Histones from the testis of the crab cancer pagurus

Histones from the testis of the crab cancer pagurus

Comp. Biochem. Physiol. Vol. 72B, pp. 393 to 399, 1982 Printed in Great Britain. 0305-0491/82/030393-07$03.00/0 ©1982 Pergamon Press Ltd HISTONES FR...

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Comp. Biochem. Physiol. Vol. 72B, pp. 393 to 399, 1982 Printed in Great Britain.

0305-0491/82/030393-07$03.00/0 ©1982 Pergamon Press Ltd

HISTONES FROM THE TESTIS OF THE CRAB CANCER PAGURUS MURIEL CHAUVIERE 1, PIERRE SAUTIERE 2, MAURICE COUPPEZ 2 and PHILIPPE CHEVAILLIER 1

1Laboratoire de Biologie Cellulaire, ERA CNRS no 400, Universit6 Paris-Val de Marne, 94010-Cr6teil, France 2Unit6 124 INSERM et Institut de Recherches sur le Cancer, Place de Verdun, BP 311, 59020 Lille, France (Received 9 November 1981) Abstract--I, Histones from testis of Cancer pagurus were extracted, and then purified by gel filtration chromatography on Biogel P10 and P60. 2. Five main fractions were characterized by their electrophoretic mobilities and their amino acid compositions. 3. A great similarity was observed between arginine-rich histones H3 and H4 from crab testis and those from calf thymus except that crab histone H3 contains only one cysteine residue. 4. Crab histones HI, H2A and H2B were significantly different from their calf thymus counterparts, differences which were occasionally observed in other marine invertebrates.

INTRODUCTION

Cancer pagurus is a marine invertebrate belonging to the class Crustacea. The D N A of this species contains approximately 25~o of light satellite, essentially a poly (dA-dT).poly (dA-dT) copolymer which is composed of 93~o alternating A and T residues and 2.7~o C + G (Skinner, 1967; Laskowsky, 1972). The presence of satellite is a common feature of crab D N A and it appears that the genome of most eukaryotes contains varying quantities of satellite or redundant D N A s (Arrighi et al., 1970; Maio, 1976; Skinner, 1977). A large number of publications has contributed to the characterization of these D N A s in Arthropods (crab, drosophila) and Mammals (mouse, monkey, guineapig, man) (Laskowsky, 1972; Sabeur et al., 1974; Maio, 1976; Skinner, 1977). The localization and biological functions of these DNAs, however, are not accurately known and remain putative. Indeed, it would appear from the studies, especially in Mammals and Drosophila (Maio, 1976), that there is a relationship between the presence of satellite DNA, constitutive heterochromatin and centromeric regions. The correlation agrees with the fact that highly repeated D N A s are not transcribed (cf. review of Kurnit, 1979). Attempts to localize the crab satellite in situ have shown that there is no preferential site within the crab interphase nucleus for A - T rich satellite D N A (Chevailtier et al., 1974). Concerning DNA-associated proteins in crabs, no study has yet been published. The purpose of the present work was to purify and characterize testis histones of Cancer pagurus and is intended to be the first step in the comparison of the structures and properties of poly d(A-T) chromatin with chromatin composed of main DNA. MATERIALS AND METHODS Cancer pagurus testes were frozen in liquid nitrogen and stored at - 8 0 ° C until use. All operations up to chromatographic purification of histones were performed at 4°C. c.a.P. 72/3a--E

393

Preparation of nuelei Testes were finely minced and homogenized in a Waring blendor with 50 mM Tris-HCl, containing 0.25 M sucrose, 3.3 mM CaCIz and 0.5 mM phenylmethylsulfonylfluoride (PhMeSO2F). The homogenate was filtered through six layers of surgical gauze and centrifuged at ll00g for 25 min. The nuclear pellet was then resuspended in the same buffer containing 2.2 M sucrose and was centrifuged at 47,000 .q for 1 hr.

