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Cha, S., Agarwal, R. P., & Parks, J. R. E. (1975) Biochem. Pharmacol. 24, 2187-2197. Czichi, U., & Lennarz, W. J. (1977) J. Biol. Chem. 252, 790 1-7904. Ericson, M. C., Gafford, J. T., & Elbein, A. D. (1977) J. Biol. Chem. 252, 7431-7433. Heifetz, A., & Elbein, A. D. (1977) J. Biol. Chem. 252, 3057-3063. Kohler, P. O., Grimley, P. M., & O’Malley, B. W. (1 969) J . Cell Biol. 40, 8-27. Lee, Y. P., & Lardy, H. A. (1965) J. Biol. Chem. 240, 1427-1436. Lehle, L., & Tanner, W. (1975) Biochim. Biophys. Acta 399, 364-3 74. Lucas, J . J . , & Levin, E. (1977) J. Biol. Chem. 252, 43 30-43 36. Mankowski, T., Sasak, W., & Cojnacki, T. (1975) Biochem. Biophys. Res. Commun. 65, 1292-1297. McSweeney, G. P. (1965) J. Chromatogr. 17, 183-185. Molnar, J., Chao, H., & Ikehara, Y. (1971) Biochim. Biophys. Acta 239, 401-409, Morrison, J. F. (1969) Biochim. Biophys. Acta 185, 269-286. Roseman, S . (1958) J. Am. Chem. SOC.80, 3166-3174.
Segal, I. H. (1975) in Enzyme Kinetics, p 320, Wiley, New York. Spiro, M. J., Spiro, R. G., & Bhoyroo, V. D. (1976) J. Biol. Chem. 251, 6400-6408. Struck, D. K., & Lennarz, W. J. (1977) J. Biol. Chem. 252, 1007-101 3. Takatsuki, A., Kohno, K., & Tamura, G. (1975) Agric. Biol. Chem. 39, 2089-2091. Takatsuki, A,, Kawamura, K., Okina, M., Kodama, Y., Ito, T., & Tamura, G . (1977) Agric. Biol. Chem. 41, 2307-2309. Tkacz, J . S., & Lampen, J. 0. (1975) Biochem. Biophys. Res. Commun. 58, 287-295. Verman, A. K., Raizada, M. K., & Schutzbach, J. S. (1977) J . Biol. Chem. 252, 7235-7242. Waechter, C. J., & Lennarz, W. J . (1976) Annu. Reu. Biochem. 45, 95-1 12. Waechter, C . J., & Harford, J. B. (1977) Arch. Biochem. Biophys. 181, 185-198. Wedgwood, J . F., Warren, C. D., & Strominger, J. L. (1974) J. Biol. Chem. 249, 6316-6324. Wolfenden, R. (1972) Acc. Chem. Res. 5, 10-18.
Isolation and Characterization of the Histone Variants in Chicken Erythrocytest Michael K. Urban,* Samuel G. Franklin, and Alfred Zweidler
ABSTRACT: Chicken erythrocyte histones 2A, 2B, and 3 can be resolved into nonallelic primary structure variants by polyacrylamide gel electrophoresis in the presence of Triton X-100. These variants were isolated and characterized by analysis of their tryptic and thermolytic peptides. The major variants of chicken H2A and H2B differ from the analogous component of calf thymus by a small number of conservative amino acid substitutions in the basic terminal regions, which
interact with DNA. This moderate rate of allelic evolution of the slightly lysine-rich histones contrasts with the complete conservatism found in the arginine-rich histones. Chicken H4 and both chicken H 3 variants are identical with their corresponding components in mammals. The amino acid substitutions distinguishing histone variants are located within the highly conserved hydrophobic regions, which are involved in histone-histone interactions.
