One important finding from our studies on ionizing radiation mutagenesis (IRM). In Escherichia coli is that IRM is not uniformly dependent on the umuDC genes as is the case for UV radiation mutagenesis. That is, there are umuDC-dependent and umuDC-independent mechanisms of IRM. We have already isolated and begun mapping xmu (X-ray mutability) mutations that block umuDC-independent IRM, and that appear to be distinct from the recA, lexA, and umuDC loci. Towards our long-term objective of understanding the molecular basis of IRM, we propose to accomplish the following specific aims: (1) to determine the number and exact location of the xmu genes using the genetic procedures of conjugation and transduction (presently there appear to be three loci); (2) to clone at least two xmu genes (during this grant period) into useful plasmids in order to identify and characterize their gene products using the maxicell technique and SDS-polyacrylamide gel electrophoresis; (3) to fuse the plasmid-carried xmu genes with a lacZ gene in order to study the regulation of xmu genes using an assay for beta-galactosidase; (4) to use the well-characterized his trp reversion system to determine the mutational spectra in the xmu strains under several irradiation conditions, and for chemical mutagens that produce specific types of DNA damage; (5) to test whether the xmu mutations inhibit induction of the recA gene, using a recA::lacZ fusion and an assay for beta galactosidase activity, and to test whether xmu mutations can be suppressed by mutations in other genes, and to test whether xmu mutations confer radiation sensitivity; (6) to determine the nucleotide sequences of xmu genes using the dideoxy-chain termination method of Sanger in order to formulate models for xmu gene action. Health Relatedness: This proposal suggests genetic and molecular biological procedures for increasing the understanding of the molecular mechanisms of IRM. Since mutagenesis and carcinogenesis are known to be linked, this research should provide valuable insights into the molecular mechanism of ionizing radiation carcinogenesis.