Molecular events initiated by ionizing radiation which result in genetic damage and carcinogenesis in mammalian cells will be investigated. We plan to construct recombinant DNA molecules which combine a portion of the bovine papilloma virus genome (which will provide a replicon and selectable marker capable of functioning in mammalian cells), a portion of E. coli plasmid pBR322 (which will provide a replicon and selectable marker capable of functioning in E. coli) and the E. coli lac i gene (which will serve as an indicator for the occurrence of specific genetic events). Structural modifications will be introduced into the lac i gene including point mutations, short tandem duplications, frameshift mutations and insertions of foreign DNA. Plasmids containing mutant i genes will be introduced into mouse C127I cells; transformed cell foci will be isolated and used for analysis of the effects of radiation-treatment on reversion to the wild type i gene phenotype. Reversion of mutant i genes will be scored by transforming the appropriate indicator bacterial strain and subsequent evaluation of colonies on plates containing chromogenic substrate. Alternatively, bacterial lysates will be assayed for lac repressor protein. Effects of ionizing radiation on reversion of the various types of lesions will be investigated (including effects of dose, dose rate and radiation quality). Inducible mutagenic or recombinogenic functions will be evaluated by fusing irradiated cells with untreated cells carrying plasmids containing mutant i genes. The ability of mutant human fibroblasts (e.g. cells from ataxia telangiectasia patients) to respond to DNA damage will be evaluated by similar fusion experiments. DNA containing radiation-induced lesions or other specific modifications will be examined by introducing this DNA into cells carrying the mutant plasmids and testing for the induction of a mutagenic or recombinogenic state. Finally, the ability of extracts from irradiated cells to cause specific genetic alterations will be determined. The long range goal of this research is to understand how cells respond to DNA damage and what role this response plays in carcinogenesis. The assay we propose holds the promise of providing specific information on molecular events which occur in intact cells.