E u c a r y o t i c chromatin is arranged into similar repeating units, nucleosomes, each consisting of ca. 200 base pairs of DNA associated with two molecules each of histones 2A, 2B, 3, and 4 and variable amounts of H1 and/or H5 (Kornberg, 1974; Olins & Olins, 1974; Oudet et al., 1975; Van Holde et al., 1974). While the nucleosomal “spacer region”, which is associated with Hl-H5, is variable between cells of the same species and different species, the nucleosomal “core region”, which contains the histone octamer (H2A, H2B, H3, H4)2 and 140 base pairs of DNA, is extremely uniform (Felsenfeld, 1978). It is therefore not surprising that H1-like histones have been found to be quite variable, while the nucleosomal core histones have been highly conserved in evolution (De Lange, 1978; De Lange & Smith, 1975; Elgin & Weintraub, 1975).
This conservatism is particularly stringent within the hydrophobic regions of the histones, which may be involved in important intermolecular interactions (Kootstra & Bailey, 1978). We have previously shown that mammalian histones 2A, 2B, and 3 can be resolved into nonallelic primary structure variants by polyacrylamide gel electrophoresis in the presence of the nonionic detergent Triton X-100 (Franklin & Zweidler, 1977; Zweidler, 1976, 1978; A. Zweidler and S. Franklin, unpublished experiments). Since the H2A and H 3 variants appear to have been preserved unchanged throughout the evolution of mammals, it was important to determine if the same variants already existed in nonmammalian species. We report here on the isolation and characterization of two variants each of chicken erythrocyte histones 2A, 2B, and 3.
From the Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, Pennsylvania 1911 1 . Received May 2, 1979. This work was supported by US.Public Health Service Grants CA-09035, CA-15135, CA-06927, and RR-05539 and an appropriation from the Commonwealth of Pennsylvania.
Materials and Methods Preparation of Cells and Isolation of Nuclei. All procedures were conducted at - 2 “ C unless specified. Blood from a White Leghorn rooster was collected from a severed jugular
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CHICKEN HISTONE VARIANTS
vein into one-sixth volume of 1% (w/v) sodium heparin in Seligmann's balanced salt solution (SBSS). Red blood cells were separated from serum by centrifugation at 1600g for 10 min, the buffy coat was removed by aspiration, and blood cells were resuspended and centrifuged twice more in SBSS. Preliminary experiments involved isolation of nuclei from cells lysed by the following different methods: 0.05% saponin in buffer A (10 mM potassium-Tris-maleate, pH 7.4, 5 mM MgC12, and 50 mM glycine); 1% Triton X-100 in buffer A; 10 mM potassium-Tris-maleate, pH 7.4, 25 mM glycine, and 1 mM CaC12 (buffer B); the method of A. Zweidler, M. Urban, and P. Goldman (unpublished experiments). All buffers also contained 1 mM TLCK' and 1 mM TPCK (proteolytic inhibitors) and 1% (v/v) TDG and 1 mM DTE (antioxidants). The last method, which was used for all subsequent nuclear isolations, includes the following: direct homogenization of -70 O C frozen cells, without thawing, in 30 mL of buffer A at 3300 rpm for 1 min in a Waring blender fitted with a foam adaptor. In all cases, the lysate was centrifuged at 300g for 5 min, the supernatant was aspirated, and the crude nuclear pellet was rehomogenized in buffer A by blending at 7000 rpm for 30 s and centrifuged as above through a 5-mL underlayer of 5% (w/v) sucrose in buffer A. This pellet was homogenized at 4000 rpm for 30 s in 1% (w/v) Triton X-100 in buffer A and centrifuged as above. The