Histones from embryos of the sea urchin Arbacia lixula

DEVELOPMENTAL

BIOLOGY

Histones

35, 115-124

from

(1973)

Embryos

of the Sea Urchin

ADOLFO RUIZ-CARRILLO* Centro

de Genetica,

Section

de Biopolimeros, Accepted

Arbacia

/ix&a’

AND JAIME PALAIJ~ Au. Glmo.

June

Fmnco

999, Barcelona-14,

Spain

I, 1973

Whole histones and histone fractions of the sea urchin, Arbacia hula, embryos have been characterized by their appearance during development and by their amino acid composition. Comparison of electrophoretic mobility of the histone fractions from hatching blastula and gastrula stage embryos demonstrates the similarity of the basic proteins at these two stages. Histones P2al and F3 of hatching embryos are very similar to those of sperm, including the presence of cysteine in F2al from both sources. Both F2al and F3 display electrophoretic heterogeneity due to acetylation, not observed in the homologous sperm histones. F2a2 from embryos has different electrophoretic mobility than that from sperm, although their amino acid compositions are very similar. The relative proportion of F2a2 increases whereas that of F3 decreases during gastrulat.ion. Slightly lysine-rich histone F2b could not be recovered from embryos by the standard methods of extraction. The very lysine-rich histone Fl of late embryos is partially phosphorylated and is remarkably different from that of sperm, notably by its higher electrophoretic mobility and lower content in arginine and proline. The significance of these results is discussed with regard to the structure and activity of chromatin.

effecters of gene modulation (for a recent review see Allfrey, 1971), as yet little is known about nucleoprotein changes that occur in developing systems. Histones have been studied in the early embryogenesis of the chicken (Agrell and Christensson, 1965; Kischer et al., 1966; Kischer and Hnilica, 1967), newt (Asao, 1969), loach (Vorobyev et al., 1969), sea urchin (Repsis, 1967; Silver and Comb, 1967; Ord and Stocken, 1968; Vorobyev et al., 1969; Hnilica and Johnson, 1970; Orengo and Hnilica, 1970; Thaler et al., 1970; Benttinen and Comb, 1971; Crane and Villee, 1971; Hill et al., 1971; Easton and Chalkley, 1972), and maturing pea seedlings (Fambrough et al., 1968). These studies have provided knowledge as to the apparent lack of histones in very early stages (Ord and Stocken, 1968; Asao, 1969; Hnilica and Johnson, 1970), their changing percent composition in the nucleus at different stages (Agrell and Christensson, 1965; Marushige and Ozaki, 1967; Thaler et al., 1970; Benttinen and Comb, 1971: Hill et al., 1971), and their differential rate of synthesis (Fambrough et al., 1968; Voro-

INTRODUCTION

Fertilization initiates the activation of the quiescent male and female gametic nuclei. The activat.ion includes the triggering of the DNA synthetic machinery (Hinegardner et al., 1964), the synthesis of heterogeneous nuclear RNA detectable by the early cleavage stages (Aronson and Wilt, 1969), and the transport of messenger RNA from the nucleus into the cytoplasm (Infante and Nemer, 1967; Kedes and Gross, 1969). These developmental changes presumably require a selective activation of nuclear genes (Marushige and Ozaki, 1967; Johnson and Hnilica, 1970). The molecules responsible for such genetic control may therefore be expected to undergo molecular change. Although nuclear proteins are considered the most likely candidates for the ‘This research was supported in part by a grant. from the U.S. Public Health Service (GM - 13645). 2 Present address: The Rockefeller University, New York, New York 10021. s Present address: Instituto de Biologia Fundamental. Universidad Autonoma de Barcelona. Barcelona-13, Spain. To whom reprint requests should be addressed. 115

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BIOLOGY

byev et al., 1969, Orengo and Hnilica, 1970; Thaler et al., 1970; Crane and Villee, 1971). None of these studies has demonstrated the occurrence of qualitative changes in individual histone fractions during the course of development. Because actively differentiating embryonic tissue represent a developmental step between gametic and somatic tissues, it is of importance to know whether or not the embryonic nucleus contains unique types of histones. The present paper deals with the fractionation and chemical characterization of histones from synchronized sea urchin, A. lixula, embryos at the swimming blastula stage, and a comparison of the histones from blastula and gastrula stages. The histones of the sperm (Palau et al., 1969) and of other tissues (Ruiz-Carrillo, unpublished) of this species have already been characterized. MATERIALS

