1] Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, USA. [2] California Institute for Quantitative Biosciences (QB3), San Francisco, California, USA.
Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts, USA.
Nat Protoc. 2014 Aug;9(8):1867-81. doi: 10.1038/nprot.2014.127. Epub 2014 Jul 10.
The quantitative analysis of genetic interactions between pairs of gene mutations has proven to be effective for characterizing cellular functions, but it can miss important interactions for functionally redundant genes. To address this limitation, we have developed an approach termed triple-mutant analysis (TMA). The procedure relies on a query strain that contains two deletions in a pair of redundant or otherwise related genes, which is crossed against a panel of candidate deletion strains to isolate triple mutants and measure their growth. A central feature of TMA is to interrogate mutants that are synthetically sick when two other genes are deleted but interact minimally with either single deletion. This approach has been valuable for discovering genes that restore critical functions when the principal actors are deleted. TMA has also uncovered double-mutant combinations that produce severe defects because a third protein becomes deregulated and acts in a deleterious fashion, and it has revealed functional differences between proteins presumed to act together. The protocol is optimized for Singer ROTOR pinning robots, takes 3 weeks to complete and measures interactions for up to 30 double mutants against a library of 1,536 single mutants.
对基因对突变之间的定量遗传相互作用的分析已被证明是描述细胞功能的有效方法，但它可能会错过功能冗余基因的重要相互作用。为了解决这一限制，我们开发了一种称为三突变分析（TMA）的方法。该程序依赖于一个查询菌株，该菌株在一对冗余或相关的基因中包含两个缺失，然后与一组候选缺失菌株杂交，以分离三突变体并测量它们的生长。TMA 的一个核心特征是询问当两个其他基因缺失时合成生病的突变体，但与单个缺失的相互作用最小。这种方法对于发现当主要因子缺失时可以恢复关键功能的基因非常有价值。TMA 还发现了由于第三种蛋白质失活并以有害方式起作用而导致严重缺陷的双突变体组合，并且揭示了假定一起起作用的蛋白质之间的功能差异。该方案针对 Singer ROTOR 销钉机器人进行了优化，需要 3 周时间完成，并针对 1536 个单突变体库测量了多达 30 个双突变体的相互作用。
