Isolation and characterization of chromatin subfractions from rat liver

Rat-liver chromatin was digested with micrococcal nuclease at low ionic strength in the presence of a low concentration of CaCl2. The nuclease digest was successfully separated into three fractions, P1, P2, and P3, by gel filtration on a column of Sepharose 2B. P1 fraction was shown to be a mixture of long fragments of partially digested chromatin by the sedimentation profile or by electrophoresis of DNA. P2 fraction contained four histones H2A, H2B, H3, and H4 in almost equal amounts, together with nonhistone protein of low molecular weight. The DNA was composed of three or four fragments less than 300 base pairs long. From the Kav value of the P2 fraction, the average size was estimated to be about 240 base pairs. On analytical ultracentrifugation, this fraction exhibited a monophasic boundary and a sedimentation value of 13.7S. P3 fraction contained nonhistone proteins which showed a molecular weight larger than that of H1 histone. The size of DNA was estimated to be less than 50 base pairs from the Kav value. Based on these results, the P2 fraction was concluded to consist of nucleosome monomer enriched in nonhistone proteins. The P3 fraction is presumably the nuclease-sensitive or internucleosome portion, which contains small amounts of nonhistone proteins.