AND

METHODS

Preparation of embryos of the sea urchin A. lixula. Sea urchins were collected on the Mediterranean coast and used the same day or kept in an aquarium with flowing sea water. Fifty to sixty females were used in each experiment. The ripe gonads were excised and transferred into individual beakers filled with 250 ml of filtered sea water. Most of the eggs were shed spontaneously or within 10 min upon gentle swirling. The suspensions of eggs were filtered through a double layer of cheesecloth and allowed to sediment for 10 min. Each sediment was resuspended and sedimented three more times by the same procedure. Each batch of eggs was fertilized by addition of two drops of fresh sperm washed and diluted 1: 10 in filtered sea water. The eggs were washed twice more to remove the excess of sperm. Only batches in which more than 95% of the eggs showed fertilization membranes were used. The fertilized eggs were filtered through four layers of cheesecloth and pooled into a 50-l cylindrical container, and filtered sea

VOLUME

35,1973

water was then added up to a final volume of 40 1. The suspension was stirred gently at 21-23°C. Embryos were allowed to develop to swimming blastula (12-13 hr) and to advanced gastrula (21-23 hr). In every experiment an aliquot was allowed to go to prism stage in order to check that development was normal. Isolation of nuclei from A. lixula embryos. All the operations were carried out at 4°C. Embryos were harvested at the desired stage of development by centrifugation at 450 g for 5 min. Pellets were resuspended in 8 vol of 1.5 M glucose, and the suspension was centrifuged at 500 g for 5 min (Hinegardner, 1962). This washing was repeated twice more. The last pellets were homogenized in 8 vol of 0.25 M sucrose-O.003 M CaCl,-0.005 M NaHSO,, pH 7.0, with an MSE tissue blender for 10 set at high speed. The homogenate was centrifuged at 1100 g for 10 min. The sediments were homogenized twice more by the same procedure. The crude nuclear pellets were washed twice with 8 vol of 0.01 A4 trisodium citrate-O. 14 M NaCl-0.005 M NaHSO,, pH 7.0, twice with 8 vol of 0.1 M Tris, pH 7.6 (Wang, 1962; Murray, 1965), and twice with 8 vol of 0.14 M NaCl. Isolation of histones from A. lixula embryos.’ Whole histones were obtained by extraction of the washed nuclear pellets with five vol of 0.25 N HCl, stirring for 2 hr at 4”. Histone fraction Fl was extracted from the washed nuclear pellet with 5 vol of 5% HClO, as described by Johns (1964). Histones F2a and F3 were extracted from the residue of the Fl extraction by addition of 5 vol of ethanol followed by two extractions with 1.25 N HCl-ethanol (1:4, v/v), stirring for 1 hr each time. The combined extracts were dialyzed against ethanol, and fractions F3 and F2a were recovered according to Johns (1964). Fraction F2b was ‘Owing to the overall similarity of sea urchin embryonic histones to those found in mammalian somatic tissues, the nomenclature of Johns (1964) is used to describe homologous histones.

RUIZ-CARRILLO

AND PALAU

Histones

extracted from the last sediment with 5 vol of 0.25 N HCI. The residue after the extraction with acid was resuspended in 3 vol of 8 M urea-HCl, pH 1.0. After stirring for 15 min, the suspension was centrifuged for 10 min at 27,000g. The supernatant was dialyzed against a large volume of 0.25 N HCl for 18 hr, and clarified by centrifugation. Six volumes of acetone were added to the supernatant, and the precipitate formed overnight was recovered and washed three times with acetone, and finally dried under vacuum. Fraction F2a was further separated into F2al and F2a2 by acetone precipitation from a hydrochloric solution according to Phillips and Johns (1965). Fraction F3 prepared according to Johns (1964) was further purified by affinity chromatography (Ruiz-Carrillo and Allfrey, 1973).

