Banting and Best Department of Medical Research, The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
Nat Biotechnol. 2011 Apr;29(4):361-7. doi: 10.1038/nbt.1832. Epub 2011 Mar 27.
Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (∼45%) of the 1,101 essential yeast genes, with ∼30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.
条件温度敏感（ts）突变是研究酵母酿酒酵母中必需基因的有价值的试剂。我们构建了 787 个 ts 菌株，涵盖了 1101 个必需酵母基因中的 497 个（约 45%），其中约 30%的基因由多个等位基因代表。所有的等位基因都整合到 S288C 常见参考菌株的天然基因组位置，并与一个 kanMX 可选择标记相连，允许通过基于合成遗传阵列（SGA）的高通量方法进行进一步的遗传操作。我们展示了两种这样的操作：440 株的条形码化，这使得化学遗传抑制分析成为可能，以及携带不同亚细胞结构荧光标记的菌株阵列的构建，这使得使用高内涵筛选对表型进行定量分析成为可能。GFP-微管标记的定量分析确定了着丝粒和凝聚素基因在纺锤体解体中的作用。这个突变体集合应该有助于广泛的系统研究，旨在理解必需基因的功能。
