| Description |
I developed new assays for homologous and illegitimate recombination. Recombination events were studied by inducing doublestrand breaks by selecting for the excision of a Tn10d-Tc element. The assays allow observation of the integration of homologous or non-homologous DNA segments into the break site, as well as measurement of the frequency and size of any accompanying deletions. The assays for double-strand break repair utilize lactose operons which are strategically placed around the chromosome. Tn10d-Tc elements are inserted into the lactose operons of MudJ elements for homologous recombination assays, and in genes prior to MudJ elements for illegitimate recombination assays. These Tn10d-Tc elements prevent lactose utilization. In my assays, repair of induced doublestrand breaks can result in lactose utilization if: 1) homologous sequences within the chromosome or on plasmids are used to repair break sites within the Mud Jelement; 2) DNA from non-homologous sequences is integrated into a break-site prior to a MudJ which contains an active promoter in the correct orientation; or 3) deletion formation occurs as the result of a breaksite prior to a MudJ, revealing a distant promoter in the correct orientation. Although all of the assays have been developed, only one illegitimate assay has been tested extensively. The results of these tests seem to indicate that both the integration of a foreign promoter and deletion formation may occur in response to the induction of double-strand breaks. Further testing and sequencing of recombinants is necessary to confirm these results. |
