PubMed Health⌕ Search

Biomedical subjects

C L Mooney

Publications and source records attributed to C L Mooney.

9 recordsLinked to original sources
PubMed1985

Correction of a nucleotide-excision-repair mutation by human chromosome 19 in hamster-human hybrid cells.

A UV-sensitive mutant line of CHO cells, UV20, was shown to be phenotypically corrected to resistance by fusion with human lymphocytes or fibroblasts. Only human chromosome 19 correlated with the DNA repair phenotype of resistant hybrid clones and their resistant or sensitive subclones. This study demonstrates the mapping of a human repair gene by direct selection of complementing hybrids in the presence of a DNA-damaging agent (mitomycin C).

Animals↗
PubMed1985

Defective DNA cross-link removal in Chinese hamster cell mutants hypersensitive to bifunctional alkylating agents.

DNA repair-deficient mutants from five genetic complementation groups isolated previously from Chinese hamster cells were assayed for survival after exposure to the bifunctional alkylating agents mitomycin C or diepoxybutane. Groups 1, 3, and 5 exhibited 1.6- to 3-fold hypersensitivity compared to the wild-type cells, whereas Groups 2 and 4 exhibited extraordinary hypersensitivity (30- to 90-fold). Mutants from Groups 1 and 2 were exposed to 22 other bifunctional alkylating agents in a rapid assay that compared cytotoxicity of the mutants to the wild-type parental strain, AA8. With all but two of the compounds, the Group 2 mutant (UV4) was 15- to 60-fold more sensitive than AA8 or the Group 1 mutant (UV5). UV4 showed only 6-fold hypersensitivity to quinacrine mustard. Alkaline elution measurements showed that this compound produced few DNA interstrand cross-links but numerous strand breaks that were revealed by proteinase treatment. Therefore, the extreme hypersensitivity of mutants from Groups 2 and 4 appeared specific for compounds the main cytotoxic lesions of which were DNA cross-links. Mutant UV5 was only 1- to 4-fold hypersensitive to all the compounds. Repair kinetics of DNA interstrand cross-link production and removal was measured by alkaline elution for AA8 and mutants UV4 and UV5 after exposure to diepoxybutane. Although the initial number of cross-links was similar for the three cell lines, during 24-h incubation, the efficiency of removal of cross-links was lowest in UV4 and intermediate in UV5. These results suggest that the different levels of sensitivity of the five complementation groups to bifunctional alkylation damage are specifically related to different efficiencies of DNA cross-link removal. The phenotype of hypersensitivity to both UV radiation and cross-link damage exhibited by the mutants in Groups 2 and 4 appears to differ from those of the known human DNA repair syndromes.

Alkylating Agents↗
PubMed1984

Repair of DNA adducts in asynchronous CHO cells and the role of repair in cell killing and mutation induction in synchronous cells treated with 7-bromomethylbenz[a]anthracene.

CHO cells of normal or UV-sensitive phenotypes were analyzed for their ability to remove DNA adducts produced by the carcinogen 7-BrMeBA. At a dose of 0.1 microM, which reduced the survival of the normal AA8 cells to approximately 90% and the mutant UV5 cells to approximately 20%, the frequency of adducts was 5-6 per 10(6) nucleotides for both cell types, and AA8 cells removed approximately 30% of the adducts in 8 h and approximately 55% in 24 h. In contrast, UV5 and mutants from four other genetic complementation groups had no significant removal. Binding of 7-BrMeBA did not vary through the cell cycle in synchronous cultures. At a dose of mutagen (0.07 microM) resulting in approximately 25% survival of asynchronous UV5, the survival of synchronous cultures rose about threefold from early G1 to early S phase and then decreased somewhat in late S/G2. At a dose (0.28 microM) producing similar survival of asynchronous cultures, AA8 cells differed qualitatively in that survival decreased progressively by 5- to 10-fold between early G1 and the early part of S, and rose steeply through late S/G2 to give a 10- to 20-fold increase. We conclude that DNA repair is the major determinant of variations in survival through the cycle in normal cells. The patterns observed are consistent with a mechanism of killing in AA8 cells in which adducts disrupt DNA replication, while in UV5 cells transcriptional blocks or other effects may govern lethality. Induced mutations at the aprt and hprt loci showed changes through the cycle in both AA8 and UV5 cells, and the patterns were not readily explainable by the action of repair.

Animals↗
PubMed1982

Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions.

Five UV-sensitive mutant strains of CHO cells representing different genetic complementation groups were analyzed for their ability to perform the incision step of nucleotide excision repair after UV exposure. The assay utilized inhibitors of DNA synthesis to accumulate the short-lived strand breaks resulting from repair incisions. After 6 J/m2, each of the mutants showed less than 10% of the incision rate of the parental AA8 cells. After 50 J/m2, the rate in AA8 was similar to that at 6 J/m2, but the rates in the mutants were significantly higher (approximately 20% of the rate of AA8). Thus by this incision assay the mutants were phenotypically indistinguishable. Each of the mutants were hypersensitive to mutation induction at both the hprt and aprt loci by a factor of 10, and in the one strain tested ouabain resistance was induced sevenfold more efficiently than in AA8 cells. Sister chromatid exchange was also induced with sevenfold increased efficiency in the two mutant strains examined. Thus, these CHO mutants resemble xeroderma pigmentosum cells in terms of their incision defects and their hypersensitivity to DNA damage by UV.

