Structure of active chromatin: isolation and characterization of transcriptionally active chromatin from rat liver

Abstract

Rat liver nuclei were isolated in low-ionic-strength buffer in the absence of bi- and multi-valent cations. Digestion of these nuclei by endogenous nuclease, micrococcal nuclease and DNase I revealed that a minor chromatin fraction was preferentially digested into poly- and oligo-nucleosomes. Southern blot hybridization with various active gene probes confirmed that these chromatin fragments represent coding and 5ƀ upstream regions of transcriptionally active chromatin. Active chromatin fragments were released selectively into the medium, with inactive chromatin remaining inside the nuclei, under the above ionic conditions. The inclusion of bivalent cations during the digestion of nuclei reversed the solubility behaviour of active chromatin. Rearrangement and exchange of histone H1 between chromatin fragments was prevented by using low-salt conditions in all steps in the absence of bivalent cations. All histones, including H1, were present in stoichiometric amounts in this active chromatin fraction. Active nucleosomes showed a lower electrophoretic mobility than bulk nucleosomes in an acrylamide/agarose composite gel in the absence of Mg2+, but were selectively bound to the gel in the presence of this ion.