Methylation and degradation of chromatin DNA in isolated rat liver cell nuclei]

Methylation of chromatin DNA in rat liver cell nuclei incubated in a medium with [3H]CH3-S-adenosyl methionine was studied. It was shown that under the given experimental conditions DNA methylation and chromatin degradation by endogenous nuclear nuclease (nucleases) with a formation of chromatin structural subunits occur simultaneously. An analysis of methylated chromatin DNA degradation products based on a number of approaches demonstrated a predominant methylation of extra-nucleosomal DNA. The data obtained suggest that chromatin of isolated nuclei contain sites with supermethylated DNA fragments incorporating not less than 400 nucleotide pairs. These sites possess an increased sensitivity to endogenous nuclease.     

Iron-induced DNA damage and synthesis in isolated rat liver nuclei.

Incubation of iron with isolated rat liver nuclei stimulated fragmentation of single-stranded DNA, incorporation of [3H]thymidine into DNA and the binding of 59Fe to DNA. FeCl2 was about twice as active as FeCl3. Lipid peroxidation took place in nuclei incubated with FeCl2, but not with FeCl3. Generation of reactive forms of oxygen was required for iron-mediated DNA damage, but evidence for direct interaction of reactive oxygen with DNA was not found. Apparent adducts of iron bound to DNA seemed to be formed by an enzymic mechanism.     

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.     

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.     

Characteristics of the DNA polymerase beta-directed unscheduled DNA synthesis in isolated nuclei from adult rat liver

Isolated nuclei from adult rat liver have been used as a model system to define several characteristics of the unscheduled DNA synthesis supported by DNA polymerase beta. Many of these characteristics have been found to reflect some catalytic properties (pH optimum, divalent cations requirement, dependence on all four deoxyribonucleoside triphosphates, apparent Km for dTTP) as well as sensitivity to various agents (differential inhibitors of eukaryotic DNA polymerases, phosphate, DNA intercalating drugs, chemical or thermal denaturation) commonly regarded as typical of DNA polymerase beta itself. Given the new picture of the enzymology of DNA repair synthesis which has recently emerged, none of the above characteristics seem to be suitable candidates as diagnostic tools of a repair polymerization process.     

Stimulation of DNA synthesis by an inositol polyphosphate-activated protein phosphatase in calcium-deprived rat liver cells

Specific stages of the prereplicative G1 phase of the cell cycle in nonneoplastic cells requires extracellular Ca2+ for successful transition. These are the G0-G1 and the G1-S transitions. A variety of agents are able to replace Ca2+ and to at least partially stimulate cells to replicate their chromosomes. One of these agonist, inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], has been demonstrated by us to also stimulate the activity of a phosphoprotein phosphatase. The addition of a purified preparation of the protein phosphatase to Ca2(+)-deprived G1-S-blocked T51B rat liver cells stimulates a rapidly responding fraction of cells to enter their S phase, and this effect is blocked by protein phosphatase inhibitors heparin and inhibitor 2.     

Calmodulin stimulates DNA synthesis by rat liver cells

Incubation in low (0.02 mM)-calcium medium prevented T51B rat liver cells from initiating DNA synthesis. Raising the calcium concentration in the medium from 0.02 to 1.25 mM caused these arrested cells to initiate DNA synthesis 1–2 hours later. The possibility of this rapid DNA-synthetic response to calcium addition being mediated through Ca-calmodulin complexes was suggested by the following observations: It was blocked by the putative Ca-calmodulin blockers chlorpromazine and trifluoperazine; the trifluoperazine-inhibited cells were stimulated by purified rat calmodulin; and purified rat calmodulin itself (10^?7 to 10^?6 moles/l) mimicked calcium action, unless the already low ionic calcium concentration in the calcium-deficient medium was reduced further by adding the specific calcium chelator EGTA.     

Nucleoside triphosphate inhibition of DNA synthesis in isolated rat hepatic nuclei

Adenosine triphosphate was found to inhibit DNA synthesis in preparations of isolated nuclei from rat liver, when added at concentrations reported in the literature to be in common use. Other nucleoside triphosphates also profoundly affect both total synthesis and the kinetics of the reaction when present in excess. These effects are not completely uniform in nuclei prepared by different methods.     

