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Biomedical subjects

J E Cleaver

Publications and source records attributed to J E Cleaver.

At least 199 records · Page 11Linked to original sources

Perturbations in simian virus 40 DNA synthesis by ultraviolet light.

Perturbations of Simian Virus 40 (SV40) DNA replication by ultraviolet (UV) light during the lytic cycle in permissive monkey CV-1 cells resemble those seen in host cell DNA replication. Formation of Form I DNA molecules (i.e. completion of SV40 DNA synthesis) was more sensitive to UV irradiation than synthesis of replicative intermediates or Form II molecules, consistent with inhibition of DNA chain elongation. The observed amounts of [3H]thymidine incorporated in UV-irradiated molecules could be predicted on the assumption that pyrimidine dimers are responsible for blocking nascent DNA strand growth. The relative proportion of labeled Form I molecules in UV-irradiated cultures rapidly increased to near-control values with incubation after 20 or 40 J/m2 of light (0.9--1.0 or 1.8--2.0 dimers per SV40 genome, respectively). This rapid increase and the failure of Form II molecules to accumulate suggest that SV40 growing forks can rapidly bypass many dimers. Form II molecules formed after UV irradiation were not converted to linear (Form III) molecules by the dimer-specific T4 endonuclease V, suggesting either that there are no gaps opposite dimers in these molecules or that T4 endonuclease V cannot use Form II molecules as substrates.

Cell Line↗

Excision repair of ultraviolet damage in mammalian cells. Evidence for two steps in the excision of pyrimidine dimers.

The incidence of pyrimidine dimer formation and the kinetics of DNA repair in African green monkey kidney CV-1 cells after ultraviolet (UV) irradiation were studied by measuring survival, T4 endonuclease V-sensitive sites, the fraction of pyrimidine dimers in acid-insoluble DNA as determined by thin layer chromatography (TLC), and repair replication. CV-1 cells exhibit a survival curve with extrapolation number n = 7.8 and Do = 2.5 J/m2. Pyrimidine dimers were lost from acid-insoluble DNA more slowly than endonuclease-sensitive sites were lost from or new bases were incorporated into high molecular weight DNA during the course of repair. Growth of CV-1 cultures in [3H]thymidine or X-irradiation (2 or 10 krads) 24 h before UV irradiation had no effect on repair replication induced by 25 J/m2 of UV. These results suggest that pyrimidine dimer excision measurements by TLC are probably unaffected by radiation from high levels of incorporated radionuclides. The endonuclease-sensitive site and TLC measurements can be reconciled by the assumption that pyrimidine dimers are excised from high molecular weight DNA in acid-insoluble oligonucleotides that are slowly degraded to acid-soluble fragments.

Cell Line↗

Decline in mutation frequency in temperature-sensitive SV40 viruses before viral DNA synthesis.

Lesions that promote reversion from a temperature-sensitive to a wild-type phenotype were induced in temperature-sensitive late mutants of SV40 virus by UV irradiation. When cultures infected with UV-irradiated temperature-sensitive mutants were grown for various times at permissive temperature (35 degrees C) and then at restrictive temperature (39 degrees C), the reversion frequency declined just before the onset of semiconservative DNA synthesis when DNA synthesis began at 32 degrees C. This can be explained by competition between reactions that lead to the onset of viral DNA synthesis and reactions that repair the lesions before the onset of viral DNA synthesis.

DNA Repair↗

Photoreactivation of thymine dimers in UV-irradiated human cells: unique dependence on culture conditions.

UV-irradiated human fibroblasts in tissue culture were exposed to photoreactivating light in an attempt to demonstrate a light-dependent loss of thymine dimers from the acid-insoluble fraction of the DNA. The only experimental conditions in which this phenomenon was observed was if the cells were grown for at least 10 days in Dulbecco's modified Eagle's minimum essential medium. Such cells lost a maximum of between 10-30% of the thymine dimers from their DNA during illumination for 1 h. When cells were grown in a variety of other media the phenomenon was not observed. The present experiments do not discriminate between true enzymatic photoreactivation and a medium dependent photosensitization phenomenon that is not enzymatic in nature.

