title: Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans.
abstract: The ARG5,6 gene from the dimorphic fungus Candida albicans was cloned by functional complementation of the arginine auxotrophy present in strain EL2 (Arg-) using a gene library constructed in the double autonomously replicating sequence vector pRM1. Sequence analysis revealed a putative 857 amino acid polypeptide (95 kDa) which showed high homology (63% protein identity) to the Saccharomyces cerevisiae ARG5,6 gene. Similarly to the S. cerevisiae gene, the C. albicans ARG5,6 gene is responsible for both the acetylglutamate kinase and acetylglutamyl-phosphate reductase activities, the second and third steps of arginine biosynthesis at the mitochondria. The C. albicans ARG5,6 gene complemented the arg6 mutation present in S. cerevisiae (strain D160-4D) on a yeast episomal plasmid using its own regulatory signals. A set of non-integrative high-efficiency plasmid vectors based on this gene marker was constructed and a null C. albicans arg5,6 delta strain was obtained using the common URA3-blaster strategy. In addition, we generated an arg5,6 delta null mutant in a single transformation event, thus improving the basic strategy for generating gene deletions in C. albicans.
authors.affiliation: Departamento de Microbiologi'a II, Facultad de Farmacia, Universidad Complutense de Madrid,Avda. Ramn y Cajal s/n, 28040 Madrid,Spain.
authors.forename: A
authors.initials: A
authors.lastname: Negredo
authors.affiliation: Departamento de Microbiologi'a II, Facultad de Farmacia, Universidad Complutense de Madrid,Avda. Ramn y Cajal s/n, 28040 Madrid,Spain.
authors.forename: L
authors.initials: L
authors.lastname: Monteoliva
authors.affiliation: Departamento de Microbiologi'a II, Facultad de Farmacia, Universidad Complutense de Madrid,Avda. Ramn y Cajal s/n, 28040 Madrid,Spain.
authors.forename: C
authors.initials: C
authors.lastname: Gil
authors.affiliation: Departamento de Microbiologi'a II, Facultad de Farmacia, Universidad Complutense de Madrid,Avda. Ramn y Cajal s/n, 28040 Madrid,Spain.
authors.forename: J
authors.initials: J
authors.lastname: Pla
authors.affiliation: Departamento de Microbiologi'a II, Facultad de Farmacia, Universidad Complutense de Madrid,Avda. Ramn y Cajal s/n, 28040 Madrid,Spain.
authors.forename: C
authors.initials: C
authors.lastname: Nombela
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D001120:Arginine / Q000096:biosynthesis*; D001483:Base Sequence; D002176:Candida albicans / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D017353:Gene Deletion; D015723:Gene Library; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D001120:Arginine; D000445:Aldehyde Oxidoreductases; C018722:N-acetyl-gamma-glutamyl-phosphate reductase; D017851:Phosphotransferases (Carboxyl Group Acceptor); C018721:acetylglutamate kinase