The objective of this proposal is to characterize nonallelic gene conversion in mammalian cells, using the SV40 genetic system as a tool. We plan to construct modified SV40 genomes in which a stretch of nonessential DNA has been replaced with a duplicate copy of a segment of the gene for T antigen, thereby creating a region of nonallelic homology. By inserting a wild type gene segment into a genome that contains a mutation in the corresponding portion of the intact gene, we can select (by plaque assay under nonpermissive conditions) for transfer of a single strand from the wild type "donor" segment to the mutant "recipient" segment. This arrangement of donor and recipient segments allows strand transfer to occur as an intramolecular event, which, we anticipate, will increase its frequency. Moreover, this arrangement allows the structures of both participants in the conversion to be examined after the event, thereby allowing us to probe the mechanistic details. We have chosen to use mutations in the viral gene for T antigen, which is required for viral replication, in order to ensure that conversion precedes replication. In this way, we can focus on cellular, as opposed to virus-specific, events and we can reduce the opportunity for intermolecular events, which would complicate the interpretation of experimental results. By transfecting such constructs into monkey cells we will address several questions about nonallelic gene conversion. 1) At what frequencies are point, insertion, and deletion mutations converted? 2) Is the transfer of single strands reciprocal or nonreciprocal? 3) What length of single strand is normally transferred? 4) How frequently is mismatch repair associated with gene conversion? 5) Does the initiation of strand transfer require a break (single- or double-stranded) in the donor segment or in the recipient segment? Clear answers to these questions would define the process of nonallelic gene conversion in transfected DNA and allow realistic models of mammalian gene conversion to be formulated. These studies form an important part of our long-range objectives of defining the recombinational capabilities of monkey cells as a model for comparison with other mammalian cells.
NIH ExPorter
NIH ExPorter