from

Sea Urchin

Embryos

117

ysis of l-2 mg of the purest samples in 6 N HCl in sealed evacuated tubes for 18 hr at llO”, by ion-exchange chromatography (Spackman et al., 1958) in an amino acid analyzer (Unichrom, Beckman). All analyses given in Table II represent an average of 2-4 determinations. Determination of the presence of cysteine in A. lixula embryo F2al. F2a1, 0.1

pmole, was allowed to react with 0.15 pmole of N-[l-‘“C]ethylmaleimide (SA 5.4 mCi/mmole, Schwarz/Mann Inc., Orangeburg, New York). The reaction was carried out according to Riordan and Vallee (1967), as described previously (Ruiz-Carrillo and Allfrey, 1973). The extent of labeling was determined after polyacrylamide gel electrophoresis of the reacted sample. A slice of the gel containing the protein was combusted in a Packard TriIsolation of histon.es from other tissues. Carb Model 305 Sample Oxidizer (Wangh A. lixula sperm whole histone was obtained et al., 1972), with a recovery of 98%. from washed sperm by extraction with 0.25 14C-carbon dioxide radioactivity was N HCl as described by Palau et al. (1969). counted in a mixture containing 5 ml of Calf thymus whole histone was obtained ethanolamine, 9 ml of methanol, and 5 ml from the washed chromatin by extraction of scintillation liquid (1.5% PPO, 0.1% with 0.25 N HCl as described by Ruiz-Carbis-MSB in toluene). Samples were rillo and Allfrey (1973). counted in a Packard Tri-Carb Model 3375 Polyacrylamide gel electrophoresis and Scintillation Spectrometer at an efficiency quantitative analysis of histones. Histone of 62%. preparations were characterized by electroThe amount of protein put on the gel was phoretic analysis in 10 x 0.6 cm, 15% determined by the method of Lowry et al. polyacrylamide gels containing 2.5 M urea (1951) using desiccated calf thymus F2al by the method of Panyim and Chalkley as standard. The percentage of cysteine (1969), as modified by Wangh et al. (1972). was then calculated from the specific activAfter electrophoresis, the destained gels ity of the protein, assuming a molecular were scanned at 615 nm in a Beckman weight of 11,300. DU-Gilford 2000 spectrophotometer Treatment of histones with alkaline equipped with a linear transport system. phosphatase. Blastula whole histone and The areas under the peaks obtained were histone fractions Fl and F2a2 were discalculated by integration of the curves solved in 0.1 M Tris. HCl (pH 8.0) at a using a DuPont Curve Resolver. Each elec- concentration of 1 mg/ml. and 2 units of E. trophoretic band was expressed as percent coli alkaline phosphatase (Type III, Sigma of the total area of all the bands in a gel. Chem. Co., St. Louis, Missouri) were The values thus obtained were corrected added per milligram of histone. The solufor differential dye binding (Johns, 1967). tions were dialyzed against 0.1 M Tris. HCl Amino acid analysis. The amino acid (pH 8.0) at 25” for 1 hr. The reactions were composition was determined after hydrolstopped by adding 100% TCA to the solu-

118

DEVELOPMENTAL

BIOLOGY

tions to a final concentration of 18%. The precipitates were washed with 18% TCA, 0.1% HCl in acetone, and acetone. The last pellets were dried under vacuum. RESULTS

The yield of the acid-soluble proteins from chromatin of swimming blastula is two to three times higher than that of gastrula embryos. The electrophoretic patterns of both whole histones (Fig. la) show very close similarities. This result is in good agreement with that of Hill et al. (1971) during the embryogenesis of S. purpuratus. Vorobyev et al. (1969) found changing electrophoretic patterns between these two stages during the development of S. dr6bachien.G embryos. However, since the major differences are in the slow-moving components, contamination by cytoplasmic basic proteins (BZckstrSm, 1966; Thaler et al., 1970) may account for such discrepancies. Five main groups of bands are present corresponding to histone fractions Fl, F3, F’2b, F2a2, and F2al in order of increasing electrophoretic mobility (see below). Despite the similarity of band patterns the proportions of the histone fractions differ in these two stages of development. The relative amount of each fraction was determined by electronic integration of the peaks obtained by densitometric tracing of whole histone gels, shown in Fig. lb. Although the affinity for dye is different among the histone fractions as shown by Johns (1967)) it was assumed that the dye

