Dissociation and reconstitution of chromatin without appreciable degradation of the proteins.

When chromatin from Novikoff hepatoma ascites cells was dissociated in 3 M NaCl – 7 M urea either at pH 6 or 8, degradation of chromosomal proteins was observed in two-dimensional gel electrophoretic patterns. This degradation was not prevented by 50 mM NaHSO₃ but was prevented by 1 mM PMSF (phenylmethylsulfonyl fluoride). Reconstitution of the chromatin components dissociated in 3 M NaCl – 7 M ure ? 0.05 M sodium acetate (pH 6.0) containing 1 mM PMSF resulted in reassociation of DNA, histones and the major nonhistone proteins (B24, B26, B33, BE, BJ, C1, C6, CG, CH, CM, C14, CP, C18, CR, CS and C25). Two-dimensional gel electrophoresis showed that although the proportion of the nonhistone proteins to histones was lower in reconstituted than in native chromatin, the template activity of the reconstituted chromatin was similar to that of native chromatin.