Genome-wide Synthetic Lethality Screens on Microarrays: probing DNA
metabolism in S. cerevisiae .
Siew Loon Ooi (1),
Marina Lee (1), Daniel Yuan (1), Arne IJpma (1),
Sharon Sookhai-Mahadeo (1), Xuewen Pan (1), Rafael Irizarry (3),
Carol W. Greider (1),
Daniel D. Shoemaker (2), Forrest A. Spencer (1), Jef D. Boeke (1)
(1) Molecular Biology and Genetics, Johns Hopkins University SOM, 725 N.
Wolfe Street, Baltimore, MD 21205, USA; (2) Rosetta Inpharmatics Inc.,
12040 115th Street Ave NE, Kirkland, WA 98034, USA; (3) Bloomberg School of Public
Health,
Johns Hopkins University, Baltimore, MD 21205, USA
The recent
completion of the set of deletion alleles of essentially all ORFs in
S. cerevisiae makes possible the analysis of populations of defined
deletion mutants using microarrays. Each mutant was assigned two unique
molecular barcodes-20-mer oligonucleotides which serve as strain
identifiers. We describe for the first time a microarray-based synthetic
lethality screen performed on 4667 haploid deletion mutants. The 'query
mutation' of interest is generated in the pools of haploid deletion
strains via direct transformation of a PCR-generated targeting
construct. The targeting construct contains a URA3 marker that
replaces part or all of the query gene, and the genomic fragments
flanking the query gene of interest. We performed synthetic lethality
screens for TLC1 , which encodes for the telomerase RNA
component, and SGS1 , which has homology to E. coli RecQ and
human BLM and WRN helicases. We identified known synthetic lethal
interactors for both TLC1 and SGS1 . We are currently
performing synthetic lethality screens for genes involved in DNA
metabolism including SIR2, SIR3, YKU70 and
YKU80 , with the goal of generating an interaction map for DNA
metabolism pathways in S. cerevisiae.
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