VOLUME

binding capacity of homologous fractions was the same at these two stages of development. As it can be seen in Table 1 the relative proportion of F2a2 increases markedly, whereas that of F3 decreases during gastrulation. A similar decrease in the arginine-rich histones (F2al and F3) has been reported by Vorobyev et al. (1969) during the gastrulation of S. dr6bachiensis embryos. This change is not due to differential extent of phosphorylation since the treatment of whole histone with alkaline phosphatase does not result in a change of the relative proportions of the different histones (results not shown). The possibility that the changing proportions of F2a2 and F3 could be accounted for by differential extractability in HCl was investigated. Treatment of the chromatin residue with concentrated urea solutions at pH 1.0, after solubilization of the histones by repeated washings with HCl, resulted in a mixture of proteins in which the histones were not the main component. The electrophoretic analysis of this fraction showed that the residual histones were present in proportions very similar to those found in the HCl extract, except for Fl that was present in lower amounts. The amino acid composition of whole histone from blastula and gastrula embryos is given in Table 2. The values are very similar to those obtained by Hill et al. (1971) in S. purpuratus, by Vorobyev et al. (1969) in S. driibachierzsis, and to those found by Kischer et al. (1966) in the early embryogenesis of chicken. As compared to

TABLE PROPORTIONS Stage Sperm Swimming Gastrula

blastula

OF HISTONE

FRACTIONS

35, 1973

PROM DIFFERENT

1 STAGES OF DEVELOPMENT

OF Arbacia

hula

Fl

F2b

F2a2

F2al

F3

41.0” zt 1.4b (48.7’) 25.0 +z 0.2 (29.9) 24.5 * 1.3 (30.1)

10.5 zt 0.3 (15.6) 17.5 * 0.7 (26.2) 16.0 + 1.0 (24.8)

14.5 + 0.5 (8.2) 16.0 + 1.0 (9.1) 25.0 zt 1.2 (14.7)

11.5 * 0.8 (10.7) 15.5 zt 0.7 (14.6) 16.0 * 0.2 (15.6)

21.5 zt 1.4 (16.9) 25.5 + 0.7 (20.2) 18.0 + 1.3 (14.7)

a Mean percentage of the areas under the peaks of the respective b Standard deviation. CMean percentage corrected for differential dye binding (Johns,

whole 1967).

histone

gel scans

(see Fig.

1).

RUIZ-CARRILLO

AND

Histones

PALALl

TABLE AMINO

ACID COMPOSITION Amino

acid

OF WHOLE Whole histone”

HISTONES

from

n Swimming b Gastrula c Moles/100 d Detected

12.8’ 2.1 8.7 6.5 5.5 5.7 9.7 4.5 8.7 11.7 5.8 0.3 1.4 4.5 7.5 2.4 2.2

Whole histoneb

13.2 1.9 8.9 7.2 5.4 6.3 9.9 4.5 9.0 11.0 5.5 0.6 1.2 4.1 6.9 2.5 1.9

119

Embryos

2

AND OF HISTONE

FRACTIONS

FROM EMBRYOS Histone

Fl Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine

Sea Urchin

29.1 1.5 2.4 4.8 6.4 4.7 6.4 5.7 4.7 21.9 2.8 0.0 0.4 2.9 4.2 1.0 0.8

OF Arbacia

lixula

fractions’

F2al 9.4 1.7 10.7 6.4 5.5 3.9 8.3 1.9 13.8 8.9 7.7 0.56 0.7 5.5 8.6 3.3 3.0

F2a2

F3

11.9 2.1 9.9 5.9 4.5 6.5 8.9 4.0 10.0 11.2 5.7 0.0 0.5 3.8 10.6 2.6 1.9

8.8 1.9 12.0 4.4 6.7 5.0 11.6 4.3 7.0 12.5 4.3 1.0 1.1 4.0 10.6 2.3 2.5

blastula stage. stage. moles total amino acid. Values are not corrected for hydrolytic losses. by the specific activity of the protein after reaction with N-ethylmaleimide-L’C.

the composition of whole histone from calf thymus, sea urchin embryonic whole histone have a lower content in basic and a higher content in acidic amino acids. A comparison of the embryonic HClsoluble histones to those of sperm (Fig. la) shows striking differences in pattern and in relative proportions (Table l), primarily in the Fl, F2b, and F2a2 fractions. Fl from blastula stage isolated with 5% HClO, shows two electrophoretic components (Fig. 2) whose mobility is significantly greater than that of sea urchin sperm and calf thymus (Fig. la). This heterogeneity is due to phosphorylation as demonstrated by treatment of the protein with alkaline phosphatase. As shown in Fig. 3, the slower electrophoretic component of Fl disappears after dephosphorylation. This finding is in good agreement with those of Sherod et al. (1970) and Balhorn et al. (1972), who found that phosphorylation of Fl occur in actively dividing systems. The

