Gene regulation during morphogenesis in Candida albicans
This thesis describes attempts to investigate the regulation of the Candida albicans hyphal-specific gene HYR1 by a functional dissection of the HYR1 promoter, protein localisation studies and analysis of HYR1 expression in C. albicans morphological mutants. Sequencing of the HYR1 promoter revealed several putative cis-acting elements within 700 bp of the determined HYR1 transcriptional start site. The possibility of using the LAC4 gene from Kluyveromyces lactis as a reporter for dissection of the HYR1 promoter in C. albicans was investigated. Expression of LAC4 in S. cerevisiae and C. albicans was driven by the C. albicans ADH1 promoter. LAC4 expression was carbon-source-dependent in Saccharomyces cerevisiae as shown by a plate assay and -galactosidase assay, and was confirmed by northern analyses which showed high levels of LAC4 mRNA. However, -galactosidase activity was not detectable in C. albicans transformants using the plate assay or the enzyme assay, and this lack of LAC4 expression was confirmed by northern analysis of the LAC4 mRNA. Preliminary Southern analysis revealed that the LAC4 sequences in S. cerevisae and C. albicans are maintained at approximately equal copy numbers between transformants. Hence LAC4 was not sufficiently sensitive to act as reporter of HYR1 expression and therefore the recently developed yEGFP gene was used for a preliminary HYR1 promoter dissection. However, the HYR1 promoter-yEGFP fusions failed to confirm a role for these elements in the regulation of HYR1 expression . Nevertheless, the hyphal-specific nature of HYR1 expression was confirmed by analysis of the HYR1-yEGFP mRNA by northern analysis. The yEGFP reporter also proved to be too insensitive for use as a reporter of HYR1 expression in C. albicans. To investigate the proposed localisation of the Hyr1p, an in-frame Hyr1-yEGFP fusion was created and expressed in C. albicans and S. cerevisiae. However, this work was inconclusive and the status of Hyr1p as a component of the hyphal cell wall remains to be confirmed.