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A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds

TitleA molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds
Publication TypeJournal Article
Year of Publication2009
AuthorsHo, CH, Magtanong, L, Barker, SL, Gresham, D, Nishimura, S, Natarajan, P, Koh, JLY, Porter, J, Gray, CA, Andersen, RJ, Giaever, G, Nislow, C, Andrews, B, Botstein, D, Graham, TR, Yoshida, M, Boone, C
JournalNature Biotechnology
Volume27
Pagination369-377
Date PublishedApr
Type of ArticleArticle
ISBN Number1087-0156
Keywords1, 2, 3-BETA-D-GLUCAN SYNTHASE, CYCLOHEXIMIDE, DELETION MUTANTS, ELONGATION-FACTOR, GENE-DELETION, MAMMALIAN PROTEIN, ORDERED ARRAYS, OVEREXPRESSION STRAINS, resistance, SACCHAROMYCES-CEREVISIAE GENOME, SPONGE THEONELLA SP
Abstract

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.

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