Improving synthetic lethal screens by regulating the yeast centromere sequence.
The synthetic lethal screen is a useful method in identifying novel genes functioning in an alternative pathway to the gene of interest. The current synthetic lethal screen protocol in yeast is based on a colony-sectoring assay that allows direct visualization of mutant colonies among a large population by their inability to afford plasmid loss. This method demands an appropriate level of stability of the plasmid carrying the gene of interest. YRp-based plasmids are extremely unstable and complete plasmid loss occurs within a few generations. Consequently, YCp plasmids are the vector of choice for synthetic lethal screens. However, we found that the high-level stability of YCp plasmids resulted in a large number of false positives that must be further characterized. In this study, we attempt to improve the existing synthetic lethal screen protocol by regulating the plasmid stability and copy number. It was found that by placing a yeast centromere sequence under the control of either inducible or constitutive promoters, plasmid stability can be significantly decreased. Hence, altering the conditions under which yeast cells carrying the plasmid PGAL1-CEN4 were cultivated allowed us to develop a method that eliminated virtually 100% of false positives and drastically reduced the time required to carry out a synthetic lethal screen.
Barbour
L
L
Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada.
Zhu
Y
Y
Xiao
W
W
D000619:Aminohydrolases / Q000235:genetics; D002262:Carboxy-Lyases / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D002503:Centromere / Q000235:genetics*; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D005574:Formate-Tetrahydrofolate Ligase / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D005804:Genes, Lethal; D005821:Genetic Techniques; D008565:Membrane Proteins; D008668:Metallothionein / Q000235:genetics; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000235:genetics; D024101:Mitochondrial Proteins; D009097:Multienzyme Complexes / Q000235:genetics; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D029701:Saccharomyces cerevisiae Proteins; D044767:Ubiquitin-Protein Ligases; D015003:Yeasts / Q000235:genetics*
C500769:CUP1-1 protein, S cerevisiae; D002352:Carrier Proteins; D005656:Fungal Proteins; C112520:MMS2 protein, S cerevisiae; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009097:Multienzyme Complexes; C492046:SLS1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C007121:copper thionein; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D008668:Metallothionein; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D044767:Ubiquitin-Protein Ligases; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae; D000619:Aminohydrolases; D002262:Carboxy-Lyases; C022378:phosphoribosylaminoimidazole carboxylase; D005574:Formate-Tetrahydrofolate Ligase