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

Nature Biotechnology volume 27, pages 369–377 (2009)Cite this article

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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References

  1. Giaever, G. et al. Genomic profiling of drug sensitivities via induced haploinsufficiency. Nat. Genet. 21, 278–283 (1999).

    Article  CAS  Google Scholar 

  2. Hoon, S. et al. An integrated platform of genomic assays reveals small-molecule bioactivities. Nat. Chem. Biol. 4, 498–506 (2008).

    Article  CAS  Google Scholar 

  3. Rine, J., Hansen, W., Hardeman, E. & Davis, R.W. Targeted selection of recombinant clones through gene dosage effects. Proc. Natl. Acad. Sci. USA 80, 6750–6754 (1983).

    Article  CAS  Google Scholar 

  4. Butcher, R.A. et al. Microarray-based method for monitoring yeast overexpression strains reveals small-molecule targets in TOR pathway. Nat. Chem. Biol. 2, 103–109 (2006).

    Article  CAS  Google Scholar 

  5. Luesch, H. et al. A genome-wide overexpression screen in yeast for small-molecule target identification. Chem. Biol. 12, 55–63 (2005).

    Article  CAS  Google Scholar 

  6. Parsons, A.B. et al. Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways. Nat. Biotechnol. 22, 62–69 (2004).

    Article  CAS  Google Scholar 

  7. Hillenmeyer, M.E. et al. The chemical genomic portrait of yeast: uncovering a phenotype for all genes. Science 320, 362–365 (2008).

    Article  CAS  Google Scholar 

  8. Hughes, T.R. et al. Functional discovery via a compendium of expression profiles. Cell 102, 109–126 (2000).

    Article  CAS  Google Scholar 

  9. Parsons, A.B. et al. Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast. Cell 126, 611–625 (2006).

    Article  CAS  Google Scholar 

  10. Douglas, C.M. et al. The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase. Proc. Natl. Acad. Sci. USA 91, 12907–12911 (1994).

    Article  CAS  Google Scholar 

  11. Justice, M.C. et al. Elongation factor 2 as a novel target for selective inhibition of fungal protein synthesis. J. Biol. Chem. 273, 3148–3151 (1998).

    Article  CAS  Google Scholar 

  12. Fried, H.M. & Warner, J.R. Molecular cloning and analysis of yeast gene for cycloheximide resistance and ribosomal protein L29. Nucleic Acids Res. 10, 3133–3148 (1982).

    Article  CAS  Google Scholar 

  13. Liu, Y.-X., Hsiung, Y., Jannatipour, M., Yeh, Y. & Nitiss, J.L. Yeast topoisomerase II mutants resistant to anti-topoisomerase agents: identification and characterization of new yeast topoisomerase II mutants selected for resistance to etoposide. Cancer Res. 54, 2943–2951 (1994).

    CAS  PubMed  Google Scholar 

  14. Kanik-Ennulat, C., Montalvo, E. & Neff, N. Sodium orthovanadate-resistant mutants of Saccharomyces cerevisiae show defects in golgi-mediated protein glycosylation, sporulation and detergent resistance. Genetics 140, 933–943 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Heitman, J., Movva, N. & Hall, M. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253, 905–909 (1991).

    Article  CAS  Google Scholar 

  16. Wiesen, K.M., Xia, S., Yang, C.-P.H. & Horwitz, S.B. Wild-type class I [beta]-tubulin sensitizes Taxol-resistant breast adenocarcinoma cells harboring a [beta]-tubulin mutation. Cancer Lett. 257, 227–235 (2007).

    Article  CAS  Google Scholar 

  17. Douglas, C.M., Marrinan, J.A., Li, W. & Kurtz, M.B. A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3-beta-D-glucan synthase. J. Bacteriol. 176, 5686–5696 (1994).

    Article  CAS  Google Scholar 

  18. Li, M.Z. & Elledge, S.J. MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules. Nat. Genet. 37, 311–319 (2005).

    Article  CAS  Google Scholar 

  19. Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002).

    Article  CAS  Google Scholar 

  20. Pierce, S.E. et al. A unique and universal molecular barcode array. Nat. Methods 3, 601–603 (2006).

    Article  CAS  Google Scholar 

  21. Tong, A.H.Y. et al. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294, 2364–2368 (2001).

    Article  CAS  Google Scholar 

  22. Winzeler, E.A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999).

    Article  CAS  Google Scholar 

  23. Gresham, D. et al. Genome-wide detection of polymorphisms at nucleotide resolution with a single DNA microarray. Science 311, 1932–1936 (2006).

    Article  CAS  Google Scholar 

  24. Käufer, N.F., Fried, H.M., Schwindinger, W.F., Jasin, M. & Warner, J.R. Cycloheximide resistance in yeast: the gene and its protein. Nucleic Acids Res. 11, 3123–3135 (1983).

