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High-throughput and sensitive assay to measure yeast cell growth: a bench protocol for testing genotoxic agents

Abstract

Intracellular metabolites and environmental agents continuously challenge the structural integrity of DNA. In the yeast Saccharomyces cerevisiae, the complete collection of open reading frame deletion mutants, in combination with powerful screening methods, allows for the comprehensive analyses of cellular responses to insult. We have developed a protocol to determine the sensitivity of growing yeast to DNA-damaging agents that is based on automatic measurements of the optical density of very small (100 μl) liquid cultures. This simple method is highly sensitive, provides quantifiable data and offers high-throughput screening capability. Starting with the treatment of cells with different doses of damaging agents, pre-prepared growing media containing 96-well plates are inoculated and cell population is automatically monitored every 10 min for 48 hours. With the aid of a multi-channel pipette, the sensitivity of a number of yeast strains to several concentrations of drug can be tested in triplicate in less then 4 hours.

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Figure 1: Exposure of wild-type, rad1Δ, rad7Δ, rad16Δ and rad26Δ cells to UV irradiation.
Figure 2: Calculation of growth parameters.
Figure 3: Schematic of the algorithm for automatic calculation of the doubling time and the lag time.

References

  1. Hoeijmakers, J.H.J. Genome maintenance mechanisms for preventing cancer. Nature 411, 366–374 (2001).

    Article  CAS  Google Scholar 

  2. Hanawalt, P.C., Ford, J.M. & Lloyd, D.R. Functional characterization of global genomic DNA repair and its implications for cancer. Mutat. Res. 544, 107–114 (2003).

    Article  CAS  Google Scholar 

  3. Friedberg, E.C. DNA damage and repair. Nature 421, 436–440 (2003).

    Article  Google Scholar 

  4. Suter, B., Auerbach, D. & Stagljar, I. Yeast-based functional genomics and proteomics technologies: the first 15 years and beyond. Biotechniques 40, 625–644 (2006).

    Article  CAS  Google Scholar 

  5. Prakash, S. & Prakash, L. Nucleotide excision repair in yeast. Mutat. Res. 451, 13–24 (2000).

    Article  CAS  Google Scholar 

  6. Hartwell, L.H. Yeast and cancer. Biosci. Rep. 24, 523–544 (2004).

    Article  Google Scholar 

  7. Rieger, K.-J. et al. Large-scale phenotypic analysis in microtitre plates of mutants with deleted open reading frames from yeast chromosome III: key-step between genomic sequencing and protein function. Methods Microbiol. 28, 205–227 (1999).

    Article  CAS  Google Scholar 

  8. Rieger, K.-J. et al. Chemotyping of yeast mutants using robotics. Yeast 15, 973–986 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Warringer, J. & Blomberg, A. Automated screening in environmental arrays allows analysis of quantitative phenotypic profiles in Saccharomyces cerevisiae. Yeast 20, 53–67 (2003).

    Article  CAS  Google Scholar 

  11. Toussaint, M. et al. A high-throughput method to measure the sensitivity of yeast cells to genotoxic agents in liquid cultures. Mutat. Res. 606, 92–105 (2006).

    Article  CAS  Google Scholar 

  12. Conconi, A. et al. Mitotic viability and metabolic competence in UV-irradiated yeast cells. Mutat. Res. 459, 55–64 (2000).

    Article  CAS  Google Scholar 

  13. 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 

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

    Article  CAS  Google Scholar 

  15. Steinmetz, L.M. et al. Systematic screen for human disease genes in yeast. Nature Genet. 31, 400–404 (2002).

    Article  CAS  Google Scholar 

  16. Warringer, J. et al. High-resolution yeast phenomics resolves different physiological features in the saline response. Proc. Natl. Acad. Sci. USA 100, 15724–15729 (2003).

    Article  CAS  Google Scholar 

  17. Dielbandhoesing, S.K. et al. Specific cell wall protein confer resistance to Nisin upon yeast cells. Appl. Environ. Microbiol. 64, 4047–4052 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Aouida, M. et al. Comparative roles of the cell wall and cell membrane in limiting uptake of xenobiotic molecules by Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 47, 2012–2014 (2003).

    Article  CAS  Google Scholar 

  19. Osterberg, M. et al. Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 103, 11148–11153 (2006).

    Article  Google Scholar 

  20. Warringer, J. & Blomberg, A. Involvement of yeast YOLI51W/GRE2 in ergosterol metabolism. Yeast 23, 389–398 (2006).

    Article  CAS  Google Scholar 

  21. Caesar, R., Warringer, J. & Blomberg, A. Physiological importance and identification of novel targets for the N-terminal acetyltransferase NatB. Eukaryot. Cell 5, 368–378 (2006).

    Article  CAS  Google Scholar 

  22. Ericson, E. et al. Genetic pleiotropy in Saccharomyces cerevisiae quantified by high-resolution phenotypic profiling. Mol. Gen. Genomics 275, 605–614 (2006).

    Article  CAS  Google Scholar 

  23. Fernandez-Ricaud, L. et al. PROPHECY — a database for high-resolution phenomics. Nucl. Acids Res. 33, D369–D373 (2005).

    Article  CAS  Google Scholar 

  24. Lehmann, A.R. Replication of damaged DNA by translesion synthesis in human cells. FEBS Lett. 579, 873–876 (2005).

    Article  CAS  Google Scholar 

  25. Sancar, A. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu. Rev. Biochem. 73, 39–85 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. K. Kobryn for critical reading of the manuscript and the Centre Génomique Fonctionelle de Sherbrooke for helpful discussions. The software was developed by J. Gervais-Bird (Génome Québec and Genome Canada). M. Toussaint is a recipient of a NSERC fellowship and this work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to A.C.

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Correspondence to Antonio Conconi.

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Toussaint, M., Conconi, A. High-throughput and sensitive assay to measure yeast cell growth: a bench protocol for testing genotoxic agents. Nat Protoc 1, 1922–1928 (2006). https://doi.org/10.1038/nprot.2006.304

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