Development of a method to reduce recessive background effects of the gene disruption library

David Alvaro(2), Michael Lisby(1), Rodney Rothstein(1). (1) Genetics and Development, Columbia University, 701 W. 168th St., New York, NY, 10032, USA; (2) 701 W 168th St, HHSC 1606, New York, NY 10032.

Relocalization of a Rad52-YFP fusion protein to subnuclear foci can be used as a marker for recombinational DNA repair. In mitotically growing cells, spontaneous foci form at a low frequency. These foci likely reflect the formation of repair centers in response to spontaneous DNA lesions. Mutations in various pathways of DNA metabolism lead to elevations in the frequency of Rad52 foci. To detect additional genes affecting DNA repair processes, we began screening the disruption library by introducing a Rad52-YFP reporter and assaying focus formation. Initial results revealed that recessive genetic factors in the library strains, independent of the documented gene disruptions, influence the Rad52 focus phenotype. To minimize background effects, a method was developed to eliminate recessive factors in the library background. This is accomplished by creating hybrid diploids homozygous for the gene deletion by mating each library strain to a W303 strain containing a conditional wild type copy of the chromosome bearing the gene disruption, which itself can be selectively lost after mating. Over 2400 diploid strains have been screened for Rad52-YFP focus levels and 44 gene deletions have been identified that exhibit significantly elevated focus levels. The genes identified in the screen reflect many pathways of DNA metabolism including replication, repair, recombination, sister chromatid cohesion, chromosome segregation, and transcription, as well as several uncharacterized genes.

Program Nr. 492C from 2004 Yeast meeting

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