Preparation of deoxyribonucleoprotein Purified nuclei were washed three times with NaC1/Cit, pH 7.2 (0.15 M NaCI, 0.015 M sodium citrate) containing 0.5mM PhMeSO2F and then with 50mM Tris (pH7.2) containing 0.35 M NaC1 and 0.5 mM PhMeSO2F. The deoxyribonucleoprotein was pelleted at 2000 g for 30 min. In some experiments deoxyribonucleoprotein was directly prepared from testes without noticeable effect on the quality of our histone preparations. Preparation of whole histone. Whole histone was extracted overnight from deoxyribonucleo protein by gentle stirring in 0.25 N HCI. The extract was clarified by filtration through Millipore filters (0.22/~m) dialyzed against distilled water and lyophylized. Whole histone was sometimes dialyzed against 0.25 N HCI and precipitated by adding 6 volumes of cold acetone (48 hr at -20°C) according to Fornells and Subirana (1977). After centrifugation, the pellet was washed three times with cold acetone and dried in vacuo. Histonefractions. Crude histone fractions were prepared according to the methods described by Johns (1964, 1967): they are designated by "f" throughout this work according to Johns' nomenclature. Arginine-rich histones were extracted from deoxyribonucleo protein by ethanol: 40~ guanidinium chloride, (3 : 1, v/v), pH 7. Fractions f2a2 and f2al were obtained by differential precipitation with 1 and 3 volumes of cold acetone, respectively. Histone fraction 13 was generally prepared according to the second procedure of Johns (1964). Deoxyribonucleo protein was extracted with 1.25 N HCI: ethanol (1:4, v/v) and, after centrifugation and filtration, fraction 13 was precipitated from the supernatant with cold acetone (0.7 vol).

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MURIEL CHAUVIEREet al.

After removal of the arginine-rich histones, the pellet of residual deoxyribonucleoprotein was extracted with 0.25 N HCI and the lysine-rich fractions fl and f2b were precipitated from the extract with 3 and 5 volumes of acetone, respectively.

were incubated with cytoplasmic extract for 2 hr at 3 7 C with 0.002~ di-isopropyl-fluorophosphate as inhibitor of proteolysis.

Purification of histone.l?action.~ The histone fractions were further purified by get filtration chromatography at room temperature. Fractions f2a2 and f2al were loaded on a Biogel P60 column (5 cm × 95 cm) equilibrated and eluted with chloroformsaturated 0.01 N HCI. Fractions 13 and f2b were applied onto a Biogel PI0 column (2.6cm × 180cm) equilibrated and eluted with chloroform-saturated 0.01 N HCt. The histone samples were dissolved in 0.1 M Tris-HC1, pH 8.2, 6 M guanidinium chloride, 5'?,,, mercaptoethanol and kept at room temperature under nitrogen for 24 hr before chromatography in order to ensure complete reduction of the disulfide bridges. Histones obtained after purification of crude 'T' fractions are designated by "H" according to the international rules.

Whole histone

RESULTS

Analytical procedures Whole histone and fractions were identified at all stages of preparation and purification by electrophoresis on polyacrylamide gels in 0.9 M acetic acid 2.5 M urea or 0.9 M acetic acid 6.25 M urea (Panyim & Chalkley, 1969). The acrylamide concentration was 15"~;. Electrophoresis on polyacrylamide slab gels containing 0.1"~, sodium dodecyl sulphate and 15°~,~, acrylamide was performed according to Laemli (1973). Amino acid analyses were performed with a Beckman Multichrom amino acid analyser. Histone samples were hydrolyzed in 6 N HC1 at 110' C for 24 and 72 hr in vacuo in the presence of one drop of 1!',~,phenol in order to prevent hydrolytic losses of tyrosine. Deacetylation O/histones Whole histone or histone fractions were deacetylated by deacetylase extracted from calf thymus cytoplasm by the method proposed by Inoue and Fujimoto (1970). Histones

