Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases

Abstract

The action of micrococcal nuclease, DNase I and DNase II on mouse TLT hepatoma chromatin revealing the periodicity of its structure as visualized by denaturing and non-denaturing gel electrophoresis, was consistent with the action of these enzymes on other chromatins. Micrococcal nuclease showed a complex subnucleosome fragment pattern based on multiples of 10 base pairs with a prominant couplet at 140/160 base pairs and the absence of the 80 base pair fragment. This couplet of the core and minimal nucleosome fragments was conspicuously present in the mononucleosomes found in the 11S fractions of a glycerol gradient centrifugation. DNase I and II produced a fairly even distribution of a 10 base pair increasing series of fragments to about 180 base pairs, a pattern also repeated in the DNA of nucleosome glycerol-gradient fractions. In limited digestions by these nucleases multinucleosomic DNA fragments are pronounced. These fragment lengths are multiples of an estimated average repeat length of nucleosome DNA of 180 base pairs. The action of the endogenous Mg/Ca-stimulated endonuclease produced only limited cuts in the hepatoma chromatin resulting primarily in multi-nucleosomic DNA fragment lengths and only upon lengthy digestion limited subnucleosomic, 10-base-pair multiple fragments are produced. The putative euchromatin-enriched fractions (50-75S) of the glycerol gradient centrifugation of autodigested chromatin, similarly, contained primarily the multinucleosomic DNA fragment lengths. These results are consistent with our previous electron microscopic demonstration that autodigested chromatin as well as the putative euchromatin-enriched fractions were composed of multi-nucleosomic chromatin segments containing a full complement of histones.

Eukaryotic ternary transcription complexes. I. The release of ternary transcription complexes of RNA polymerases I and II by the endogenous nucleases of rat liver nuclei

“…In the light of these findings, the associated release of RNA polymerases and part of the nuclear DNA in the autodigestion technique invites speculation that the released DNA may be enriched for transcribed sequences. A preliminary report confirming this has already been published (34) and indirect evidence from other groups has suggested that hepatic endogenous nucleases may preferentially digest transcriptionally active chromatin (27,35). A more detailed analysis of the DNA sequences released in our system is in preparation.…”

Tellurite-induced oxidative stress leads to cell death of murine hepatocarcinoma cells

“…To this end, Transplantable Liver Tumor (TLT) cells-a murine hepatocarcinoma cell line-were cultured in the absence or in the presence of tellurite and biochemical and morphological determinations were carried out in order to determine the extent and type of cell death. TLT cells have been previously used to assess both in vivo and in vitro anticancer cytotoxic effects (Duerksen and Connor 1978;Taper et al 1987;Verrax et al 2004;Crokart et al 2005;Benites et al 2008). Experimental tests included lactate dehydrogenase (LDH) leakage (cell survival), ATP levels (functional ability of cells), and caspase-3 (DEVDase) activity and cleavage of poly(ADPribose)polymerase (PARP) to assess the nature of the cell demise.…”