pellet was then suspended in buffer B. Histone Extraction. After washing in buffer B, pelleted nuclei were extracted with 0.27 M NaCl-10 mM trisodium ethylenediaminetetraacetate (EDTA), pH 7.2, and centrifuged at 9000g for 10 min. The pellet was suspended in 1 M NaCl, centrifuged as above, made 0.25 N in HCl, and recentrifuged to remove acid-insoluble material. The histone extract was judged to be free of cytoplasmic and nuclear membrane protein by polyacrylamide gel electrophoresis (Jackson, 1976a,b). Alternatively, nuclei in buffer B were digested for 15 min at - 2 OC with 30 units/mL micrococcal nuclease (Sigma), frozen at -70 "C, quick-thawed in 2 mM EGTA, and then extracted with 1 M NaC1, 0.25 M HCl, 1% TDG, and 100 pg/mL protamine sulfate (Sigma) in order to ensure quantitative recovery of all histone components. The acid-soluble proteins were precipitated by the addition of one-fifth volume of 120% (w/v) trichloroacetic acid (C13AcOH). Pellets were washed with 20% C1,AcOH and 0.4% (v/v) HCl in acetone and with acetone alone (with 1% TDG) by resuspension and centrifugation and dried in a vacuum desiccator. Isolation of Histone Variants. After removal of H1 and H5 from the histone extract with 5% (v/v) perchloric acid (Johns & Diggle, 1969), the remaining proteins were dissolved in 8 M urea in 0.1 M sodium acetate buffer, pH 4.85, and fractionated at room temperature by ion-exchange chromatography on a 1.5 X 25 cm carboxymethylcellulose column (Whatman, CM-52) equilibrated with the above buffer containing 4 M urea. Elution was carried out with the following concentrations of guanidinium chloride (Research plus) in the equilibrium buffer: 1.8% (200 mL), a linear gradient of 1.8-2.5% (200 mL), and finally a 5% wash (100 mL). Individual histone components were isolated from the above fractions enriched in individual histone classes by preparative polyacrylamide gel electrophoresis in the presence of Triton X-100 (A. Zweidler and S. Franklin, unpublished experiments). I Abbreviations used: TPCK, L- 1-tosyl-2-phenylalanyl chloromethyl ketone (Sigma); TLCK, L- 1-tosyllysylchloromethyl ketone (Sigma); DTE, dithioerythritol (Pierce); TDG, thiodiglycol (Pierce).
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Gel Electrophoresis. Protein fractions were analyzed on 12% polyacrylamide gels containing 6 mM Triton X-100 and 3-7.5 M urea (Zweidler, 1978) and on 15% polyacrylamide gels with 2.5 or 6 M urea (Panyim & Chalkley, 1969). Electrophoresis was also performed in 18% polyacrylamide0.1% NaDodS04 gels according to Laemmli (1970). Cyanogen Bromide Cleavage. Typically, an excess of cyanogen bromide in 100 pL of 70% (v/v) formic acid, flushed with nitrogen, was used to cleave 50 pg of protein at room temperature for 12 h. The reaction mixture was lyophilized, dissolved in 100 pL of doubly distilled water, and relyophilized. Amino Acid Analysis. Ampules were cleaned in chromic-sulfuric acid and rimed with 1% (v/v) TDG and 5% (v/v) mercaptoacetic acid before use. Samples were hydrolyzed under nitrogen in constant-boiling 6 N HC1, that was distilled from ninhydrin, for 24 h at 110 OC. For performic acid oxidation of cysteine, a mixture of 10 volumes of 30% (v/v) hydrogen peroxide and 88% (v/v) formic acid was permitted to stand at room temperature for 1 h. This mixture was then reacted with the protein sample for 4 h at 0 OC, lyophilized, and hydrolyzed as above. Hydrolysates were lyophilized, dissolved in 0.1 M sodium citrate buffer, pH 2.2 (Pierce), and analyzed in a Durrum D-500 amino acid analyzer. Analysis was performed in triplicate and normalized to cohydrolyzed histone standards