Barbour L, Zhu Y, Xiao W
Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada.
Genome. 2000 Oct;43(5):910-7.
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.
合成致死筛选是一种用于鉴定在与感兴趣基因的替代途径中发挥作用的新基因的有用方法。目前酵母中的合成致死筛选方案基于菌落分区测定法，该方法通过突变菌落无法承受质粒丢失，从而能够在大量群体中直接观察到它们。这种方法要求携带感兴趣基因的质粒具有适当水平的稳定性。基于YRp的质粒极其不稳定，几代之内就会完全丢失质粒。因此，YCp质粒是合成致死筛选的首选载体。然而，我们发现YCp质粒的高稳定性导致大量假阳性结果，必须进一步进行鉴定。在本研究中，我们试图通过调节质粒稳定性和拷贝数来改进现有的合成致死筛选方案。结果发现，通过将酵母着丝粒序列置于诱导型或组成型启动子的控制下，质粒稳定性可显著降低。因此，改变携带质粒PGAL1-CEN4的酵母细胞的培养条件，使我们开发出一种方法，该方法几乎消除了100%的假阳性结果，并大幅缩短了进行合成致死筛选所需的时间。
