Luck, et a l . , 1958). Chromatography on Amberlite separates the histone components into three histone : l y s i n e - r i c h histone I; s l i g h t l y - l y s i n e r i c h histones IIb1 and IIb2; and arginine-rich histones III and IV. Figure 5, shows the f r a c t i o n a t i o n of cleavage embryo C and D f r a c t i o n s on Amberlite. There i s an i n i t i a l broad peak of r a d i o a c t i v i t y (fractions 20-70) that should include l y s i n e - r i c h histone I and other contaminating basic proteins. The second radioactive peak (fractions 71-120) eluted by the guanidine hydrochloride gradient was due to the presence of the s l i g h t l y -lysine r i c h histones IIb1 and IIb2. F i n a l l y , the l a s t radioactive peak (fraction 121-160) eluted in the region where the arginine-rich histones III and IV were expected to appear. Amberlite chromatography of fr a c t i o n s C and D from stage 42 embryos yielded a s i m i l a r elution pattern as seen in figure 6. The s i m i l a r i t y of the Amberlite f r a c t i o n a t i o n p r o f i l e s for both labeled cleavage and swimming tadpole histones suggested that there was no q u a l i t a t i v e change i n the pattern of histone synthesis throughout the early development of Xenopus l a e v i s embryos. In order to i d e n t i f y the newly synthesized radioactive proteins i n the Amberlite peaks of cleaving (figure 5) and stage 42 embryos (figure 6) as histones, we f i r s t electrophoresed them on one gel and then co-electrophoresed them with an unlabelled histone marker from trout t e s t i s (I, Ilb1, or I I I , a g i f t from Dr. G. Dixon) or histone IV from pea cn a second gel. By co-electrophoresis we mean that each radioactive protein i n the 34 Figure 5. Amberlite Chromatography Of Radioactive Histones Synthesized By Cleavage Embryos , Stages 1-9, Of Xenopus Laevis. Basic proteins (5-8 mg), fraction C and D from CM-cellulose chromatography, were applied to a 0.6 x 55 cm column and eluted with a 50 ml linear gradient of 8-1495 guanidine hydrochloride in 0.1 H sodium phosphate, pH 6.8. This gradient was followed by a 4095 guanidine hydrochloride wash (Luck, et a l . , 1958) to elute the arginine-rich histones III and IV. Fractions of 0.35 ml were collected from a column over a three day period at room temperature and radioactive assays using the f i l t e r disc technique of Bollum (1968) were performed as described in figure 1. 35 Figure 6. Amberlite Chromatography Of Radioactive Histones Synthesized By Swimming Embryos, Stage 42, Of Xenopus Laevis. Conditions were the same as indicated in figure 5. 3 5a 5376 A 11,053 i i i i i i i i 1 1 1 1— 20 60 100 140 FRACTION NUMBER 36 Amberlite peaks was run together with a non-radioactive marker on the same gel. Both the densitometric tracing of the amidoblack stained band and the r a d i o a c t i v i t y pattern of the gel were then determined. As seen i n figure 7, densitometric tracings indicated that the i n i t i a l Amberlite radioactive peak from stage 42 embryos co-electrophoresed with the l y s i n e - r i c h histone I from trout t e s t i s , although other contaminants were also present. In t h i s gel and i n each of the others in figure 7, s u f f i c i e n t amounts of radioactive histones from the Amberlite peaks of swimming embryos were applied to the gels to obtain densitometric tracings of t h e i r amidoblack stained bands as well as t h e i r r a d i o a c t i v i t y p r o f i l e . As the two patterns coincided, only the densitometric tracings at 660 nm for the stained bands are shown for both the radioactive proteins from the Amberlite peaks and the same proteins co-electrophoresed with non-radioactive histone markers. The second peak co-electrcphoresed with the s l i g h t l y l y s i n e - r i c h histone IIb1 from trout t e s t i s . We presume that the s l i g h t l y l y s i n e - r i c h histone IIb2 i s also present in t h i s f r a c t i o n . F i n a l l y , i n the 40% guanidine hydrochloride wash, electrophoresis showed two bands. The slower migrating band co-electrophoresed with arginine-rich histone III from trout and the faster moving band with histone IV from pea. There was s t i l l a low l e v e l of contaminating proteins i n t h i s region. These may represent a c i d i c proteins liberated by the abrupt increase i n the guanidine hydrochloride concentration as described by Stellwagen and Cole (1968). Densitometric tracings were also obtained from acrylamide 37 Figure 7. Tracings From acrylamide Gels Of amberlite Peaks For Stage 42 Embryos,figure 6. Conditions were the same as those indicated in figure 2. Densitometric tracings at 660 nm both for proteins from the amberlite peaks and histones used as markers are indicated by solid lines. (1) Top gel: electrophoresis of the lysine-rich peak. Bottom gel: co-electrophoresis of this peak with lysine-rich histone I from trout as a marker. (2) Top gels: electrophoresis of the slightly lysine-rich histone peak. Bottom gel : co-electrophoresis of this peak with slightly lysine-rich histone IIb1 from trout. (3) Top gel: electrophoresis of the arginine-rich peak. Bottom gel: co-electrophoresis of this peak with arginine-rich histone III from trout. (4) Top gel: electrophoresis of the arginine rich histone peak. Bottom gel: co-electrophoresis of this peak with arginine-rich histone IV from pea. 37a 38 gels of the radioactive Amberlite peaks from cleavage embryos (figure 5) co-electrophoresed with unlabeled histone markers i n order to i d e n t i f y these newly synthesized proteins as histones, As seen i n figure 8, each of the major classes of histones present i n these embryos was synthesized during t h i s early period of development preceeding gastrulation. S u f f i c i e n t amounts of the l y s 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 histones were obtained from the Amberlite peaks of cleavage embryos to obtain densitometric tracings of their amidoblack stained bands as well as their r a d i o a c t i v i t y p r o f i l e . As the two patterns coincided we have shown only the densitometric tracings i n part 1 and part 2 of figure 8. However, d i l u t i o n of the radioactive proteins i n the arginine-rich histone peak from the Amberlite column onto several gels greatly decreased their staining with amidoblack. we therefore used autoradiography to establish the i d e n t i t y of these labeled histones by t h e i r co-electrophoresis with unlabeled arginine-rich histones III (trout) and IV (pea) , respectively. The histone f r a c t i o n from stage 42 and cleavage embryos that had been separated on Amberlite chromatography and further characterized by amino acid analysis. The amino acid compositions of the three Amberlite peaks from stage 42 embryos are shown i n table 7 where they are compared to newt embryo and c a l f thymus histones. The f i r s t peak, Xenppus la e v i s histone I , i s shown to have the highest lysine content of the separated histones. This high lys i n e to arginine r a t i o i s c h a r a c t e r i s t i c of the l y s i n e - r i c h 3 9 Figure 8. Tracings From acrylamide Gels Of Amberlite Peaks For Cleavage Embryos From Figure 7. Conditions and fraction numbers were the same as indicated in figure 2 and 7, respectively. Autoradiograms of the radioactive proteins from the Amberlite peaks are indicated by dashed lines in (3) and (4) densitometric tracings at 660 nm. Both the radioactive proteins and the non-radioactive histones used as markers are indicated by solid lines. (1) Top gel: electrophoresis of the lysine-rich histone peak. Bottom gel: co-electrophoresis of this peak with lysine-rich histone I from trout as a marker. (2) Top gel: electrophoresis of the slightly lysine-rich histone peak. Bottom gel: co-electrophoresis of this peak with slightly lysine-rich histone IIb1 from trout. (3) co-electrophoresis of the radioactive arginine-rich histone peak with unlabeled histone III from trout. Both tracings are from the same gel. (4) co-electrophoresis of the radioactive arginine-rich histone peak with unlabeled histone IV from pea. Both tracings are from the same gel. 39a 40 Table 7. Amino Acid Composition Of Histones From Stage 42 Embryos Compared With Those Of Stage 20 Hewt Embryos And Calf Thymus Histone Fractions. X. laevis Histones (Mol %) Newt Histones' (Mol %) ^ ( M q. % ) Amino Acid I II III + IV Fl Flla Fill* Fib Fllb F i l l ' Lys 18.8 14.1 10.4 18.6 10.5 9.3 26.2 13.5 9.3 His 0.2 3.4 2.7 2.6 3.6 3.1 0.2 2.8 1.6 Arg 1.3 7.5 8.7 1.7 3.8 5.4 2.6 7.9 12.8 Asp 4.7 6.7 7.6 4.7 6.3 6.9 2.5 5.6 4.4 Thr 4.3 3.9 5.1 3.4 5.8 6.6 5.4 5.2 7.3 Ser 4.3 6.9 5.6 14.1 13.0 10.2 6.5 7.8 4.1 Glu 4.4 5.2 11.0 8.6 8.6 7.5 4.3 8.7 9.8 Pro . 