Electrophoresis in acidic gels in b o t h 2.5 and 6.25 M urea separated crab whole histone into five major b a n d s which were c o m p a r e d with h o m o l o g o u s histones from calf thymus run under the same conditions (Fig. 1, gels 1-4). Histone H1 exhibited a higher cathodic mobility than its m a m m a l i a n h o m o logue and showed at least three subfractions on sodium dodecyl sulphate potyacrylamide gel (Fig. 1, gel 6). Cancer payurus histones H2A and H2B were well resolved in b o t h 2.5 a n d 6 . 2 5 M urea-gels, whereas calf histones were not separated in 6.25 M urea. Crab histone H2B migrated faster than crab histone H2A. O n sodium dodecyl sulphate polyacrylamide gel crab histone H2A and H2B comigrated (Fig. 1, gel 6). Histones H3 and H4 b o t h contain three components. Deacetylation assays revealed that in b o t h cases the two faint anodic b a n d s were m o n o and diacetylated histones (data not shown).

Aryinine-rich histones f 3 . Crude histone fraction 13, obtained by ethanolHCI extraction from c h r o m a t i n according to the method 2 of Johns (1964), is heterogeneous (Fig. 2(B), gel 2). Histone H3, major c o m p o n e n t of this fraction, is c o n t a m i n a t e d by histones H1 and H2A and a very faint band migrating at the position of H2B. Histone H3 was further purified by exclusion chromatography on Biogel P I 0 (Fig. 2(A), 2B: gel 4). Histone H3 could also be obtained after c h r o m a t o g r a p h y of

i~ ! ~i~ ~i!~i!i i! ~

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Fig. 1. Analytical gel electrophoresis of whole histone from calf thymus (gels 1, 3, 5) and from crab testis (gels 2, 4, 6) in three different conditions: Gel 1, 2:0.9 M acetic acid, 2.5 M urea, 15~o polyacrylamide: gels 3,4:0.9 M acetic acid, 6.25 M urea, 15~, polyacrylamide; gels 5,6:0.1 ~o dodecylsulphate, 15"~,, polyacrylamide slab gel (Laemli et al., 1973). Gels 1-4 were stained overnight with Coomassie brilliant blue G 250 (Blakesley et al., 1977); gels 5 and 6 were stained with Coomassie blue R 250.

Crab testis histones 2B

the determination of the amino acid composition of protein X may lead to its identification. Pure histone H4 was recovered after gel filtration of histone fraction f2al on Biogel P60 (Fig. 3(A)). The comparison of the amino-acid compositions of crab histones H3 and H4 with their calf homologues (Table 1) shows that there are no significant differences. We like to point out, however, that crab histone n 3 contains one more serine and one less cysteine residue than calf histone H3. This suggests a substitution of one cysteine by one serine residue, as already noted in different species (Hooper et al., 1973; Brandt et al., 1974). f2a2. This fraction was shown to be a mixture of histones H2A and H3 and, to a lesser extent, histone H2B (Fig. 4(B), gel 2). The elution profile of the different proteins is shown in Fig. 4(A). Peak 2 corresponds to pure histone H3 while peak 1 contains a mixture of histones H2A and H2B. Indeed the molecular weights of these two proteins are similar, which renders their separation by gel filtration difficult. The ascending part of peak 1 contains pure H2A while its descending part is a mixture of H2A and H2B. The amino acid composition of Cancer pagurus histone H2A is somewhat different from that of its calf histone counterpart. The number of serine residues is

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0.2

I 300

2A

H2B! 400

395

i

l 500

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Fig. 2. Chromatography of Cancer pagurus histone fraction 13 on Biogel P10 (200-400 mesh). (A) Elution pattern of 40 mg of the histone 13 dissolved in 1.5 ml 6 M guanidinium chloride, 0.1 M Tris-HCl, pH 8.2, 5% mercaptoethanol and applied to a column (180cm x 2.6cm) equilibrated and eluted with 0.01 N HCI. The flow rate was 18 ml/hr and two ml-fractions were collected. (B) Electrophoretic control of fractions obtained after BiogelPl0 chromatography of histone fraction 13 (Fig. 2(A)) on 0.9 M acetic acid, 2.5M urea, 15~o polyacrylamide gels. Gels were stained with Coomassie brilliant blue G 250. Gel 1: whole histone from crab testis; gel 2: histone fraction 13; gel 3: histone H I ; g e l 4: histone H3.

