Scientific article
English

Development of an integrative transformation system for the opportunistic pathogenic yeast Candida lusitaniae using URA3 as a selection marker

Published inYeast, vol. 21, no. 2, p. 95-106
Publication date2004
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.

Keywords
  • Amino Acid Sequence
  • Base Sequence
  • Blotting
  • Southern
  • Candida/enzymology/genetics
  • Cloning
  • Molecular
  • Conserved Sequence
  • DNA
  • Fungal/chemistry/genetics
  • Fungal Proteins/genetics
  • Guanine Nucleotide Exchange Factors/genetics
  • Molecular Sequence Data
  • Mutagenesis
  • Insertional
  • Phylogeny
  • Polymerase Chain Reaction
  • Saccharomyces cerevisiae Proteins/genetics
  • Sequence Alignment
  • Transformation
  • Genetic
Citation (ISO format)
FRANÇOIS, Fabienne et al. Development of an integrative transformation system for the opportunistic pathogenic yeast Candida lusitaniae using URA3 as a selection marker. In: Yeast, 2004, vol. 21, n° 2, p. 95–106. doi: 10.1002/yea.1059
Main files (1)
Article (Published version)
accessLevelRestricted
Identifiers
ISSN of the journal0749-503X
124views
0downloads

Technical informations

Creation26/10/2020 15:04:00
First validation26/10/2020 15:04:00
Update time14/11/2024 14:51:12
Status update14/11/2024 14:51:12
Last indexation14/11/2024 14:54:50
All rights reserved by Archive ouverte UNIGE and the University of GenevaunigeBlack