5.7 5.9 4.9 7.0 4.2 3.2 9.1 4.7 3.8 Gly 8.3 5.9 9.1 11.9 13.1 12.3 7.3 8.2 8.7 Ala 11.2 12.9 8.1 14.9 10.6 10.7 24.2 11.5 11.7 Cys/2 0 0.6 0 0 0 :;p 0 0 Val 6.2 ; 6.9 6.5 5.1 5.8 6.7 • 4.0 6.7 5.8 Met 0.8 1.3 1.8 0.4 0.5 1.1 0.1 0.8 1.2 He 2.1 4.4 4.8 1.6 4.4 5.4 1.2 4.5 5.4 Leu 6.0 7.4 8.4 3.9 6.8 8.2 5.0 8.6 8.6 Tyr 0.3 3.5 3.0 0.3 0.9 0.7 0.7 3.0 2.4 Phe 1.2 3.5 3.9 1.3 2.0 2.9 0.6 1.3 2.5 B/A 2.2 2.1 1.2 1.7 1.9 1.2 4.3 1.7 1.6 Lys/Arg 10.5 1.9 1.2 10.8 2.8 1.7 10.1 1.7 0.8 " Asao, 1969.b Represents combined histones HI -f IV.e Rasmussen et al., 1962. 4^ O 0> 41 histones. However, the t o t a l mole percent of alanine and proline are lower than would be expected for histone I, as seen i n c a l f thymus. This i s probably due to comtamination by other basic proteins that are eluted i n the l y s i n e - r i c h peak, as shown in figure 8. The vamino acid composition of the second peak, II, combined f r a c t i o n containing both s l i g h t l y l y s i n e - r i c h histones IIb1 and IIb2, i s similar" to known values for c a l f thymus. This f r a c t i o n also compares with that of newt histone I l a except for the higher serine and lysine content and the greater lysine to arginine r a t i o shown i n the newt. The t h i r d peak, eluted in the 40% gradient on Amberlite, appears to be c h a r a c t e r i s t i c for the arginine-rich histones III plus IV, having a low lysine to arginine r a t i o . Also, the presence of cysteine i s noted i n our arginine-rich f r a c t i o n . Cysteine i s absent in a l l histones except for histone III (Johns, 1971). The presence of tryptophan was measured i n each peak by the colormetric assay method of Opienska-Blauth, 1963. There was no tryptophan present i n these peaks, the absence of tryptophan i s another c h a r a c t e r i s t i c feature of histones. As noted e a r l i e r , the l y s i n e - r i c h histone peak from stage 42 embryos (figure 7) was contaminated with other basic proteins after Amberlite chromatography. This may have been due to the synthesis of basic proteins, such as ribosomal protiens, which i s known to occur during t h i s period of development in Xenojjus l a e v i s (Hallberg and Brown, 1968). To further characterize the l y s i n e - r i c h histones i n t h i s region and to separate them from other basic proteins, we re-chromatographed the i n i t i a l 42 Amberlite peak on a second amberlite column using a shallow gradient, as described by Kinkade and Cole (1966) , and Bustin and Cole, (1968). We can see i n figure 9 that t h i s gradient separated the contaminating proteins from the l y s i n e - r i c h histones, the l a t t e r emerging as three labeled peaks s t a r t i n g at 9.4% i n the guanidine hydrochloride gradient. Bustin and Cole (1968) have shown that there i s a s i m i l i a r microheterogeneity of l y s i n e - r i c h histone I in rabbit and c a l f thymus as well as rabbit and chicken l i v e r using t h i s method. Re-chromatography of the l y s i n e - r i c h histone peak of cleavage embryos on a shallow gradient also showed a separation of histone I from basic protein contaminants. 43 Figure 9. Chromatography Of The Lysine-Bich Histone Peak From Stage 42 Embryos On Amberlite Using A Shallow Gradient. Basic proteins (10mg) from the f i r s t peak of the original Amberlite chromatography of stage 42 embryos were eluted on a 0.6x55 cm column with a 200 ml linear gradient of 7-14% guanidine hydrochloride in 0.1 H sodium phosphate, pH 6.8, according to the method of Bustin and Cole, 1968. Fractions of 0.35 ml were collected over an 8 day period at room temperature. Badioactive assays were done as described in figure 1. The arrows point to the 3 lysine-rich peaks that began eluting at 9.4% in the guanidine hydrochloride gradient. T5 — » 70 CO > O ~2 C P M X 1 0 7 0 , 3 5 m l F R A C T I O N CO o Cn A Y A N Cn CO O O O Z c m O TO \ t i i t i t •t t K3 N O CO O O m— t t I I I I t t t i t l °/o G U A N I D I N E H Y D R O C H L O R I 44 DISCUSSION Chromatographic and electrophoretic analyses indicate that the major classes of histones (I,II,III and IV) are synthesized during cleavage (stages 1-9) and i n swimming tadpoles (stages 37-42) periods of development in Xenopus l a e v i s . Thus, Xeno_p_us la e v i s embryos appear to synthesize the f u l l complement of histones throughout their early development and do not acquire q u a l i t a t i v e l y unique "cleavage histones" as had been suggested by some cytochemical experiments (Horn, 1962). In these experiments we have been looking at histone synthesis i n t o t a l c e l l extracts. Since histones may be defined as "basic proteins that at some time are associated with DNA" (Murray,1964), we must qualify our use of the term histone i n t h i s regard. We have been observing newly synthesized basic proteins i n a t o t a l c e l l extract that appear to be histones by three d i f f e r e n t c r i t e r i a : electrophoresis, Amberlite chromatography and amino acid analysis. The data presented here represent the f i r s t biochemical evidence for histone synthesis both before and after gastrulation in an amphibian. We did not use nuclear preparations i n this i n i t i a l study for two reasons. F i r s t , nuclear preparations from early amphibian embryos are generally poor, due to large f r a g i l e nuclei and to extensive cytoplasmic yolk and pigment granule contamination. Second, i n e a r l i e r experiments, Berlowitz and B i r n s t i e l (1967) isolated a p a r t i a l l y p u r i f i e d , * 1*C02-labeled nuclei preparation from swimming embryos of Xenojpjos laevis and extracted histones d i r e c t l y from t h i s preparation. Using 45 amberlite chromatography, they demonstrated peaks corresponding to the synthesis of major histone f r a c t i o n s in swimming tadpoles, although they did not characterize these f r a c t i o n s by gel electrophoresis or amino acid analysis. Berlowitz and B i r n s t i e l found that the nuclei in the anucleolate mutant of Xenopjas laevis contained markedly d i f f e r e n t amounts of i n d i v i d u a l histones than did nuclei of tailbud embryos at comparable stages. This conclusion may be subject to doubt, however, as t h e i r nuclear preparations may have contained varying degrees of cytoplasmic contamination. In fact, the data of Hallberg and Brown (1969) on the anucleolate mutant of X^HSEHS .l3.