A Single-Transformation Gene Function Test in Diploid Candida albicans

Abstract

ABSTRACT 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.