New transposons to generate GFP protein fusions in Candida albicans.
Transposon elements are important tools for gene function analysis, for example they can be used to easily create genome-wide collections of insertion mutants. Transposons may also carry sequences coding for an epitope or fluorescent marker useful for protein expression and localization analysis. We have developed three new Tn5-based transposons that incorporate a GFP (green fluorescent protein) coding sequence to generate fusion proteins in the important fungal pathogen Candida albicans. Each transposon also contains the URA3 and Kan(R) genes for yeast and bacterial selection, respectively. After in vitro transposition, the insertional allele is transferred to the chromosomal locus by homologous recombination. Transposons Tn5-CaGFP and Tn5-CaGFP-URA3::FLIP can generate C-terminal truncated GFP fusions. A URA3 flipper recycling cassette was incorporated into the transposon Tn5-CaGFP-URA3::FLIP. After the induction of Flip recombinase to excise the marker, the heterozygous strain is transformed again in order to obtain a GFP-tagged homozygous strains. In the Tn5-CaGFP-FL transposon the markers are flanked by a rare-cutting enzyme. After in vitro transposition into a plasmid-borne target gene, the markers are eliminated by restriction digestion and religation, resulting in a construct coding for full-length GFP-fusion proteins. This transposon can generate plasmid libraries of GFP insertions in proteins where N- or C-terminal tagging may alter localization. We tested our transposon system by mutagenizing the essential septin CDC3 gene. The results indicate that the Cdc3 C-terminal extension is important for correct septin filament assembly. The transposons described here provide a new system to obtain global gene expression and protein localization data in C. albicans.
Dias
Marcos Vinicios Salles
MV
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. dos Bandeirantes 3.900, Ribeirão Preto, SP, Brazil.
Basso
Luis Roberto
LR
Coelho
Paulo Sergio Rodrigues
PS
D019677:Artificial Gene Fusion / Q000379:methods*; D002176:Candida albicans / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000096:biosynthesis; D004251:DNA Transposable Elements; D005656:Fungal Proteins / Q000096:biosynthesis; D049452:Green Fluorescent Proteins / Q000235:genetics*; D016254:Mutagenesis, Insertional; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014170:Transformation, Genetic
D018797:Cell Cycle Proteins; D004251:DNA Transposable Elements; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins