Biochemical Genomics Linking Genes and Activities

连接基因和活性的生化基因组学

• 批准号: 6794229
• 负责人: Eric M. Phizicky
• 金额: $ 15.75万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-09 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6794229
• 关键词: RNA splicing; Saccharomyces cerevisiae; affinity chromatography; biochemistry; calcium binding protein; endopeptidases; enzyme activity; functional /structural genomics; fungal genetics; fungal proteins; fusion gene; gene expression; genetic library; genetic manipulation; genetic techniques; glutathione transferase; linkage mapping; membrane activity; membrane proteins; open reading frames; palmitates; pheromone; polymerase chain reaction; protein purification

DESCRIPTION (Applicant's Abstract): A rapid, sensitive and widely applicable biochemical genomics approach has recently been developed to identify genes from the yeast Saccharomyces cerevisiae that specify biochemical activities. To this end, an available genomic set of ORFs (open reading frames) was used to construct an array of 6144 individual yeast strains, each expressing a different yeast open reading frame (ORFs) fused at its N-terminus to glutathione S-transferase (GST). To identify ORF-associated activities, strains were grown in defined pools and GST-ORFs were purified; then pools were assayed for activities, and active pools were deconvoluted to identify the source strain and GST-ORF associated with activity. In this way 14 different activities have been linked to a specific GST-ORF, including five activities that modify proteins or process RNA, four activities that can act on small molecules, and five activities that bind DNA or modulate DNA binding of other proteins. In principle this biochemical genomics approach can be used to identify the GST-ORF associated with any detectable activity, provided that it is functional, solubilized during extraction, and purifies with other required components. This approach is rapid; starting with the pools of purified GST-ORFs, it takes about two weeks to identify an ORF-associated activity. It is also sensitive because the purified GST-ORF pools can be assayed for hours. The goal of this proposal is to enhance the repertoire of this biochemical genomics approach in two ways: First, the number of biochemically functional ORF fusions will be expanded by making a C-terminal ORF-fusion library (since a large number of ORFs are not functional as N-terminal fusions, including many membrane proteins), and by adding several hundred ORFs currently not in the library. With these ORF-fusion strains, virtually every gene in yeast will be amenable to this biochemical genomics approach. Second, this approach will be extended to membrane-associated proteins, which comprise as many as 30 percent of the proteins in yeast, and are historically more difficult to purify. Using a variety of known activities, we will develop methods to purify and assay pools of membrane-associated ORF-fusions. Then we will apply these methods to two activities, which have not previously been linked to ORFs: (1) an enzyme catalyzing the attachment of palmitate to proteins, and (2) a protease responsible for degradation of the yeast mating pheromone a-factor. Application of these techniques to other organisms, including humans and pathogens, will greatly accelerate biochemical analysis and can be used to rapidly identify drug targets.

描述（申请人的摘要）：快速，敏感且广泛适用 最近已经开发了生化基因组学方法来鉴定基因 来自酿酒酵母的酵母糖含量，该酿酒酵母指定生化活动。到 这端，使用了一组可用的基因组ORF（开放式阅读框） 构造一个6144个单独的酵母菌菌株的阵列，每个酵母菌菌株表达 在其N末端融合的不同酵母开放阅读框（ORF） 谷胱甘肽S-转移酶（GST）。确定与ORF相关的活动，应变 在定义的池中生长，并纯化GST-ORF；然后分析池 用于活动和活跃的池进行反驳以识别来源 与活动相关的应变和GST-ORF。以这种方式14不同 活动已与特定的GST-ORF链接，包括五个活动 改变蛋白质或过程RNA，四个可以对小的活动 分子和五个结合DNA或调节其他DNA结合的活性 蛋白质。原则上，这种生化基因组学方法可用于 确定与任何可检测活动相关的GST-ORF 功能性，在提取过程中溶解，并与其他所需的净化 成分。这种方法很快；从纯化的池开始 GST-ORF，确定与ORF相关的活性大约需要两个星期。它 也很敏感，因为可以测定纯化的GST-ORF池数小时。 该提议的目的是增强这种生化的曲目 基因组学以两种方式方法：首先，生化功能的数量 ORF融合将通过制作C末端ORF融合库来扩展（因为 大量的ORF不起作用，作为N末端融合，包括许多 膜蛋白），通过添加当前不在 图书馆。使用这些ORF融合菌株，酵母中的每个基因几乎都是 适合这种生化基因组学方法。第二，这种方法将是 扩展到与膜相关的蛋白，该蛋白占多达30％ 酵母中蛋白质的蛋白质，历史上很难净化。使用 各种已知活动，我们将开发净化和分析的方法 与膜相关的ORF合并池。然后，我们将将这些方法应用于 两项以前尚未与ORF相关的活动：（1）酶 催化棕榈酸盐的附着在蛋白质上，（2）蛋白酶 负责降解酵母交配信息素A因子。应用 在包括人类和病原体在内的其他生物的这些技术中， 大大加速生化分析，可用于快速识别 药物靶标。