Chromatin fragments containing bovine 1.715 g ml-1 satellite DNA. Purification by chromatography on malachite green DNA affinity resin.

Abstract

Chromatin fragments containing bovine 1.715 g ml-1 satellite DNA (1.715 satellite chromatin) were purified in order to test for differences between satellite chromatin and unfractionated chromatin. The final purification procedure involved digestion of isolated nuclei with Eco RI restriction endonuclease, solubilization of a fraction of the chromatin by lysis of the nuclei at low ionic strength, and chromatography of the soluble chromatin fragments on malachite green DNA affinity resin. Digestion of chromatin within steer kidney and thymus nuclei by Eco RI reached limits at which 22% and 37%, respectively, of the potential sites for the enzyme in the 1.715 satellite chromatin were cleaved. A test of the 1.715 g ml-1 satellite DNA extracted from digested thymus nuclei showed it was not significantly nicked at Eco RI sites. Fractionation of soluble chromatin fragments produced by Eco RI digestion on the basis of size using either sucrose gradient centrifugation or chromatin gel electrophoresis yielded 1.715 satellite chromatin of 60-80% purity. However, chromatography of the soluble chromatin fragments on columns of malachite green resin gave 1.715 satellite chromatin of higher purity and in greater amounts than could be obtained with the sucrose gradients or chromatin gels. Using malachite green resin, hundreds of micrograms of 1;715 satellite chromatin could be obtained with a purity that was usually at least 95% as determined by melting of the extracted DNA.