title: Development of an integrative transformation system for the opportunistic pathogenic yeast Candida lusitaniae using URA3 as a selection marker.
abstract: 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.
authors.affiliation: Laboratoire des Sciences Végétales, Faculté de Pharmacie, Université René Descartes-Paris 5, Paris 75006, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: François
authors.forename: Florence
authors.initials: F
authors.lastname: Chapeland-Leclerc
authors.forename: Jean
authors.initials: J
authors.lastname: Villard
authors.forename: Thierry
authors.initials: T
authors.lastname: Noël
mesh_terms: 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
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; C121632:GEA2 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; D029701:Saccharomyces cerevisiae Proteins