§.y-i§ indicated that anucleolate embryos synthesized histones to almost the same extent (13%) as did the normal stage 42 embryos. Our procedure of i s o l a t i n g histones from a t o t a l c e l l homogenate, rather than from p a r t i a l l y p u r i f i e d n u c l e i , and subsequently separating out contaminating basic proteins, p a r t i c u l a r l y ribosomal proteins, attempted to overcome some of these d i f f i c u l t i e s . The only other biochemical investigation of histone synthesis i n the early development of an amphibian i s that of Asao (1969,1970), who injected labeled amino acid mixtures into female newts before mating and separated the labeled histones by Amberlite chromatography on a micro scale. In contrast to our findings that synthesis of the major classes of histones in X§S2ES§ liSli§ 3icl not change q u a l i t a t i v e l y throughout early development, Asao found that there was no accumulation of labeled histones in the nuclei of the Japanese newt, Triturus 46 pyrrhoqaster , before early qastrulation. He also observed that arginine-rich histones predominated over l y s i n e - r i c h ones in lat e blastula and early gastrula and, quite suprisingly, that the dorsal l i p region was r e l a t i v e l y lacking in histones at late b l a s t u l a . This suggested to Asao that the template a c t i v i t y of DNA was not repressed by these basic proteins during early development i n the newt. One explanation for this difference between our observations and Asao's i s that we may be looking at both cytoplasmic synthesis and nuclear accumulation of labeled histones. Asao (1969) did look at a fr a c t i o n he ca l l e d "cytoplasmic" basic proteins. However, th i s i s a misnomer as th i s f r a c t i o n consisted mostly of yolk granules and some other cytoplasmic components that, according to Asao, remained as a surface layer a f t e r centrifugation of the nuclei. This would not correspond to the whole c e l l extract examined in this work. There i s a p o s s i b i l i t y that some of the histones included in t h i s i s o l a t i o n procedure are not embryonic . Baltus, et a l . , (1968) have reported the presence of double stranded DNA i n yolk p l a t e l e t s from amphibian embryos. This low molecular weight DNA (20 X 106 daltons) i s complexed to the phospholipids ins yolk, and associated with i t are basic proteins. These protein are basic i n composition (20% basic residues) with a very high serine content (31-39%). The percentage of basic residues implies that t h i s may be a histone-like protein, while the high serine content can be explained by the extraction procedure (1.8H NaCl at 100°C) which would free serine from phosphoserine present i n yolk. This DNA and associated protein i s probably 47 maternal i n o r i g i n . When phosphoproteins are synthesized i n the l i v e r they may complex with free nucleohistone (Rudeck and Wallace, 1968) and after transport in complex form to the ovaries they can be converted into yolk p l a t e l e t s during v i t e l l o g e n e s i s . However, since the work presented in t h i s thesis has been based on histone synthesis, not t o t a l histone content, any maternal yolk histone contamination i s of l i t t l e consequence. The histone to DNA r a t i o i n c e l l s or i n the t o t a l embryo was not calculated in these experiments for several reasons.' F i r s t , for true quantitation, histones must always be p u r i f i e d from chromatin isol a t e d from nuclei which could not be done in Part I of t h i s thesis. Any other means of i s o l a t i o n leads to contamination by yolk and other basic proteins in the c e l l such as ribosomal proteins. Second, the amount of DNA present i n the c e l l s of Xengpus embryos at any given stage i s s t i l l a point of controversy at t h i s time. The reports indicate a wide range for DNA due to technical d i f f i c u l t i e s in studying the egg and the large amounts of storage materials and other constituents compared to the low DNA content. There i s a large amount of mitochondrial DNA (Dawid, 1965) and yolk p l a t e l e t DNA (Brachet, 1968) which constitutes at least 300 to 500 times the d i p l o i d DNA complement of somatic c e l l s . Third, the d i f f e r e n t procedures used in i s o l a t i n g cytoplasmic DNA (Dawid, 1965; Harcq, et a l . , 1968) lead to differences in the reported amounts of DNA present. Fourth, i f the r a t i o s are to be calculated then one must have a synchronous population of eggs . Owing to the 48 labeling procedures used and the laying time of the mating frogs, eggs may vary by as much as 2 to 3 hours in development in a given batch. Biochemical studies have shown that histcnes are synthesized during early embryogenesis i n the sea urchin (Johnson and H n i l i c a , 1971). Kedes, et a l . , (1969) and Lindsay and tJemer (1969) used d i f f e r e n t i a l labeling with 1 * C - l y s i n e and 3H-tryptophan to detect basic proteins which are d e f i c i e n t in tryptophan, and hence are probably histones rather than ribosomal proteins (Borun, et a l . , 1967). Kedes, et a l . , (1969) have estimated that about 25% of the t o t a l protein synthesized during cleavage in sea urchins may be histones whereas we obtain a figure of 12% cleavage i n Xenogus . Further proof that histones are newly synthesized af t e r f e r t i l i z a t i o n i n sea urchin has been obtained by Hoav and Nemer (1971). These workers studied the function of the '^-polysomes" that make up 73% of the polysomes i n the 200 c e l l b l a stula stage. Characterization of nascent proteins on these l i g h t polysomes by electrophoresis and by chromatography on amberlite showed them to be histones. Our observations on Xenopus laevis embryos indicate that there are no q u a l i t a t i v e changes i n the synthesis of the major classes of histones during early development in this amphibian. These newly synthesized histones appear to be the same as histones present in adult tissues of Xenopus l a e v i s as well as histones p u r i f i e d from other organisms, as shown by electrophoresis and amino acid analysis. Recently Claycomb and V i l l e e (1972) have isolated proteins from Xenojaus laevis embryos 49 that bind to a c a l f thymus DMA-cellulose column. They have shown that there i s synthesis of proteins in the molecular weight range for histones during cleavage and blastula stages, a finding that agrees with our data. 50 Part 2. Nuclear Accumulation Of Newly Synthesized Histones During Early Development Of Xenopus Laevis. « 51 INTRODUCTION PART II The previous work described here (Thesis Part 1; Byrd and Kasinsky, 1973; Kasinsky and Byrd, 1972) demonstrated that a l l classes of histones were synthesized throughout development and that there were no q u a l i t a t i v e changes in the types of histones synthesized. These histones appeared to be similar to those present i n swimming tadpoles as well as i n adult tissues of Xenopus. Destree, et a l . , 1972 have succeeded i n demonstrating the presence of histones in nuclear preparations of Xenopus at early stages of development, i n accord with our own findings on histone synthesis during t h i s period. However, a discrepancy arises between the findings in Xeno^us and the work of Asao (1969,1970,1972) on the Japanese newt Tr i t u r u s . Asao has not been able to detect either histone synthesis or nuclear accumulation by chromatographic means in the pre-gastrula newt embryo. To demonstrate that newly synthesized histones are accumulated i n the pre-gastrula nucleus, we have extracted r e -labeled histones from nuclear chromatin preparations of Xenojaus embryos. The experiments discussed in t h i s portion of the thesis indicate that these embryonic histones are s i m i l a r to those found i n adult Xenogus l i v e r , as shown by acrylamide gel electrophoresis and amino acid analysis. We have also u t i l i z e d Actinomycin D pulse labeling experiments to demonstrate that newly synthesized histones are made almost e n t i r e l y from newly synthesized RNA rather than maternal mRNA. 52 METHODS AND MATERIALS 5. MATING OF FROGS AND LABELING OF XENOPUS LAEVIS EMBRYOS l§i25£us la e v i s embryos were obtained from mating adults stimulated