University School of Biotechnology, G.G.S. Indraprastha University.
University School of Biotechnology, G.G.S. Indraprastha University;
J Vis Exp. 2022 Sep 13(187). doi: 10.3791/63967.
Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype. This paper describes a protocol for carrying out this 'multicopy suppression'-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.
鉴定遗传相互作用是一种通过深入了解基因与其他基因的功能关系以及组织成生物途径和过程来破译基因功能的有力工具。尽管大多数遗传筛选最初是在酿酒酵母中开发的，但裂殖酵母提供了一个互补的平台来进行这些遗传筛选。鉴定遗传相互作用的常用方法之一是在功能丧失突变体中过量表达来自全基因组高拷贝数质粒文库的克隆，然后选择能够抑制突变体表型的克隆。本文描述了在裂殖酵母中进行这种基于“多拷贝抑制”的遗传筛选的方案。该筛选有助于鉴定与裂殖酵母中 Ell1 转录延伸因子缺失相关的遗传毒性应激敏感表型的多拷贝抑制子。该筛选通过将查询 ell1 缺失突变体菌株转化为高拷贝数裂殖酵母 cDNA 质粒文库，并在含有 4-硝基喹啉 1-氧化物 (4-NQO) 的 EMM2 平板上选择抑制剂启动，4-NQO 是一种诱导遗传毒性的化合物。随后，从两个入围的抑制子克隆中分离质粒，并通过限制性内切酶消化释放插入 DNA。释放插入 DNA 片段的质粒被重新转化到 ell1 缺失菌株中，以确认这些抑制子质粒克隆在存在 4-NQO 和其他遗传毒性化合物的情况下恢复 ell1 缺失突变体生长的能力。显示出删除表型挽救的那些质粒被测序以鉴定负责抑制 ell1 删除相关遗传毒性应激敏感表型的基因。
