ABSTRACT
We have isolated the Candida albicans HIS4 (CaHIS4) gene by complementation of a his4-34 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 2514 bp, whose translation product shares a global identity of 44% and 55% to the His4 protein homologs of S. cerevisiae and Kluyveromyces lactis, respectively. Analysis of CaHIS4 sequence suggests that, similarly to S. cerevisiae HIS4, it codes for a polypeptide having three separate enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase) which reside in different domains of the protein. A C. albicans his4 strain is complemented with this gene when using a C. albicans-S. cerevisiae-Escherichia coli shuttle vector, thus enabling the construction of a host system for C. albicans genetic manipulation. In addition, upstream of the sequenced CaHIS4 sequence, we have found the 3'-terminal half of a gene encoding a PEX5-like protein.
Subject(s)
Candida albicans/genetics , DNA, Fungal/genetics , Fungal Proteins/genetics , Genes, Fungal/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Saccharomyces cerevisiae Proteins , Transcription Factors/genetics , Alcohol Oxidoreductases , Amino Acid Sequence , Aminohydrolases , Base Sequence , Candida albicans/chemistry , Cloning, Molecular , DNA, Fungal/chemistry , DNA, Fungal/isolation & purification , Genetic Complementation Test , Molecular Sequence Data , Mutation , Peroxisome-Targeting Signal 1 Receptor , Pyrophosphatases , Restriction Mapping , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Candida albicans/genetics , Cloning, Molecular/methods , Genes, Fungal , Genetic Vectors , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Genetic Complementation Test , Molecular Sequence DataABSTRACT
The karotype of Candida albicans 1001, a pathogenic isolate, presents two additional chromosome bands when compared with C. albicans 1006 strain. These two bands were a 2600 kb chromosome located between chromosome group 1-R and chromosome 2 (named chromosome 2*) and a 710 kb small chromosome, called snc due to its similarity in size to the supernumerary chromosome in strain WO-1. A comparison of karyotypes of strains 1001, 1006 and WO-1 has enabled us to conclude that chromosomes 2 and 7 are involved in such a reorganization giving rise to the new chromosome bands of strain 1001. We describe a tentative physical map of C. albicans 1001 based on the previously outlined map of C. albicans strain 1006.
