Molecular cloning and genetic mapping of the PET494 gene of Saccharomyces cerevisiae

Summary The activity of the nuclear gene PET494 is required to allow expression of the yeast mitochondrial gene oxi2. To aid the study of the mechanism of action of PET494 we have isolated this gene from yeast DNA. A clone bank of yeast DNA fragments in a yeast-E. coli shuttle vector was screened by transformation for a plasmid able to complement the pet494-1 amber mutation. A complementing plasmid was obtained that contained a unique 4.4 kb yeast sequence. This 4.4 kb sequence contains the PET494 gene. Integration of a plasmid containing it into chromosomal DNA by homologous recombination, and subsequent genetic analysis, demonstrated that the 4.4 kb fragment was tightly linked to the pet494-1 mutation. In addition, the corresponding 4.4 kb sequence isolated from a pet494-1 mutant failed to complement the mutation. A 2 kb fragment, subcloned from the original plasmid retained the ability to complement the mutation. The pet494-1 mutation maps to chromosome XIV between rna2 and lys9, approximately 2.4 cm from lys9.