The objective of this research is to determine the extent to which extramutational events, such as mitotic recombination, gene conversion, gene inactivation, multilocus deletions, and chromosome non-disjunction or chromosomal rearrangements, are involved in the expression of recessive mutations in cultured mammalian cells. We propose to develop and utilize several model systems that will facilitate investigation of these genetic processes at the molecular level. Our ultimate goal is a clear understanding of the cellular processes and mechanisms involved in the generation and expression of heritable variation in mammalian somatic cells. A system to facilitate the detection and analysis of targeted homologous recombination events will be developed using a series of CHO APRT deletion or insertion mutants. This system will be used to study targeted recombination of transfected or microinjected APRT gene sequences with the endogenous CHO APRT locus; to determine the relative frequencies of targeted integration and gene conversion events, the effect of various-sized deletions or insertions in the target gene sequence on recombination frequencies, and whether transcribed genes are better targets for directed homologous recombination than are non- transcribed genes. Another system, employing CHO cell hybrids constructed between different, non-overlapping APRT deletion mutants, will be used to assay spontaneous or induced frequencies of interallelic mitotic recombination between endogenous APRT alleles in their normal chromosomal environment. A third system will examine the generation of homozygosity or hemizygosity for recessive mutations in cultured cells by genetic events such as mitotic recombination, chromosome nondisjunction or multilocus deletions that are undetectable in conventional CHO and V-79 mutagenesis assays. Other studies will focus on the effect of chromosomal environment on the relative susceptibility of a gene to mutation, deletion, or recombination.
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