These experiments aim to develop systems for the analysis of mutation in human cells at the DNA sequence level. The approach combines the powerful techniques of bacterial molecular genetics with recombinant DNA methodologies. The thoroughly studied bacterial gene lacI is the target for mutaton and is moved in and out of human cells on vectors that can replicate in both bacterial and mammalian cells. After mutagenesis in human cells, the gene is returned to E. coli, where mutations are detected and are analyzed by rapid genetic mapping and DNA sequencing. The system will be used to study carcinogen-induced mutations at the sequence level and also spontaneous mutations such as insertions of transposable elements. Furthermore, the experiment will be done in cell lines from patients having hereditary diseases associated with DNA repair deficiencies and a high frequency of cancer. The approach should permit the study of the molecular biology of mutation in human cells and its relation to carcinogenesis.