Stimulation of DNA synthesis in rat liver and kidney by cold acclimation.

Exposure of adult Wistar female rats for 7 days to 4 degrees C leads to a marked increase in the weight of the liver and kidney, caused by an increased content of DNA and an increased number of cells in these organs. The weight as well as the DNA content of the cross-striated muscle do not change appreciably. Acclimation of the warmblooded rat to cold stimulates mitosis indirectly in cells capable of division, similarly as it stimulates directly the mitotic activity in mouse and human cells cultured and adapted to cold in vitro.     

Formation of deoxyribonucleoside 5'-monophosphates dependent on DNA synthesis in nuclei isolated from regenerating rat liver

Hydrolysis of deoxyribonucleoside 5'-triphosphate, resulting in deoxyribonucleoside 5'-monophosphate formation dependent on DNA synthesis, was observed in nuclei isolated from regenerating rat liver. The intensity of the hydrolysis in nuclei varied at different times after partial hepatectomy, showing its maximum at 48 h. The rates of DNA synthesis altered corresponding to the intensities of hydrolysis. Proportionality between decrease in DNA synthesis and decrease in dNMP production was also observed in nuclei treated with various inhibitors of DNA synthesis. The formation of dNMP was detected with the four DNA substrates, indicating no involvement of specific dNTPase . Although regenerating nuclei contained a nonspecific dNTPase activity that can cause release of dNMP , this activity was independent of DNA synthesis and not inhibited by inhibitors of DNA synthesis. These results indicated that regenerating liver nuclei had two different activities for dNMP production; one is DNA synthesis-dependent, and the other is a non-specific dNTPase activity. This paper has focused on the former activity.     

Decreased ribonucleic acid synthesis in isolated rat liver nuclei during starvation

1. Isolated nuclei from starved rats showed a lowered incorporation of [(14)C]UMP into RNA. 2. The Mg(2+)-dependent incorporation was decreased by 30% after 1 day of starvation, but incorporation in the presence of Mn(2+) and ammonium sulphate decreased only after longer periods of starvation. 3. RNA synthesis by nuclei in the presence of excess of added RNA polymerase was unchanged after 1 day of starvation and was inhibited by 20% after 4 days. 4. The capacity of nuclei to bind actinomycin D was unchanged after 1 day and was decreased by 20% after 4 days of starvation.     

Inhibition of DNA synthesis by isolated liver nuclei from frog virus 3 infected mice

Incorporation of [³H] dTTP into acid insoluble material by liver nuclei from mice infected for 3 hours with 5 × 10⁸ PFU of Frog Virus 3 was markedly decreased. The incorporation was noticeably stimulated by the addition of calf thymus DNA. It has been verified that the inhibition of incorporation by the nuclei from infected animals was not related to an increase of deoxyribonuclease activity and that the product of the reaction showed the properties of DNA.     

Three-dimensional distribution of DNase I-sensitive chromatin regions in interphase nuclei of embryonal carcinoma cells

In situ nick-translation allows the visualization of nuclease-sensitive chromatin regions in interphase nuclei. We have analyzed the three-dimensional (3-D) distribution of DNase I-sensitive regions of chromatin in nuclei from mouse P19 embryonal carcinoma cells by making optical sections using confocal scanning laser microscopy. In undifferentiated as well as embryonal carcinoma cells differentiated in vitro, DNase I-sensitive regions of chromatin are observed as discrete spots in the nucleus. These spots represent clusters of DNase I-sensitive sites. By optical sectioning, we show that these spots are preferentially, but not exclusively, localized at the nuclear periphery. No differences were observed in the spatial distribution of DNase I-sensitive sites in P19 EC cells or the differentiated P19 END-2 cells. Furthermore, we did not observe differences in the distribution of DNase I-sensitive chromatin regions during the cell cycle. These findings indicate, at least for P19 mouse embryonal carcinoma cells and their differentiated derivative END-2, that the compartmentalization of DNase I-sensitive chromatin regions is a general characteristic of the nucleus, independent of cell cycle stage or differentiation state. Since evidence has been presented that DNase I-sensitive sites are associated with actively transcribed chromatin, our results indicate that active transcribing chromatin is compartmentalized, preferentially in the periphery of the nucleus.