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Exclusion chromatography of the acid-extracted histones was done at room temperature on Bio-Gel P-60 as described previously [21]. Cation-exchange chromatography was performed on CM-cellulose (Whatman CM-52) in a 0.05 M acetic acid/Tris buffer in 6 M urea at pH 5.3 using a linear NaCl gradient [lo]. Histone H2B(z, was purified by reverse-phase chromatography on a preparative Beckman/Ultrapore C3 column (1 x 25 cm) using a 0-70% acetonitrile gradient in 0.05 M heptafluorobutyric acid. Peptides were separated on FBondapak/Waters (0.4 x 30 cm) or an Ultrapore/Beckman (0.4 x 7.6 cm) column using either trifluoroacetic acid or heptafluorobutyric acid as counter-ion and a linear 0-70% acetonitrile gradient. Analytical proceduresPolyacrylamide gel electrophoresis was performed in acetic acid/ 2.5 M urea gels [23] in the presence of SDS [24] or the non-ionic detergent Triton X-100 [25].Proteins were hydrolyzed in 5.7 M HCl, 0.025% (mass/vol.) phenol in evacuated and sealed glass vials at 110°C for 24 h. Peptides were hydrolyzed in 5.7 M HCI, 0.025% phenol (mass/vol.) (200 p1) by the gas-phase procedure at 150°C for 90 min in a screw-capped 50 ml vial with a teflon sliding valve [26]. Amino acids were quantified after ion-exchange chromatography and post-column detection with hypochlorite and o-phthalaldehyde [27] (Waters HPLC amino acid analyzer). No corrections for incomplete hydrolysis or the destruction of amino acids were made. All amino acid composition data are based on duplicate analysis of the protein and peptides generated from at least two isolation procedures. SequencingPreliminary sequence studies automated N-terminal degradation on the unfractionated wheat histone H2Bs were done at the 100-nmol level on a spinning cup sequencer as described previously 1281. For all other automated degradations a gas-phase sequencer was used as described previously [29]. The identity and purity of the HPLC fractions were determined by a manual gas-phase sequencing procedure on glass-filter discs [30]. All prominent peaks were subjected to Nterminal sequence analysis and the reported peptides sequenced at least twice (supplement Figs 2, 3).Cyanogen bromide cleavage. Histone H2B was cleaved in 70% formic acid by the addition of a 150 molar excess of CNBr 1311. The reaction mixture was diluted in H20 and lyophilized.Slaphylococcus aureus V8 protease (Miles) digestion was performed on approximately 1 mg protein in a 0.05 M Tris buffer pH 18. Hayashi, H., Nomoto, M. & Iwai, K. (1980) 7.8 at 37°C at an enzymc/substrate mass ratio of 1:20 [32]. The digest was centrifuged and subsequently fractionated by HPLC. The digestion of the N-terminal region (CNZ) at pH 4.0 was often incomplete and occurred predominantly between a Glu-Lys at position 17.Endoproteinase Arg-C (Boehringer) digestion was executed on approximately 1 mg protein in a 1% (niassjvol.) NaHC03 buffer pH 8.0 at 37°C at an enzyme/substrate ratio of 1 : 50 [33]. The digest was centrifuged and subsequently fractionated by HPLC.Cleavage in dilute acid was performed in 0.0...

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...