Biochemical Genomics Linking Genes and Activities

连接基因和活性的生化基因组学

基本信息

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
项目类别：
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

项目摘要

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）：一种快速、灵敏且广泛适用的方法最近开发了生化基因组学方法来识别基因来自具有特定生化活性的酿酒酵母。到为此，使用了可用的 ORF（开放阅读框）基因组集构建了 6144 个酵母菌株的阵列，每个酵母菌株表达一个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、菌株在指定池中生长并纯化 GST-ORF；然后对池进行分析活动，并对活动池进行解卷积以确定来源菌株和 GST-ORF 与活性相关。这样就有14种不同的活动已与特定 GST-ORF 相关联，包括五项活动修饰蛋白质或加工 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用于识别与任何可检测活动相关的 GST-ORF，前提是它具有功能性，在提取过程中溶解，并与其他所需的物质一起纯化成分。这种方法速度很快；从纯化池开始 GST-ORF，识别 ORF 相关活性大约需要两周时间。它也很敏感，因为纯化的 GST-ORF 库可以分析数小时。该提案的目标是增强这种生化物质的功能基因组学方法有两种：第一，生化功能的数量 ORF 融合将通过制作 C 端 ORF 融合文库来扩展（自大量 ORF 不具有 N 端融合功能，包括许多膜蛋白），并通过添加目前不在图书馆。有了这些 ORF 融合菌株，几乎酵母中的每个基因都将被适合这种生化基因组学方法。其次，这种方法将扩展到膜相关蛋白，其中高达 30% 酵母中的蛋白质，并且历史上更难以纯化。使用各种已知的活性，我们将开发纯化和测定的方法膜相关 ORF 融合池。然后我们将应用这些方法以前未与 ORF 关联的两种活性：(1) 酶催化棕榈酸酯与蛋白质的附着，以及（2）蛋白酶负责酵母交配信息素α因子的降解。应用这些技术对其他生物体，包括人类和病原体的影响，将大大加速生化分析，可用于快速鉴定药物靶点。