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Systematic exploration of essential yeast gene function with temperature-sensitive mutants

Nature Biotechnology volume 29, pages 361–367 (2011)Cite this article

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Abstract

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (∼45%) of the 1,101 essential yeast genes, with ∼30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)–based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.

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Figure 1: Profiling the temperature sensitivity of ts strains.
Figure 2: Zaragozic acid rescues bet2-1 and cdc43-2 ts phenotype.
Figure 3: High-content screening of the ts allele collection identifies abnormal spindle morphology associated with cohesin and condensin mutants.
Figure 4: Relationship between cohesin, condensin and chromosomal passenger complexes (CPC).

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Acknowledgements

We thank the members of the Boone, Andrews and Bloom laboratory for their input and discussions. We thank S. Biggins (Fred Hutchinson Cancer Research Center) for her insights on cohesin and condensin components. We thank M. Zackrisson (University of Gothenburg) for statistical support. The Sli15-6A-GFP construct was a gift from E. Schiebel (University of Heidelberg) and the Mad1-NLS construct was a gift from R. Wozniak (University of Alberta). We also thank Jennifer Reginold for manual inspection of HCS images. F.J.V. was supported by a postdoctoral fellowship from the Best Foundation. B.A. and C.B. were supported by Genome Canada through the Ontario Genomics Institute as per research agreement 2004-OGI-3-01 and the Canadian Institutes of Health Research (MOP-97939).

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Author notes

  1. Zhijian Li and Franco J Vizeacoumar: These authors contributed equally to this work.

Authors and Affiliations

  1. Banting and Best Department of Medical Research and Department of Molecular Genetics, The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada

    Zhijian Li, Franco J Vizeacoumar, Sondra Bahr, Jingjing Li, Renqiang Min, Zhen-Yuan Lin, Anastasia Baryshnikova, Hong Lu, Zhun Yan, Ke Jin, Sarah Barker, Guri Giaever, Corey Nislow, Michael Costanzo, Anne-Claude Gingras, Zhaolei Zhang, Brenda Andrews & Charles Boone

  2. Department of Cell and Molecular Biology, Göteborg University, Göteborg, Sweden

    Jonas Warringer & Anders Blomberg

  3. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

    Frederick S Vizeacoumar & Alessandro Datti

  4. Department of Computer Science and Engineering, University of Minnesota, Minneapolis, Minnesota, USA

    Benjamin VanderSluis, Jeremy Bellay & Chad L Myers

  5. FoldRx Pharmaceuticals Inc., Cambridge, Massachusetts, USA

    Michael DeVit, James A Fleming & Chris Bulawa

  6. Department of Biology, University of North Carolina, Chapel Hill, North Carolina, USA

    Andrew Stephens, Julian Haase & Kerry Bloom

  7. Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy

    Alessandro Datti

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  1. Zhijian Li
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Contributions

Z.L., F.J.V., C.B. and B.A. conceived and designed the experiments. Z.L. and S.B. constructed the ts strains. J.W. and A.B. conducted and analyzed liquid growth profiling experiments. Z.L., F.J.V., F.S.V., J.L., R.M., K.J. and Z.Z. generated and analyzed the HCS data. M.C., A.B., B.V., J.B. and C.L.M. generated and analyzed the data. Z.Y.L. and A.C.G. performed mass spectrometry analyses. Z.L., Z.Y., M.D., J.A.F., C.B., G.G. and C.N. performed the chemical genetic experiments. Z.L., F.J.V., A.S., J.H. and K.B. performed the analysis on cohesin and condensin complexes. H.L., Z.Y., S.B., A.D., G.G., C.N., C.B., M.C., F.S.V., A.D., A.G., Z.Z. and K.B. provided technical support/reagents/materials/analysis tools. Z.L., F.J.V., M.C., B.A. and C.B. wrote the paper.

Corresponding authors

Correspondence to Brenda Andrews or Charles Boone.

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Li, Z., Vizeacoumar, F., Bahr, S. et al. Systematic exploration of essential yeast gene function with temperature-sensitive mutants. Nat Biotechnol 29, 361–367 (2011). https://doi.org/10.1038/nbt.1832

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