by p i t u i t a r y injections of hormone and were dejelled by cysteine hydrochloride as described i n Part 1, Methods and Materials. A. **C-Labeled Embryos Embryos were labeled as described i n Part 1. Batches of 500 embryos were labeled either f o r 6 hours aft e r f e r t i l i z a t i o n (stages 1-9) or for 6 hours at stage 37-38 and 6 hours at stage 40-41 for swimming tadpoles. Embryos were labeled i n small disposable tissue culture flasks (50ml) containing 100^Ci of Na 1 4C03 (spe c i f i c a c t i v i t y ca. 50 rnCi/mmol, Schwarz/Mann) in modified Holtfreter's s o l u t i o n . Labeling was stopped by cooling the embryos to 4° C and then nuclei were prepared immediately. B. 3 H-Lysine Labeled Embryos Embryos were prepared for labeling by p a r t i a l l y d i s s o c i a t i n g them as described by Landesman and Gross (1968) th i s method makes the embryos permeable to large molecules such as lys i n e and Actinomycin D . Kutsky (1950) has shown previously that amphibian embryos are impermeable to amino acids under normal in vivo conditions. The di s s o c i a t i o n medium was 53 made up of (4.36 gm of NaCI, 0.18 gm of KCl, 0.089 gm of Na2HPOU, 0.019 gm of KH2P04, 2.13 gm of NaHC03, 0.27 gm of Na2S04 and d i s t i l l e d water to one l i t e r ) combined with ethylene diamine t e t r a a c e t i c acid (EDTA) disodium s a l t (0.002M). Nine hundred dejelled embryos were placed i n 500 ml of th i s solution and were dissociated within 60-90 minutes at room temperature. Dissociation causes the blastomeres to lose t h e i r normal c e l l to c e l l contact; however, they are s t i l l contained within the v i t e l l i n e membrane. After d i s s o c i a t i o n the embryos were rinsed in complete medium (dissociation medium with 0.22 gm CaCl2, 0.263 gm MgCl»6water and 5 gm serum albumin, bovine). Up to 300 embryos were then placed i n 2-1 ml of complete medium in tissue culture f l a s k s as described before in Part 1. Solutions of 3H-lysi n e (specific a c t i v t i y 55 Ci/mmole, New England Nuclear) 20 £i/ml and and Actinomycin D (20t/Lg/ral, Sigma Chemical Corp.) were added d i r e c t l y to the fl a s k s and gently shaken on a Eberbach shaker i n a fume hood for 2 to 3 hours at room temperature. 6. ISOLATION OF NUCLEI A. Method I Nuclei from 1500-2000 embryos were isolated as described by Arms, 1970. These embryos were homogenized in 80 ml of 0.1M sucrose-0.003M CaC12 i n a glass Bounce homogenizer and centrifuged at 600 g for 5 minutes . The r e s u l t i n g p e l l e t was resuspended three times and washed with 80 ml of 0.25M sucrose-54 0.001M MgCl2 at 600g for 5 minutes. after centrifugation, the pe l l e t was resuspended i n 0.5 ml of 0.25M sucrose-0.001H MgC12, layered over a discontinuous sucrose gradient of 2.4M , 2.0M, 1.7M, 1.2M, and 0.8M sucrose containing 0.001M MgC12. Nuclei were separated by centrifugation at 150,000 g for 75 minutes in a Spinco SW 50.1 rotor. B . Method I I Nuclei from embryos and adult l i v e r were isol a t e d using the method developed by Destree, et a l . , 1972. Tissue (15 gm) or embryos (2000) were homogenized d i r e c t l y i n 160 ml of 2.4 M sucrose, 3mM CaCl2, 5mM Tris-HCl, pH 8.0 containing 0.5% Triton X-100 and 0.05M sodium metabisulfite. The homogenate was centrifuged for 4 hours at 108,000 g i n a SW 27 Spinco rotor and the nuclei were pelleted. 7. I S O L A T I O N O F C H R O M A T I N Chromatin from embryo and adult l i v e r nuclei was obtained using a modified method of Shaw and Huang , 1970. Nuclear p e l l e t s from 30 gm of wet tissue or 6000 embryos were suspended in 40 ml of 0.075M NaCl-0.024M EDTA, pH 8.0 and centrifuged at 7700 g for 15 minutes. This step was repeated three more times. The r e s u l t i n g p e l l e t was suspended in 40 ml of 0.005M T r i s , pH 8.0 and centrifuged at 7700 g for 10 minutes. This wash was repeated once and followed by two washes of 0.01M T r i s , 0.002M T r i s , and 0.0004M T r i s , pH 8.0. The chromatin from the nuclear 55 p e l l e t was then allowed to swell overnight in 100 ml of d i s t i l l e d water, pH 8.0. 8. EXTRACTION OF HISTONES Histones were extracted from chromatin solutions by adding 2N H2SOU to a f i n a l concentration of O.UN H2S04. The solution was then vortexed, homogenized in a Bounce glass homogenizer and s t i r r e d at 4° C for one hour. The solution was then centrifuged at 10,000 g for 30 minutes, the p e l l e t was resuspended i n 0.4N H2S04 and s t i r r e d again for one hour and centrifuged as before. The supernantants were collected at -20° CC. Absolute ethanol was added to a f i n a l volume of 3:1 , ethanol/supernantant. After the histone-sulfate complex had precipitated overnight, i t was collected by centrifugation at 10,000 g for 30 minutes and vacuum dried. 9. CHROMATOGRAPHY A. Carboxymethylcellulose Histone f r a c t i o n s were p u r i f i e d on CM-cellulose (Whatman CM-32) prepared as described i n Part 1 methods. The histones were dissolved i n 1 ml of urea buffer (8M urea, 0.001M d i t h i o t h r e i t i o l , 0.05M sodium acetate, pH 5.5) and washed into a 1X25 cm column with 100 ml of buffer to elute non-adhering 56 proteins. The histones were eluted with a stepwise gradient of 0.3M NaCl in urea buffer. The histone f r a c t i o n was then dialyzed against d i s t i l l e d water and lyophylized. B. Amberlite Chromatography Amberlite resin was prepared by the method of luck, et a l . , (1958) as described in Part 1. After e q u i l i b r a t i o n , the resin was suspended in 8% (w/v) guanidine hydrochloride in 0.1H sodium phosphate buffer, pH 6.8. Columns were packed with a i r pressure. A l i n e a r gradient of 8-14% guanidine hydrochloride was used to separate histones from each other. After the gradient was completed 40% guanidine hydrochloride was added to elute the f i n a l adhering protein. 10. PAPER ELECTROPHORESIS OF LABELED HISTONES Cleavage embryos were labeled as described in methods. One mg of **C labeled whole histone was digested in one ml of 6N HC1 for 24 hours in vacuo at 120° C. An aliquot (50^) was applied to a sheet of Whatman No. 3MM paper for high voltage electrophoresis at pH 6.5 i n pyridine/acetic acid/water (3:1:1) at 62 volts/cm for 30 minutes i n a varsol cooled apparatus. Known marker amino acids were applied as standards. After electrophoresis the paper was dried. The radioactive amino acids were detected by cutting 1cm s l i c e s of the paper and counting them in toluene base s c i n t i l l a t i o n f l u i d . A duplicate run on the same paper was stained n 1% nihydrin/acetone to 57 i d e n t i f y the labeled spots. 11. FRACTIONATION OF HISTONES Lysine r i c h histone I was isol a t e d from Xengpus embryos and adult tissue by extraction of chromatin with 5% TCA, a procedure that s o l u b i l i z e s the l y s i n e - r i c h histone I (DeNooij and Westenbrink, 1962). A. Adult Liver Histones IIb1 and IIb2 i s o l a t e d by Amberlite chromatography were separated from each other using the procedure of Palau and Butler (1966) by s e l e c t i v e extraction with organic solvents. Histones III and IV were isolated by Amberlite chromatography and were rechromatographed on Biogel P-60 ( i t was prepared as described by the Biorad Laboratory catalogue). Fractions from Amberlite were dialyzed against 0.1M g l a c i a l acetic acid, lyophilyzed and redissolved in 0.01N HCl. After the histones were dissolved i n 1ml 0.01N HCl, they were washed into a 1X170cm column and eluted with 0.01 N HCl. Fractions of 6 ml were colle c t e d at a flow rate of 1 ml/hr. This chromatography separated histones III from IV. 58 B. Embryonic Histones Embryonic histones were fractionated on Amberlite columns as described in Part 1 Methods. 