Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions

Disruption of newly identified genes in the pathogen Candida albicans is a vital step in determination of gene function. Several gene disruption metho ds described previously employ long regions of homology flanking a selectab le marker. Here, we describe disruption of C. albicans genes with PCR produ cts that have 50 to 60 bp of homology to a genomic sequence on each end of a selectable marker. We used the method to disrupt two known genes, ARG5 an d ADE2, and two sequences newly identified through the Candida genome proje ct, HRM101 and ENX3, HRM101 and ENX3 are homologous to genes in the conserv ed RIM101 (previously called RIM1) and PacC pathways of Saccharomyces cerev isiae and Aspergillus nidulans. We show that three independent hrm101/hrm10 1 mutants and two independent enx3/enx3 mutants are defective in filamentat ion on Spider medium. These observations argue that HRM101 and ENX3 sequenc es are indeed portions of genes and that the respective gene products have related functions.