Improving synthetic lethal screens by regulating the yeast centromere sequence

被引:5
作者
Barbour, L [1 ]
Zhu, Y [1 ]
Xiao, W [1 ]
机构
[1] Univ Saskatchewan, Dept Microbiol & Immunol, Saskatoon, SK S7N 5E5, Canada
关键词
synthetic lethal screen; yeast; centromere; inducible promoter; MRE11;
D O I
10.1139/gen-43-5-910
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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 P-GAL1-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.
引用
收藏
页码:910 / 917
页数:8
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