CLONING OF THE CANDIDA-ALBICANS HIS1 GENE BY DIRECT COMPLEMENTATION OF A CANDIDA-ALBICANS HISTIDINE AUXOTROPH USING AN IMPROVED DOUBLE-ARS SHUTTLE VECTOR

ARS2 and ARS3 are two Candida albicans (Ca) DNA fragments with autonom ous replicating activity that have been shown to promote non-integrati ve genetic transformation of both Ca and Saccharomyces cerevisiae (Sc) . We have developed several shuttle vectors based on either ARS fragme nt, or the combination of both, and using the CaURA3 gene as a selecti on marker. The combination of ARS2 and ARS3 fragments in a single vect or did not increase transformation frequencies but improved the stabil ity of transformant plasmids in Ca cells, so that the degree of intrac ellular recombination was reduced. A Ca genomic DNA library was constr ucted on the double-ARS vector, pRM1, to be used for direct cloning in Ca by complementation of the histidine auxotrophy of strain CA9. By s creening this library, we cloned CaHIS1, the Ca gene that encodes ATP phosphoribosyl transferase, one of the enzymes that participates in hi stidine 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 st andard Ca genetic map. Our results represent the first demonstration o f 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.