as a correction for hydrolytic losses. Peptide Mapping [According to Franklin & Zweidler (1977) and A. Zweidler and S . Franklin (Unpublished Experiments)]. Histones were digested with trypsin in 0.1 M ammonium bicarbonate, pH 8.1 (ratio of 1:50 enzyme to substrate, added twice), for 4 h at 37 OC. The samples were lyophilized, suspended in electrophoresis buffer (see below), and centrifuged to recover the tryptic core. Thermolytic digestion of the core (ratio of 1:500 enzyme to substrate) was in 0.1 M ammonium acetate buffer, pH 7.0, for 2.5 h at 37 OC. Electrophoresis for both tryptic and thermolytic peptides was in pyridine-acetic acid-1 -butanol-water (1: 1:2:36), pH 4.7, for 1 h at 600 V on Avicell microcrystalline cellulose plates. Ascending chromatography was for 3-4 h in pyridine-acetic acid-1-butanol-water (10:3:15: 12). Visualization was with fluorescamine. Peptides were eluted with constant-boiling 6 N HCl and hydrolyzed for amino acid analysis as described above. Results The electrophoretic resolution of the chicken histones can be significantly improved over the acid-urea system of Panyim & Chalkley (1969) by the addition of Triton X-100 to polyacrylamide gels (Figure l). By using the optimum concentration of urea and Triton X-100, two subfractions each of chicken erythrocyte histones 2A, 2B, and 3 can be resolved. Since some of the histone components occur in very small relative amounts, it was necessary to eliminate cytoplasmic contaminants as much as possible. For this purpose, the nuclei were not isolated by the standard methods of cell lysis with detergents but rather by shearing off the cell membranes and cytoplasmic layer in a modified Waring blender, allowing the complete exclusion of air (A. Zweidler, M. Urban, and P. Goldman, unpublished experiments), followed by low-speed centrifugation through a denser underlayer to separate the nuclei from cytoplasmic debris. If globin is used as a marker for cytoplasmic contamination, this method is the most efficient in obtaining "clean" nuclei (Figure 2). In order to prevent complications due to degradation and partial oxidation of methionine residues (Zweidler, 1978), it was essential to isolate
3954
BIOCHEMISTRY
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Table I: Comparison of the Amino Acid Composition of Chicken and Mammalian Histone Variantsa amino acid composition (mol %) 2A.2
2A.I ~~
ch
Calf
2R.1 .~
~~
calf
ch
calf
2R.2 . ~ .
ch
mouse
ch
3.1.calf
3.2.ch
3.3. ch
Asx
6.2 7.0 6.2 6.3 4.8 4.7 4.8 4.7 3.7 3.6 3.8 Thr 3.9 3.1 3.1 3.3 6.4 7.4 6.4 7.5 7.4 7.5 7.6 Ser 3.1 3.4 3.9 3.9 11.2 11.3 12.0 11.1 3.7 4.2 4.7 Glx 9.3 8.6 9.3 9.6 8.0 8.0 8.0 8.0 11.1 10.6 11.2 PI0 3.9 3.9 3.9 4.0 4.8 4.8 4.8 4.8 4.4 4.8 4.4 10.8 10.4 10.8 11.1 5.6 5.7 4.8 4.8 5.2 4.9 5.8 G~Y 10.4 13.1 13.2 14.3 13.2 10.4 9.8 10.4 13.3 13.1 Ala 13.1 0.7 1.5 0 0 0 0.7 0 0 0 0 0 Cys 4.5 4.4 4.3 Val 7.2 6.1 7.2 6.4 6.2 6.2 6.2 6.4 1.5 1.5 1.6 1.6 1.5 Met 1.0 1.6 0.8 0.8 0 0 5.2 5.4 5.3 4.8 5.3 4.5 [le 5.3 4.8 4.7 4.7 4.7 8.6 8.9 9.0 4.8 4.7 4.8 4.8 11.6 11.4 Leu 12.4 12.5 1.9 2.2 2.2 4.0 3.8 4.0 4.0 2.3 2.3 2.3 2.2 Tvr Pie 0.8 0.8 0.8 0.8 1.6 1.5 1.6 1.6 3.0 3.0 2.8 His 3.1 2.3 3.1 2.3 2.4 2.4 2.4 2.4 1.5 1.5 1.6 16.0 16.4 9.6 11.2 10.9 10.7 16.0 16.7 9.7 10.0 LYS 10.9 9.3 9.3 9.3 6.4 6.2 6.4 6.1 13.3 13.9 13.0 '4% 9.3 Analysis was performed in triplicate and normalized to cohydralyzed calf histone standards (De Lange et al., 1972; Hayashi & Iwai, 1971; Hnilica et al., 1970; Ishikawa et al., 1972; Sauti& et al., 1975) as a correction for hydrolytic losses and is expressed as mole percent.
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