3B

0.6

0.4

0.2 H4

histone fraction f2a2 on Biogel P60 (Fig. 4(A)) but in this case, the yield was much lower. f 2 a l . Histone H4 was the main component of this fraction as shown by analytical electrophoresis (Fig. 3(B), gel 2). Histones H2A and H2B were present in small amounts in that fraction, together with an unknown material, termed X, which has not been identified yet. This protein which is located between H2B and diacetylated H4 does not appear to be histone H4 with a higher degree of acetylation and does not seem to be a digestion product of a histone, since care was taken during the preparations (use of a proteolytic inhibitor, dialysis against HCI) (Eickbush et al., 1976; Fornells and Subirana, 1977) to prevent the appearance of such compound. Band X may be a high mobility group protein or, in this differentiating tissue, a specific protein appearing at a given step of gametogenesis, for instance, a specific histone variant. Only

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I 1100

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1200 H

I 1500

I 1600

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Fig. 3. Chromatography of Cancer payurus histone fraction f2al on Biogel P60 (100-200mesh). (A) Elution pattern of 40mg of the histone fraction f2al dissolved in 1.5 ml 6 M guanidinium chloride, 0.1 M Tris-HCl, pH 8.2, 5~o mercaptoethanol and applied to a column (95cm x 5cm) equilibrated and eluted with 0.01 N HC1. The flow rate was 30 ml/hr and 3 ml fractions were collected. (B) Polyacrylamide gel electrophoresis of fractions obtained after Biogel P60 chromatography of histone fraction f2al (Fig. 3(A)). The electrophoretic control was performed as in Fig. 2(B). Gel l: whole histone from crab testis; gel 2: histone fraction f2al ; gel 3: histone H4.

16.88

1.7

0.8 4.1 0.5 0.5 28.7

4.1

2.0 5.4 6.7 3.4 10.1 6.9 25.1

H2B

2.1 l

5.65(7) 1.38t2) 6.03(8) 5.66(7) 3.67(5) 1.61(2) 15.05(19) 2.31(3) 6.86(9j 128 129

5.26(7) 6.12(8j 11.32(14 15) 8.34(11) 2.60(3) 5.64(7) 12.50(16)

Cancer pagurus

2.5

9 2 6 6 5 2 20 3 8 125

6 8 14 10 6 7 13

Calf thymus

H2A

1.09

6.90(8) 0.62(1) 4.36(5) 11.52(14) 2.07(3) 0.86(1) 9.92(12) 1.60(2) 8.73(11) 120-121

7.04(8) 2.68(3) 6.19(7-8) 10.15(2) 4.33(5) 11.88(14) 11.15(14)

Cancer pagurus

1.16

6 16 3 1 14 4 12 129

8

8 5 4 12 5 14 17

Calf thymus

H3

0.72

3.98(5) 7.37(10) 4.55(6) 11.56(15) 4.50(6) 5.52(7) 13.09(18) 0.48(1) 4.56(6) 1.05(2) 5.17(7) 9.20(12) 2.14(3) 2.92(4) 9.22(13) "1 1.43(2) 13.50(18) 135

Cancer pagurus

0.72

5 10 5 15 6 7 18 2 6 2 7 12 3 4 13I~l 2 18 135

Calf thymus

H4

0.78

8.14(8) 1.02(1) 5.56(6) 8.03(8) 3.75(4) 2.00(2) 10.64(11) ~ 2.01(2) 13.74(14) 101 102

