The complete gene disruption library of the yeast Saccharomyces cerevisiae has recently become available. In many ways, this resource will facilitate genome-wide analysis and the main aim of this proposal is the development of methods to enhance the utility of this resource. For one specific aim, we will develop a novel donor yeast strain to permit mating-based plasmid transfer for rapid screening of the disruption library with any plasmid-based reporter assay. Such an approach will facilitate the identification of all genes that affect any particular process for which a reporter assay can be designed. The second specific aim is to develop reagents to allow in vivo excision of a plasmid based gene disruption for integration into the genome of any laboratory strain background. The disruption construct is designed to permit recycling of the selectable marker via direct repeat recombination to allow additional rounds of gene disruptions using the same approach. In conjunction with the mating-based plasmid transfer, this will allow rapid construction of new deletion strain libraries. The proposal can be divided into the following two specific aims: ? ? (1) Create a universal donor strain to be used in conjunction with the current (or any future) yeast gene disruption library to permit the facile introduction of any reporter plasmid into the set of disruptions via kar-mediated plasmid transfer. ? ? (2) Develop methods to permit the transfer of gene disruptions into any yeast genetic background by activating a plasmid-based gene disruption cassette in vivo. ? ? The methods outlined here are not specific to the yeast genome; they can be applied to any sequenced genome. For example, gene disruptions can be constructed in any transformable species for which a sequenced genome is available (e.g., Candida or one of many bacterial strains). Successful completion of these aims will greatly expand the methods available in the molecular geneticist's tool kit.
