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

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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Figure 1: Construction of the MoBY-ORF library by homologous recombination in yeast.
Figure 2: Identifying a recessive spontaneous drug-resistant mutant by MoBY-ORF complementation cloning.
Figure 3: Mapping a drug-resistant mutant by MoBY-ORF complementation cloning and yeast tiling microarrays.
Figure 4: MOA analysis of theopalauamide and stichloroside.
Figure 5: Theopalauamide and theonellamide represent a novel class of sterol-binding compound.

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Acknowledgements

We thank A. Smith and L. Heisler for advice on the data analysis of barcode microarray and A. Ward for technical assistance with the YTM experiments. We thank B. Sheikh for primer design, diagnostic digest predictions and sequencing scripts. We thank K. Toufighi for data analysis of liposome leakage experiments. Theonellamide was a kind gift from S. Matsunaga (Graduate School of Agricultural and Life Sciences, The University of Tokyo). L.M. was supported by a Canadian Institutes of Health Research Doctoral Research Award. R.J.A. was supported by a research grant from NSERC. C.N. was supported by NHGRI (MOP-84305). G.G. was supported by NHGRI (MOP-81340). D.B. was supported by NIGMS Center for Quantitative Biology (GM071508) and R01 (GM046406). T.R.G. was supported by National Institutes of Health (GM62637). M.Y. and S.N. were supported by an Energy and Industrial Technology Development Organization (NEDO) project on development of basic technology to control biological systems using chemical compounds. C.B. was supported by Genome Canada through the Ontario Genomics Institute as per research agreement 2004-OGI-3-01 and Canadian Institutes of Health Research agreement number MOP-57830.

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Contributions

C.H.H. was involved in MoBY-ORF construction, carried out experiments, data analysis and interpretation of cloning drug-resistant mutants, carried out the genetic and cell biological experiments to characterize the MOAs of theopalauamide and stichloroside and wrote the manuscript; L.M. was involved in MoBY-ORF construction and wrote the manuscript; S.L.B. was involved in MoBY-ORF construction, sequencing and functional studies of the MoBY-ORF library, and wrote the manuscript; D.G. carried out all the experiments and data analysis of the YTM experiments, and wrote the manuscript; S.N. carried out all the experiments and data analysis of the in vitro ergosterol binding experiment, prepared fluorescently-labeled theonellamide A, and wrote the manuscript; P.N. carried out experiments and data analysis of the liposome leakage experiments, and wrote the manuscript; J.L.Y.K. provided computational support for MoBY-ORF validation, database construction and sequencing analysis; J.P. purified theopalauamide and stichloroside; C.A.G. purified theopalauamide and stichloroside; R.J.A. purified theopalauamide and stichloroside, and edited the manuscript; G.G. provided data analysis of cloning drug-resistant mutants with MoBY-ORF complementation using barcode microarray, and edited the manuscript. C.N. provided data analysis of cloning drug-resistant mutants with MoBY-ORF complementation using barcode microarray, and edited the manuscript; B.A. edited the manuscript; D.B. was involved in YTM analysis and edited manuscript; T.R.G. provided data analysis of all of the liposome leakage experiments, and wrote the manuscript; M.Y. provided data analysis and interpretation of the theopalauamide and theonellamide results, and wrote the manuscript; C.B. conceived and planned the construction of the MoBY-ORF library, provided data analysis and interpretation of the results, and wrote the manuscript.

Corresponding authors

Correspondence to Minoru Yoshida or Charles Boone.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 5 and Supplementary Methods (PDF 1755 kb)

Supplementary Table 1

Primers and restriction digest results for all MoBY-ORF clones are based on the 1st May 2005 freeze of the S288C genome sequence. (XLS 4992 kb)

Supplementary Table 2

Primers used to amplify the barcode/KanMX4 cassettes. (XLS 74 kb)

Supplementary Table 3

Sequencing analysis of MoBY-ORF library clones. (XLS 1632 kb)

Supplementary Table 4

Functional complementation of temperature-sensitive mutants with MoBY-ORF library clones. (XLS 87 kb)

Supplementary Table 6

Affymetrix TAG4 microarray MoBY-ORF barcode annotation. (XLS 665 kb)

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Ho, C., Magtanong, L., Barker, S. et al. A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds. Nat Biotechnol 27, 369–377 (2009). https://doi.org/10.1038/nbt.1534

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