Project description:Compared to other model organisms and despite the clinical relevance of the pathogenic yeast Candida albicans, no comprehensive analysis has been done to provide experimental support of its in silico-based genome annotation. Here we have undertaken a genome-wide experimental annotation to accurately uncover the transcriptional landscape of the pathogenic yeast C. albicans using strand-specific high-density tiling arrays. RNAs were purified from cells growing under conditions relevant to C. albicans pathogenicity, including biofilm, lab-grown yeast and serum-induced hyphae as well as cells isolated from the mouse caecum. This work provides a genome-wide experimental validation for a large number of predicted ORFs for which transcription had not been detected by other approaches. Additionally, we identified more than 2000 novel transcriptional segments, including new ORFs and exons, non-coding RNAs (ncRNA) as well as convincing cases of antisense gene transcription. We also characterized the 5’- and 3’-untranslated regions (UTR) of expressed ORFs, and established that genes with long 5’UTRs are significantly enriched in regulatory functions controlling filamentous growth. Furthermore, we found that genomic regions adjacent to telomeres harbor a cluster of expressed ncRNAs. To validate and confirm new ncRNA candidates, we adapted an iterative strategy combining both genome-wide occupancy of the different subunits of RNA polymerases I, II and III, and expression data. This comprehensive approach allowed the identification of different families of ncRNA. In summary, we provide a comprehensive expression atlas that covers relevant C. albicans pathogenic developmental stages in addition to a discovery of new ORF and non-coding genetic elements.
Project description:Compared to other model organisms and despite the clinical relevance of the pathogenic yeast Candida albicans, no comprehensive analysis has been done to provide experimental support of its in silico-based genome annotation. Here we have undertaken a genome-wide experimental annotation to accurately uncover the transcriptional landscape of the pathogenic yeast C. albicans using strand-specific high-density tiling arrays. RNAs were purified from cells growing under conditions relevant to C. albicans pathogenicity, including biofilm, lab-grown yeast and serum-induced hyphae as well as cells isolated from the mouse caecum. This work provides a genome-wide experimental validation for a large number of predicted ORFs for which transcription had not been detected by other approaches. Additionally, we identified more than 2000 novel transcriptional segments, including new ORFs and exons, non-coding RNAs (ncRNA) as well as convincing cases of antisense gene transcription. We also characterized the 5’- and 3’-untranslated regions (UTR) of expressed ORFs, and established that genes with long 5’UTRs are significantly enriched in regulatory functions controlling filamentous growth. Furthermore, we found that genomic regions adjacent to telomeres harbor a cluster of expressed ncRNAs. To validate and confirm new ncRNA candidates, we adapted an iterative strategy combining both genome-wide occupancy of the different subunits of RNA polymerases I, II and III, and expression data. This comprehensive approach allowed the identification of different families of ncRNA. In summary, we provide a comprehensive expression atlas that covers relevant C. albicans pathogenic developmental stages in addition to a discovery of new ORF and non-coding genetic elements. We have undertaken a genome-wide experimental annotation to accurately uncover the transcriptional landscape of the pathogenic yeast C. albicans using strand-specific high-density tiling arrays. RNAs were purified from cells growing under conditions relevant to Candida albicans pathogenicity, including biofilm, lab-grown yeast and serum-induced hyphae as well as cells isolated from the mouse caecum. We also adapted a strategy in which genome-wide occupancy of different subunits of RNA polymerases (RNAP) I, II and III, is combined with expression data to annotate ncRNAs resulting from real transcriptional events. For this purpose we have performed ChIP-chip of subunits that represent the three RNAP machines in C. albicans cells growing in rich media (YPD) at 30°C. In this study, we performed peak detection only for RNA Polymerase III (Rpc82p). All detected peaks and their genomic features are included as a supplementary file on the Sample record (GSM561024).
Project description:Total genomic DNAs of J118, CdUM4b, the pseudotetraploid hybrid strain HBT1 and the progeny hybrid strain HBP1 were isolated. In an attempt to determine the number of copies of individual chromosomes, we used comparative genome hybridization (CGH) analysis using specially designed whole genome microarrays representing C. albicans and C. dubliniensis genomes. The arrays contained 60 mer probes that fell in three categories either C. albicans specific or C. dubliniensis specific or common to both. The specific probes were expected to work as internal controls when hybridized to parental genomic DNA alone. The arrays were individually hybridized to Cy3-labeled genomic DNA from either of C. albicans (J118), C. dubliniensis (CdUM4b) parents or the somatic psudotetraploid hybrid strain, HBT1 and the pseudodiploid progeny hybrid HBP1. After experimentally determining species-specific probes and common probes, the ratios of intensity on the HBT1 hybrid array to those of either of the parents were calculated. The copy number was determined for the entire chromosomes by comparing individual probe intensities from the pseudotetraploid to either diploid parent genome DNA intensities.
Project description:Candida albicans and Candida dubliniensis are closely related species displaying differences in virulence and genome content, therefore providing potential opportunities to identify novel C. albicans virulence genes. C. albicans gene arrays were used for comparative analysis of global gene expression in the two species in reconstituted human oral epithelium (RHE). C. albicans (SC5314) showed upregulation of hypha-specific and virulence genes within 30 min postinoculation, coinciding with rapid induction of filamentation and increased RHE damage. C. dubliniensis (CD36) showed no detectable upregulation of hypha-specific genes, grew as yeast, and caused limited RHE damage. Several genes absent or highly divergent in C. dubliniensis were upregulated in C. albicans. One such gene, SFL2 (orf19.3969), encoding a putative heat shock factor, was deleted in C. albicans. ΔΔsfl2 cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of SFL2 into the ΔΔsfl2 strain. Moreover, SFL2 overexpression in C. albicans triggered hyphal morphogenesis. Although SFL2 deletion had no apparent effect on host survival in the murine model of systemic infection, ΔΔsfl2 strain-infected kidney tissues contained only yeast cells. These results suggest a role for SFL2 in morphogenesis and an indirect role in C. albicans pathogenesis in epithelial tissues.
Project description:Candida albicans and Candida dubliniensis are closely related species displaying differences in virulence and genome content, therefore providing potential opportunities to identify novel C. albicans virulence genes. C. albicans gene arrays were used for comparative analysis of global gene expression in the two species in reconstituted human oral epithelium (RHE). C. albicans (SC5314) showed upregulation of hypha-specific and virulence genes within 30 min postinoculation, coinciding with rapid induction of filamentation and increased RHE damage. C. dubliniensis (CD36) showed no detectable upregulation of hypha-specific genes, grew as yeast, and caused limited RHE damage. Several genes absent or highly divergent in C. dubliniensis were upregulated in C. albicans. One such gene, SFL2 (orf19.3969), encoding a putative heat shock factor, was deleted in C. albicans. ΔΔsfl2 cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of SFL2 into the ΔΔsfl2 strain. Moreover, SFL2 overexpression in C. albicans triggered hyphal morphogenesis. Although SFL2 deletion had no apparent effect on host survival in the murine model of systemic infection, ΔΔsfl2 strain-infected kidney tissues contained only yeast cells. These results suggest a role for SFL2 in morphogenesis and an indirect role in C. albicans pathogenesis in epithelial tissues.