Deoxyribonucleoproteins and the tissue-specific restriction of the deoxyribonucleic acid in chromatin.

Abstract

Although histones appear necessary for the restriction of DNA (Bonner et al. 1968), their lack of tissue and species specificity point to the necessary presence of other factors in chromatin that are required for the highly organ-specific genetic restriction of the DNA. The present paper analyses the type of restriction of the DNA in native chromatin by analysing the type of restriction in hybrid chromatins composed of portions of the dissociated products of the chromatins from two different organs. Techniques for the isolation of chromatin, the RNA synthesis in vitro, the isolation ofRNA formed in vitro from chromatin template and the DNARNA hybridization have been reported elsewhere (Spelsberg & Hnilica, 1970a). Briefly, isolated rat (male Sprague-Dawley) liver and thymus nuclei (Blobel & Potter, 1966) were extracted three times with 80mM-NaCl-20mM-EDTA, pH 6.3, then once with 0.3m-NaCl and twice with 0.01 x SSCt with a Teflon homogenizer. Each extraction was followed by centrifugation at 4000gav. for 20min. The RNA synthesis in vitro was carried out for 4h. In each reaction 200,ug ofDNA or 600-1000,ug ofchromatin DNA was used as a template with 400-500 units of RNA polymerase from Micrococcus luteus (Nakamoto, Fox & Weiss, 1964). Each reaction mixture contained 400,umol of tris-HCl buffer, pH 8.0, 25pumol of MgCl2, 10,umol of MnCl2, 30,umol of 2-mercaptoethanol, 4,tmol each of GTP, ATP and CTP, 2.35,umol of [3H]UTP (50,uCi/,mol; from Schwarz BioResearch Inc., Orangeburg, N.Y., U.S.A.), 10,tmol of spermidine phosphate and 50,ug of bentonite. The reactions were carried out in the presence of 0.1M-NaCl in a final volume of 5.0rml at room temperature. After incubation the reaction mixture was made 0.3M with respect to KCI, incubated for 20min and centrifuged at 2000g for 10min. This procedure releases 85-95% of the