Research abstract
Article abstract
Nature Biotechnology 27, 369 - 377 (2009)
Published online: 6 April 2009 | doi:10.1038/nbt.1534
A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds
Cheuk Hei Ho1,2,9, Leslie Magtanong1,2,9, Sarah L Barker1,2,9, David Gresham3, Shinichi Nishimura4,5, Paramasivam Natarajan6, Judice L Y Koh1,2, Justin Porter7, Christopher A Gray7, Raymond J Andersen7, Guri Giaever1,2,8, Corey Nislow1,2, Brenda Andrews1,2, David Botstein3, Todd R Graham6, Minoru Yoshida4 & Charles Boone1,2,4
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.
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
- Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, USA.
- Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama, Japan.
- Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
- Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA.
- Department of Chemistry, Earth & Ocean Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
- Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
- These authors contributed equally to this work.
Correspondence to: Charles Boone1,2,4 e-mail: charlie.boone@utoronto.ca
Correspondence to: Minoru Yoshida4 e-mail: yoshidam@riken.jp
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