The fungal pathogen Candida albicans is naturally diploid, and current gene disruption strategies require two successive transformations. We describe here a genetic construct (UAU1) for which two copies may be selected. Insertion of UAU1 into one genomic site, after a single transformation, allows selection for segregants with two copies of the insertion. Major classes of segregants are those carrying homozygous insertion mutations and allelic triplications, which have two insertion alleles and a wild-type allele. Thus nonessential and essential genes may be distinguished rapidly through PCR tests for homozygosis and triplication. We find that homozygous mutations may be isolated at three nonessential loci (ADE2, RIM20, and YGR189), while only allelic triplications were found at two essential loci (SNF1 and CDC28). We have unexpectedly isolated homozygous mutants with mutations at CDC25; they are viable but defective in filamentation on serum-containing medium. The UAU1 cassette is thus useful to assess rapidly the essentiality of C. albicans genes.Candida albicans is an opportunistic fungal pathogen. It has been of experimental interest for two main reasons. First, it is a significant pathogen that causes oral mucosal infections, vaginitis, nosocomial bloodstream infections, and a variety of deep tissue infections (26). Therefore, many experimental studies have focused on pathogenesis, drug resistance, and analysis of prospective drug targets. Second, it is a distant cousin of the most well-characterized unicellular eukaryote, Saccharomyces cerevisiae, so that the function of a C. albicans gene may be suggested by its role in S. cerevisiae. Yet, surprisingly, C. albicans may use gene products and regulatory pathways in novel ways (4,16,17,28,34,37). The contrast between C. albicans and S. cerevisiae can provide unique insight into regulatory mechanisms, interpathway relationships, and general aspects of eukaryotic biology.Molecular genetics has played an increasingly prominent role in studies of C. albicans, particularly with the partial genomic sequence as a tool for gene discovery. However, genetic methods are cumbersome for two reasons (30). First, C. albicans strains are diploid (or of higher ploidy) and there is no meiotic division, so that gene disruption mutants must be constructed through two successive transformations. Second, with current methods, disruption of one allele may be straightforward, but disruption of the second allele is infrequent if the second disruption construct is homologous to the first. These problems can make it difficult to construct a homozygous mutant and to determine whether a gene is essential for growth.We describe here a genetic strategy that circumvents these difficulties. It yields homozygous insertion mutations after a single transformation. The strategy provides a rapid test for essential genes and should thus accelerate drug target validation. In addition, the strategy can provide a preliminary assessment of mutant phenotypes, and it lends itself to large-scale analysi...

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