Yeast Barcoders: a chemogenomic application of a universal donor-strain collection carrying bar-code identifiers

Abstract

The ability to perform complex bioassays in parallel enables experiments that are otherwise impossible because of throughput and cost constraints. For example, highly parallel chemical-genetic screens using pooled collections of thousands of defined Saccharomyces cerevisiae gene deletion strains are feasible because each strain is bar-coded with unique DNA sequences. It is, however, time-consuming and expensive to individually bar-code individual strains. To provide a simple and general method of barcoding yeast collections, we built a set of donor strains, called Barcoders, with unique bar codes that can be systematically transferred to any S. cerevisiae collection. We applied this technology by generating a collection of bar-coded 'decreased abundance by mRNA perturbation' (DAmP) loss-of-function strains comprising 87.1% of all essential yeast genes. These experiments validate both the Barcoders and the DAmP strain collection as useful tools for genome-wide chemical-genetic assays.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Strategy used to bar-code DAmP strains.
Figure 2: Growth rate of DAmP strains.
Figure 3: Drug sensitivity assay.
Figure 4: Genome-wide profiles of strain sensitivity to selected compounds.

References

  1. 1

    Hensel, M. et al. Simultaneous identification of bacterial virulence genes by negative selection. Science 269, 400–403 (1995).

  2. 2

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

  3. 3

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

  4. 4

    Jo, W.J. et al. Identification of genes involved in the toxic response of Saccharomyces cerevisiae against iron and copper overload by parallel analysis of deletion mutants. Toxicol. Sci. 101, 140–151 (2008).

  5. 5

    Brass, A.L. et al. Identification of host proteins required for HIV infection through a functional genomic screen. Science 319, 921–926 (2008).

  6. 6

    Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006).

  7. 7

    Mazurkiewicz, P., Tang, C.M., Boone, C. & Holden, D.W. Signature-tagged mutagenesis: barcoding mutants for genome-wide screens. Nat. Rev. Genet. 7, 929–939 (2006).

  8. 8

    Giaever, G. et al. Chemogenomic profiling: identifying the functional interactions of small molecules in yeast. Proc. Natl. Acad. Sci. USA 101, 793–798 (2004).

  9. 9

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

  10. 10

    Lum, P.Y. et al. Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes. Cell 116, 121–137 (2004).

  11. 11

    Shoemaker, D.D., Lashkari, D.A., Morris, D., Mittmann, M. & Davis, R.W. Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy. Nat. Genet. 14, 450–456 (1996).

  12. 12

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

  13. 13

    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).

  14. 14

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

  15. 15

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

  16. 16

    Baganz, F., Hayes, A., Marren, D., Gardner, D.C. & Oliver, S.G. Suitability of replacement markers for functional analysis studies in Saccharomyces cerevisiae. Yeast 13, 1563–1573 (1997).

  17. 17

    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).

  18. 18

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

  19. 19

    Dwight, S.S. et al. Saccharomyces genome database (SGD) provides secondary gene annotation using the Gene Ontology (GO). Nucleic Acids Res. 30, 69–72 (2002).

  20. 20

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

  21. 21

    St Onge, R.P. et al. Systematic pathway analysis using high-resolution fitness profiling of combinatorial gene deletions. Nat. Genet. 39, 199–206 (2007).

  22. 22

    Lee, W. et al. Genome-wide requirements for resistance to functionally distinct DNA-damaging agents. PLoS Genet. 1, e24 (2005).

  23. 23

    Gadsden, M.H., McIntosh, E.M., Game, J.C., Wilson, P.J. & Haynes, R.H. dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae. EMBO J. 12, 4425–4431 (1993).

  24. 24

    Game, J.C. Yeast cell-cycle mutant cdc21 is a temperature-sensitive thymidylate auxotroph. Mol. Gen. Genet. 146, 313–315 (1976).

  25. 25

    Hardman, J.G., Limbird, L.E. & Gilman, A.G. (eds.) Goodman and Gilman's The Pharmacological Basis of Therapeutics 10th edn. (McGraw-Hill, New York, 2001).

  26. 26

    Scherf, U. et al. A gene expression database for the molecular pharmacology of cancer. Nat. Genet. 24, 236–244 (2000).

  27. 27

    Ross-Macdonald, P. et al. Large-scale analysis of the yeast genome by transposon tagging and gene disruption. Nature 402, 413–418 (1999).

  28. 28

    Mnaimneh, S. et al. Exploration of essential gene functions via titrable promoter alleles. Cell 118, 31–44 (2004).

  29. 29

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

  30. 30

    Uetz, P. et al. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000).

Download references

Acknowledgements

We thank J. Horecka, N. Berbenetz and M. Urbanus for comments on the manuscript. This work is supported by grants from the US National Institute of Health and Canadian Institutes of Health Research to G.G. (MOP-81340) and to C.N. (MOP-84305), and from Genome Canada (to C.B. and B.J.A.).

Author information

Z.Y. performed all bar-coding experiments and analysis and wrote this paper. M.C., B.J.A., J.P. and C.B. designed and created the original DAmP collection. L.E.H. did the data analysis. F.K. sequenced all bar codes. G.G. and C.N. designed the study, analyzed the data and wrote the paper.

Correspondence to Corey Nislow.

Ethics declarations

Competing interests

F.K. is employed by Prognosys Biosciences, Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1, Supplementary Tables 1–5, Supplementary Methods (PDF 530 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Yan, Z., Costanzo, M., Heisler, L. et al. Yeast Barcoders: a chemogenomic application of a universal donor-strain collection carrying bar-code identifiers. Nat Methods 5, 719–725 (2008) doi:10.1038/nmeth.1231

Download citation

Further reading