FIGURE 2.

Separation, identification, and post-translational modification of H3 histones. A, separation of H3 variants, co-eluted from reversed phase HPLC, in an acid urea (AU) gradient gel with Triton X-100 from 0 to 10 mm where H3 variants co-electrophorese under AU conditions at 0 mm Triton. The slant in electrophoretic mobility across the gradient, marked by methylene blue at the discontinuous running front, by nonhistone proteins in this partially purified HPLC H3 pool and by two guidance lines, results from the glycerol gradient used to stabilize the gradient gel (16). The characteristic high affinity for Triton of core histone H3 is modulated by differences of exposed side chains within the histone fold of the H3 variants (Fig. 1B) (48, 49). The broken line marks the optimal concentration of 6 mm Triton X-100 in 8 m urea and 1 m acetic acid for the separation of the H3 variants of U. maydis. B, pattern of H3 variants in AUT gels, pulse-labeled in vivo with [³⁵S]methionine for 20 min at 0.1 mCi/liter (lanes A and B) or with [³H]lysine for 30 min at 0.4 mCi/liter (lanes C and D) or chased for 24 h in fresh SD medium into stationary phase (lanes E and F). Coomassie-stained gel lanes are marked by c, and 33-day fluorographic exposures are marked by f. Nonacetylated and hexa-acetylated H3 species are marked by 0 and 6, respectively. C, pattern of histone H3 variants in logarithmically growing cultures, incubated with 4 mCi/liter [³H]acetic acid for 5 min (lanes A and B) or 20 min (lanes C and D). Coomassie-stained gel lanes are marked by c, and 18-day fluorographic exposures are marked by f. Nonacetylated through hexa-acetylated H3 species are marked by numbers 0–6, respectively.