Peculiarities of nuclear genome transcription in the early stages of liver regeneration. Complex nature of the activating effect of the cytoplasmic regenerating factor on transcription in isolated rat liver nuclei

The cytosol fractions from resting rat liver and from the liver 1 hour after partial hepatectomy which were not adsorbed on DEAE-cellulose were subjected to gel filtration with a view of elucidating the mechanism of its activating effect on the RNA-synthesizing ability of isolated nuclei of resting liver. The activating effect of this fraction is a complex one and includes both specific and non-specific factors. A subfraction typical for regenerating liver was detected, which activates RNA elongation in a synthesizing system with isolated nuclei of resting liver.     

Effect of cytosol on RNA-synthesizing ability of isolated nuclei of rat hepatocytes

The addition of cytozol to isolated nuclei of the liver is accompanied by a rise in the amount of a new synthetizing RNA and changes in its nucleotide composition. An increase in the intensity of [3H] ATP incorporation at the end of incubation evidences for polyadenylation of the reaction product. Under the effect of cytozol 30-32% of RNA synthetized de novo is released. Heating of cytozol (50 degrees C, 15 min) leads to a loss of its ability to activate RNA synthesis by isolated nuclei.     

DNA degradation in isolated rat liver nuclei

Purified rat liver nuclei were incubated at 4°C. After different intervals of incubation aliquots of the nuclear suspension were taken and DNA was extracted by a common SDS-phenol-chloroform procedure. The fractionation of DNA by agar gel electrophoresis revealed large DNA fragments. It was shown that the well-known DNA degradation to monomers and its multiples is preceeded by an earlier breakdown of DNA into characteristic large fragments.     

Histone binding to isolated rat liver nuclei

Calf thymus histone H3 bound irreversibly to the isolated rat liver nuclei. The rate and extent of binding was a function of the incubation period and the concentration of both H3 and nuclei, but independent of the temperature. The binding was saturable and was inhibited by simultaneous presence of various histones. Approximately 94% of the bound H3 was associated with nuclear membrane fraction.     

Characterization of ribonuclease P isolated from rat liver cytosol.

Rat liver ribonuclease P was isolated from a cytosolic fraction and shown to have optimal activity in the presence of 1 mM MgCl2 and 150-200 mM KCl using Escherchia coli pre-tRNA(Tyr) as substrate. In cesium sulfate isopycnic density gradients, the enzyme had a buoyant density of 1.36 g/ml, indicating that it is a ribonucleoprotein complex. Analysis of the RNAs in the enzyme sample purified through two successive Cs2SO4 density gradient steps revealed the copurification of two major species of RNA (RRP1 and RRP2) along with several less abundant RNAs. Rat liver ribonuclease P activity was insensitive to micrococcal nuclease pretreatment. However, the nuclease-treated preparations contained several incompletely degraded RNA species that may have been sufficient to support the ribonuclease P activity. When RNase A was substituted for micrococcal nuclease, the ribonuclease P activity was diminished by greater than 90%, suggesting the requirement for an RNA subunit for activity.     

Initiation of RNA synthesis in isolated rat liver nuclei

Isolated rat liver nuclei were incubated under conditions when RNA polymerase I or RNA polymerase II was preferentially active. It was shown that [gamma-32P] ATP and [gamma-32P] GTP were incorporated into phenol extractable, TCA-precipitable material. RNase, actinomycin D, heparin and, in the case of RNA-polymerase II, alpha-amanitine inhibited precursor incorporation. These data are interpreted as evidence in favour of the initiation of RNA synthesis in isolated rat liver nuclei.     

Effect of cytosol on DNA synthesis in isolated HeLa cell nuclei

Cytosol obtained by centrifugation of cytoplasm from synchronized S-phase HeLa cells at 200 000 × g for 30 min had a stimulatory effect on the rate and extent of DNA synthesis in isolated nuclei. The cytosol preserved the ability of isolated nuclei to initiate early nascent intermediates (primary DNA pieces). The stimulatory activity was partially separated from the DNA polymerase activity present in the cytosol.     

Effect of vitamin E on transcription in isolated nuclei and rat liver chromatin in normal status and in E-hypovitaminosis]

The effect of alpha-tocopherol on the RNA-polymerase activity in isolated rat nuclei and chromatin from normal and E-deficient rats and the possible role of tocopherol-binding proteins in this process were studied. Some differences in the RNA-polymerase activities of the nuclei were found; however, in vitro added alpha-tocopherol had no effect on the level of the label incorporation into RNA. No effect of alpha-tocopherol on this process was observed after addition of cytosol either. Analysis of chromatins from normal and E-deficient rats revealed no differences in their RNA-polymerase activities. In vitro added alpha-tocopherol increased the RNA-polymerase activity of normal (but not of vitamin E-deficient) rats. Some differences in the RNA-polymerase activities were noted after addition to the incubation medium of the Triton X-100-solubilized nuclear fraction specifically binding alpha-tocopherol. This effect was enhanced in the presence of exogenous alpha-tocopherol. The susceptibility of chromatin from normal and E-deficient rats to DNAse I hydrolysis was also found to be different. It was concluded that vitamin E can influence the RNA-polymerase activity of the nuclei and chromatin as well as the chromatin structure and that alpha-tocopherol-binding proteins are necessary for the vitamin E effect on the RNA-polymerase activity to be manifested.