In order to understand the genetic basis of X-ray and EMS resistance, we propose to clone the human DNA sequence coding for these phenotypic properties and to characterize its structure and activity. EM9 cells are a mutant Chinese hamster ovary cell line that is hypersensitive to killing by X-rays and alkylating agents. EM9 cells are approximately 1.4 times more sensitive to X-rays and 17 times more sensitive to killing by EMS (evaluated by Do values) than the parental Chinese hamster ovary line (AA8). Alkaline elution analysis of repair in EM9 cells shows them to be deficient in the ability to repair single strand breaks in DNA. By DNA mediated gene transfer, we have produced transgenic EM9 cells that contain human DNA and display an enhanced resistance to X-ray and EMS. The objective of the research proposed here is to clone and characterize the putative human repair gene responsible for the reversion of the EM9 cells. To accomplish this objective, we propose to generate and screen cosmid libraries from revertant transformant cell lines containing human DNA. Cosmids will be screened by hybridization techniques for human DNA and the marker gene (PSV2neo). Cosmids that are screened as positive, and therefore potentially containing the repair gene, will be further screened by DNA mediated gene transfer into EM9 cells for phenotypic activity. The structure of the cloned human sequences will be analyzed by restriction mapping. The phenotypic activity of sensitive cells containing the repair gene will be analyzed by cell survival, and DNA single and double strand break repair capability will be determined by alkaline and neutral elution, respectively. Our preliminary work suggests that there is a human gene that confers X-ray resistance to a sensitive hamster cell line. The proposed research will provide a more intimate understanding of resistance to X-ray cell killing in man.
| Barrows, L R; Paxton, M B; Kennedy, K A et al. (1991) Characterization of revertants of the CHO EM9 mutant arising during DNA transfection. Carcinogenesis 12:805-11 |
| Barrows, L R; Borchers, A H; Paxton, M B (1987) Transfectant CHO cells expressing O6-alkylguanine-DNA-alkyltransferase display increased resistance to DNA damage other than O6-guanine alkylation. Carcinogenesis 8:1853-9 |
| Spiro, I J; Barrows, L R; Kennedy, K A et al. (1986) Transfection of a human gene for the repair of X-ray- and EMS-induced DNA damage. Radiat Res 108:146-57 |