5.11(5) 7.70(8j 1.34(1-2) 6.75(7) 1.16(1) 16.03(16) 7.05(7)

Cancer paourus

0.78

9 1 6 8 4 2 11t~ 2 14 102

5 7 2 6 1 17 7

Calf thymus

Calf thymus histone HI (Hnilica, 1972) and crab testis histone amino acid values are expressed as moles per cent of all amino acids. In brackets values are the assumed numbers of amino acids per mole for crab histones. Amino acid values for calf thymus histones H2A (Sautiere et ul., 1974), H2B (lwai et al., 1970). H3 (DeLange et al., 1972) and H4 (DeLange et al.. 1969) are expressed as number of residues per mole. ~"~ Average of at least two analyses. ~h~ Values for threonine and serine were obtained by linear extrapolation to zero hydrolysis time. ~' Determined as cysteic acid after performic oxidation. .L~ 72 h hydrolysis values. "~ Including E-N-methyllysinc.

Asp 3.24 Thr (b) 4.20 Ser (b) 4.36 Glu 5.57 Pro 8.81 Gly 4.95 Ala 23.62 Cys/2 {c) Val (d) 5.74 Met 0.33 lie (d) 2.28 Leu (d) 5.49 Tyr 1.78 Phe 1.63 Lys 24.32 His 0.79 Arg 2.89 Total number of residues Lys/Arg 8.41

Amino acids

H 1 (unfractionated) Cancer Calf pagurus thymus

Table 1. Amino acid compositions of histones from ('ancer pagurus testis (a)

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Crab testis histones

f2b. The major component of this fraction was histone H2B, slightly contaminated by anodic components and by histones H1 and H3 (Fig. 5B, gel 3). Further purification of historic H2B on Biogel P10 was rather easy (Fig. 5). This protein differs from its calf thymus homologue in several respects. Its molecular weight is apparently lower since it migrates more rapidly during electrophoresis. It is soluble in neutral or acidic alcoholic solutions and so is extracted with all arginine-rich histone fractions. The amino acid composition of crab histone H2B is compared with calf thymus histone H2B in Table 1. The crab and calf histones H2B differ by higher quantities of alanine and isoleucine in the former, partially compensated by a reduced valine content. The major difference involves a very low proline content. A slight decrease of the lys/arg ratio is also observed.

4B

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H2A

H2B

i i

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397

I I 1300

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Fig. 4. Chromatography of Cancer pagurus histone fraction f2a2 on Biogel P60 (10~200 mesh). (A) Elution profile of crab histone fraction f2a2; same experimental conditions as in Fig. 3A. (B) Electrophoretic analysis of fractions obtained after gel filtration chromatography of histone fraction f2a2 on Biogel P60 (Fig. 4(A)). Same conditions as in Fig. 2(B). Gel 1: whole histone from crab testis; gel 2: histone fraction f2a2; gel 3: histone H2A.

higher than in the calf protein, while the threonine content is lower. The most important differences are the presence of methionine and the quantity of histidine: 1.6~, opposed to 3.1% in calf thymus. We may also note the overall decrease of the number of basic and neutral amino acids (Ala, Ile, Leu) in crab testis.

DISCUSSION

The five major histones from Cancer pagurus testis were purified and partially characterized by conventional methods. The electrophoretic migration and amino acid composition of the most conserved histones H3 and H4

A230nm

5B

0.0

0.0

L ysine-rich histones

f l . The selective extraction of histone fraction fl by 5% perchloric acid (method I of Johns, 1964) from the Cancer payurus chromatin could not be used because of the poor extraction yield of protein. Moreover, fraction fl, obtained either with the method 2 of Johns (1964) or after 0.25 N HC1 extraction of the residual pellet whose f2a fraction had been previously extracted (1967), was found to contain histone H1 as major component together with non-histone proteins. All our attempts to isolate pure H1 from fraction fl, either by gel filtration or ion exchange chromatography was unsuccessful. In fact, histone H1 was found to be present in small amounts in fraction 13 and was isolated in pure form from this fraction (Fig. 2(A), (B): gel 3). The amino acid composition of unfractionated crab histone H1 is given in Table 1. This composition is characterized by high amounts of alanine and lysine which together account for about 50% of the total number of residues. The most striking differences observed with calf thymus histone H1 are the presence of histidine and methionine residues and a higher content in aromatic residues.

