Repeated Use of Gal1 for Gene Disruption in Candida Albicans

A technique which has the potential to allow repeated use of the same selectable marker to create gene disruptions in Candida albicans has been developed. In this approach, originally described for Saccharomyces cerevisiae, the selectable marker is flanked by direct repeats. Mitotic recombination between these repeats leads to elimination of the selectable marker. A module in which the GALq1 gene is flanked by direct repeats of the bacterial CAT gene was constructed and used to disrupt one copy of the URA3 gene in a gal1 mutant. Gal- revertants were selected by plating on 2-deoxy-D-galactose (2DOG). The frequency of 2DOG-resistant colonies recovered was 20 times higher than that obtained with a similar construct not flanked by direct repeats. Of these, 20% had lost the GAL1 gene by recombination between the direct repeats. The GAL1 gene was used again to disrupt the remaining wild-type copy of the URA3 gene of one of these gal1 isolates, resulting in a stable ura3 mutant. This technique should be generally applicable to derive homozygous gene disruptions in this diploid organism.