A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds.
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.
Cheuk Hei
CH
Ho
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.
Leslie
L
Magtanong
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.
Sarah L
SL
Barker
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, USA.
David
D
Gresham
Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama, Japan.|Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Shinichi
S
Nishimura
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA.
Paramasivam
P
Natarajan
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.
Judice L Y
JLY
Koh
Department of Chemistry, Earth & Ocean Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
Justin
J
Porter
Department of Chemistry, Earth & Ocean Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
Christopher A
CA
Gray
Department of Chemistry, Earth & Ocean Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
Raymond J
RJ
Andersen
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.|Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
Guri
G
Giaever
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.
Corey
C
Nislow
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.
Brenda
B
Andrews
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, USA.
David
D
Botstein
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA.
Todd R
TR
Graham
Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama, Japan.
Minoru
M
Yoshida
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.|Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama, Japan.
Charles
C
Boone
D003001:Cloning, Molecular / Q000379:methods*; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods* / Q000639:trends*; D016366:Open Reading Frames / Q000235:genetics*