phosphorylation of Fl amounts to 14% of the total Fl. As judged both by densitometry and protein recovery during extraction, the relative proportion of Fl in the blastula nucleus is about one-half the proportion in which the very lysine-rich histone is found in the sperm nucleus (Palau et al., 1969). This shift in relative amount corresponds to a change from more than two-fifths to about one-fifth of the histone moiety. The one-fifth value is close to that usually found in mammalian somatic tissues. Embryonic Fl has an amino acid composition (Table 2) which is distinctly different from that of sperm Fl (Palau et al., 1969) particularly by the lower content in arginine and proline and the higher content in acidic amino acids. The reproducibility of the electrophoretic pattern and amino acid composition of different preparations, as well as the use of histone-protease inhibitor (Panyim et al., 1968) during the isolation of nuclei argue against the possibility of this

120

DEVELOPMENTAL

BIOLOGY

VOLUME

35, 1973 F2b

0b F2b

Calf thymus

Fl

A. //;uu/a sperm .-_“~.-_-

I A //iu/u

blostula

FI

i

F2bF2a2

;

i I Fi h

A. /ixu/a

F2b F2a2

I(

I

gastruta

\

FIG. 1. A comparison of histones from calf thymus and different developmental stages of Arbacia lixula. (a) Polyacrylamide gel electrophoretic patterns of whole histone from calf thymus, A. Zixuln sperm and embryos at hlastula and gastrula stages. (b) Densitometric tracings of the corresponding whole histone gels described in (a).

histone being a degradation product of a sperm-like Fl. Orengo and Hnilica (1970) reported a comparable histone in the hatching embryos of S. purpuratus. Embryonic histones F2al and F2a2, obtained from F2a, show good electrophoretic purity (Fig. 2) whereas F3 prepared accordconing to Johns (1964) was consistently taminated by FZa2. Both fractions were

separated by affinity chromatography (Ruiz-Carrillo and Allfrey, 1973). A solution of reduced crude fraction F3 in 0.1 M sodium phosphate buffer (pH 6.0) was ppassed through a column containing chloromercuribenzoate bound to Sepharose 4B. Histone F3 was bound to the organomercurial while histone F2a2 was not retained. Histone F3 eluted with 0.5 A4

RUESCARRILLO

/

/

AND PALAU

1/ !i 1: j!

FIG. 2. Polyacrylamide gel electrophoresis of whole histone and histone fractions from Arbacia LU&I embryos at blastula stage. (a) Whole histone; (b) to (e) histone fractions Fl, F3, F2a2, and F2a1, respectively.

e3

‘-a

b

FIG. 3. Phosphorylation of histone Fl from Arbacia lixula embryos. (a) Histone Fl; (b) histone Fl after treatment with Escherichia coli alkaline phosphatase. Note the disappearance of the slow moving component of the doublet. Additional bands are due to the enzyme.

Histones

from

Sea Urchin

Embryos

121

cysteine shows a high degree of purity (Fig. 2). Histones F2al and F3 have the same electrophoretic mobilities (Figs. la and 2) and amino acid composition (Table 2) as those of sperm (Palau et al., 1969) and calf thymus. The microheterogeneity of electrophoretic bands of blastula F2al and F3 is due to different extents of acetylation of those histones (Wangh et al., 1972; RuizCarrillo and Allfrey, 1973). It is interesting to note that despite the evolutionary stability that the sequence of the arginine-rich et al., histones demonstrate (DeLange 1969a, b, 1972) F2al from sea urchin embryos contains cysteine, as detected by reaction with N-ethylmaleimide-l-l%. This amino acid was also found to be present in the F2al from A. lixula sperm (Subirana, 1971; Wangh et al., 1972), but is absent in the F2al from all nonechinoid animals studied thus far. Histone F2a2 from blastula and gastrula stages has a different electrophoretic mobility from that of sperm (Fig. la) although its amino acid composition does not differ significantly (Palau et al., 1969). The electrophoretie mobility and amino acid composition of embryonic F2a2 are closer to the homologous calf thymus histone than to that of sperm. The heterogeneity shown by this fraction (Fig. 2) is probably due to contamination with F2b, as judged by its mobility, since treatment with alkaline phosphatase does not produce any change in the relative proportion of both bands. When saline-washed chromatin was extracted with 0.25 N HCl, a fraction whose electrophoretic mobility resembles that of calf thymus F2b, but differs from that. of sperm F2b, was extracted along with the other histones (Fig. la). Nevertheless, no comparable histone could be isolated from blastula embryos by the method of fractionation employed. Since the method 1 of Johns (1964) involves an extraction of the nuclear sediment with perchloric acid, previous to the extraction of F2b, the possibility that this protein was irreversibly in-

