A Yeast Overexpression Array: Synthetic Dosage Lethality and the Identification of New Pho85 Substrates.
Richelle Sopko (1), Nicolle Preston (2), Dongqing Huang (1), Gordon Chua (2), Kristine Willis (1), Mike Snyder (3), Charlie Boone (2), Brenda Andrews (1)
(1) Medical Genetics, University of Toronto, 1 Kings College Circ, Toronto, ON, M5S 1A8, Canada;
(2) Banting and Best Department of Medical Research, University of Toronto, Toronto ON, Canada;
(3) Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut
We constructed a plasmid-based yeast (S288C) array comprised of ~5800 yeast strains. Each strain carries a high copy plasmid containing a unique S. cerevisiae gene, tagged with GST-HisX6, under the control of the GAL1 promoter. We identified 843 genes that reduce cellular fitness when overexpressed; we have phenotypically characterized strains expressing these toxic genes using transcriptional profiling, fluorescence microscopy, and drug sensitivity and resistance screening. We have also combined our overexpression array with synthetic genetic array (SGA) analysis which enables the high throughput examination of gene overexpression in a mutant background; this concept is referred to as synthetic dosage lethality (SDL). We used our array to screen a strain deleted for the Pho85 cyclin-dependent protein kinase for SDL interactions. All known substrates of Pho85 are negatively regulated by phosphorylation; we rationalized that overexpression of negatively regulated targets may be detrimental in the absence of Pho85 and that SDL may be a valid means to identify kinase substrates. From this screening, we identified 67 genes that when overexpressed in pho85delta cause slow growth, including 5 known targets, 17 genes involved in polarized growth, and several genes involved in vacuolar function and cell cycle regulation. Systematic SDL may provide a general means to examine other negatively regulated systems such as protein degradative pathways.
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