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

Zhijian Li; Franco J. Vizeacoumar; Sondra Bahr; Jingjing Li; Jonas Warringer; Frederick S. Vizeacoumar; Renqiang Min; Benjamin Vandersluis; Jeremy Bellay; Michael Devit; James A. Fleming; Andrew Stephens; Julian Haase; Zhen Yuan Lin; Anastasia Baryshnikova; Hong Lu; Zhun Yan; Ke Jin; Sarah Barker; Alessandro Datti; Guri Giaever; Corey Nislow; Chris Bulawa; Chad L. Myers; Michael Costanzo; Anne Claude Gingras; Zhaolei Zhang; Anders Blomberg; Kerry Bloom; Brenda Andrews; Charles Boone

doi:10.1038/nbt.1832

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

Zhijian Li, Franco J. Vizeacoumar, Sondra Bahr, Jingjing Li, Jonas Warringer, Frederick S. Vizeacoumar, Renqiang Min, Benjamin Vandersluis, Jeremy Bellay, Michael Devit, James A. Fleming, Andrew Stephens, Julian Haase, Zhen Yuan Lin, Anastasia Baryshnikova, Hong Lu, Zhun Yan, Ke Jin, Sarah Barker, Alessandro DattiGuri Giaever, Corey Nislow, Chris Bulawa, Chad L. Myers, Michael Costanzo, Anne Claude Gingras, Zhaolei Zhang, Anders Blomberg, Kerry Bloom, Brenda Andrews, Charles Boone

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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.

Original language	English (US)
Pages (from-to)	361-367
Number of pages	7
Journal	Nature biotechnology
Volume	29
Issue number	4
DOIs	https://doi.org/10.1038/nbt.1832
State	Published - Apr 2011

Bibliographical note

Funding Information:
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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Li, Z., Vizeacoumar, F. J., Bahr, S., Li, J., Warringer, J., Vizeacoumar, F. S., Min, R., Vandersluis, B., Bellay, J., Devit, M., Fleming, J. A., Stephens, A., Haase, J., Lin, Z. Y., Baryshnikova, A., Lu, H., Yan, Z., Jin, K., Barker, S., ... Boone, C. (2011). Systematic exploration of essential yeast gene function with temperature-sensitive mutants. Nature biotechnology, 29(4), 361-367. https://doi.org/10.1038/nbt.1832

Li, Z, Vizeacoumar, FJ, Bahr, S, Li, J, Warringer, J, Vizeacoumar, FS, Min, R, Vandersluis, B, Bellay, J, Devit, M, Fleming, JA, Stephens, A, Haase, J, Lin, ZY, Baryshnikova, A, Lu, H, Yan, Z, Jin, K, Barker, S, Datti, A, Giaever, G, Nislow, C, Bulawa, C, Myers, CL, Costanzo, M, Gingras, AC, Zhang, Z, Blomberg, A, Bloom, K, Andrews, B & Boone, C 2011, 'Systematic exploration of essential yeast gene function with temperature-sensitive mutants', Nature biotechnology, vol. 29, no. 4, pp. 361-367. https://doi.org/10.1038/nbt.1832

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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.",

author = "Zhijian Li and Vizeacoumar, {Franco J.} and Sondra Bahr and Jingjing Li and Jonas Warringer and Vizeacoumar, {Frederick S.} and Renqiang Min and Benjamin Vandersluis and Jeremy Bellay and Michael Devit and Fleming, {James A.} and Andrew Stephens and Julian Haase and Lin, {Zhen Yuan} and Anastasia Baryshnikova and Hong Lu and Zhun Yan and Ke Jin and Sarah Barker and Alessandro Datti and Guri Giaever and Corey Nislow and Chris Bulawa and Myers, {Chad L.} and Michael Costanzo and Gingras, {Anne Claude} and Zhaolei Zhang and Anders Blomberg and Kerry Bloom and Brenda Andrews and Charles Boone",

note = "Funding Information: 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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AU - Vizeacoumar, Frederick S.

AU - Min, Renqiang

AU - Vandersluis, Benjamin

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AU - Devit, Michael

AU - Fleming, James A.

AU - Stephens, Andrew

AU - Haase, Julian

AU - Lin, Zhen Yuan

AU - Baryshnikova, Anastasia

AU - Lu, Hong

AU - Yan, Zhun

AU - Jin, Ke

AU - Barker, Sarah

AU - Datti, Alessandro

AU - Giaever, Guri

AU - Nislow, Corey

AU - Bulawa, Chris

AU - Myers, Chad L.

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AU - Gingras, Anne Claude

AU - Zhang, Zhaolei

AU - Blomberg, Anders

AU - Bloom, Kerry

AU - Andrews, Brenda

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N2 - 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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Systematic exploration of essential yeast gene function with temperature-sensitive mutants

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