12. ELECTROPHORESIS Histones were analyzed on polyacrylamide gels according to the method of Panyim and Chalkley (1969). This i s a modification of the procedure of Bonner, et a l . , (1968) described in Part 1 Methods. The difference i s that the TEMED i s mixed with 43.2% acetic acid rather than 48 ml of KOH (IN) and 17.2 ml of acetic acid as described by Bonner, et a l . , 1968. Both solutions are diluted to 100 ml with d i s t i l l e d water. Samples (100 g) were applied to 15% acrylamide gels (0.5X7.5)cm containing 6.25 M urea, pH 3.2. Samples were incubated in 0.1M d i t h i o t h r e i t o l for one hour at 37° C. Electrophoresis was carried out at 1.75 mA/gel for 4 hours at room temperature a f t e r pre-electrophoresis to remove persulfate. Gels were stained in 0.1% amido black in 0.9N acetic acid and 20% ethanol. The destained gels were scanned at 660 nm i n a G i l f o r d model 2400 spectrophotometer. Radioactive assays were done as described in Methods Part 1. 59 1 3 . AMINO ACID ANALYSIS Samples of the various histones were hydrolyzed i n vacuo at 1 1 0 ° C for 24 hours i n 6N HCl. After evaporation to dryness, the hydrolysates were dissolved in d i s t i l l e d water and the i r amino acid composition determined on a Technicon amino acid analyser. 60 RESULTS Nuclear accumulation of newly synthesized histones was examined i n pre- and post-gastrula embryos. Nuclei were iso l a t e d from Xenopus laevis embryos using two d i f f e r e n t procedures (Arms, 1970; Destree, et a l . , 1972). The acid soluble histones extracted by both procedures were highly contaminated by other basic proteins and further p u r i f i c a t i o n was necessary. The chromatin preparation of Shaw and Huang (1970) yielded a soluble histone preparation, from embryonic nuclei prepared by the method of Destree, et a l . , (1972) when followed by chromatography of the acid-soluble histones on CM-c e l l u l o s e urea columns as described in methods and materials. P u r i f i e d histone f r a c t i o n s were run on polyacrylamide gels according to the method of Panyim and Chalkley, 1969. These gels, shown in Figure 10, indicate that a l l the major classes of histones were present i n nuclei from both pre- and post-gastrula embryos and were similar to those found i n adult somatic tissue. The major classes of histones in nuclei of the embryos were newly synthesized, as can be seen by the incorporation of radioactive l 4C02 into the histone bands. In order to distinguish between histone synthesis and side chain modification of pre-existing histones, such as acetylation (Candido and Dixon, 1972), **C labeled histones were acid hydrolyzed and the resulting amino acids subjected to paper electrophoresis. Figure 11 indicates that the 1*C02 label was actually being incorporated into the amino acid residues of the 61 Figure 10. Disc Electrophoresis Of Histones From Xenopus Embryos And Adult Liver On 15% Acrylamide Gels According To The Method Of Panyim And Chalkley, 1969. Acid-soluble histones were electrophoresed on 15% acrylamide gels made 6.25M with urea (Panyim and Chalkley, 1969). The final pH before electrophoresis was 3.2. Gels were pre-electrophoresed for 3.5 hours to remove persulfate. Samples of **C-labeled histone were incubated in 0.1M dithiothrietiol, 0.9N glacial acetic acid, 10M urea for 1 hour at 37° CC. Proteins (100^g samples) were run at 1.75 mA/gel for four hours and stained in 0.1% amido black in 0.9N acetic acid and 20% ethanol. Gels were destained in a diffusion destainer and scanned at 660nm (solid line) in a Gilford model 2400 spectrophotometer. Radioactive assays (dotted line) were performed by incubating 0.5mm gel slices in 0.1 ml of hydrogen peroxide (30%) at 50° CC for 1 hour. After incubation Aquasol (New England Nuclear Corp.) was added and counting was performed on a Onilux II-A scintillation counter. (A) adult liver (B) stage 42 swimming embryos (C) cleavage embryos 61a cm 62 Figure 11. Paper Electrophoresis Of an Amino Acid Hydrolysate From **C-Labeled Histones Of Cleavage Xenopus Embryos. Cleavage embryos were labeled as described in Methods and Materials. A one mg sample of **C-labeled whole histone was digested in 6H HCl for 24 hours in vacuo. An aliquot (50^ g) was applied to a sheet of Whatman 3MM f i l t e r paper for high voltage electrophoresis at pH 6.5 for 30 minutes at 3500 volts. Known marker amino acids were applied as standards. One-half of the sheet was stained in 1% nihydrin/acetone and the other half was dried. The radioactive amino acids in the dried half were detected by cutting 1 cm slices of the dried paper and counting the slices in toluene scintillation fluid. (A) . Glutamic acid and lysine. (B) . Aspartic acid and arginine. (C) . Methionine and phenylalanine. (D) . Hydrolyzed histones from nuclei of cleavage embryos. orange g t asp B C D ©< Y '00. a. 7 5 . 50. 25. z! 8! ' 1 5 ' ' ' ' To ' '"' ' I ' ' 1 1 1 DISTANCE me thy! green lys arg met, phe I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 — 5 10 15 20 c m ) QJ 63 newly synthesized histones. A considerable portion of the **C02 l a b e l entered the glutamic and aspartic acid residues, as had been observed by Cohen (1954). Some label also appeared in the basic amino acid residues. Neutral amino acids were not separated by t h i s method, although **C label could also be found i n the amino acid mixture near the o r i g i n . Thus, the appearance of label i n a variety of amino acid residues indicated that we were observing the biosynthesis of histones, not merely histones whose side chains had been modified by 1*C-labeled acetate groups. To determine i f these newly synthesized histones present in embryonic nuclei are sim i l a r in amino acid composition to those in adult somatic tissue, the acid-soluble histones from adult and embryonic sources were fractionated as described i n Methods and Materials and amino acid analysis -performed on these f r a c t i o n s . Calf thymus histones were used as a standard for comparison with Xenopus proteins. The res u l t s of the amino acid analyses are shown in Tables 8 and 9. In Table 8 res u l t s for histones from nuclei of adult Xenopus l i v e r have been compared to values obtained for c a l f thymus (Johns, 1971) . Besides t h e i r b a s i c i t y , which i s exceeded only by the protamines, each histone f r a c t i o n has pa r t i c u l a r c h a r a c t e r i s t i c s that i d e n t i f y i t . Lysine-rich histone I i n both c a l f thymus and Xenopus l i v e r had a high content of lysine, alanine and proline with l i t t l e h i s t i d i n e . The s l i g h t l y l y s i n e -r i c h histones Ilb1 and IIb2 in c a l f thymus and Xenopus laevis which showed a high leucine and serine content respectively, 64 Table 8. amino Acid Composition of Histones From Xenopus Liver Nuclei And Calf Thymus From Johns, 1971. \ C a l f Thyir. Anino I I l b l a c i d s l y s 26.8 10.2 h i s t r a c e 3.1 ar g 1.3 9.4 asp 2.5 6.6 t h r 5.6 3.9 s e r 5.6 3.4 g l u 3.7 9.8 pro 9.2 4.1 g i y 7.2 10.8 a l a 24.3 12.9 v a l 5.4 6.3 cys/2 0.0 t r a c e met 0.0 t r a c e i l e 1.5 3.9 l e u 4.5 12.4 t y r 0.9 2.2-phe 0.9 0.9 B/A 4.6 1.4 H i s t o n e s (Hoi %) IIb2 I I I 14.1 9.0 10.2 2.3 1.7 2.2 6.9 13.0 12.8 5.0 4.2 5.2 6.4 6.8 6.3 10.4 3.6 2.2 3.7 11.5 6.9 4.9 4.6 1.5 5.9 5.4 14.9 10.8 13.3 7.7 7.5 4.4 8.2 t r a c e 1.0 t r a c e 1.5 1.1 1.0 5.1 5.3 5.7 4.9 9.1 8.2 4.0 2.2 3.S 1.6 3.1 2.1 1.7 1.6 2.2 Xer.orros l i v e r h i s t o n e s (Mol %) I I l b l I l b Z I I I IV 24.0 14.0 17.0 7.7 13.4 0.5 2.2 2.4 1.6 2.2 1.7 9.2 9.1 14.0 13.2 2.S 5.2 6.0 5.6 6.2 3.4 4-1 6.3 • 7.6 4.7 8.6 6.5 7.9 5.8 2.7 5.5 7.9 7.5. 10.3 6.4 10.3 4.4 4-5 3.8 2.6 4.8 9.0 8.7 7.3 12.0 23.4 10.2 10.6 11.0 7.1 6.5 7.2 5.5 5.3 6.5 - - 0.5 -t r a c e 5.1 2.8 2.0 1.2 1.1 4.8 4.6 5.9 5.9 5.3 9.8 5.7 9.2 8.6 1.0 2.0 3.3 2.7 3.9 0.9 2.7 1.6 4.0 3.6 3.2 1.5 2.1 . 