It is our objective to define the pathways by which DNA molecules exchange information in somatic cells. We plan to exploit the excellent SV40 genetic system to analyze these pathways in detail in monkey kidney cells. We propose to construct a series of modified SV40 genomes in which the intron in the T antigen gene is replaced with nonSV40 DNA. Because the intron is dispensible, we can use these genomes to examine legitimate and illegitimate exchange pathways. The modified SV40 genomes will be linked in precise ways into defined partial dimers, so that we can study the exchange processes as intramolecular events for maximum sensitivity. We will design these partial dimers so that they must rearrange their information content along different pathways in order to produce viable recombinants. In this way we will analyze legitimate and illegitimate exchange processes separately and in competition, test the importance of single-stranded regions in promoting exchanges, and evaluate the potential for post-exchange repair of mismatches in presumptive heteroduplex intermediates. These studies represent the development of model systems for investigating exon-shuffling, genetic exchange at replication forks, and gene conversion, respectively. By comparing the capacity of differnt molecular constructs to produce viable progeny after transfection and microinjection, we can assess quantitatively the relative likelihood of various pathways of information exchange. By analyzing individual recombinants by restriction analysis and nucleotide sequencing, we can gain additional information about the potential mechanisms involved. Ultimately these studies and future ones should generate a probabalistic network of potential information exchanges in monkey cells as a model for comparison with other somatic cells.
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