0.4

0.2

i i 300

5A

I ; 400

i

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Fig. 5. Chromatography of Cancer pagurus histone fraction f2b on biogel Pl0 (200-400 mesh). (A) Elution pattern of crab histone fraction f2b on Biogel Pl0; experimental procedure was similar as indicated in Fig. 2A. (B) Electrophoretic control of fractions obtained after gel filtration chromatography of histone fraction f2b on Biogel P10 (Fig. 5(A)); same conditions as in Fig. 2(B). Gel 1: whole histone from crab testis; gel 2: histone fraction f2b; gel 3: histone H2B.

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MURIEL CHAUVIEREet al.

Recent reports have shown the nucleosomal strucare similar to those from other species, except that ture of AT- or GC-satellite-rich chromatin and even histone H3 from crab testis contains only one cysteine residue instead of two in calf thymus taken as a refer- of reconstituted chromatin with synthetic poly d(A T) ence. Cysteine observed in histone H4 from Echinoand poly d(G-C) (Bokhon'Ko et al., 1976; Musich et derms (Subirana, 1971; Strickland et al., 1974; Woual., 1977: Simpson et al., 1979). Moreover, results ters-Tyrou et al., 1974; VanHoutte-Durand et al., obtained with reconstituted chromatin from poly 1977) and in some other species such as Palaemon d(A T) or poly d(G-C) and histones seem to show that the structure of the inner D N A of core particles serratus (Sellos et al., 1979) was not found after perdepends on the nature of the D N A (Simpson et al., formic oxidation of crab histone H4. 1979). The use of biological materials of the same The three other histories, as in other species, vary origin, poly d(A T) and histones from Cancer pa~lurus. more or less when compared to the homologous hisshould enable us to determine the respective roles tones from calf thymus. It is noteworthy that most differences in the compositions of histones H1, H2A played by these two components in the variations of and H2B are also observed, although to various chromatin fine structure. extents, in other marine invertebrates. The presence of methionine and histidine in histone H1 appears to be Acknowledffements Wc wish to thank Mrs M. J. Dupire a common feature of several marine invertebrates for expert technical assistance and Mrs D. Tesson for the such as Asterias rubens, Psammechinus miliaris, Parepreparation of the manuscript. This work was supported chinus anyulosus (Echinoderms), (VanHoutte-Durand by the Centre National de la Recherche Scientifique (ATP et al., 1977: Wouters-Tyrou, 1977: Strickland et al., "Chromatine'" no. 2875 et 2888). 1980), Sipunculus nudus (Sipunculid), (Mazen et al., 1976) and Palaemon serratus (Crustacea), (Sellos et al., REFERENCES 1979). In these species as in Cancer payurus, the lys/ arg ratio is lower than in Mammalian histones H1. ARRIGHI F. E., MANDEL M., BERGENDAHL J. & Hsu J. Histones H2B from crab, shrimp and Sipunculus (1970) Buoyant densities of DNA of mammals. Biochem. (Mazen et al., 1978) all show an increase in the Genet. 4, 367-376. amount of leucine and isoleucine and a decrease in BLAKESLEY R. W. & BOEZI J. A. (1977) A new staining technique for proteins in polyacrylamide gels using Coothe amounts of valine and proline compared to calf massie Brilliant Blue G250. Anal. Biochem. 82, 58(~582. thymus histone H2B. The variations in the quantities BOKHON'KO A. & REEDER R. H. (1976) The subunil strucof arginine and lysine in crab and star-fish are much ture of mouse satellite chromatin. Biochem. Biophys. Res. lower than in the shrimp and in the Sipunculus. HisCommun. 