Construction and real-time RT-PCR validation of Candida albicans PALS-GFP reporter strains and their use in flow cytometry analysis of ALS gene expression in budding and filamenting cells.
The gene encoding yeast-enhanced green fluorescent protein (GFP) was placed under control of ALS gene promoters in Candida albicans. The PALS-GFP reporter strains were validated using various techniques including a new real-time RT-PCR assay to quantify ALS gene expression. The PALS-GFP reporter strains were grown in media that promoted yeast or germ tube forms, and the resulting fluorescence was measured by flow cytometry. In addition to results that indicate differences in ALS gene expression due to growth medium, growth stage and developmental programme, new data show large differences in transcriptional level among the ALS genes. Expression of ALS1 was associated with transfer of the PALS1-GFP strain to fresh growth medium. ALS3 expression increased markedly when germ tubes were visible microscopically and ALS7 expression exhibited a transient peak between 2 and 3 h following inoculation into fresh YPD medium. Transcription from the ALS1 and ALS3 promoters was strongest among those tested and contrasted markedly with the weaker promoter strength at the ALS5, ALS6, ALS7 and ALS9 loci. These weaker transcriptional responses were also observed using real-time RT-PCR measurements on wild-type C. albicans cells. Assuming a positive correlation between transcriptional level and protein production, these results suggest that some Als proteins are abundant on the C. albicans cell surface while others are produced at a much lower level.
Green
Clayton B
CB
Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA.
Zhao
Xiaomin
X
Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA.
Yeater
Kathleen M
KM
Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA.
Hoyer
Lois L
LL
Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA.
D001483:Base Sequence; D002176:Candida albicans / Q000235:genetics* / Q000254:growth & development; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
C093538:ALS1 protein, Candida albicans; C113620:ALS3 protein, Candida albicans; D017931:DNA Primers; D004271:DNA, Fungal; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins