The primary structure of the histone H2A₍₂₎ type from wheat germ

Abstract

The histone H2A,,, type from wheat germ comprises at least two highly homologous isohistones with 151 amino acid residues. Microheterogeneity occurs mainly at the N-terminal and C-terminal regions, These isohistones have both N-terminal(7 amino acid residues) and C-terminal(l5 amino acid residues) extensions relative to calf thymus histone H2A.Histones are essential for the ordered packing of DNA into nucleosomes and higher-order structures [I]. The widespread occurrence of isohistones (for review see [2, 31) suggests that nucleosomes are heterogeneous with respect to their histone content. In animal systems, different sets of the non-allelic histone variants are expressed coordinately at different stages of development and cell differentiation [3, 41. Less is known about the structural variation of plant histones, and there is no evidence yet on the existence or otherwise of a developmental program , and the other, H2AC2,, with blocked N-termini exhibiting both N-terminal and C-terminal extensions. Each type comprises at least two highly homologous species which only becomes apparent on sequencing. Neither HPLC, ion-exchange chromatography nor Triton X-IOO/PAGE has succeeded in separating the closely related iso-histones within each type from each other. We report here the characterization of the structure of the H2A,,, type isohistone from wheat germ. MATERIALS AND METHODS Isolation of histone H2A,,,Wheat germ (Triticum aestivum) was obtained from Sasko Mills (Rondebosch, Cape). This material contained at least 80% of the cultivar T4. Chromatin was isolated essentially according to the method of Johns and Butler [9] with some modifications as described previously [6].Exclusion chromatography of histones was performed at room temperature on a column (4.5 x 80 cm) of Bio-Gel P-60 (100-200 mesh) as described previously buffer (pH 5.30) containing 6 M urea. A linear NaCl gradient was used to elute the fractions (Fig. 1 a). After desalting, the H2A[,,-containing fraction was further purified on a Bio-Gel P-60 column equilibrated with 20 mM HC1, 50 mM NaCl as described previously [7]. The eluant contained in addition, 10 mM sodium bisulphite, 1 mM phenylmethylsulfonylfluoride and 1 mM N-a-tosyl-L-lysylchloromethane, solubilized first in a small volume of propan-1-01. Analytical proceduresPAGE was performed either in acid/urea/Triton X-100 gels according to Zweidler [9] or in SDS according to Laemmli [lo]. Amino acid compositions were determined after 24 h hydrolysis in 5.7 M HCl containing 0.025% (massivol.) phenol. No correction factors for incomplete hydrolysis or destruction of amino acids were applied.Cleavage of the acetylated N-terminal methionine residue with CNBr [ll] was performed as described previously [6]. Peptides were generated through enzymatic cleavage employing trypsin (after prior citraconylation of the isohistone), endoproteinase Lys-C, and Staphylococcus aureus V8 protease as described previously for isohistone H2A,,, [7]. The peptides generated through ezymatic cleavages were fractionated by g...