New Methods for Using Gene Disruption Libraries

描述（由申请人提供）：酿酒酵母的完整基因破坏文库最近已经可用。在许多方面，这一资源将促进全基因组分析，本提案的主要目的是开发方法来提高这一资源的效用。为了一个特定的目标，我们将开发一种新的供体酵母菌株，以允许基于交配的质粒转移，以便用任何基于质粒的报告基因检测快速筛选破坏文库。这种方法将有助于识别影响任何特定过程的所有基因，可以为此设计报告基因分析。第二个具体目标是开发试剂，以允许在体内切除基于质粒的基因破坏，以便整合到任何实验室菌株背景的基因组中。破坏结构的设计允许通过直接重复重组来回收可选择的标记，从而允许使用相同的方法进行额外的基因破坏。结合基于配对的质粒转移，这将允许快速构建新的缺失菌株文库。该建议可分为以下两个具体目的：

DESCRIPTION (provided by applicant): 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.