    Article  Google Scholar 

  25. Vézina, C., Kudelski, A. & Sehgal, S.N. Rapamycin (AY-22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active principle. J. Antibiot. (Tokyo) 28, 721–726 (1975).

    Article  Google Scholar 

  26. Brown, E.J. et al. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369, 756–758 (1994).

    Article  CAS  Google Scholar 

  27. Chiu, M.I., Katz, H. & Berlin, V. RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex. Proc. Natl. Acad. Sci. USA 91, 12574–12578 (1994).

    Article  CAS  Google Scholar 

  28. Sabatini, D.M., Erdjument-Bromage, H., Lui, M., Tempst, P. & Snyder, S.H. RAFT1: A mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 78, 35–43 (1994).

    Article  CAS  Google Scholar 

  29. Coval, S.J., Puar, M.S., Phife, D.W., Terracciano, J.S. & Patel, M. SCH57404, an antifungal agent possessing the rare sodaricin skeleton and a tricyclic sugar moiety. J. Antibiot. (Tokyo) 48, 1171–1172 (1995).

    Article  CAS  Google Scholar 

  30. Hauser, D. & Sigg, H.P. Isolierung und abbau von sordarin. 1. Mitteilung über sordarin. Helv. Chim. Acta 54, 1178–1190 (1971).

    Article  CAS  Google Scholar 

  31. Capa, L., Mendoza, A., Lavandera, J.L., Gomez de las Heras, F. & Garcia-Bustos, J.F. Translation elongation factor 2 is part of the target for a new family of antifungals. Antimicrob. Agents Chemother. 42, 2694–2699 (1998).

    Article  CAS  Google Scholar 

  32. Drotschmann, K. et al. Mutator phenotypes of yeast strains heterozygous for mutations in the MSH2 gene. Proc. Natl. Acad. Sci. USA 96, 2970–2975 (1999).

    Article  CAS  Google Scholar 

  33. Krepkiy, D. & Miziorko, H.M. Identification of active site residues in mevalonate diphosphate decarboxylase: implications for a family of phosphotransferases. Protein Sci. 13, 1875–1881 (2004).

    Article  CAS  Google Scholar 

  34. Schuldiner, M. et al. Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123, 507–519 (2005).

    Article  CAS  Google Scholar 

  35. Muhlrad, D. & Parker, R. Aberrant mRNAs with extended 3′ UTRs are substrates for rapid degradation by mRNA surveillance. RNA 5, 1299–1307 (1999).

    Article  CAS  Google Scholar 

  36. Yan, Z. et al. Yeast Barcoders: a chemogenomic application of a universal donor-strain collection carrying bar-code identifiers. Nat. Methods 5, 719–725 (2008).

    Article  CAS  Google Scholar 

  37. Bolard, J. How do the polyene macrolide antibiotics affect the cellular membrane properties? Biochim. Biophys. Acta 864, 257–304 (1986).

    Article  CAS  Google Scholar 

  38. Arneson, P.A. & Durbin, R.D. Studies on the mode of action of tomatine as a fungitoxic agent. Plant Physiol. 43, 683–686 (1968).

    Article  CAS  Google Scholar 

  39. Simons, V. et al. Dual effects of plant steroidal alkaloids on Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 50, 2732–2740 (2006).

    Article  CAS  Google Scholar 

  40. Keukens, E.A.J. et al. Dual specificity of sterol-mediated glycoalkaloid induced membrane disruption. Biochim. Biophys. Acta 1110, 127–136 (1992).

    Article  CAS  Google Scholar 

  41. Kitagawa, I., Kobayashi, M., Inamoto, T., Yasuzawa, T. & Kyogoku, Y. The structures of 6 antifungal oligoglycosides, stichloroside-a1, stichloroside-a2, stichloroside-b1, stichloroside-b2, stichloroside-c1, and stichloroside-c2, from the sea-cucumber stichopus-chloronotus (brandt). Chem. Pharm. Bull. (Tokyo) 29, 2387–2391 (1981).

    Article  CAS  Google Scholar 

  42. Matsunaga, S., Fusetani, N., Hashimoto, K., Walchli, M. & Theonellamide, F. A novel antifungal bicyclic peptide from a marine sponge Theonella sp. J. Am. Chem. Soc. 111, 2582–2588 (1989).

    Article  CAS  Google Scholar 

  43. Matsunaga, S. & Fusetani, N. Theonellamides A-E, cytotoxic bicyclic peptides, from a marine sponge Theonella sp. J. Org. Chem. 60, 1177–1181 (1995).

