Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene

Abstract

cdc28, one of several genes required for cell division in the yeast Saccharomyces cerevisiae, has been isolated on recombinant plasmids. A recombinant plasmid pool containing the entire yeast genome was constructed by partial digestion of yeast DNA with the four-base recognition restriction endonuclease Sau3A to give the equivalent of random fragments, size selection on sucrose gradients, and introduction of the fragments into the yeast vector YRp7 by use of the homology of Sau3A ends with those generated in the vector by cleavage with BamHI. Recombinant plasmids capable of complementing cdc28 mutations were isolated by transformation of a cdc28ts strain and selection for clones capable of growth at the restrictive temperature. Plasmids responsible for complementing the cdc28ts phenotype were shown to recombine specifically with the chromosomal cdc28 locus, confirming the identity of the cloned sequences. In addition, one of the recombinant plasmids was capable of complementing a mutation in tyr1, a gene genetically linked to cdc28. This method of gene isolation and identification should be applicable to all yeast genes for which there are readily scorable mutants.