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PubMed1979

Metabolism of O6-alkyldeoxyguanosines and their effect on removal of O6-methylguanine from rat liver DNA.

O6-Methyldeoxyguanosine and O6-ethyldeoxyguanosine are weak inhibitors (of approximately equal potency) of the removal of O6-methylguanine from methylated DNA by a rat liver enzyme in vitro. When administered to rats, O6-ethyldeoxyguanosine retarded the removal from liver DNA of the O6-methylguanine which had been produced by pretreatment with dimethylnitrosamine, but the effect was short lived. O6-Methyldeoxyguanosine was much less effective. When cells in culture were grown in a medium containing radioactive O6-methylguanine or O6-methyldeoxyguanosine there was negligible incorporation of the methylated base into DNA, but substantial conversion to guanine which was incorporated. When these substances were injected into rats after partial hepatectomy, a very small incorporation of O6-methylguanine into DNA apparently occurred. Both O6-ethyldeoxyguanosine and O6-methyldeoxyguanosine were dealkylated by rat liver extracts, but the methylated derivative was metabolized much more rapidly. O6-Methylguanosine and O6-ethylguanosine were also dealkylated by rat liver extracts, but the corresponding bases were not attacked. This reaction was probably carried out by the adenosine deaminase in the extracts because it could be prevented by addition of erythro-9-(2-hydroxy-3-nonyl)adenine, a potent adenosine deaminase inhibitor, and could also be effected by purified calf intestinal adenosine deaminase. The Km for the demethylation of O6-methyldeoxyguanosine by calf intestinal adenosine deaminase was comparable to that for adenosine, whereas the Km for O6-ethyldeoxyguanosine was ten times greater. The V for O6-methyldeoxyguanosine was about 11% that for adenosine, but that for O6-ethyldeoxyguanosine was only 0.3%. The higher Km and the slower V for O6-ethyldeoxyguanosine may contribute to the slower dealkylation of this nucleoside by liver extracts and could account for its greater effect on slowing O6-methylguanine excision from DNA in vivo.

Adenosine Deaminase↗
PubMed1978

[Reaction between dexamethasone-receptor complexes and isolated nuclei from Zajdela hepatoma and rat liver cells].

Temperature-activation of the hormone-receptor complex (HRC) was shown to be necessary to ensure its translocation from cytoplasm to nucleus both in the rat liver and hepatoma. Hepatoma nuclei bind 20 times less HRC derived from homologous hepatoma cytosol (0.15 pmol/mg DNA), but twice as much (5.6 pmol/mg DNA) of HRC from heterologous liver cytosol, as compared with the binding of HRC from normal liver cytosol by liver nuclei (3 pmol/mg DNA), Ka of HRC with the acceptor sites in hepatoma and liver nuclei were found to be practically of the same order of magnitude. The above findings suggest an inhibition of cytosol-nucleus translocation of HRC from the cytosol of hepatoma cells as a possible cause of the nonresponsiveness of the latter to the hormone.

Animals↗
PubMed1977

DNA synthetic capabilities of differentiating sperm cells.

Spermatogenic cells separated by velocity sedimentation were analysed by a micro-procedure for differentiation-associated changes in DNA synthetic capabilities. DNA-dependent DNA polymerase activity is maximal in premeiotic and meiotic cells, sequentially declines in progressively more differentiated spermiogenic cells to a minimum value in testicular spermatozoa which is 1/14 of the maximum. No further decrease of activity is observed during the subsequent process of sperm cell maturation and, at the end-differentiated state, the potential of sperm cells for DNA synthesis is demonstrated by the presence of substantial activities of thymidine and thymidylate kinases as well as DNA polymerase activity, as determined by in vitro assay, are polymerase. Although levels of DNA polymerase activity, as determined by in vitro assay, are negatively correlated with the state of differentiation, the findings support the hypothesis that, in this cell system, DNA synthetic enzymes may not be limiting factors in the control of DNA synthesis.

Animals↗
PubMed1979

Metabolic activation of 3-amino-5H-pyrido[4,3-b]indole, a highly mutagenic principle in tryptophan pyrolysate, by rat liver enzymes.

3-Amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), a mutagenic principle in tryptophan pyrolysates, binds to DNA after metabolic activation by rat liver enzymes. The enzymes for activation of Trp-P-2 were found in both microsomes and the cytosol. The reaction required NADPH and ATP, metabolic and was inhibited by 7,8-benzoflavone. Considerable binding was observed with only microsomes as enzyme source, but further addition of cytosol enhanced the binding, enhancement depending on the amount of cytosol added. Inducers for microsomal mixed-function oxidases induced activating enzyme(s) for Trp-P-2, 3-methylcholanthrene being most effective, followed by polychlorinated biphenyls and then phenobarbital.

Animals↗
PubMed1979

Soluble factors from liver and hepatomas which inhibit [3H]thymidine incorporation into DNA of Novikoff hepatoma cells.

The nature of soluble factors from liver and hepatomas which inhibit [3H]thymidine incorporation into DNA was studied in Novikoff hepatoma cells. The decreased activity in hepatoma preparations was due to loss of a high-molecular-weight heat-labile factor. Although this factor cochromatographed with arginase activity on Sephadex G-150, it does not appear to result from this activity as judged by the failure of arginine to prevent the inhibitory effect on [3H]thymidine incorporation. Both liver and hepatomas contained a heat-stable factor with inhibitory activity. Studies with ethanol-soluble material suggested that the action was not solely attributable to the presence of unlabeled thymidine, since the apparent molecular weight was too high and since the factor(s) inhibited [3H]leucine incorporation into protein in addition to inhibiting [3H]thymidine incorporation in DNA.