    Article  CAS  Google Scholar 

  44. Bewley, C.A. & Faulkner, D.J. Theonegramide, an antifungal glycopeptide from the Philippine lithistid sponge theonella swinhoei. J. Org. Chem. 59, 4849–4852 (1994).

    Article  CAS  Google Scholar 

  45. Ott, R.G. et al. Flux of sterol intermediates in a yeast strain deleted of the lanosterol C-14 demethylase Erg11p. Biochim. Biophys. Acta 1735, 111–118 (2005).

    Article  CAS  Google Scholar 

  46. Bagnat, M. & Simons, K. Cell surface polarization during yeast mating. Proc. Natl. Acad. Sci. USA 99, 14183–14188 (2002).

    Article  CAS  Google Scholar 

  47. Jones, G.M. et al. A systematic library for comprehensive overexpression screens in Saccharomyces cerevisiae. Nat. Methods 5, 239–241 (2008).

    Article  CAS  Google Scholar 

  48. Shendure, J. & Ji, H. Next-generation DNA sequencing. Nat. Biotechnol. 26, 1135–1145 (2008).

    Article  CAS  Google Scholar 

  49. Kahvejian, A., Quackenbush, J. & Thompson, J.F. What would you do if you could sequence everything? Nat. Biotechnol. 26, 1125–1133 (2008).

    Article  CAS  Google Scholar 

  50. Deutschbauer, A.M. et al. Mechanisms of haploinsufficiency revealed by genome-wide profiling in yeast. Genetics 169, 1915–1925 (2005).

    Article  CAS  Google Scholar 

  51. Sopko, R. et al. Mapping pathways and phenotypes by systematic gene overexpression. Mol. Cell 21, 319–330 (2006).

    Article  CAS  Google Scholar 

  52. Smith, V., Botstein, D. & Brown, P.O. Genetic footprinting: a genomic strategy for determining a gene's function given its sequence. Proc. Natl. Acad. Sci. USA 92, 6479–6483 (1995).

    Article  CAS  Google Scholar 

  53. Jorgensen, P. et al. High-resolution genetic mapping with ordered arrays of Saccharomyces cerevisiae deletion mutants. Genetics 162, 1091–1099 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Rogers, B. et al. The pleiotropic drug ABC transporters from Saccharomyces cerevisiae. J. Mol. Microbiol. Biotechnol. 3, 207–214 (2001).

    CAS  PubMed  Google Scholar 

  55. Rancati, G. et al. Aneuploidy underlies rapid adaptive evolution of yeast cells deprived of a conserved cytokinesis motor. Cell 135, 879–893 (2008).

    Article  CAS  Google Scholar 

  56. Dunham, M.J. et al. Characteristic genome rearrangements in experimental evolution of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 99, 16144–16149 (2002).

    Article  CAS  Google Scholar 

  57. Gresham, D. et al. The repertoire and dynamics of evolutionary adaptations to controlled nutrient-limited environments in yeast. PLoS Genet. 4, e1000303 (2008).

    Article  Google Scholar 

  58. Williams, D.E. et al. Dominicin, a cyclic octapeptide, and laughine, a bromopyrrole alkaloid, isolated from the Caribbean marine sponge Eurypon laughlini. J. Nat. Prod. 68, 327–330 (2005).

    Article  CAS  Google Scholar 

  59. Butcher, R.A. & Schreiber, S.L. A microarray-based protocol for monitoring the growth of yeast overexpression strains. Nat. Protoc. 1, 569–576 (2006).

    Article  CAS  Google Scholar 

  60. Pierce, S.E., Davis, R.W., Nislow, C. & Giaever, G. Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures. Nat. Protoc. 2, 2958–2974 (2007).

    Article  CAS  Google Scholar 

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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.

Author information

Author notes

  1. Cheuk Hei Ho, Leslie Magtanong and Sarah L Barker: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Cheuk Hei Ho, Leslie Magtanong, Sarah L Barker, Judice L Y Koh, Guri Giaever, Corey Nislow, Brenda Andrews & Charles Boone

  2. Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada

    Cheuk Hei Ho, Leslie Magtanong, Sarah L Barker, Judice L Y Koh, Guri Giaever, Corey Nislow, Brenda Andrews & Charles Boone

  3. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, USA

    David Gresham & David Botstein

  4. Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama, Japan

    Shinichi Nishimura, Minoru Yoshida & Charles Boone

  5. Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan

    Shinichi Nishimura

  6. Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA

    Paramasivam Natarajan & Todd R Graham

  7. Department of Chemistry, Earth & Ocean Sciences, University of British Columbia, Vancouver, British Columbia, Canada

    Justin Porter, Christopher A Gray & Raymond J Andersen

  8. Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada

    Guri Giaever

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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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