Cloning of the Candida albicans HIS1 gene by direct complementation of a C. albicans histidine auxotroph using an improved double-ARS shuttle vector.
ARS2 and ARS3 are two Candida albicans (Ca) DNA fragments with autonomous replicating activity that have been shown to promote non-integrative genetic transformation of both Ca and Saccharomyces cerevisiae (Sc). We have developed several shuttle vectors based on either ARS fragment, or the combination of both, and using the CaURA3 gene as a selection marker. The combination of ARS2 and ARS3 fragments in a single vector did not increase transformation frequencies but improved the stability of transformant plasmids in Ca cells, so that the degree of intracellular recombination was reduced. A Ca genomic DNA library was constructed on the double-ARS vector, pRM1, to be used for direct cloning in Ca by complementation of the histidine auxotrophy of strain CA9. By screening this library, we cloned CaHIS1, the Ca gene that encodes ATP phosphoribosyl transferase, one of the enzymes that participates in histidine biosynthesis. The deduced protein, CaHis1p, is 60.6% identical (73% similar) to ScHis1p (EC 2.4.2.17). The cloned gene is the first auxotrophic gene marker mapped to fragment I of chromosome 5 in the standard Ca genetic map. Our results represent the first demonstration of a direct cloning system in the opportunistic fungus Ca that does not require the use of an intermediate host such as Sc for plasmid rescue. This system could be used for the isolation of any gene affected in Ca mutants displaying a selectable or identifiable phenotype.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002176:Candida albicans / Q000235:genetics*; D002874:Chromosome Mapping; D003001:Cloning, Molecular / Q000379:methods*; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008969:Molecular Sequence Data