New Methods for Using Gene Disruption Libraries

使用基因破坏文库的新方法

• 批准号: 6823438
• 负责人: Rodney J. ROTHSTEIN
• 金额: $ 6.15万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-20 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6823438
• 关键词: Saccharomyces cerevisiae; abstracting; biomedical resource; biotechnology; field study; fungal genetics; gene mutation; genetic library; genetic manipulation; genetic markers; genetic recombination; genetic strain; genetic techniques; method development; nucleic acid sequence; plasmids; polymerase chain reaction; reporter genes; restriction endonucleases

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.

描述(由申请人提供)：酿酒酵母的完整基因中断文库最近已可用。在许多方面，这一资源将促进全基因组分析，这项提议的主要目的是开发方法来提高这一资源的效用。为了一个特定的目的，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，以任何基于质粒的报告实验快速筛选中断文库。这种方法将有助于识别影响任何特定过程的所有基因，可以为这些过程设计报告分析。第二个具体目标是开发试剂，允许在体内切除基于质粒的基因中断，以便整合到任何实验室菌株的基因组中。中断结构的设计允许通过直接重复重组回收可选标记，以允许使用相同方法进行额外的几轮基因中断。与基于交配的质粒转移相结合，这将允许快速构建新的缺失菌株文库。这项建议可分为以下两个具体目标： (1)创建一个通用的供体菌株，与当前(或任何未来)酵母基因中断文库结合使用，以允许通过KAR介导质粒转移将任何报告质粒轻松地引入到中断集合中。 (2)开发方法，通过在体内激活基于质粒的基因中断盒，允许将基因中断转移到任何酵母遗传背景中。 这里概述的方法并不是酵母基因组所特有的；它们可以应用于任何已测序的基因组。例如，基因中断可以在任何可转化物种中构建，这些物种可以获得测序基因组(例如念珠菌或许多细菌菌株中的一种)。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。

项目成果

(Z)-Ethyl 2-hydroxy-imino-2-(4-nitro-benz-yl)ethanoate.

(Z)-2-羟基-亚氨基-2-(4-硝基-苯甲基)乙酸乙酯。

• DOI: 10.1107/s1600536809052386
• 发表时间: 2009
• 期刊: Acta crystallographica. Section E, Structure reports online
• 作者: Caracelli,Ignez;Trindade,AntonioC;Moran,PauloJS;Hinoue,Luciana;Zukerman-Schpector,Julio;Tiekink,EdwardRT
• 通讯作者: Tiekink,EdwardRT