2.0 2.3 cn 65 Table 9 . Amino Acid Composition o f Histones From Nuclei Of Late Blastula Xenopus Embryos. 65a A m i n o a c i d s l y s his a r g a s p t h r - s e r g l u p r o c y s / 2 g i y a l a v a l m e t i l e l e u t y r p h e B / A I 1 9 . 0 0 . 3 3 . 1 5 . 9 2 . 6 1 6 . 0 2 . 8 1 0 . 9 4 . 1 1 0 . 5 4 . 9 t r a c e 0 . 8 3 . 1 0 . 6 0 . 4 2 . 6 I I b l + I I b 2 1 7 . 0 2 . 5 7 . 4 7 . 7 3 . 5 1 2 . 9 7 . 6 7 . 1 6 . 9 9 . 6 3 . 8 2 . 1 4 . 6 5 . 8 2 . 6 1 . 6 1 . 8 I I I + I V 8 . 0 2 . 5 1 1 . 0 5 . 1 5 . 2 4 . 5 1 4 . 1 5 . 0 0 . 4 8 . 6 8 . 4 5 . 2 2 , 5 3 . 5 7 . 2 1 . 7 4 . 1 0 . 8 66 although the value for serine in Xenopus la e v i s histones IIb2 was somewhat low. The arginine-rich histones III and IV were also s i m i l a r in c a l f thymus and Xenopus l i v e r . Histone III from both sources contained cysteine, a c h a r a c t e r i s t i c feature of t h i s histone, while histone IV had a high glycine l e v e l . On the basis of these data, we can see that Xenopus l i v e r histones are si m i l a r to those from c a l f thymus. Table 9 represents the amino acid analysis for Xenopus blastula histones obtained from nuclei. The amino acid composition of histones in blastula embryos can be compared to those in adult l i v e r by averaging the values for Ilb1 and IIb2 in Table 8. The arginine-rich histones III and IV can also be compared by t h i s method. It i s apparent from Tables 8 and 9 that the amino acid composition of the s l i g h t l y l y s i n e - r i c h histones and the arginine-rich histones are similar i n nuclei from late blastula embryos and adult l i v e r . However, seme major differences in amino acid composition should be noted. One major discrepancy i s the high serine value and low alanine i n histone I of the blastula embryo. The other major difference can be seen i n the lower r a t i o of basic to a c i d i c amino acids in the blastula histones. These differences probably arise from contamination of the histone with phospholipids or nonhistone proteins during their i s o l a t i o n from yolk laden embryos. Destree, et a l . , (1973) have found that chromatin preparations from blastula or gastrula Xenopus embryos may be contaminated with other proteins as shown by the i r absorption spectra when compared to neurula or erythrocyte chromatin preparations. When 67 the data of Table 9 are compared with the amino acid compositions of swimming tadpole histones obtained from whole homogenates (see Table 7, Part 1) i t i s apparent that there i s a good agreement. Thus, sim i l a r histone fractions can be p u r i f i e d from Xenop_us embryos either from whole homogenates or d i r e c t l y from nuclear chromatin. F i n a l l y , the synthesis of histones from RNA template prior to gastrulation was examined in order to distinguish the role of maternal and newly synthesized mRNS. In these experiments, histone synthesis in cleaving embryos was studied i n the presence and absence of Actinomycin D, a drug that i n h i b i t s RNA synthesis. As the embryos are permeable to neither t h i s drug nor to amino acids, they must be p a r t i a l l y dissociated i n order to examine the incorporation of 3H-lysine into newly synthesized histones. A technique for dis s o c i a t i n g Xjgjjojjus embryos involves the incubation of embryos in modified Steam's medium ccntaininq 0.002M EDTA, as described by Landesman and Gross, 1968. After d i s s o c i a t i o n , the c e l l s are contained within the v i t e l l i n e membrane but they do not have normal c e l l to c e l l contact for several hours. Under these conditions the embryos become highly permeable both to labeled amino acids and to Actinomycin C Although t h i s treatment i s a rather harsh , i t does allow detectable d i f f e r e n t i a t i o n and histogenesis (Landesman and Gross, 1969; Abe and Yamana, 1971). Si b l i n g embryos (stages 5-6) were divided into two batches of 900 each and incubated with 3H-lysine ( s p e c i f i c a c t i v i t y 55 Ci/ramole) at a concentration of 20 Ci/ml for 3 hours. One group 68 had Actinomycin D added to a f i n a l concentration of 20^g/ml to stop s i g n i f i c a n t RNA synthesis (Landesman and Gross, 1969). Abe and Yamada (1971) found that Actinomycin D l e v e l s as low as 0.4 ^g/ml inhibited a l l RNA synthesis. Figures 12 and 13 show the elution patterns of histones extracted from these labeled embryos. There was incorporation of label into a l l major classes of histones. When Actinomycin D was added to early blastulae at stage 5 to 6 and the embryos are labeled for three hours, there was a 20 f o l d decrease i n the amount of synthesis as compared to that observed in the controls. This observation suggests that prior to gastrulation, from stages 5 to 6, histones are translated almost e n t i r e l y off newly synthesized mRNA, thus maternal RNA probably plays a small r o l e , i f any, in the synthesis of histones during t h i s period of development. 69 Figure 12. amberlite Chromatography Of 3H-Labeled Histories Synthesized By Cleaving Xenopus Embryos (stages 5-8) In The Presence Of Actinomycin D. After partial dissociation of the blastomeres within the vi t e l l i n e membrane, 900 cleaving embryos were incubated in complete Stearn»s medium as described by Landesman and Gross, 1968. Actinomycin D (20^g/ml) and 3H-lysine (20^Ci/ml; specific activity 55 Ci/mmole; New England Nuclear) were added to the incubation medium. Histones were prepared as described in methods and materials. Basic proteins from 900 embryos were applied to a 0.3 X 20 cm column of Amberlite and eluted with a 16 ml linear gradient of 8-11% guanidine hydrochloride in 0.1M phosphate buffer^pH 6.8. This gradient was followed by a f i n a l wash of 40% guanidine hydrochloride to elute the arginine-rich histones. Fractions of 4 drops were collected from a column over a 48 hour period and radioactive assays were performed using the f i l t e r disc technique of Bollum, 1968. 69a F R A C T I O N N O . 70 Figure 13. amberlite Chromatography Of 3H-Lysine Histones Synthesized By Control Embryos. Conditions were the same as described in figure 12 except that Actinomycin D was not added in this experiment. 70 71 D I S C U S S I O N The preceding experiments were addressed to the question: are histones synthesized and incorporated into the chromatin prior to gastrulation in the amphibian? And i f so, are each of the major classes of histones present i n the nucleus i n the early embryo and are they similar to those found i n l a t e r stages of development or in adult somatic tissue? The work described in Part 1 revealed that the 5 major classes of histones are synthesized in t o t a l c e l l homogenates i n Xenopus embryos throughout early development. From that observation we have assumed that histones are synthesized i n the cytoplasm and then transported to the nucleus where they are incorporated into chromatin. However there i s disagreement i n the l i t e r a t u r e concerning the time lag of transport of newly synthesized histones from the cytoplasm to the nucleus. Pulse-chase experiments on the i n t r a c e l l u l a r transport of protamine, a s p e c i a l class of histones extremely enriched in arginine, shows that protamine i s transported to the nucleus i n trout t e s t i s within 1-2 minutes of synthesis ( l i n g , et a l . , 1969). However Johnson and H n i l i c a (1971) present evidence that histones are synthesized during early cleavage i n sea urchins but may be held in the cytoplasm for several c e l l cycles before becoming incorporated into chromatin. Thus, i n our previous work u t i l i z i n g t o t a l homogenates, we may have been observing cytoplasmic histone synthesis but not nuclear accumulation. The present study indicates that t h i s i s not the case; newly synthesized histones are incorporated into nuclear chromatin. 