Cassettes for the PCR-mediated construction of regulatable alleles in Candida albicans.
The recent availability of genome sequence information for the opportunistic pathogen Candida albicans has greatly facilitated the ability to perform genetic manipulations in this organism. Two important molecular tools for studying gene function are regulatable promoters for generating conditional mutants and fluorescent proteins for determining the subcellular localization of fusion gene products. We describe a set of plasmids containing promoter-GFP cassettes (P(MET3)-GFP, P(GAL1)-GFP, and P(PCK1)-GFP), linked to a selectable nutritional marker gene (URA3). PCR-mediated gene modification generates gene-specific promoter, or gene-specific promoter-GFP, fusions at the 5'-end of the gene of interest. One set of primers can be used to generate three strains expressing a native protein of interest, or an amino-terminal GFP-tagged version, from three different regulatable promoters. Thus, these promoter cassette plasmids facilitate construction of conditional mutant strains, overexpression alleles and/or inducible amino-terminal GFP fusion proteins.
Gerami-Nejad
Maryam
M
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.
Hausauer
Danielle
D
McClellan
Mark
M
Berman
Judith
J
Gale
Cheryl
C
D000483:Alleles; D019677:Artificial Gene Fusion; D001483:Base Sequence; D002176:Candida albicans / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D005800:Genes, Fungal; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics
D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins