EXPRESSION OF GENETIC VARIATION IN CULTURED CELLS

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Recombination plays an important role in both the generation and limitation of genetic diversity in living organisms. It represents one of the major mechanisms for the generation and expression of somatic mutations, and has been shown to be involved in many types of tumorigenesis (retinoblastoma, etc.). Paradoxically, in normal cells, the same recombinational pathways may provide important mechanisms for the preservation of genomic stability and the repair of DNA. At present, little is known about these recombinational mechanisms in mammalian cells The long-term goal of this project is to understand the cellular processes and molecular mechanisms involved in the generation and expression of heritable variation in mammalian somatic cells. A unique set of mutant cell lines and systems for the analysis of homologous recombination at an endogenous gene locus-the Chinese hamster adenine phosphoribosyltransferase (APRT) locus, will be used to address questions regarding several fundamental aspects of homologous recombination in mammalian somatic cells. The specific aims of this proposal are: (i) to investigate the effects of length of shared sequence homology and internal sequence heterologies on the frequency of targeted recombination in mammalian cells, using a series of different length APRT gene fragments as donor sequences for targeted correction of a three-basepair deletion at the endogenous APRT locus; (ii) to examine the effects of transcription of target gene and donor sequences on the frequency of targeted and direct-repeat recombination in mammalian cells, by employing targeted gene replacement/ integration strategies to obtain cell lines in which the endogenous APRT gene has been replaced with an APRT coding sequence driven by a heterologous, inducible metallothionein promoter, (iii) to examine the effects of cell cycle phase on the frequency of targeted homologous recombination at the endogenous APRT locus in Chinese hamster cells, by assaying targeted recombination frequencies in elutriationsynchronized populations; and (iv) to utilize gene targeting techniques to study intrachromosomal recombination in a 170 kilobasepair genomic region flanking the endogenous APRT locus. It is our hope that such studies will provide considerable insight into the mechanisms of recombination in mammalian somatic cells.