The use of rodent mutant cell lines with specific repair defects for the study of aberration induction, and for identification and characterization of human repair genes

Author

Gloria Ann Greer

Date of Award

5-1991

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Sankar Mitra

Committee Members

Michael Fry, Al McFee, Julian Preston

Abstract

In order to study the mechanism of induction of mutations and chromosome aberrations by ionizing radiations, it is particularly useful to have available radiation-sensitive mutants. While several X-ray sensitive rodent cell lines are available, they have been selected rather non-specifically. I determined that selection for resistance to the DNA replication/repair inhibitor cytosine arabinoside (ara-C) would enable me to produce a set of X-ray sensitive mutant cell lines that would be defective in the resynthesis step in excision or recombination repair. Such mutant cells could also be used for the isolation and characterization of human DNA repair genes. In particular. it was predicted that the repair gene defective in individuals with ataxia telangiectasia (AT) might be amenable to study with ara-C resistant (X-ray sensitive) mutants, since additional studies in our laboratory had shown that AT cells were resistant to ara-C. In the long term, it is hoped that determining the specific defect in AT might lead to an understanding of the possible role of defective repair in tumor induction and/or progression. The general approach used to isolate ara-C resistant CHO cell mutants was to treat cells with EMS and select in increasing concentrations of ara-C. Although several mutants have been isolated, one in particular, AraC^R213, has been subject to the most extensive studies. It was selected largely because it shows the greatest sensitivity to X rays. Survival curves show that AraC^R213 cells are hypersensitive to the killing effect of X rays with an LD_{10 of 2.5 Gray as compared to the wild-type cells that had an LD10 of 6 Gray. An analysis of the cell cycle showed that the S-phase is about 2 hours longer in the mutant than in the parental cell line. Further, the mutant showed an increased frequency of X-ray induced chromosomal aberrations in the G1 and G2 stages of the cell cycle compared to wild-type frequencies. There was no increase in sister chromatid exchange levels. All of these observations in AraC^R213 are very similar to those made with AT cells in our and other laboratories. Even more importantly, complementation analysis of AraC^R213 x AT hybrid cells indicated that the gene responsible for X-ray sensitivity of AT was also mutated in the mutant cell. Thus, AraC^R213 appears to have a phenotype and probably genotype that is very similar if not exactly the same as those of AT. This makes it quite different from other X-ray sensitive cells that have been isolated in other laboratories. Procedures are now underway to identify the "repair" gene that appears to be defective in both AT and AraC^R213 cells. The hamster mutant has been transfected with DNA from wild-type human cells by electroporation. Ten primary transformants were isolated that showed wild-type repair after irradiation and one of these was used to generate a secondary transformant. A human-specific oligonucleotide primer was used to PGR amplify Alu sequences in the hamster transformants. Southern blot analysis of the PGR product using the human Alu fragment from BLUR8 plasmid verified the presence of human Alu DNA.}

Recommended Citation

Greer, Gloria Ann, "The use of rodent mutant cell lines with specific repair defects for the study of aberration induction, and for identification and characterization of human repair genes. " PhD diss., University of Tennessee, 1991.
https://trace.tennessee.edu/utk_graddiss/11119