Biochemical Genomics Linking Genes and Activities

连接基因和活性的生化基因组学

• 批准号: 6536489
• 负责人: Eric M. Phizicky
• 金额: $ 37.97万
• 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 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6536489
• 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（申请人摘要）：一种快速、灵敏和广泛适用的 生物化学基因组学方法最近已发展到确定基因 来自指定生化活性的酿酒酵母。到 为此，使用一组可用的ORF（开放阅读框）基因组， 构建6144个单独的酵母菌株的阵列，每个酵母菌株表达 不同的酵母开放阅读框（ORF）在其N-末端融合， 谷胱甘肽S-转移酶（GST）。为了鉴定ORF相关活性， 在确定的池中生长并纯化GST-ORF;然后测定池 对活动和活动池进行去卷积，以确定源 菌株和与活性相关的GST-ORF。14种不同的 这些活动与特定的GST-ORF相关联，包括五项活动 修饰蛋白质或加工RNA的四种活动， 分子和五种结合DNA或调节DNA结合其他 proteins.原则上，这种生物化学基因组学方法可用于 鉴定与任何可检测活性相关的GST-ORF，前提是 是功能性的，在提取过程中溶解，并与其他所需的纯化 件.这种方法是快速的;从纯化的 GST-ORF，需要大约两周的时间来鉴定ORF相关的活性。它 也是敏感的，因为纯化的GST-ORF库可以被测定数小时。 这项提议的目的是增强这种生化反应的能力， 基因组学的方法有两种：一是生物化学功能的数量 ORF融合体将通过制备C-末端ORF-融合体文库来扩增（因为 大量ORF不具有N-末端融合的功能，包括许多 膜蛋白），并通过添加数百个ORF，目前没有在 图书馆有了这些ORF融合菌株，几乎酵母中的每一个基因都将被 这一生物化学基因组学的方法。第二，这种方法将 扩展到膜相关蛋白，其中包括多达30%的 酵母中的蛋白质，并且历史上更难纯化。使用 各种已知的活动，我们将开发方法来纯化和测定 膜相关ORF融合物的池。然后我们将这些方法应用于 两种以前没有与ORF联系在一起的活性：（1）酶 催化棕榈酸酯与蛋白质的连接，和（2）蛋白酶 负责酵母交配信息素A因子的降解。应用 将这些技术应用于其他生物，包括人类和病原体， 大大加快了生化分析的速度， 药物靶点