A comprehensive strategy enabling high-resolution functional analysis of the yeast genome

Abstract

Functional genomic studies in Saccharomyces cerevisiae have contributed enormously to our understanding of cellular processes. Their full potential, however, has been hampered by the limited availability of reagents to systematically study essential genes and the inability to quantify the small effects of most gene deletions on growth. Here we describe the construction of a library of hypomorphic alleles of essential genes and a high-throughput growth competition assay to measure fitness with unprecedented sensitivity. These tools dramatically increase the breadth and precision with which quantitative genetic analysis can be performed in yeast. We illustrate the value of these approaches by using genetic interactions to reveal new relationships between chromatin-modifying factors and to create a functional map of the proteasome. Finally, by measuring the fitness of strains in the yeast deletion library, we addressed an enigma regarding the apparent prevalence of gene dispensability and found that most genes do contribute to growth.

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Figure 1: A library of hypomorphic alleles of essential yeast genes constructed using the DAmP approach.
Figure 2: A flow cytometry–based technique for high-precision growth rate measurements.
Figure 3: Growth rate measurements of deletion and DAmP yeast strains by the flow cytometry–based technique.
Figure 4: Utility of DAmP strains for identification of drug targets.
Figure 5: Highly sensitive growth rate measurements enable resolution of previously inaccessible classes of genetic interactions.
Figure 6: Functional dissection of the 26S proteasome.

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Acknowledgements

We acknowledge J. Hill and E. Rodriguez for help with strain construction; C.-S. Chin for flow cytometry software development; J. Newman for an introduction to high-throughput flow cytometry; R. Tsien (University of California, San Diego) for a construct encoding dTomato; B. Toyama for assistance with graphics; C. Boone, G. Giaever and C. Nislow for communication of results before publication; and V. Denic, J. Hollien, J. Ihmels, N. Ingolia, J. Newman and members of the Weissman laboratory for helpful discussion and critical reading of the manuscript. This work was supported by funding from the Howard Hughes Medical Institute (J.S.W.), the Fannie and John Hertz Foundation (D.K.B.), the US National Science Foundation (D.K.B) and the Larry L. Hillblom Foundation (D.M.C.).

Author information

Correspondence to Jonathan S Weissman.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1–2, Supplementary Note, Supplementary Methods (PDF 1848 kb)

Supplementary Data 1

List of primers used in this study. (XLS 425 kb)

Supplementary Data 2

Growth rate measurements for flow cytometry assay validation. (XLS 88 kb)

Supplementary Data 3

DAmP library growth rates measured by flow cytometry. (XLS 185 kb)

Supplementary Data 4

Deletion library growth rates measured by flow cytometry. (XLS 476 kb)

Supplementary Data 5

Growth rate measurements used for genetic interaction data. (XLS 85 kb)

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Breslow, D., Cameron, D., Collins, S. et al. A comprehensive strategy enabling high-resolution functional analysis of the yeast genome. Nat Methods 5, 711–718 (2008) doi:10.1038/nmeth.1234

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