A one-hybrid system for the analysis of transcription factors in Candida albicans
This thesis describes the design and construction of a one-hybrid system for the analysis of transcriptional regulators in C. albicans. In Saccharomyces cerevisiae, Escherichia coli LexA fusions have been used to examine the mechanisms of action of transcription factors by targeting them artificially to reporter genes containing the EcLexA operator. However, the EcLexA ORF is unlikely to be functional in C. albicans because it contains twelve CUG codons, which are decoded as serine instead of leucine in this fungus. Therefore we used the Staphylococcus aureus lexA ORF, which lacks CUG codons, to develop an equivalent tool for C. albicans. SaLexA is used in combination with a StlacZ reporter carrying the SalexA operator sequence. The SaLexA system was validated using two transcription factors: CaNrg1 and CaGcn4. The SaLexA-CaNrg1 fusion repressed the reporter in C. albicans confirming that CaNrg1 is a repressor. Also, the SaLexA-CaGcn4 fusion activated the reporter confirming that CaGcn4 is an activator. Following these successful validation experiments, the one-hybrid system was used to show that CaNrg1-mediated repression is dependent upon the Sssn6-Tup1 co-repressor complex. The system was also used to examine the transcriptional activities of the C. albicans APSES proteins Efg1 and Efh1, which contribute to the regulation of morphogenesis in C. albicans. Efg1 was shown to act as a transcriptional repressor, whereas Efh1 acts as a transcriptional activator.