Isolation of chromatin fragments rich in non-histone protein after endonuclease digestion of rat liver nuclei

Detergent-washed rat liver nuclei, prepared in the presence of a protease inhibitor, were incubated for up to 60 min at 37 °C. The action of endonucleases produced chromatin fragments which could be removed from the nuclei by extraction with 8 M urea 50 mM phosphate, pH 7.6, 15% of the total nuclear DNA being extracted. No DNA could be detected in this extract after incubation of nuclei at 4 °C. The chromatin fragments were sedimented by centrifugation at 90000 g or by chromatography on Sepharose 4B. The DNA fragment sizes were similar to those found in nucleosome particles but the protein/DNA ratio was approx. 5.3:1. The nuclei were prelabelled with [³H]tryptophan and 60% of the label present in the 8 M urea extract was found to sediment with the chromatin fraction. SDS polyacrylamide gel electrophoresis of the latter showed the presence, in addition to histones, of at least 25 polypeptide species of tightly bound non-histone proteins with molecular weights in excess of 30000.