Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms.
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.
Department of Cellular and Developmental Biology, Harvard University, Cambridge, MA 02138.
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Kranz
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Holm
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D005785:Gene Conversion; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D005838:Genotype; D009154:Mutation; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D012689:Sequence Homology, Nucleic Acid