Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

A general life-death selection strategy for dissecting protein functions

Nature Methods volume 6, pages 813–816 (2009)Cite this article

Abstract

Clonal selection strategies are central tools in molecular biology. We developed a general strategy to dissect protein functions through positive and negative clonal selection for protein-protein interactions, based on a protein-fragment complementation assay using Saccharomyces cerevisiae cytosine deaminase as a reporter. We applied this method to mutational or chemical disruption of protein-protein interactions in yeast and to dissection of the functions of an allosterically activated transcription factor, Swi6.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Development and characterization of the OyCD PCA.
Figure 2: Dissecting transcriptional activity of Swi6.

Similar content being viewed by others

References

  1. Reguly, T. et al. J. Biol. 5, 11 (2006).

    Article  Google Scholar 

  2. Pelletier, J.N., Campbell-Valois, F.X. & Michnick, S.W. Proc. Natl. Acad. Sci. USA 95, 12141–12146 (1998).

    Article  CAS  Google Scholar 

  3. Michnick, S.W., Ear, P.H., Manderson, E.N., Remy, I. & Stefan, E. Nat. Rev. Drug Discov. 6, 569–582 (2007).

    Article  CAS  Google Scholar 

  4. Mahan, S.D., Ireton, G.C., Knoeber, C., Stoddard, B.L. & Black, M.E. Protein Eng. Des. Sel. 17, 625–633 (2004).

    Article  CAS  Google Scholar 

  5. Hartzog, P.E., Nicholson, B.P. & McCusker, J.H. Yeast 22, 789–798 (2005).

    Article  CAS  Google Scholar 

  6. Wei, K. & Huber, B.E. J. Biol. Chem. 271, 3812–3816 (1996).

    Article  CAS  Google Scholar 

  7. Fang, F., Hoskins, J. & Butler, J.S. Mol. Cell. Biol. 24, 10766–10776 (2004).

    Article  CAS  Google Scholar 

  8. Korkegian, A., Black, M.E., Baker, D. & Stoddard, B.L. Science 308, 857–860 (2005).

    Article  CAS  Google Scholar 

  9. Block, C., Janknecht, R., Herrmann, C., Nassar, N. & Wittinghofer, A. Nat. Struct. Biol. 3, 244–251 (1996).

    Article  CAS  Google Scholar 

  10. Porter, S.W. & West, A.H. Biochim. Biophys. Acta 1748, 138–145 (2005).

    Article  CAS  Google Scholar 

  11. Bahler, J. Annu. Rev. Genet. 39, 69–94 (2005).

    Article  CAS  Google Scholar 

  12. Andrews, B.J. & Moore, L.A. Proc. Natl. Acad. Sci. USA 89, 11852–11856 (1992).

    Article  CAS  Google Scholar 

  13. Sedgwick, S.G. et al. J. Mol. Biol. 281, 763–775 (1998).

    Article  CAS  Google Scholar 

  14. Remy, I., Wilson, I.A. & Michnick, S.W. Science 283, 990–993 (1999).

    Article  CAS  Google Scholar 

  15. Tarassov, K. et al. Science 320, 1465–1470 (2008).

    Article  CAS  Google Scholar 

  16. Pelletier, J.N., Campbell-Valois, F.X. & Michnick, S.W. Proc. Natl. Acad. Sci. USA 95, 12141–12146 (1998).

    Article  CAS  Google Scholar 

  17. Block, C., Janknecht, R., Herrmann, C., Nassar, N. & Wittinghofer, A. Nat. Struct. Biol. 3, 244–251 (1996).

    Article  CAS  Google Scholar 

  18. Giaever, G. et al. Nature 418, 387–391 (2002).

    Article  CAS  Google Scholar 

  19. Rollins, C.T. et al. Proc. Natl. Acad. Sci. USA 97, 7096–7101 (2000).

    Article  CAS  Google Scholar 

  20. Ralser, M., Goehler, H., Wanker, E.E., Lehrach, H. & Krobitsch, S. Biotechniques 39, 165–166, 168 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank H. Bussey (McGill University) for providing yeast strains and p41XGal1 plasmids, B. Andrews (Donelly Centre, University of Toronto) for plasmids pBA487 and pBA251 and Swi6 antibodies, T. Clackson and V. Rivera (Ariad) for pC4EN-FM3, M.J. Booth for constructing p415Gal-Linker-OyCD-F[2], and M. Malleshaiah and C. Chan for comments. This research was supported by grants from the Canadian Institutes of Health Research (MOP-152556) and the Canada Research Chairs Program to S.W.M. P.H.E. acknowledges the Faculté des etudes supérieurs de l'Université de Montréal for scholarships.

Author information

Authors and Affiliations

  1. Département de Biochimie, Université de Montréal, Montréal, Québec, Canada

    Po Hien Ear & Stephen W Michnick

Authors
  1. Po Hien Ear
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stephen W Michnick
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.H.E. and S.W.M. designed the experiments, analyzed the results and wrote the manuscript. P.H.E. performed the experiments.

Corresponding author

Correspondence to Stephen W Michnick.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Tables 1–3 (PDF 4330 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ear, P., Michnick, S. A general life-death selection strategy for dissecting protein functions. Nat Methods 6, 813–816 (2009). https://doi.org/10.1038/nmeth.1389

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1389

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing