New Methods for Using Gene Disruption Libraries

描述（由申请人提供）：酵母酿酒酵母（Saccharomyces cerevisiae）的完整基因破坏文库最近已可用。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制定方法，提高这一资源的效用。对于一个特定的目标，我们将开发一种新的供体酵母菌株，允许交配为基础的质粒转移的破坏库的快速筛选与任何质粒为基础的报告分析。这样的方法将有助于鉴定影响任何特定过程的所有基因，可以设计报告基因测定。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，以整合到任何实验室菌株背景的基因组中。破坏构建体被设计为允许选择标记通过直接重复重组再循环，以允许使用相同方法进行额外轮次的基因破坏。结合基于交配的质粒转移，这将允许快速构建新的缺失菌株文库。该提案可分为以下两个具体目标： (1)创建通用供体菌株以与当前（或任何未来）酵母基因破坏文库结合使用，以允许通过kar-mediated质粒转移将任何报告质粒容易地引入到破坏组中。 (2)开发通过在体内激活基于质粒的基因破坏盒将基因破坏转移到任何酵母遗传背景中的方法。 这里概述的方法不是特定于酵母基因组;它们可以应用于任何测序的基因组。例如，基因破坏可以在任何可转化的物种中构建，对于这些物种，测序的基因组是可用的（例如，念珠菌或许多细菌菌株之一）。这些目标的成功实现将极大地扩展分子遗传学家工具箱中可用的方法。

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.