Regulation of the Aspergillus nidulans cytochrome C gene : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand

Abstract

The filamentous fungus Aspergillus nidulans has been genetically and biochemically well-characterised and thus provides an attractive model for studies on the regulation of eukaroytic gene expression. This study was undertaken to investigate the factors affecting respiratory function in A. nidulans. Due to the central role of cytochrome c in oxidative respiration, this study was focused on the cytochrome c gene and primarily upon how oxygen availability affects its expression. The Aspergillus nidulans cytochrome c gene (eyeA) appears to be transcriptionally activated in response to oxygen availability (Raitt et al., 1994). In the yeast S. cerevisiae, oxygen availability activates its cytochrome c genes via a heme-activated protein HAP1, which binds to the promoter region of each gene (Pfiefer et al., 1989). Since heme is only synthesised in the presence of oxygen, activity of the HAP1 protein serves as an intracellular signal of oxygen availability. In the upstream region of the cycA gene, a sequence with homology to the S. cerevisiae HAP1 binding site was present (Raitt, 1992). To determine the significance of the putative HAP1 binding site and the role of other promoter sequences in the A. nidulans cycA gene, a promoter-reporter vector was constructed. However, upon sequencing of the cycA promoter in the reporter vector, a sequencing error was discovered in the published cycA gene by Raitt et al. (1994) which affected the position of the major translational start site. Further examination of the cycA sequence also revealed a possible undetected intron (Intron I). To determine the number of introns in the cycA gene, RT- PCR was performed on cycA RNA. Sequencing of the RT-PCR amplified products showed that the previously undetected intron (Intron I) was present, and that the cycA gene contains three instead of two introns as published by Raitt et al. (1994). Since the published ATG start site was located within Intron I, a new translational start site was proposed. The major consequences of these changes to the cycA gene was that the putative HAP1 site was now located within the coding region of the gene, and therefore could not be a regulatory element. In addition only 247 bp of cycA promoter sequence remained cloned for analysis. To obtain additional promoter sequence, an A. nidulans genomic library was screened with a BamHI cDNA probe containing 224 bp of the 5' region of the cycA gene. Three positive clones were obtained, of which λLM9 and λLM5 were identical, and λLM19 was an overlapping clone with λLM9 and λLM5. Restriction enzyme and Southern blott analysis of the two overlapping cycA clones, revealed that 2.1 kb EcoRI fragments from both clones contained the 5' region. The 2.1 kb EcoRI fragment from λLM9 was cloned into pUC18 and sequenced. The completed upstream sequence of the A. nidulans cytochrome c gene was obtained, and putative regulatory signals including the HAP1 binding site were found, and compared with published promoter sequences which regulate the expression of respiratory-encoding genes from yeast.