The long term objective of the research described in this proposal is to understand the mechanisms by which various agents mutate living cells. The approach is to start with a mutagenic agent for which there is at east some information concerning the products induced in the DNA. This agent is used to mutagenize the cI gene of lambda phage, either as a prophage incorporated in the genome of the Escherichia coli host cell, as free phage in solution, or as isolated phage DNa in solution. The host cells are wild-type or have defined genetic defects affecting DNA replication and repair. The mutations produced are characterized by determining the induced changes in DNA base sequence. In some cases, the changes are inferred from reversion of mutants, from a collection of several hundred, of known sequence. In some cases, mutations induced in short synthetic polynucleotides; will be studied; for technical reasons these synthetic pieces will probably first be incorporated in the lacZ(alpha) gene in M13 phage. The results are used to determine mechanisms of mutagenesis. The agents to be studied in the period covered by this proposal are ultraviolet light, ionizing radiation, the base analog bromouracil, perhaps some agent which forms bulky adducts to DNA such as the aminopyrenes and, probably, some other agent which may come to our attention at a later time. Also, additional data will be collected, from this lab and from others now sequencing mutations in the lambda cI gene, on the utility of this system for studies in mutagenesis.
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