Gene isolation by complementation in Candida albicans and applications to physical and genetic mapping

We have isolated three genes, ARG57, SER57, and LYS1, on the basis of their function in Candida albicans. A C. albicans transformation vector containing the C. albicans URA3 gene, a Candida ARS sequence, and a portion of the Saccharomyces cerevisiae 2 microns circle containing the replication origin was constructed. Clones from genomic libraries in this vector were isolated by direct complementation of the auxotrophies in strain 1006 (arg57 ser57 lys1 ura3 MPA1). Transformants typically contain two to four plasmids in a mixed tandem multimer. A scheme to resolve mixed multimers into monomers in vivo by transformation of S. cerevisiae with Candida transformant DNA selecting Ura+ transformants was devised. Monomeric plasmids were then isolated by transformation of Escherichia coli with the S. cerevisiae transformant DNA. These were retested by transformation of strain 1006 to identify the specific plasmid that complemented the auxotrophy. The chromosomal locations of the genes were determined by hybridization to C. albicans chromosomes separated on contour-clamped homogenous electric field gels. We used these locations to assess the stability of individual C. albicans chromosomes in parasexual genetic analysis. The Lys(+)-complementing clone was shown to be LYS1 by complementation of S. cerevisiae lys1 mutants. These cloned genes help to align the Candida physical and genetic maps and provide additional markers for the transformation system.