Animals↗

Repair replication and sister chromatid exchanges as indicators of excisable and nonexcisable damage in human (xeroderma pigmentosum) cells.

Alkylating agents cause damage to DNA which can be repaired by exicsion repair (repair replication) and which induces sister chromatid exhcanges (SCEs). During early times after exposure (within 5 hr) normal and xeroderma pigmentosum (XP) cells perform similar amounts of repair replication. At later times (after 20 hr) normal cells have completed repair replication but XP cells continue at a lower rate. This slowly repaired component of alkylation damage appears to be related to the higher frequencies of SCEs induced by alkylating agents in XP cells compared to normal cells. Therefore, whereas repair replication is an indicator of exicsed damage, SCEs may be indicators of unexcised damage and may show better correlations with the potentially mutagenic and carcinogenic effects of exposure to enbironmental agents.

Alkylation↗

Induction of thioguanine- and ouabain-resistant mutants and single-strand breaks in the DNA of Chinese hamster ovary cells by 3H-thymidine.

Cultured Chinese hamster cells were labeled with 6-3H-thymidine or 5-methyl-3H-thymidine and allowed to accumulate damage from 3H decays for various periods of time while frozen. The frequencies of cells resistant to 6-thioguanine or ouabain and the amount of DNA damage (i.e., number of single-strand breaks) were determined and compared with the mutation frequencies resulting from X and ultraviolet light irradiation. Whereas 3H decays and X rays made only 6-thioguanine-resistant mutants, ultraviolet light made both 6-thioguanine- and ouabain-resistant mutants. 3H decays originating at the 6 position were two to three times as effective as decays at the 5-methyl position in making drug-resistant mutants, but decays at both sites were equally effective in making single-strand breaks. Mutants and strand breaks produced by beta irradiation of the nucleus probably are the same irrespective of the site of the decay in thymine; these results indicate that the local transmutation effects of 3H decay produce more mutations when they occur at the 6 position than at the 5-methyl position.

Animals↗

Metabolism of caffeine to nucleic acid precursors in mammalian cells.

Caffeine is rapidly metabolized in human and mouse cells in culture by de-methylation: within 1-3 h of exposure to millimolar concentrations of labeled caffeine, more than 90% of the pool consists of labeled products of metabolism and less than 10% is still caffeine. The methyl groups seem to be transferred and used in the de novo synthesis of thymine, guanine, and adenine to nucleic acids. Normal fibroblasts, Lesch-Nyhan fibroblasts, xeroderma pigmentosum fibroblasts, HeLa cells, wild type mouse cells, and adenine phosphoribosyl-transferase-deficient mouse cells all seem to metabolize caffeine similarly.

Animals↗

Defective thymine dimer excision by cell-free extracts of xeroderma pigmentosum cells.

Crude extracts of normal human diploid fibroblasts and of human peripheral blood lymphocytes excise thymine dimers from purified ultraviolet-irradiated DNA, or from the DNA presumably present as chromatin in unfractionated cell-free preparations of cells that had been labeled with [3H]thymidine. Extracts of xeroderma pigmentosum cells from complementation groups A, C, and D also excise thymine dimers from purified DNA, but extracts of group A cells do not excise dimers from the DNA of radioactively labeled unfractionated cell-free preparations.

Cell Line↗

Sister chromatid exchange in xeroderma pigmentosum cells that are defective in DNA excision repair or post-replication repair.

The formation of sister chromatid exchanges has been postulated to depend upon the action of DNA repair enzymes. Our experiments with various human cell lines show that the yield of sister chromatid exchanges is within normal limits in both excision-repair-defective and post-replication-repair-defective cells from the autosomal recessive disease, xeroderma pigmentosum. These results indicate that hypotheses invoking known DNA repair processes to acconnt for the recombination of sister chromatids are inadequate and that the exact enzymatic processes are as yet unknown.

Cell Line↗