122

DEVELOPMENTAL BIOLOGY

solubilized in the chromatin was considered. Therefore, to avoid the treatment with perchloric acid, method 2 of Johns (1964) was used. However the HCl extraction, after HCl-ethanol treatment, yielded, besides Fl, a protein fraction in trace amounts that when analyzed by electrophoresis appeared to be a mixture of all the histones, in which the F2b was not the main component. To test whether the F2b insolubility was due to complex formation with other chromatin components by electrostatic interactions, the final residue after HCl extraction in Johns’ method 1 (1964) was extracted with urea-HCl at pH 1.0. The electrophoretic pattern of the proteins thus extracted showed that they were a mixture of histones that were not fully solubilized in the previous extractions and of other nonhistone proteins. The relative percentage of the F2b in that mixture was again too low to account for all the protein of this fraction seen in the pattern of whole histone (Table 1). As pointed out by Sonnenbichler and Nobis (1970), the low pH treatment of chromatin produces a partial insolubilization of histones, therefore it is possible that owing to the acid conditions used in this work the F2b was selectively insolubilized. No attempt has been made to separate the F2b from whole histone because of the small amounts of material available. DISCUSSION

The results presented here show that embryonic histones display quantitative changes during development, and that they differ qualitatively and quantitatively from those of the male gamete nuclei. In general, the embryonic histones resemble those of mammalian somatic tissues more than they resemble sperm histones. Kischer and Hnilica (1967) reported that postgastrula embryonic chick whole histones are poorer in lysine, arginine, and alanine as compared to the histones of more developed embryos. A similar situa-

VOLUME 351973

tion was found by Asao (1969) during the development of the newt. Although we do not find such a high content in serine and glycine, embryonic sea urchin whole histones are higher in acidic and lower in basic amino acids. It is possible that the histones are solubilized associated with small amounts of serine-rich acidic proteins. The lower yield of whole histone from gastrula as compared to that of blastula stage is in agreement with the results of Marushige and Ozaki (1967) and Hill et al. (1971), who found that the ratio of nonhistone nuclear proteins to histones increases during blastulation. The arginine-rich histones, F2al and F3, show a considerable extent of acetylation whereas the homologous histones from sperm are not acetylated. This change probably reflects the high synthetic activity of the nucleus during embryonic development. The very lysine-rich histone, Fl, presents the most dramatic changes of all the embryonic histones with respect to that of sperm. This protein is presumably made shortly before blastulation (Orengo and Hnilica, 1970). In earlier stages it is present in very low proportions (Benttinen and Comb, 1971) if at all, since perchloric acid extracts proteins that are rich in serine (Orengo and Hnilica, 1970; Ruiz-Carrillo, unpublished observations). The phosphorylation of Fl is probably correlated with the high activity in DNA synthesis of the embryos (Balhorn et al., 1972). Littau et al. (1965) have suggested that the very lysine-rich histones have a tendency to form extended conformations to cross-link complexes of DNA with other histones. This view is supported by recent findings that the Fl histones are not involved in the supercoiling of the DNA in chromatin (Richards and Pardon, 1970), and that they interact with the DNA only by the amino and carboxyl terminal regions (Boublick et al., 1971). The low content in proline of blastula Fl may thus