72 although the rate of incorporation from possible cytoplasmic pools remains to be examined. There i s also disagreement i n the l i t e r a t u r e concerning the presence of histones in the nuclei of pre-gastrula amphibian embryos. Asao (1969,1972) has reported an absence of histones prior to gastrulation from the nuclei of the Japanese newt. Destree, et a l . , (1972), have demonstrated that histones are present i n the nuclei of pre-gastrula Xenopus embryos; however, they did not show whether these histones were newly synthesised. The experiments reported here indicate that dividing cleavage nuclei take up radioactive l a b e l into each of the major classes (Figure 10) and that these histones are s i m i l a r to those in l a t e r stages of development as well as i n an adult somatic tis s u e . Since new histones would be required for rapidly dividing tissues (Stellwagen and Cole, 1969), these r e s u l t s support our o r i g i n a l hypothesis that histones would be synthesized prior to gastrulation during t h i s period of rapid DNA synthesis and c e l l d i v i s i o n . We could not examine histone synthesis in the very early stages of cleavage (stages 1-4, Nieukoop and Faber, 1956) because the time lapse from f e r t i l i z a t i o n to early blastula i s approximately 3 hours whereas the method used to detect synthesis requires a longer incubation period. Secondly, egg laying by the females during mating occurs over a prolonged period, r e s u l t i n g in a population of eggs d i f f e r i n g from each other by a few hours in age. If there are any unusual basic proteins accumulated i n the nucleus during the f i r s t few cycles 73 a f t e r f e r t i l i z a t i o n , our experimental protocol would net detect them. Asao (1969,1972) could not detect the presence of histones in the newt embryo prior to early gastrulation. Arginine-rich histones were the f i r s t to appear followed by the other classes as development proceeded. Re obtained these data by chromatography of labeled acid-soluble proteins on Amberlite. The discrepancy between these r e s u l t s and our data and those of Destree, et' a l . , (1972) may ari s e from technical d i f f i c u l t i e s of i s o l a t i n g uncontaminated nuclei from early yolk-laden embryos. Our e a r l i e r work and that of Destree, et a l . , (1972) shewed that extensive care must be taken to remove l i p i d and phospholipid contaminants in histone preparations from cleavage embryos. In these procedures sodium metabisulfite i s used to i n h i b i t proteolysis i n the chromatin preparations and Triton X-100 to y i e l d cleaner nuclear preparations free from cytoplasmic contaminants. Tata, et a l . , 1972, showed that there was a marked drop in the phospholipids of rat l i v e r nuclei after treatment with the detergent Triton X-100. Secondly, Asao (1969) noted the appearance of a s p e c i f i c class of histones from pre-gastrula embryos, the arginine-rich histones, that eluted i n the H0% guanidine hydrochloride gradient. This may r e s u l t from contamination of t h i s elution peak with other basic proteins, an a r t i f a c t reported by Stellwagen and Cole, (1969), rather than the actual presence of histones. Therefore, the discrepancy between our findings and those of Asac may the result of yolk contamination i n pre-gastrula embryos rendering Asao's histone 74 f r a c t i o n s insoluble prior to gastrulation. The twenty f o l d decrease i n the amount of histone synthesis in the presence of actinomycin D implies that histones are made almost e n t i r e l y from newly synthesized mRNA ater stages 5 to 6. The 5% residual incorporation i n the presence of actinomycin D could be the result of incomplete i n h i b i t i o n or to translation of mRNA transcribed i n the embryo prior to addition of actinomycin D. However, abe and Yamana (1971) have shown that a concentration as low as 0.4^g/ml of Actinomycin D w i l l i n h i b i t a l l classes of measurable ana synthesis i n Xenopus embryos using the same conditions described here. Further, Davidson, et a l . , 1968, have shown that there i s not a s i g n i f i c a n t production of newly synthesized RNA i n Xenopus before mid-blastula. This would imply that the proteins synthesized are translated from maternal mRNA. This residual synthesis may also be due to the trans l a t i o n of maternal mRNA synthesized during oogenesis. However, the o v e r a l l e f f e c t s of Actinomycin D must also be considered before t h i s intrepretation can be made. There are three major r e s u l t s that occur when sea urchins are treated with high lev e l s (H^g/ml) of Actinomycin D at f e r t i l i z a t i o n (Gross and Cousineau, 1964): Actinomycin D powerfully i n h i b i t s RNA synthesis; c e l l d i v i s i o n proceeds, although with some slowing in rate ; while v i s i b l e d i f f e r e n t i a t i o n and amino acid incorporation into protein are unchanged. After f e r t i l i z a t i o n differences appear i n the sedimentation patterns of polyribosomes of normal and Actinomycin D treated sea urchin embryos which r e f l e c t changes in the amounts of newly 75 synthesized and maternal templates avaible for t r a n s l a t i o n . Kedes and Gross (1969) have shown that by early cleavage and swimming blastula Actinomycin D treated embryos are able to assemble between 50-60% of, the polyribosomes accumulated by untreated animals. Other workers (Infante and Werner, 1967) found that continuous exposure of sea urchin embryos to Actinomycin D (25 y^g/ml) does not block formation of polyribosomes before and after f e r t i l i z a t i o n . This re s u l t i s consistent with the idea that protein synthesis i s independent of new RNA synthesis before and after f e r t i l i z a t i o n . After t h i s early period the formation of the s-polysome region, that transcribes the mRNA of histones, i s i n h i b i t e d by Actinomycin D. Therefore the generation of these polysomes i s dependent in part upon the synthesis of new RNA. But actual protein synthesis by pre-existing RNA i s not d i r e c t l y i n t e r f e r r e d with by Actinomycin D. The work of Crippa and Gross (1968) on Xenopus embryos has shown that at the beginning of gastrulation maternal mRNA s t i l l represents a s i g n i f i c a n t amount of the t o t a l RNA present. Our data from Amberlite chromatography of Actinomycin D treated embryos means that newly translated histones are made almost e n t i r e l y off newly synthesized mRNA after stages 5 to 6. This indicates that i f maternal RNA i s s t i l l present at t h i s stage, i t plays a limited role, i f any, i n the synthesis of histones. The only developmental system in which the synthesis of histones and i t s relati o n s h i p to maternal mRNA has been extensively studied i s the sea urchin embryo. Although there i s some disagreement among investigators, i t i s apparent that the 76 spectrum of proteins synthesized during early sea urchin embryogenesis i s mainly independent of embryonic gene function much as i n the amphibian case (Gross,1969). Kedes et a l . , 1969, have shown the biosynthesis of acid soluble nuclear proteins in cleaving sea urchin embryos. These proteins have a high lysine to tryptophan r a t i o which would suggest that these newly synthesized proteins are histones. Actinomycin B in h i b i t e d the accumulation of small polysomes and blocked most of the histone-l i k e protein synthesis, suggesting that most of these histones are made from maternal mRNA. Johnson and Hn i l i c a (1971) showed that sea urchin blastulae raised in the continuous presence of Actinomycin D contain nearly the same histone pattern as controls raised without the drug, demonstrating that histones are translated during early develcpement from maternal mRNA. Similar r e s u l t s have also been observed i n sea urchins by Crane and V i l l e e (1971) who fi n d that there i s a reduction of the synthesis of l y s i n e - r i c h histone in the presence of Actinomycin D. Moav and Nemer (1971) have concluded that the s-polysomes are the s i t e of histone synthesis, but in contrast to the previous workers they f e e l that t h i s synthesis i s promoted almost e n t i r e l y by newly synthesized mRNA. In the case of Xenopus development, our findings show that blastulae synthesize the bulk of histones from newly synthesized mRNA. On the whole, these observations on histone synthesis and the role of maternal template i n pre- and post-gastrula Xenopus development are in general agreement with many of the findings in sea urchins embryos. 