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    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
    (Massey University, 1997) McGlynn, Linda Joy
    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.
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    Construction of a novel fungal gus expression plasmid, and its evaluation in Aspergillus nidulans : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University
    (Massey University, 1996) McGowan, Tania Louise
    A GUS expression plasmid, pFunGus, was constructed containing a multi-cloning site for the insertion of gene regulatory elements, to be used in fungal reporter gene studies. A derivative of pFunGus (pFG-gpd) was constructed by the insertion of the gpdA promoter (glyceradehyde-3-phosphatc dehydrogenase) into the multi-cloning site of pFunGus for the assessment of the plasmid's transformation and expression properties in Aspergillus niduans. The correct construction of pFunGus and pFG-gpd was verified by analytical restriction digests and by its property of GUS expression in A. nidulans. The plasmid was integrated into the A. nidulans genome via cotransformation with the phleomycin resistance plasmid, pAN8-l. Transformation frequencies of between 3 and 250 transformants per µg of pAN8-l DNA were obtained. Initial screening for cotransformation yielded no pFG-gpd transformants. Attempts to improve cotransformation frequencies by optimisation of cotransformation conditions were unsuccessful. However, large scale screenings of transformants lead to cotransformants being isolated at a very low cotransformation frequency. Approximately 0.45% of pAN8-l transformants possessed the GUS phenotype. The eight pFG-gpd transformants obtained were analysed by Southern hybridisation. Six out of the eight transformants had a single copy integration. Of the remaining two transformants, one had three copies integrated at separate locations, one of which was disrupted, and the other had four copies integrated as tandem repeats, one of which was disrupted. All the transforming DNA appeared to be integrated ectopically. The physiology of the transformants was assessed by dry weight increase, colony extension and total protein content. These showed that the transformants biology was not significantly compromised by the transforming DNA. Finally, high levels of GUS expression were observed in all pFG-gpd transformants and the GUS expression per copy of the GUS expression cassette integrated into the genome was constant. These results showed that the transformed gene copy number determined the levels of gene activity rather than the position of integration in the genome. Overall these results demonstrate the potential application of the versatile GUS expression plasmid, pFunGus for reporter gene studies in filamentous fungi.
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    Genetics and physiology of respiration in Aspergillus nidulans : a thesis presented in partial fulfilment of the requirements for the degree of Doctor in Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2001) Gardiner, Rebecca Elizabeth
    Obligate aerobes such as Aspergillus nidulans primarily use the classical respiratory pathway for ATP production. However, the use of alternative energy-producing processes in A. nidulans was first speculated upon when cytochrome c-deficient strains were observed to be viable upon fermentable carbon sources. It was therefore postulated that the cycA- strains of A. nidulans may use fermentation and an alternative respiratory pathway to compensate for the non-functioning cytochrome c-dependent pathway. Characterisation of the A. nidulans cytochrome c-deficient strains was carried out. The growth parameters for strain A68 were consistent with other cytochrome c mutants; the strain grew more slowly than the corresponding wildtype strain on fermentable carbon sources, and produced higher levels of ethanol. Spectral analysis confirmed the lack of detectable levels of cytochrome c in the cycA- strains, and decreased levels of cytochrome c oxidase, consistent with the non-functioning cytochrome c-dependent respiratory pathway. The presence of a hemoglobin-like molecule in the cycA- and cycA+ strains was determined by CO binding assays. Inhibitor studies determined the presence of an alternative respiratory pathway in cycA- and cycA+ strains of A. nidulans. An active cytochrome c-dependent pathway was found to be present in the cycA+ strains, yet absent from the cycA- strains. Results also suggested the presence of a putative third terminal oxidase in the cycA- and cycA+ strains. Increased levels of b-type heme observed in the redox spectra of the cycA- strains were suggested to be associated with the putative third terminal oxidase. Therefore, the cycA- strains of A. nidulans appear to use fermentation and the alternative respiratory pathway to compensate for the non-functioning cytochrome c-dependent pathway. The putative hemoglobin molecule identified in these strains may also function as a terminal oxidase, in addition to the putative third terminal oxidase. PCR amplification with