A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds.
A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds.
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DOI：
10.1038/nbt.1534
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发表时间：
2009-04
影响因子：
46.9
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中科院分区：
文献类型：
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我们提出了一种酵母化学基因组学方法，旨在确定基因突变时赋予耐药性，从而提供有关化合物的作用模式的见解。我们开发了分子条形码化的酵母开放阅读框（MoBY-ORF）文库，其中由其天然启动子和终止子控制的每个基因与两个独特的寡核苷酸条形码一起被克隆到基于着丝粒的载体中沿着。MoBY-ORF资源具有许多遗传和化学遗传应用，但在这里，我们专注于使用互补策略克隆突变耐药基因的野生型版本，并同时用条形码微阵列测定所有转化体的适应性。互补克隆通过使用全基因组酵母平铺微阵列的突变检测进行验证，该微阵列鉴定了与耐药突变体相关的独特多态性。我们使用MoBY-ORF文库鉴定了几种耐药突变体的遗传基础，并在该分析中发现了一类新的甾醇结合化合物。
We present a yeast chemical-genomics approach designed to identify genes that when mutated confer drug resistance, thereby providing insight about the modes of action of compounds. We developed a molecular barcoded yeast open reading frame (MoBY-ORF) library in which each gene, controlled by its native promoter and terminator, is cloned into a centromere-based vector along with two unique oligonucleotide barcodes. The MoBY-ORF resource has numerous genetic and chemical-genetic applications, but here we focus on cloning wild-type versions of mutant drug-resistance genes using a complementation strategy and on simultaneously assaying the fitness of all transformants with barcode microarrays. The complementation cloning was validated by mutation detection using whole-genome yeast tiling microarrays, which identified unique polymorphisms associated with a drug-resistant mutant. We used the MoBY-ORF library to identify the genetic basis of several drug-resistant mutants and in this analysis discovered a new class of sterol-binding compounds.