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R J Reynolds

Publications and source records attributed to R J Reynolds.

At least 19 recordsLinked to original sources
PubMed1992

Electroporation-mediated gene transfer efficiency is reduced by linear plasmid carrier DNAs.

Carrier DNA has generally been found to stimulate DNA-mediated gene transfer of Chinese hamster ovary (CHO) cells by calcium phosphate coprecipitation. In studies employing electroporation, however, we observed that linear plasmid DNA was inhibitory to the transfection of CHO cells. This unexpected result prompted us to explore the effects of various types and forms of plasmid, cosmid, and chromosomal DNAs on transfection efficiencies. Both carrier DNA form and type were found to influence transfection efficiencies. Circular and linear forms of plasmid carrier DNA had opposite effects: circular plasmids increased and linear plasmids decreased transfection efficiencies. These effects were independent of homology with the selected plasmid and are probably independent of homologous recombination mechanisms. Bacterial genomic DNA failed to stimulate transfection, while calf thymus and cosmid DNA consisting primarily of human sequences stimulated transfection significantly. Our results have importance for plasmid-based experiments in mammalian cells such as those involving the induction of interplasmid homologous recombination.

Animals↗
PubMed1990

Transcription stimulates homologous recombination in mammalian cells.

Transcription stimulates homologous recombination in Saccharomyces cerevisiae and has been implicated in the control of recombinational events during the development of mammalian immune systems. Here, we describe a plasmid-based system in which an inducible promoter from the mouse mammary tumor virus is located upstream of heteroallelic neomycin genes carried on two plasmids. Pairs of plasmids are introduced into Chinese hamster ovary cells by electroporation, and recombination is monitored by scoring colonies resistant to the aminoglycoside G418. When transcription is induced with dexamethasone, a synthetic glucocorticoid hormone, and double-strand breaks are introduced at mutation sites, recombination is stimulated sixfold over noninduced levels. Inducing transcription in circular substrates or in substrates cleaved at sites distant from the mutations has no detectable effect on recombination between neomycin genes. Results are presented that are consistent with the observed stimulation of recombination occurring before plasmids integrate into the cellular DNA. Our results are discussed in relation to molecular models for extrachromosomal recombination in mammalian cells.

Alleles↗
PubMed1988

Effect of duration of exposure to benzo(a)pyrene diol-epoxide on neoplastic transformation, mutagenesis, cytotoxicity, and total covalent binding to DNA of rodent cells.

We examined the effect of different durations of exposure (20 sec to 24 hr) to (+/-) 7-beta,8 alpha-dihydroxy-9 alpha, 10 alpha -epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE I) on the induction of transformation in C3H/10T 1/2 cells and of mutations to 6-thioguanine resistance in Chinese hamster ovary cells (CHO), as well as on BPDE I-DNA binding in these two cell lines. A 20-sec exposure of the cells to BPDE I was sufficient to induce mutations and morphological transformation in vitro. However, the transformation frequency in CH3 mouse-embryo-derived 10T 1/2 cells increased twofold and the frequency of mutations in CHO cells sixfold when the exposure time to BPDE I was increased from 20 sec to 8 h. Cytotoxicity increased under similar conditions. A large number of BPDE I-DNA adducts were formed in both cell lines within the first 15-min of exposure of the cells to this ultimate carcinogen. The total covalent binding did not increase with longer than 15-min incubation times. These results suggest that in addition to its covalent binding to DNA, BPDE I may influence other cellular mechanism(s) that are responsible for the initiation of transformation and mutagenesis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1987

DNA sequence dependence of closely opposed cyclobutyl pyrimidine dimers induced by UV radiation.

Treatment of UV-irradiated DNAs with Micrococcus luteus pyrimidine dimer-DNA glycosylase results in the formation of double-strand breaks due to cleavage at closely opposed pyrimidine dimers. To determine if the induction of closely opposed dimers is significantly affected by DNA nucleotide sequence, end-labeled DNA fragments of known nucleotide sequence were UV irradiated, incubated with pyrimidine dimer-DNA glycosylase, and analyzed by electrophoresis through nondenaturing polyacrylamide gels. Distinct bands of increased electrophoretic mobility were observed, indicating that bifilar cleavage had occurred with greater probability at specific sites in each DNA sequence. In vitro enzymatic photoreactivation of dimers prior to treatment with pyrimidine dimer-DNA glycosylase prevented the appearance of bands. DNA sequence analysis revealed the presence of closely opposed runs of pyrimidines at sites of more frequent bifilar cleavage. Our results indicate that the induction of closely opposed dimers occurs with greater probability at specific sites in DNA sequences and that such sites are characterized by the presence of closely opposed pyrimidine runs.