Animals↗
PubMed1977

Evidence for nuclear and DNA binding forms of the receptor-glucocorticoid complex from hepatoma tissue culture cells.

Binding to DNA associated with cellulose has been used to investigate the receptor-glucocorticoid complex isolated from a line of rat hepatoma tissue culture cells. The amount of activated complex that bound to DNA was approximately half that which bound to nuclei. Additional results suggest the existence of two forms of the activated glucocorticoid receptor-steroid complex in about equal amounts: one form binds only to nuclei and the other binds to DNA and nuclei. The two forms also differ in their stability, with the DNA/nuclei binging form being relatively labile. The binding of either form to the appropriate acceptor is reduced by cytosol inhibitors by the same mechanism.

Carcinoma, Hepatocellular↗
PubMed1976

Nuclear binding of progesterone in hen oviduct. Role of acidic chromatin proteins in high-affinity binding.

The multiple classes of binding sites for the progesterone-receptor complex in hen oviduct muclei were found to be of chromatin origin. The highest-affinity, and presumably most physiologically important class, is localized in oviduct chromatin and contains approx. 6000-10000 sites per nucleus. None of these sites is detected in spleen chromatin. Two new techniques were used for assaying rapidly the binding of steroid-receptor complexes to soluble deoxyribonucleoproteins in vito. The extent of high-affinity binding by the nucleo-acidic protein fraction from spleen chromatin is as great as that by the nucleo-acidic protein from oviduct chromatin. Consequently the tissue-specific nuclear binding of the progesterone receptor is found not to be a consequence of the absence of the nuclear binding sites (acceptors) from chromatin of non-target tissue (spleen), but rather a result of complete masking of these sites. In the target-tissue (oviduct) chromatin, approx. 70% of the high-affinity acceptor sites are also masked. Acidic proteins, and not histones, appear to be responsible for the masking of these acceptor sites. In addition, acidic proteins represent (or at least are an essential part of) these high-affinity sites in the oviduct nucleus. Pure DNA displays a few high-and many low-affinity binding sites. In support of previous work with immature chicks, the acidic protein fraction of the nucleo-acidic results thus support the hypotheis that protein complexed with DNA, and not DNA alone, represent the high-affinity binding sites for the steroid-receptor complexes in nuclear chromatin. The lower-affinity classes of binding sites may represent DNA and/or other nuclear components.

Animals↗
PubMed1978

Adenovirus DNA-directed transcription of 5.5S RNA in vitro.

A cell-free system developed from human KB cells was used to transcribe 5.5S RNA from deproteinized adenovirus DNA in vitro. The cell-free RNA synthesis is dependent upon exogenous templates, ribonucleoside triphosphates, and cell-free postmitochondrial supernatant of human KB cells. The synthesis of 5.5S RNA is inhibited only by high levels of alpha-amanitin; therefore it is carried out by RNA polymerase III. The rate of synthesis was linear for at least 2 hr, indicating reinitiation. The 5.5S RNA synthesized in vitro is similar to the corresponding in vivo RNA in size, sequence, and coding region on adenovirus type 2 DNA. In this report is demonstrated in vitro synthesis of a facsimile of an in vivo transcript directed by deproteinized DNA in a mammalian cell-free postmitochondrial supernatant system.

Adenoviruses, Human↗
PubMed1977

Chick-embryo DNA polymerase gamma. Identity of gamma-polymerases purified from nuclei and mitochondria.

The level of DNA polymerase gamma as compared to DNA polymerases alpha and beta has been determined in chick embryo by means of specific tests: the amount of gamma-polymerase in the 12-day-old chick embryo reaches about 15% of the total polymerase activity. This enzyme is mainly localized in nuclei and mitochondria, where it represents the prevailing if not the unique DNA polymerase activity. The mitochondrial DNA polymerase gamma is likely to be associated with the internal membrane or the matrix of this organelle since it is not removed by digitonin treatment. The gamma-polymerases have been purified from chick embryo nuclei and mitochondria 500-700 times by means of DEAE-cellulose, phosphocellulose and hydroxyapatite chromatographies. The purified mitochondrial DNA polymerase gamma is closely related to the homologous enzyme purified from the nuclei of the same cells. So far, they cannot be distinguished on the basis of their sedimentation, catalytical properties and response to inhibitors or denaturating agents. The purified gamma enzymes are distinct from the chick embryo DNA polymerases alpha and beta and are not inhibited by antibodies prepared against the latter enzymes. The nuclear and mitochondrial gamma-polymerases do not respond to the oncogenic RNA virus DNA polymerase assay with natural mRNAs.

Animals↗
PubMed1975

Effects of extracts of rat tissues on the action of bleomycin.

The cell sap (105 000 times g supernatant) of various tissues of rats caused DNA degradation in the presence of bleomycin. The activity was fractionated into two peaks by column chromatography on Sephadex G-25. The activity in Peak A (excluded fraction) appeared to be due to some proteinaceous entity, while that recovered in Peak B (retarded fraction), constituting about 90% of the total activity, seemed to be due to ascorbic acid, judging by results of further gel filtration and the effect of treatment with ascorbate oxidase. Incubation of bleomycin with Peak A or B caused loss of the ability of the antibiotic to degrade DNA. It is proposed that the action of bleomycin on DNA, and its inactivation by tissue extracts, depend, at least in part, on the presence of ascorbic acid.

Animals↗