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Nat Biotechnol. 2009 Apr;27(4):369-77. doi: 10.1038/nbt.1534. Epub 2009 Apr 6.

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

Author information

1
Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
2
Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
3
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, USA.
4
Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama, Japan.
5
Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
6
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA.
7
Department of Chemistry, Earth & Ocean Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
8
Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
#
Contributed equally

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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PMID:
19349972
PMCID:
PMC3856559
DOI:
10.1038/nbt.1534
[Indexed for MEDLINE]
Free PMC Article

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