Bacterial Proteins↗
PubMed1987

Induction and repair of closely opposed pyrimidine dimers in Saccharomyces cerevisiae.

Pyrimidine dimer-DNA glycosylase activity prepared from Micrococcus luteus has been used to develop an enzyme-sensitive site assay for the detection and quantification of closely opposed pyrimidine dimers in the nuclear DNA of UV-irradiated yeast. With this assay, closely opposed dimers were found to be induced as a linear function of dose from 0 to 200 J/m2 (254 nm). Closely opposed dimer frequencies decreased during the incubation of UV-irradiated, excision repair-proficient cells under liquid-holding conditions in the dark and during post-irradiation exposure of excision-deficient cells to photoreactivating light. Incubation of excision-deficient cells in the dark had no effect on the frequency of closely opposed dimers for up to 16 h. These results indicate that closely opposed dimers in UV-irradiated yeast are subject to repair by enzymatic photoreactivation and/or by dark-repair processes dependent, at least in part, upon functions necessary for normal excision repair. The genetic and biochemical implications of these results are discussed.

DNA Damage↗
PubMed1986

A sensitive, enzymatic assay for the detection of closely opposed cyclobutyl pyrimidine dimers induced in human diploid fibroblasts.

A sensitive, enzymatic assay has been developed for the detection of closely opposed cyclobutyl pyrimidine dimers induced in UV-irradiated human diploid fibroblasts. In this assay closely opposed dimers are detected as bifilar enzyme-sensitive sites. Single-strand incisions are made at the positions of individual pyrimidine dimers through the action of M. luteus pyrimidine dimer-DNA glycosylase. Incisions at closely opposed dimers, effectively expressed as double-strand breaks, are quantified from the resulting reduction in DNA double-strand molecular weight as determined by velocity sedimentation through neutral sucrose density gradients. The stability of the bacteriophage lambda cos site under our reaction conditions indicates that opposed incisions must be relatively close to be expressed as a double-strand break. The dose response for the induction of bifilar enzyme-sensitive sites in mammalian cells was found to be complex but can be approximated by a function that increases as the 1.2-1.4 power of UV dose. The frequency of bifilar enzyme-sensitive sites observed decreased during postirradiation incubation of excision-repair-proficient human diploid fibroblasts with less than 20% still detectable at 24 h after irradiation with 5 J/m2 (254 nm). By comparison, over 80% of the bifilar enzyme-sensitive sites induced in fibroblasts assigned to xeroderma pigmentosum complementation group A remained detectable 24 h after irradiation. The implications of these results for models addressing the induction and repair of closely opposed pyrimidine dimers are discussed.

Cells, Cultured↗
PubMed1986

Repair of closely opposed cyclobutyl pyrimidine dimers in UV-sensitive human diploid fibroblasts.

An enzyme-sensitive site assay has been used to examine the fate of closely opposed pyrimidine dimers (bifilar enzyme-sensitive sites) in fibroblasts from individuals afflicted with various genetic disorders that confer increased cellular sensitivity to UV radiation. The disappearance of bifilar enzyme-sensitive sites was found to be normal in cells from individuals with Fanconi's anemia, Cockayne's syndrome, dyskeratosis congenita and the variant form of xeroderma pigmentosum. The rate of bifilar enzyme-sensitive site removal in XP cells assigned to complementation group C was reduced by an amount similar to that observed for the repair of isolated dimers. Our results indicate that the initiation of repair at closely opposed dimers is slow in XP-C cells but normal in all other cells examined.

Cell Line↗
PubMed1986

Bifilar enzyme-sensitive sites in ultraviolet-irradiated DNA are indicative of closely opposed cyclobutyl pyrimidine dimers.

