Mutation at the adenine phosphoribosyl transferase locus occurs at a higher than expected frequency in both Chinese hamster ovary cells and mouse L-cells. As a result of 2D gel analysis of mutant proteins from both heterozygotes and homozygotes a model is presented hypothesizing a two step mechanism for the production of mutants in eukaryotic cells, one step of which involves gene inactivation. The mechanism of gene inactivation will be studied using a series of DNA probes constructed from a CH-APRT gene pBR322 clone. Wild type APRT DNA will be cloned into bacteriophage M13, and this phage used as a template for the isolation of mutant DNA. Using the M13 dideoxy-system we shall sequence mutant and wild-type DNA. This will be accompanied by S1-nuclease digestion of hybrid DNA to locate alterations in DNA sequences. The E. coli apt gene has been cloned, and will be used to transform APRT- strains of mammalian cells. These can then be used to study transformation and in vitro mutagenesis. HPLC analysis of peptides from human and rodent APRT will continue.
