CLONING BY FUNCTION - AN ALTERNATIVE APPROACH FOR IDENTIFYING YEAST HOMOLOGS OF GENES FROM OTHER ORGANISMS

被引:140
作者
KRANZ, JE
HOLM, C
机构
关键词
Drosophila DNA topoisomerase II; heterologous system; inducible galactose promoter; plasmid dependence; Saccharomyces cerevisiae;
D O I
10.1073/pnas.87.17.6629
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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. and 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.
引用
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页码:6629 / 6633
页数:5
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