Incubation of UV-irradiated DNA with pyrimidine dimer-DNA glycosylase in cell-free lysates prepared from Micrococcus luteus results in the appearance of double-strand breaks. It has previously been assumed that such double-strand breaks result from cleavage at closely opposed dimers. We have used hybrid molecules of bacteriophage T7 DNA comprised of two unirradiated strands, two UV-irradiated strands, or one unirradiated and one UV-irradiated strand to test this hypothesis. Bifilar cleavage was observed only with molecules consisting of two irradiated strands and no bifilar cleavage was observed after the monomerization of pyrimidine dimers by enzymatic photoreactivation. Our results indicate that at least 80% of the double-strand breaks result from cleavage at closely opposed dimers and that the induction of dimers in one strand does not influence the induction of dimers at closely opposed positions in the complementary strand of a DNA double helix.

Carbon Radioisotopes↗
PubMed1985

Induction of in vitro transformation by near-u.v. light and its interaction with beta-propiolactone.

The ability of u.v.-A light (320-400 nm) to induce cellular transformation in vitro and to modify chemical carcinogen-induced cellular transformation was investigated in BALB/c 3T3 cell cultures. When administered as a series of nontoxic exposures, u.v.-A alone was found to induce cellular transformation as a linear function of the numbers of u.v-A exposures. Possible interactions of u.v.-A with environmentally encountered chemical carcinogens were studied by examining the effects of u.v.-A light exposures on cellular transformation in cells exposed to the direct acting carcinogen, beta-propiolactone (BPL), an alkylating agent, with a standard initiation/promotion protocol. Twenty-four hours after a single treatment with 2.5 micrograms/ml of beta-propiolactone, cells were exposed to 3.0 kJ/m2 of u.v.-A light. U.v.-A exposures were repeated weekly for up to 5 weeks, after which cells were fixed, stained and dishes were scored for type III transformed foci. Weekly exposures to u.v.-A alone for 5 weeks induced approximately 3 foci/dish. Treatment with BPL alone induced approximately 1 focus/dish (background was 0.17 foci/dish). A combination of the two treatments resulted in a marked increase in the yield of transformed foci/dish, with the u.v.-A enhancement increasing with increasing numbers of exposures (approximately 10 foci/dish after a single exposure to BPL and five u.v.-A exposures). These results suggest a synergistic interaction between BPL and subsequent u.v.-A exposures in the induction of in vitro neoplastic transformation.

Animals↗
PubMed1985

Sequence specificity of DNA cleavage by Micrococcus luteus gamma endonuclease.

DNA fragments of defined sequence have been used to determine the sites of cleavage by gamma-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus gamma endonuclease and analyzed on high resolution, denaturing, polyacrylamide gels. Gamma endonuclease was found to cleave irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to gamma radiation.

Base Sequence↗
PubMed1979

Gentamicin failure in staphylococcal bacteremia.

Gentamicin has been relied upon in some antibiotic combinations against Staphylococcus aureus, should it be present. Reported is a case of Staphylococcus aureus bacteremia treated inadvertently by gentamicin, and the gentamicin failed.

Drug Resistance, Microbial↗
PubMed1978

Removal of pyrimidine dimers from Saccharomyces cerevisiae nuclear DNA under nongrowth conditions as detected by a sensitive, enzymatic assay.

A sensitive and quantitative procedure for the detection of pyrimidine dimers in yesast nuclear DNA is described. The assay employs dimer-specific, endonuclease activities from Micrococcus luteus together with DNA sedimentation through calibrated, alkaline sucrose gradients to detect endonuclease-induced, single-strand breaks. Breaks were induced in a dose-dependent manner from 0 to 80 J m-2 at 254 nm and in numbers equivalent to the numbers of dimers induced by similar doses (Unrau et al., Biochim. Biophys. Acta, 312 (1973) 626--632). This procedure also allows the use of [6-3H] uridine to label cellular nucleic acids, but dose not require extensive DNA purification to eliminate concomitantly labeled RNA. Endonuclease-sensitive sites in the wild-type, haploid strain S288C, after irradiation with 5 J m-2 (254 nm), were removed in less than 5 min when cells were incubated in buffer (pH 7.0) at 28 degrees C. After irradiation with doses from 30 to 100 Jm-2 site removal in S288C required longer postirradiation incubations and was about 90% complete. In a radiation-sensitive strain carrying the mutant allele rad4-3 the number of endonuclease-sensitive sites remained constant for 6 h after irradiation with 5 Jm-2. The retention of sites in this strain indicates that it is defective in the excision of pyrimidine dimers.

Centrifugation, Density Gradient↗