Development of an integrative transformation system for the opportunistic pathogenic yeast Candida lusitaniae using URA3 as a selection marker.
The nucleotide sequence of the URA3 gene encoding orotidine-5'-phosphate decarboxylase (OMP DCase) of the human opportunistic pathogen yeast Candida lusitaniae was determined by degenerate PCR and chromosome walking. Deduced amino acid sequence showed strong homologies (59-85% identity) with OMP DCases of different Saccharomycetales and allowed identification of the known conserved domains. Very close upstream from the URA3 gene, the 3'-end of a gene encoding a Gea2-like protein was identified. A non-revertible C. lusitaniae ura3 mutant was selected on the basis of 5-fluoroorotic acid resistance. The mutation was a single point mutation resulting in the amino acid substitution D95V in a highly conserved domain, and in a concomitant EcoRV restriction site polymorphism. The mutant strain was successfully transformed to prototrophy following electroporation with the URA3 gene cloned in an integrative vector, with frequencies of 100-200 transformants per micro g of DNA. Southern blot analysis revealed that almost all transformants were derived from homologous recombination events at the resident locus. The GeneBank Accession No. for C. lusitaniae URA3 gene is AF450297.
François
Fabienne
F
Laboratoire des Sciences Végétales, Faculté de Pharmacie, Université René Descartes-Paris 5, Paris 75006, France.
Chapeland-Leclerc
Florence
F
Villard
Jean
J
Noël
Thierry
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D016415:Sequence Alignment; D014170:Transformation, Genetic
D004271:DNA, Fungal; D005656:Fungal Proteins; C121632:GEA2 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; D029701:Saccharomyces cerevisiae Proteins