degenerate primers was carried out to confirm the presence of an AOX gene in cycA+ and cycA- strains of A. nidulans. The product of the AOX gene is likely to function as a terminal oxidase in the alternative pathway. A comparison of fungal and plant AOX protein sequences was cauied out. A conserved cysteine residue which has been implicated in dimer formation and pyruvate regulation was found to be absent from the fungal sequences. A preliminary expression study of the A. nidulans AOX gene was carried out by RT-PCR. The putative regulatory elements identified within the A. nidulans AOX gene promoter are also located within the promoters of other respiratory-related genes (eg. A. nidulans cycA) and genes involved in reducing the formation of reactive oxygen species (ROS) (eg. A. nidulans catA-C and sod1). This implies similar mechanisms of regulation, which may be controlled in a coordinated manner. A functional analysis of the A. nidulans cycA gene promoter was carried out to identify important regulatory elements. Reporter constructs containing cycA-lacZ, fusion genes were transformed into A. nidulans. Although integrated at the argB locus, the constructs had very low levels of lacZ expression (with the exception of the positive control). A number of parameters were investigated, and a 'promoter switch' experiment commenced, however the cause of the faulty cycA-lacZ expression system was not determined. Functional expression of the A. nidulans cycA gene promoter in yeast was also attempted, as regulatory mechanisms controlling cytochrome c expression are believed to be analogous in A. nidulans and Saccharomyces cerevisiae. Reporter constructs containing cycA-lacZ fusion genes were transformed into S. cerevisiae, with resulting low β-galactosidase activity, similar to the results in A. nidulans. The wildtype strain of A. nidulans from which the cycA gene promoter fragment was amplified was shown by spectral analysis to have low levels of cytochrome c, in comparison to other wildtype strains of A. nidulans. Therefore, it is possible that the low levels of lacZ expression from the cycA-lacZ fusion genes may be representative of the level of cycA expression in that strain. However, low levels of lacZ expression were also observed for the S. cerevisiae positive control, indicating that the expression system was not working properly. Therefore, the expression of the A. nidulans cycA gene promoter in S. cerevisiae could not be assessed in this study.
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    Transformation and gene targeting in Aspergillus nidulans : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North
    (Massey University, 1996) Bird, Dianne Maria
    Transformation of a haploid Aspergillus nidulans pyrG auxotrophic strain (1-85) was optimised for the vector pGM32 containing the heterologous Neurospora crassa pyr4 gene. The resulting uracil-independent transformants could be classified into two main groups based on morphology. The minority were morphologically very similar to the parental strain, easily purified and mitotically stable. The majority (10 times more frequent) were irregular in shape and shown to be heterokaryons that could not be resolved into transformed homokaryons. Analysis of the transformant types suggested regulation of multiple copies of the gene for OMPdecase (pyr4 and pyrG) resulted in the titration/inactivation of essential trans-acting factors. The heterokaryon state was therefore a requirement for the survival of transformants containing multiple copies of the integrated vector. The effect of altering the conditions of transformation on the efficiency of gene targeting in filamentous fungi was studied. The A. nidulans niaD and amdS genes, both involved in nitrogen source utilisation, were selected as target loci. Insertional inactivation vectors were constructed (based on pGM32) and parameters shown to have an effect on the targeting frequency at the niaD locus were subsequently tested at the amdS locus. A dramatic difference in targeting was observed between the niaD and amdS loci with targeting of niaD being much more efficient than amdS for the parameters tested. The level of gene targeting using circular DNA was found to correlate with the size of the homologous segment at both loci. Similarly the level of targeting was shown to increase at both loci when vectors were linearised within the region of homology. Unexpectedly the level of targeting was unaltered at the niaD locus when transcription was induced at different stages in the transformation procedure. Likewise targeting was unaffected by altering the amount of DNA in the reaction mix. The regeneration temperature, however, did appear to have an effect on targeting, with enhanced targeting observed at the lower temperature. Gene replacement by transformation was used to disrupt the cycA gene in diploid and haploid A. nidulans strains. The first completely deficient cyc mutant in a filamentous fungus was isolated and shown to be non-lethal. Haploidisation analysis of the diploid transformant localised the chromosomal position of cycA to chromosome I.