70, 146-152. tones H2A from the four above species all contain BRANDT W. F., STRICKLANDW. N. & VON HOLT C. (1974) methionine. Moreover, in histones H2A from CrustaThe primary structure of histone F3 from shark erythrocea and Sipunculus, the amount of serine is higher cytes. Febs Lett. 40, 349-352. than in calf thymus H2A but the opposite is noted for CHEVAILLIER PH., DE RECONDO A.-M. & GEUSKENS M. histidine. However, the amino acid compositions of (1974) Deoxyribonucleic acid of the crab Cancer pagurus. lII. Intracellular localization of poly d(A-T) of Cancer histories H2A and H2B from crab testis were quite pagurus by hybridization in situ. Exp. Cell Res. 86, different from those of Echinoderms such as Psamme383-391. chinus miliaris (Wouters et al., 1978: Strickland et at., 1978) and Parechinus anyulosus (Strickland et al., DELANGE R. J., FAMBROUGHD. M., SMITH E. L. & BONNER J. (1969) Calf and pea histone IV. II. The complete 1980; Strickland et al., 1977). amino acid sequence of calf thymus histone IV : presence In conclusion, it does not appear that there is a of eN-acetyllysine. J. Biol. Chem. 244, 319-334. specific basic protein whose existence may be corre- DELANGE R. J., HOOFERJ. A. & SMrrn E. L. (1972) Comlated with that of satellite DNA, which exists in large plete amino-acid sequence of calf-thymus histone Ill. Proc. Natn. Acad. Sci. 69, 882 884. quantities in Cancer pa~turus. Nevertheless. a reserve can be made in that one protein (X) whose electro- EICKBUSCHT. H., WATSON D. K. & MOUDRIANAKISE. N. (1976) A chromatin-bound proteolytic activity with phoretic mobility is slightly greater than that of hisunique specificity for histone H2A. Cell 9, 785 792. tone H2B, was not still identified. Actually attempts FORNELLSM. & SUBmANAJ, A. (1977) Specific degradation to purify protein X are realized in order to determine of histones HI and H3. Biochem. Biophys. Res. Commun. its amino acid composition and to check if it can be 78, 217 221. classified either as a species- or as an organ-specific HOOPER J. A., SMITH E. L., SOMMER K. R. & CHALKLEY R. protein. Histones present in the nucleosome core (1973) Histone III. IV. Amino acid sequence of histone exhibit the general features of these proteins, i.e. relaIII of the testes of the Carp, Letiobus buhalus, J. Biol. Chem. 248, 3275-3279. tive stability of the compositions of histones H3 and H4 and variability of histones H2A and H2B in com- HNILICA L. S. (1972) The Structure and Biological Function~ of Histones, p. 7, CRC Press Inc., West Palm Beach, parison to other species with some analogy with Florida. several other marine invertebrates. The determination INOUE A. & FUJIMOTOD. (1970) Histone deacetylase from of the primary structure of these Cancer pagurus hiscalf thymus. Biochim. biophys. Acta 220, 307 316. tones will be interesting both for the study of chroma- IWAI K., ISHIKAWAK. & HAYASH1H. (1970) Amino-acid tin structure and from an evolutionary standpoint. sequence of slightly lysine-rich histone. Nature 226, The localization of mutation sites should lead to the 1056-1058. demonstration of their possible influence on histone- JOHNS E. W. (1964) Preparative methods for histone fractions for calf thymus. Biochem. J. 92, 55-59. histone and histone-DNA interactions and consequently explain their eventual effects on chromatin JOHNS E. W. (1967) A method for the selective extraction of histone fractions F2al and F2a 2 from calf thymus deoxystructure which is very heterogeneous in crabs conribonucleoprotein. Biochem. J. 105, 611-614. cerning DNA composition.

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