Animals↗
PubMed1982

A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency of sister-chromatid exchange.

A mutant of CHO cells (strain EM9) previously isolated on the basis of hypersensitivity to killing by ethyl methanesulfonate (EMS) is approx. 10-fold more sensitive than the parental line, AA8, to killing by both EMS and MMS. It is also hypersensitive to killing by other alkylating agents (ethyl nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine), X-rays, and ultraviolet radiation. The production and repair of DNA single-strand breaks (SSB) were studied using the technique of alkaline elution of DNA from filters. After exposure to 4 Gy of X-rays at 0 degrees C and subsequent incubation at 25 degrees C, SSB were repaired within 12 min in AA8, but little repair occurred in EM9. Similarly, with doses of EMS or MMS that produced comparable numbers of SSB in AA8 and EM9 at the end of a 10-min exposure, repair of SSB occurred more rapidly in AA8 than in EM9, suggesting that individual SSB are longer lived in EM9. EM9 was found to be hypersensitive also to the induction of mutations and sister-chromatid exchanges (SCE) by EMS; per unit dose the mutant had twice as many mutations to thioguanine resistance, 3 times as many mutations to azaadenine resistance, and a 7-fold enhancement in SCE, compared to AA8. Moreover, the baseline frequency of SCE in the mutant was extraordinarily high, i.e., 8.6 +/- 0.6 vs. 107 +/- 5 SCE/cell for AA8 and EM9, respectively, with 10 microM BrdUrd in the medium. The high SCE frequency in EM9 did not vary significantly with BrdUrd concentration over the range examined from 2.5 to 20 microM, and the percentage of 5-bromouracil substitution in the DNA was the same in EM9 and AA8 under these conditions. These data, however, do not rule out the possibility that the high SCE frequency in EM9 is a consequence of an altered sensitivity to incorporated BrdUrd. Thus, EM9 may carry a pleiotropic mutation affecting some function in DNA replication and/or DNA repair and causing the variety of phenotypic properties described in this study.

Animals↗
PubMed1981

Genetic diversity of UV-sensitive DNA repair mutants of Chinese hamster ovary cells.

Mutant lines of Chinese hamster ovary cells that show hypersensitivity to killing and mutagenesis by UV light were analyzed by genetic complementation analysis to determine whether defects in different gene loci might underlie a common cellular phenotype,. To facilitate rapid screening of mutant clones, a procedure was devised that allowed presumptive complementation to be assessed on the basis of the frequency of UV-resistant cells after fusion by polyethylene glycol. Four classes were identified among 44 clones tested. By using drug-resistance markers for selection of hybrid cells in crosses between UV mutant and wild type, a mutant from each of the four classes was shown to behave as phenotypically recessive. Hybrids were also isolated from crosses between each of the pair combinations of the four mutants. All such hybrids were relatively resistant to UV killing, providing confirmation of the complementation classes. When mutants representing the four UV-complementation classes were tested with the polyaromatic hydrocarbon 7-bromomethylbenz(a)anthracene, complementation was again seen for all pair combinations. These results suggest that each class of mutants represents a biochemical defect that plays a common role in the repair of both UV-induced and chemically induced lesions in the DNA.

Animals↗
PubMedDate not supplied

Genetic complementation between UV-sensitive CHO mutants and xeroderma pigmentosum fibroblasts.

The purpose of this study was to determine the feasibility of doing complementation analysis between DNA-repair mutants of CHO cells and human fibroblasts based on the recovery of hybrid cells resistant to DNA damage. Two UV-sensitive CHO mutant lines, UV20 and UV41, which belong to different genetic complementation groups, were fused with fibroblasts of xeroderma pigmentosum in various complementation groups. Selection for complementing hybrids was performed using a combination of ouabain to kill the XP cells and mitomycin C to kill the CHO mutants. Because the frequency of viable hybrid clones was generally less than 10(-6) and the frequency of revertants of each CHO mutant was approximately 2 X 10(-7), putative hybrids required verification. The hybrid character of clones was established by testing for the presence of human DNA in a dot-blot procedure. Hybrid clones were obtained from 9 of the 10 different crosses involving 5 complementation groups of XP cells. The 4 attempted crosses with 2 other XP groups yielded no hybrid colonies. Thus, a definitive complementation analysis was not possible. Hybrids were evaluated for their UV resistance using a rapid assay that measures differential cytotoxicity (DC). All 9 hybrids were more resistant than the parental mutant CHO and XP cells, indicating that in each case complementation of the CHO repair defect by a human gene had occurred. 3 hybrids were analyzed for their UV-radiation survival curves and shown to be much more resistant that the CHO mutants but less resistant than normal CHO cells. With 2 of these hybrids, sensitive subclones, which had presumably lost the complementing gene, were found to have similar sensitivity to the parental CHO mutants. We conclude that the extremely low frequency of viable hybrids in this system limits the usefulness of the approach. The possibility remains that each of the nonhybridizing XP strains could be altered in the same locus as one of the CHO mutants.

Animals↗