Analysis of highly purified satellite DNA containing chromatin from the mouse

Abstract

A purification scheme for satellite DNA containing chromatin from mouse liver has been developed. It is based on the highly condensed state of the satellite chromatin and also takes advantage of its resistance to digestion by certain restriction nucleases. Nuclei are first treated with micrococcal nuclease and the satellite chromatin enriched 3-5 fold by extraction of the digested nuclei under appropriate conditions. Further purification is achieved by digestion of the chromatin with a restriction nuclease that leaves satellite DNA largely intact but degrades non-satellite DNA extensively. In subsequent sucrose gradient centrifugation the rapidly sedimenting chromatin contains more than 70% satellite DNA. This material has the same histone composition as bulk chromatin. No significant differences were detected in an analysis of minor histone variants. Nonhistone proteins are present only in very low amounts in the satellite chromatin fraction, notably the HMG proteins are strongly depleted.

Reconstitution of mononucleosomes: characterization of distinct particles that differ in the position of the histone core

“…Histone analysis was done by SDS polyacrylamide gel electrophoresis (18% acrylamide, 0.45% bisacrylamide) as described in ref. 19.…”

Purification of Proteins Associated with Specific Genomic Loci

“…During the past 25 years, various chromatin isolation strategies have been pursued to establish locus-specific protein composition (Boffa et al, 1995; Ghirlando and Felsenfeld, 2008; Griesenbeck et al, 2003; Jasinskas and Hamkalo, 1999; Workman and Langmore, 1985; Zhang and Horz, 1982). While each achieved enrichment of the targeted region, none gave material of sufficient amount and purity to allow identification of bound factors.…”

A repetitive DNA-directed program of chromosome packaging during mitosis

“…It was speculated that Mg 2+ is a trigger of the condensation-decondensation transition of chromosomes during mitosis (Jerzmanowski and Staron, 1980). Cations are known to regulate the compact state of tandem repeats in heterochromatins (Horvath and Hörz, 1981; Zhang and Horz, 1982). The Ca 2+ concentration is higher on the AT-rich axis of mitotic chromosomes, probably binding to the minor groove of the AT-rich sequence (Strick et al, 2001a).…”

“…As such, cation concentration increase during mitosis is thought to stimulate the formation of new RPs and stabilize preexisting RPs. Ca 2+ binds satellite repeats more strongly than bulk chromatins (Zhang and Horz, 1982). While Ca 2+ is enriched on the axis, the Mg 2+ concentration is higher mainly in the halo of mitotic chromosomes (Strick et al, 2001a).…”