Fractionation of hen oviduct chromatin into transcriptionally active and inactive regions after selective micrococcal nuclease digestion

Abstract

This report describes a simple method for the preparation of transcriptionally active and inactive chromatins from the hen oviduct. Oviduct nuclei were incubated with micrococcal nuclease until 2–3% of the DNA was rendered acid-soluble. The nuclear suspension was centrifuged following digestion. The resulting first supernatant fraction (1SF) contained chromatin particles released from intact nuclei. The nuclei in the sediment were lysed and centrifuged again to yield a pellet and a second supernatant fraction (2SF). DNA extracted from the 1SF and 2SF were hybridized to radioactive DNA complementary to purified ovalbumin mRNA for quantitation of ovalbumin genes. The results demonstrated that 1SF DNA was 5–6 fold enriched in the ovalbumin gene as compared to total nuclear DNA, whereas 2SF DNA was 5–6 fold depleted in this transcribed sequence. The fractionation procedure is related to transcriptional activity since nonexpressed genes such as the gene for ovalbumin in hen liver and the genes for globin in the oviduct were not preferentially localized in 1SF DNA. Electrophoretic analysis of the DNA in the active fraction (1SF) revealed a single band which corresponded to mononucleosome-length DNA (145–200 base pairs). Larger molecular weight oligonucleosome fragments were observed in the 2SF. To determine whether the fractionation of the ovalbumin gene was related to DNA fragment length, 1SF and 2SF DNA were combined and separated into four size classes by preparative electrophoresis. Hybridization studies revealed that the concentration of the ovalbumin coding sequence in mononucleosome-length DNA was 5 fold greater than in undigested DNA, and 16 fold greater than in the fraction containing DNA fragments >1200 bp in length. The concentration of transcriptionally inactive genes in mononucleosome-length DNA, in contrast, was equivalent to their concentration in the larger molecular weight DNA fragments. These results suggest that micrococcal nuclease selectively excises nucleosomes from oviduct chromatin containing the ovalbumin gene. This selectivity permits the recovery of mononucleosomes in the 1SF which are enriched in the ovalbumin gene and oligonucleosomes in the 2SF which are depleted in this sequence.