Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms.
Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms.
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按功能克隆：识别其他生物体基因的酵母同源物的另一种方法。
DOI：
10.1073/pnas.87.17.6629
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发表时间：
1990
影响因子：
11.1
通讯作者：
Holm,C
中科院分区：
文献类型：
--
作者：
Kranz,JE;Holm,C
对细胞生理学和结构的研究已经确定了许多有趣的蛋白质，这些蛋白质可以通过使用酵母遗传学的力量进行功能分析。不幸的是，用两种最常用的方法--DNA杂交和抗体交叉反应--鉴定同源酵母基因往往很困难。我们描述了一种基于蛋白质功能本身来鉴定酵母同源物的策略。这种按功能克隆的策略包括首先鉴定依赖于表达克隆的外源基因的质粒的酵母突变体。然后通过突变缺陷的互补来克隆相应的酵母基因。为了检测质粒依赖性，使用Koshland等人的殖民地颜色测定[Koshland，D.，肯特，J.C. & Hartwell，L. H.（1985）Cell 40，393-403]。在本文中，我们测试了这种方法的可行性，使用良好的表征系统的DNA拓扑异构酶II在酵母中。我们表明，（i）质粒依赖性很容易识别;（ii）筛选有效地分离了所需基因中的突变;（iii）可以通过筛选逆转质粒依赖性表型来克隆该基因的野生型酵母同源物。我们的结论是，克隆功能可以用来分离酵母同源的必需基因在其他生物体中确定。
Studies of cell physiology and structure have identified many intriguing proteins that could be analyzed for function by using the power of yeast genetics. Unfortunately, identifying the homologous yeast gene with the two most commonly used approaches--DNA hybridization and antibody cross-reaction--is often difficult. We describe a strategy to identify yeast homologs based on protein function itself. This cloning-by-function strategy involves first identifying a yeast mutant that depends on a plasmid expressing a cloned foreign gene. The corresponding yeast gene is then cloned by complementation of the mutant defect. To detect plasmid dependence, the colony color assay of Koshland et al. [Koshland, D., Kent, J. C. & Hartwell, L. H. (1985) Cell 40, 393-403] is used. In this paper, we test the feasibility of this approach using the well-characterized system of DNA topoisomerase II in yeast. We show that (i) plasmid dependence is easily recognized; (ii) the screen efficiently isolates mutations in the desired gene; and (iii) the wild-type yeast homolog of the gene can be cloned by screening for reversal of the plasmid-dependent phenotype. We conclude that cloning by function can be used to isolate the yeast homologs of essential genes identified in other organisms.