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