1. The complete amino acid sequence (145 residues) of histone variant H2A,,, from wheat germ Triticum 2. The sequence of histone variant H2A(,, differs from the homologous calf histone in 61 amino acid positions. aestivum cultivar T 4 has been established from Edman degradation of large overlapping fragments.These differences include an extension of H2A,,, by 19 amino acids at its carboxyl end.Histones in general are considered to play a structural rather than a regulatory role in chromatin [l], however the widespread occurrence of histone variants (isohistones), (for review see [2,31) suggests that variants may modulate the conformation of nucleosomes, and in this way modify their susceptibility to regulatory factors. We have established previously the presence of a number of isohistones of the H2A class in wheat embryos [2, 41. We are reporting now the complete structure of one of these isohistones. This structure constitutes a new histone type in that its C-terminal region is extended beyond the size hitherto considered typical for core histones of that group.
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 T 4. Chromatin was isolated essentially according to the method of Johns and Butler [5] with some modifications as described previously [4].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). Approximately 200 mg of total acid-extracted (0.2 M H2S04) histone was dissolved in 10 ml of 6 M urea containing 2% (v/v) 2-mercaptoethanol, loaded onto the column and eluted with 0.02 M HCI (Fig. 1 a).Cation-exchange chromatography was carried out using LKB CM-Trisacryl M equilibrated with 0.05 M sodium acetate adjusted to pH 5.00 with HC1. A linear NaCl gradient was used to elute the H2A(,,-enriched fraction B (Fig. 1 b). Fraction B was then further purified by passage over a BioGel P-60 column equilibrated with 0.02 M HCl, 0.05 M NaCl as described previously [6]. The eluant contained in addition, 10 mM sodium bisulfite, 1 mM PhMeS02F and 1 mM Natosyllysylchloromethane, solubilized first in a small volume of propan-1-01 (Fig. 1 c). The H2A(,,-containing fraction was sometimes recycled over the P-60 column if there was still some contamination evident on gel electrophoresis.
Analytical proceduresPolyacrylamide gel electrophoresis (PAGE) employing the detergent Triton X-100 was performed in glass tubes (9 x 0.5 cm) [7, 81.Histone samples were hydrolysed under vacuum for 24 h at 110°C in constant-boiling HCl containing 0.025% (w/v) phenol as antioxidant unless otherwise stated. The analyses were done with either ninhydrin methodology (Beckman 119 automatic analyser) or using hypochlorite and o-phthalaldehyde post-column fluorescence detection (Waters Associates HPLC amino acid analyser [9]).Fragmentation of H2A(,, N-Bromosuccinimide (SucNBr) cleavage [lo] was performed as described previously [ll]. The increase...
