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(申请人摘要)：一种快速、灵敏和广泛适用的方法 生化基因组学方法最近被用来鉴定基因 从指定生化活性的酵母酿酒酵母中提取。至 为此，一组可用的ORF(开放阅读框架)基因组被用来 构建一个由6144个酵母菌株组成的阵列，每个酵母菌株都表达一个 不同的酵母开放阅读框架(ORF)在其N端融合为 谷胱甘肽S转移酶(GST)。鉴定ORF相关活性、菌株 在定义的池中培养，纯化GST-ORF；然后对池进行检测 对于活动，对活动池进行反卷积以确定来源 菌株和GST-ORF与活性相关。就这样，14个不同的 活动与一项具体的商品及服务税-开放源码框架挂钩，包括五项活动 修饰蛋白质或处理RNA，这四种活动可以作用于小分子 分子，以及结合DNA或调节其他DNA结合的五种活性 蛋白质。原则上，这种生化基因组学方法可以用来 确定与任何可检测活动相关联的GST-ORF，前提是 是功能性的，在提取过程中可溶解，并根据其他要求进行净化 组件。这种方法是快速的；从提纯的 GST-ORF，需要大约两周的时间才能确定与ORF相关的活动。它 也是敏感的，因为纯化的GST-ORF池可以进行数小时的检测。 这项提议的目标是增强这种生化反应的能力。 基因组学方法有两个方面：一是生化功能的数量 将通过制作C-末端ORF-融合库来扩展ORF融合(因为 大量的ORF不能作为N-末端融合发挥作用，包括许多 膜蛋白)，并通过添加数百个目前不在 图书馆。有了这些ORF融合菌株，酵母中的几乎每一个基因都将 服从于这种生化基因组学方法。其次，这种方法将是 延伸到膜相关蛋白，其含量高达30% 酵母中的蛋白质，历史上更难提纯。vbl.使用 已知的各种活性，我们将制定方法进行纯化和检测 膜相关ORF融合池。然后我们将把这些方法应用于 两种以前未与ORF关联的活性：(1)一种酶 催化棕榈酸酯与蛋白质的结合，以及(2)一种蛋白酶 负责酵母菌交配信息素a因子的降解。应用 将这些技术推广到其他有机体，包括人类和病原体，将 大大加快了生化分析的速度，可用于快速识别 毒品目标。