Systematic Genetic Analysis with Conditional Estradiol-Regulated Gene Expression Alleles in Yeast

酵母复杂的分子遗传学方法使其成为研究基本细胞功能的理想模型系统。该计划包括构建一个新的全基因组酵母突变体库，其中约6000个不同的基因被置于可诱导启动子的控制下，这意味着我们可以通过在生长介质中添加或移除诱导剂来开启或关闭基因的表达。这个新的突变体集合，我们称之为Z3集合，有许多应用，因此它将推动世界各地许多不同实验室的研究。我们对这一新的突变集合特别感兴趣，因为它将使我们能够跟踪特定基因关闭时细胞如何死亡。 有趣的是，只有~1000个酵母基因是必需的，这样当它们被关闭或删除时，细胞不会生长或分裂，其他~5000个基因是不必要的，即使它们被删除或关闭，细胞也仍然活着。我们认为大多数基因都不是必需的，因为细胞不受遗传和环境干扰的影响，因此它们进化了备用基因和途径来弥补单个基因的缺陷。在另一个项目中，我们小组测试了所有可能的酵母双突变株的致死表型。虽然我们现在知道许多基因对共同作用来控制细胞的生命，但在机制层面上，我们并不确切地知道为什么细胞依赖于这两个基因。我们将使用新的Z3收集来研究一组精选的酵母必需基因和致死双突变的末端表型。我们已经组装了许多诊断标记物，使我们能够跟踪通路和细胞隔间的功能。我们预计，不同的末端表型轨迹将为我们提供关于基因及其相应的通路如何相互连接的新知识，从而为我们提供关于细胞如何工作的有价值的新见解。

The sophisticated molecular genetic methods available with yeast, makes this organism an ideal model system for studying fundamental cellular functions. The proposed project involves the construction of a new genome-wide collection of yeast mutants, in which each one of its ~6000 different genes is placed under the control of an inducible promoter, which means we can turn the expression of the gene on or off, simply by including or removing an inducer from the growth medium. This new mutant collection, which we refer to as the Z3 collection, has numerous applications and thus it will fuel research in numerous different labs around the world. We are particularly interested in this new mutant collection because it will allow us to track how cells die when particular genes are turned off. Interestingly, only ~1000 yeast genes are essential, such that the cell will not grow or divide when they are turned off or deleted, the other ~5000 genes are nonessential and the cell remains alive even when they are deleted or shut off. We think most genes are nonessential because cells are buffered from genetic and environmental perturbations and thus they have evolved backup genes and pathways to compensate for defects in a single gene. In another project, our group has tested all possible yeast double mutants for a lethal phenotype. While we now know many gene pairs work together to control cellular life, at a mechanistic level, we do not know exactly why the cell depends on both genes. We will use the new Z3 collection to investigate the terminal phenotype of a select set of yeast essential genes and lethal double mutants. We have assembled a number of diagnostic markers that allow us to follow the function of pathways and cellular compartments. We anticipate that different terminal phenotype trajectories will provide us with new knowledge of how genes and their corresponding pathways are connected to one another and thus will provide us with valuable new insights on how cells work.