RUIZ-CARRILLOAND PALAU

Histones

influence its structure and binding to the DNA (Bradbury et al., 1967; Olins et al., 1968) and therefore result in a less tight packing of the chromatin. It is interesting to note that is is not until just before blastulation that the structure of the chromatin becomes more diffuse (RunnstrGm, 1967) and its stainability by Hale staining and susceptibility to DNase increases (Immers et al., 1967). Furthermore, studies by Johnson and Hnilica (1970) have shown that the template availability for RNA polymerase of nuclei isolated from egg and cleavage embryos remain unaffected after trypsinization, whereas that of sperm and blastula became more available. These observations suggest that the DNA-protein interaction is much weaker after blastulation than before, and the presence of an Fl histone poor in proline and the acetylation of the argininerich histones may be involved in these events. We wish to express our thanks to J. Colom and R. Martin for skillful technical assistance. We are also grateful to Dr. L. Wangh and Dr. V. G. Allfrey for critical revision of the manuscript. A.R-C. is grateful to Dr. J. A. Subirana for the opportunity to carry out experiments in his laboratory. A.R-C. acknowledges a scholarship from the Ministerio de Education y Ciencia. REFERENCES AGRELL, I. P. S., and CHRISTENSSON,E. G. (1965). Changes of histone composition in the developing chick embryo. Nature (London) 207, 638-640. ALLFREY, V. G. (1971). In “The Histones and Nucleohistones” (D. M. P. Phillips, ed.), pp. 241-294. Plenum, London. ARONSON,A. I., and WILT, F. H. (1969). Properties of nuclear RNA in sea urchin embryos. Proc. Nat. Acad. Sci. U.S. 62, 186-193. k5A0, T. (1969). Behaviour of histones and cytoplasmic basic proteins during embryogenesis of the Japanesse newt, Triturus pyrrhogaster. Exp. Cell Res. 68, 243-252. BKCKSTR~;M, S. (1966). Distribution of basic proteins in centrifuged sea urchin eggs. Exp. Cell Res. 43, 578-582. BALHORN.R., BORDWELL,J., SELLERS,L., GRANNER,D., and CHALKLEY, R. (1972). Histone phosphorylation

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and DNA synthesis are linked in synchronous cultures of HTC cells. Biochem. Biophys. Res. Commun. 46, 1326-1333. BENT~INEN, L. C., and COMB, D. G. (1971). Early and late histones during sea urchin development. J. Mol.

Biol.

57. 355-358.

BOUBLICK, M., BRADBURY,E. M., CRANE-ROBINSON,C., and RATr’LE, H. W. (1971). Proton magnetic resonance studies of the interactions of histones Fl and F2b with DNA. Nature (London) New Biol. 229, 149-150. BRADBURY,E. M., CRANE-ROBINSON,C., GOLDMAN, H., R.UTL.E, H. W. E., and STEPHENS, R. M. (1967). Spectroscopic studies of the conformations of histones and protamines. J. Mol. Biol. 29, 507-523. CRANE,C. M., and VILLEE, C. A. (1971). The synthesis of nuclear histones in early embryogenesis. J. Biol. Chem. 246, 719-723. DELANGE, R. J., FAMBROUGH,D. M., SMITH, E. L., and BONNER,J. (1969a). Calf and pea histone IV. II. The complete amino acid sequence of calf thymus histone IV; presence of e-N-acetyllysine. J. Biol. Chem. 244, 319-334. DELANGE, R. J., FAMBROUGH,D. M., SMITH, E. L., and BONNER, J. (1969b). Calf and pea histone IV. III. Complete amino acid sequence of pea seedling histone IV; Comparison with the homologous calf thvmus histone. J. Biol. Chem. 244, 5869-5679. DELNGE, R. J., HOOPER, J. A., and SMITH, E. L. (1972). Complete amino acid sequence of calf thymus histone III. hoc. Nat. Acad. Sci. U.S. 69, 882-884. EASTON, D., and CHALKLEY, R. (1972). High resolution electrophoretic analysis of the histones from embryos and sperm of Arbacia punctulata. Exp. Cell Res. 72, 502-508.

FAMBROUGH, D. M., FUJIMARA, F., and BONNER, J. (1968). Quantitative distribution of histone components in the pea plant. Biochemistry 7, 575-585. HILL, R. J., POCCIA, D. L., and DOTY, P. (1971). Towards a total macromolecular analysis of sea urchin embryo chromatin. J. Mol. Biol. 61,445-462. HINEGARDNER,R. T. (1962). The isolation of nuclei from eggs and embryos of the sea urchin. J. Cell Biol.

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HINEGARDNER,R. T., RAO, B., and FELDMAN, D. E. (1964). The DNA synthetic period during early development of the sea urchin egg. Exp. Cell Res. 36, 53-61. HNILICA, L. S., and JOHNSON,A. W. (1970). Fractionation and analysis of nuclear proteins in sea urchin embryos. Exp. Cell Res. 63. 261-270. IMMERS, J., MARKMAN, B., and RUNNSTRBM,J. (1967). Nuclear changes in the course of development of the sea urchin studied by means of Hale staining. Exp. Cell

Res. 47, 425-442.

INFANTE,A. A., and NEMER, M. (1967). Accumulation

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