Isolation and characterization of the DNA fraction of rat liver chromatin which binds polylysine

Abstract

The structure of eukaryotic chromatin has been investigated by isolating and analyzing the "accessible" DNA fraction of rat liver chromatin. This DNA fraction has been isolated by titrating the chromatin with the protese-resistant D isomer of polylysine to bind the "accessible" DNA sites. After removal of chromosomal proteins by digestion with pronase, all DNA not protected from attack by bound polylysine was removed by digestion with DNase. Even after exhaustive treatment with pronase and DNase approximately 30% of the chromatin DNA remains resistant to nuclease attack. Analysis of the isolated DNA shows it to be mainly double-stranded with an average size of 200-250 base pairs. The DNA is slightly A-T rich and contains both repetitive and "single-copy" nuleotide sequences. The results suggest that there are extensive regions in chromatin where the DNA is not tightly complexed with protein. Furthermore, the DNA of these regions is similar in gross properties to the DNA of the total genome.

Transcription of the nonrepeated fraction of "accessible" DNA in rat liver chromatin.

“…All of our results as well as those of Axel et al (23) are based on the assumption that accessible DNA is real and can be isolated by polylysine binding. In a previous paper (14) we summarized the reasons for believing this assumption to be reasonable. If, however, polylysine can migrate from its original binding sites to different DNA molecules during the isolation procedure, it is possible that an essentially random population of DNA sequences could be produced and this process would lead to exactly the results that we and Axel et al find.…”

“…The chromatin was purified by washing in buffers of low ionic strength and sonicated briefly as described (9). Isolation of Accessible DNA: Accessible DNA was isolated from chromatin by utilizing poly-D-lysine binding as described previously (14). In short poly-D-lysine-HBr (avg.…”

“…Chromosomal proteins were then removed by digestion with pronase, and all DNA not bound to polylysine was removed by digestion with DNase I. Polylysine was then removed by dissociating the polylysine-DNA complex in 2M NaCl and separating the mixture of DNA and polylysine on a colun of hydroxyapatite (HAP). The validity of the isolation procedure and the physiochemical properties of the DNA preparation are reported elsewhere (14). The DNA was further treated to remove a small fraction (s 15%) of single-stranded DNA routinely found in accessible DNA preparations.…”

“…Isolation of Whole Chromatin DNA: Whole chromatin DNA was isolated from chromatin as described previously (14). The purified DNA was extensively sonicated to reduce the fragment length to the same size as that of the accessible DNA preparation (200-250 base pairs).…”