77 CONCLUDING REMARKS Histone synthesis during the early embryogenesis of Xjsnopus la e v i s has been examined. Part 1 of t h i s thesis has shown that there are no q u a l i t a t i v e changes i n the synthesis of the major classes of histones during early development i n Xenopus l a e v i s embryos. These newly synthesized histones represent a l l 5 major classes and appear to be the same as histones present in adult somatic tissue as well as histones p u r i f i e d from other organisms, as shown by electrophoresis and amino acid analysis. Part 2 of t h i s thesis has demonstrated that these newly synthesized histones are accumulated in the nucleus both pre-and post-gastrula. The incorporation of i*C02 into newly synthesized nuclear histones has been examined. It was found that the 1 * C - l a b e l was incorporated into the histones, not merely acetylation of basic side chains with **C-acetate. It has also been shown that a l l classes of histones were synthesized almost e n t i r e l y from newly synthesized messenger prior to gastrulation. This work corroborates much of the evidence that has been gathered on histone synthesis i n sea urchin embryos, the only other developmental system i n which synthesis of these basic nuclear proteins has been examined. Recent experiments by Cohen, et a l . , (1973) have shown that a l l f i v e histone classes are found i n cleaving sea urchin embryos (16-32 c e l l stage). Also the r a t i o of the t o t a l amount of histone to DNA remains approximately constant from cleavage to prism stages with some variations i n the subcomponents of histone I and l i b . E a r l i e r 78 work by Kedes, et a l . , (1969), Johnson and Hnilica (1972), Crane and V i l l e e (1972), and Moav and Nemer (1971) showed that histones were synthesized during the early period of development in sea urchins. These observations can also be extended to the other invertebrate embryos. Cohen, et a l . , (1973) have evidence to prove that a l l major classes of histones are found in P.roso£hila preblastoderm embryos (0-2 hours). Thus, i t would appear that a l l classes of histones are present during cleavage in embryos from several phyla this i n turn would argue that unique classes of histones do not have a major r o l e i n s p e c i f i c gene regulation during early development. ft reasonable intrepretation of these r e s u l t s would be that the histones function mainly in a s t r u c t u r a l capacity i n chromatin. Histones may function as regulators of gene a c t i v i t y during early development may be through the presence of histone modifications rather than synthesis of q u a l i t a t i v e l y unique forms of histones (A possible exception may reside in the microheterogeneity of the l y s i n e - r i c h histone, Balhorn, et a l . , 1972). Poupko, et a l . , (1972) indicated that there may be a cor r e l a t i o n between gene a c t i v a t i o n cr phosphorylation and acetylation of histones during cleavage i n Rana. However, such modifications are now thought to a l t e r the binding of histones to DNA i n chromatin rather than regulate histone function (Dixon, 1972). There remain, however, investigated with regard d i f f e r e n t i a t i n g or developing several areas to the role systems. These s t i l l to be of histones in systems to be 79 studied are characterized by extreme changes i n l e v e l s of PNA, DNA, and protein synthesis as erythroblastosis, spermatogenesis, and cleavage in embryos. The problem examined i n the f i r s t two systems i s the metabolism of DNA-bound histone. Erythroblastosis i s characterized by an active erythroid c e l l s eries i n which very active DNA, RNA and protein synthesis occurs. These c e l l s d i f f e r e n t i a t e to form non-dividing c e l l s in which the l e v e l s of RNA and protein synthesis gradually decrease in the nucleated erythrocyte (Williams, 1971). It has been shown by Appels and Wells (1972) that ^ C - l a b e l e d amino acids are incorporated into a l l histone species, however, i n non-dividing polychromatic erythrocytes only histone V, s p e c i f i c to nucleated avian erythroctyes, was labeled. Pulse-chase experiments demonstrated that the decay of DNA-bound histone was s i m i l i a r to the rate at which i t was i n i t i a l l y incorporated. Spermatogenesis i s another d i f f e r e n t i a t i n g system to study with regard to histone synthesis and turnover. Dixon and his coworkers (see review, 1972) have extensively studied spermatogenesis in trout testes during which histcnes c h a r a c t e r i s t i c of the somatic c e l l are ultimately displaced from their t i g h t combinations with DNA. These histones are replaced with protamines, highly arginine-rich polypeptides. This replacement r e s u l t s in packing of the sperm DNA into a much tighte r configuration that t o t a l l y supresses gene expression. This work points out that, in d i f f e r e n t i a t i n g systems, the dynamic state of histone synthesis and the attachment and the release of histones from DNA may be more important than the 80 o v e r a l l l e v e l of histone synthesis. Therefore, i f the synthesis and turnover of histone bound to DNA were examined i n the frog embryo i t would help considerably i n further defining the role of histones i n chromatin. F i n a l l y , one important point to consider when examing histone synthesis i s not merely the possible functional or s t r u c t u r a l role that these nuclear proteins may have, but the relationship between histone synthesis and t o t a l protein synthesis during a p a r t i c u l a r period of development. As has been mentioned previously, the patterns and types of proteins synthesized during cleavage and l a t e r stages in the amphibian embryo remains to be resolved (Gurdon, 1967). The work done so far i n t h i s f i e l d has concentrated on the synthesis of t o t a l protein, rather than on the synthesis of s p e c i f i c classes of proteins (Ecker and Smith, 1971; Malacinski, 1972). Thus the present work on histone synthesis i s the f i r s t substantial study of the synthesis of a p a r t i c u l a r c l a s s of proteins during the cleavage period of amphibian development. 81 REFERENCES Abe, H. and Yamana, K, 1971. The synthesis of 5 s RNA and i t s regulation during early embryogenesis of Xenopus l a e v i s . Biochim. Biophys. Acta.. 213:392-406. A l l f r e y , V. G. 1971. Functional and metabolic aspects of DNA associated protein, i n Histones and Nucleohistongs , Johns, E. (ed.). Plenum Press, New York. Arms, K. 1971. DNA synthesis by isolated embryonic nuclei of Xenopus. Devel. Biol,. 26:497-502. Appels, R. and Wells, J. R. E. 1972. Synthesis and turnover of DNA bound histone during maturation of avian red blood c e l l s . J . Hoi. 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