Preparation of soluble chromatin and specific chromatin fractions with restriction nucleases

Abstract

By digestion of rat liver nuclei with EndoR HaeIII, EndoR EcoRI, and EndoR Bam and subsequent lysis of the nuclei approx. 90%, 40%, and 45%, respectively, of the chromatin were solubilized. The plateau values of solubilization are in agreement with a model in which the chromatin strands are crosslinked and/or attached to a supporting structure. The distribution of DNA lengths in the soluble and insoluble chromatin fractions were determined. According to digestion experiments with restriction nucleases rat liver DNA contains highly repetitive sequences, some of which are arranged in tandem repeats of 95 and 380 nucleotide pairs, respectively. With EndoR EcoRI chromatin containing the repetitive RNA was preferentially solubilized and, by subsequent sucrose gradient centrifugation, purified to about 90%. The useful properties of chromatin prepared by the specific action of restriction nucleases are discussed.

Age‐related analysis of EcoRI generated satellite DNA‐containing chromatin of rat liver

“…This may be attributed to the presence of high salt (>100mM) of monovalent cations (Na ÷ and K 4) in the digestion buffer which suppresses the endogenous endonuclease activity [17].…”

Higher‐Order Structures of Chromatin in Solution

“…It should be emphasized that in these studies ofchromatin higher-order structures, we are investigating the properties and conformational states of pieces of chromatin dissected out from chicken erythrocyte nuclei by brief nuclease digestion. Whether the additional constraints of the incorporation of chromatin into loops [1,2] or domains [3,4] modifies these conformational states remains to be shown.…”

“…In its most contracted form the diameter of the hydrated supercoil has been found from the radial distribution function to be 34 nm. Models for the arrangements of core particles in the 34-nm supercoil are discussed.There is now growing evidence that the DNA in metaphase chromosomes and in the interphase nucleus is organised into discrete loops which contain some 30000-90000 basepairsofDNA [1,2]or inchromatin domains of average size 34000 bases of DNA [3,4]. The DNA of these domains or loops when complexed with chromosomal proteins in inactive regions of chromatin and in metaphase chromosomes is highly compacted and several orders of chromatin structure must exist above the linear array of nucleosomes.…”

Relationship between DNA fragmentation and apoptosis in the programmed cell death in the rat prostate following castration

“…Such nuclear pellets from intact and castrated animals were next extracted with 1.5 mM EDTA-3 mM EGTA, pH 7.4 media (i.e., 7 ml/gm starting weight of prostatic tissue) for 30 min at 4°C. This media has been demonstrated to solubilize DNA of < 1,000 bp size [38]. The samples were next centrifuged at 9,OOOg x 20 min (4°C) to separate the < 1,000 bp size molecular weight fragments extracted into the supernatant from high molecular weight DNA remaining in the resulting pellet.…”