PCR-mediated one-step deletion of targeted chromosomal regions in haploid Saccharomyces cerevisiae
PCR-mediated one-step deletion of targeted chromosomal regions in haploid Saccharomyces cerevisiae
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DOI：
10.1007/s00253-008-1609-9
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发表时间：
2008-09-01
影响因子：
5
通讯作者：
Harashima, Satoshi
中科院分区：
文献类型：
--
作者：
Sugiyama, Minetaka;Nakazawa, Toshimasa;Harashima, Satoshi
染色体重排，特别是染色体缺失，已被开发为基因组功能分析的重要资源。为了便于这种分析，我们应用了一种以前开发的方法，染色体分裂的直接删除设计的内部或末端染色体区域携带许多非必需基因的单倍体酿酒酵母。该方法，聚合酶链反应（PCR）介导的染色体缺失（PCD），由一个两步PCR和一个删除事件的转化。在本文中，我们表明，PCD方法有效地删除内部区域在一个单一的转换。在通过该方法缺失的6个染色体区域中，以高效率成功地消除了含有10至19个非必需基因的5个区域（长度为16至38kb）。随后发现染色体XIII上未被删除的一个靶向区域含有酵母生长所必需的序列。虽然该区域中有14个基因被报道为非必需基因，但这些非必需基因之间可能发生合成致死相互作用。表型分析表明，四个缺失菌株仍然表现出正常的生长，而可能的合成生长缺陷，观察到在另一个菌株窝藏19个基因缺失的染色体XV。这些结果表明，PCD方法是一个有用的工具，删除基因，并分析其功能在确定的染色体区域。
Chromosome rearrangements, especially chromosomal deletions, have been exploited as important resources for functional analysis of genomes. To facilitate this analysis, we applied a previously developed method for chromosome splitting for the direct deletion of a designed internal or terminal chromosomal region carrying many nonessential genes in haploid Saccharomyces cerevisiae. The method, polymerase chain reaction (PCR)-mediated chromosomal deletion (PCD), consists of a two-step PCR and one transformation per deletion event. In this paper, we show that the PCD method efficiently deletes internal regions in a single transformation. Of the six chromosomal regions targeted for deletion by this method, five regions (16 to 38 kb in length) containing 10 to 19 nonessential genes were successfully eliminated at high efficiency. The one targeted region on chromosome XIII that was not deleted was subsequently found to contain sequences essential for yeast growth. While 14 individual genes in this region have been reported to be nonessential, synthetic lethal interactions may occur among these nonessential genes. Phenotypic analysis showed that four deletion strains still exhibited normal growth while possible synthetic growth defects were observed in another strain harboring a 19-gene deletion on chromosome XV. These results demonstrate that the PCD method is a useful tool for deleting genes and for analyzing their functions in defined chromosomal regions.