Genetics - Theses
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ItemNo Preview AvailableCloning and analysis of the acuG, idpA and maeA genes involved in carbon metabolism in felamentous fungus Aspergillus nidulans(University of Melbourne, 2002)This thesis describes work on cloning and analysis of the three genes encoding enzymes of carbon catabolism from the filamentous fungus Aspergillus nidulans. NADP-dependent isocitrate dehydrogenase enzymes catalyze the decarboxylation of isocitrate to 2-oxoglutarate accompanied by the production of NADPH. In mammals two different genes encode mitochondrial and cytoplasmic/peroxisomal located enzymes, while in Saccharomyces cerevisiae three separate genes specify compartment specific enzymes. In A. nidulans a single gene, idpA, is shown to specify a protein with a high degree of identity to mammalian and S. cerevisiae enzymes. Two idpA transcripts were identified and two transcription start points were determined by sequencing cDNA clones and by 5'RACE. The shorter transcript was found to be inducible by acetate and by fatty acids while the longer transcript was present in higher amounts during growth in glucose containing media. The longer transcript is predicted to encode a polypeptide containing an N-terminal mitochondrial targetting sequence as well as a C-terminal tripeptide (ARL). The shorter transcript is predicted to encode a polypeptide lacking the mitochondrial targetting signal but retaining the C-terminal sequence. Immunoblotting using antibody raised against S. cerevisiae Idp1p detected two polypeptides consistent with these predictions. The functions of the predicted targetting sequences were confirmed by analysis of transformants containing fluorescent protein fusion constructs. Using anti-Idp1p antibodies, protein localisation to mitochondria and peroxisomes was observed during growth on glucose while cytoplasmic and peroxisomal localisation was found upon acetate or fatty acid induction. Therefore it has been established that by the use of two transcription start points a single gene is sufficient to specify localisation of NADP-dependent isocitrate dehydrogenase to three different cellular compartments in A. nidulans. A deletion of the idpA gene was generated and the phenotype indicates a possible role in providing NADPH for protection against oxidative stress. The single acuG gene encodes fructose-1,6-bisphosphatase, a crucial enzyme in gluconeogenesis. It has been shown to be under strong control by CreA mediated carbon catabolite repression. A significant effect is also exerted by endogenous induction in carbon starvation conditions. This pattern of relatively weak regulation of acuG in A. nidulans is very different to the strong regulation of FBP1 gene expression and FBP activity in S. cerevisiae which acts on the level of transcription, mRNA stability and glucose inactivation of the protein. The putative novel regulators AcuK and AcuM were found to play a key role in fructose-1,6-bisphosphatase regulation. These novel proteins may be global gluconeogenic regulators in A. nidulans as they have been found to affect the regulation of other gluconeogenic enzymes: acuF encoding PEPCK, acuN encoding enolase (M.J. Hynes, personal communication) and maeA encoding malic enzyme (see below). This pattern of regulation allows gluconeogenesis to occur during growth on any carbon source metabolised via TCA cycle intermediates in A. nidulans. The malic enzyme plays an intermediary role between the TCA cycle and gluconeogenesis. Two genes with different cofactor specificity have been identified in A. nidulans and work was concentrated on the maeA gene encoding a conserved NADP-dependent malic enzyme. Two closely located starts of transcription were detected by 5'RACE and transcripts showed strong regulation by carbon sources with strong gdhB101 dependent induction by sources of glutamate. A deletion of the maeA gene was generated and the phenotype indicates a possible role in NADPH generation and providing pyruvate for acetyl-CoA synthesis to maintain carbon flux and operation of the TCA cycle during growth on compounds metabolised via the cycle.
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ItemRegulation of amidase genes in Aspergillus nidulans : mechanisms of nitrogen metabolite repression(University of Melbourne, 2002)The filamentous fungus Aspergillus nidulans can utilise a wide variety of amides as the sole nitrogen source. The expression of the required catabolic enzymes is tightly regulated in response to nitrogen availability. The global mechanism responsible for this response is termed nitrogen metabolite repression, and is mediated by the GATA zinc finger activator protein AreA. During nitrogen limitation, AreA activates the expression of genes required for the utilisation of alternative nitrogen sources. If a more easily assimilated source such as ammonium is present changes in areA mRNA stability and interaction with the negatively acting NmrA protein prevent AreA activation of catabolic gene expression. This thesis describes the isolation of three genes involved in amide utilisation in A. nidulans and an analysis of their regulation. The fmdS gene encodes a formamidase unlike the characterised acetamidase of A. nidulans, and is required for the hydrolysis of formamide. The gmdA and bzuA genes are required for the utilisation of benzamide as a nitrogen source, and encode general amidase and benzoate para-hydroxylase, respectively. Like the acetamidase, the general amidase belongs to the amidase signature family of enzymes. In addition to benzamide, the general amidase also mediates the hydrolysis of a variety of long chain amides. BzuA is required to prevent benzoate toxicity following the hydrolysis of benzamide by GmdA. The transcription of both fmdS and gmdA is highly regulated by AreA-mediated nitrogen metabolite repression, with a low level of expression during growth on preferred nitrogen sources such as ammonium and increased levels during growth on an alternative source such as alanine. In addition, this activation was shown to be enhanced during nitrogen starvation, defining an additional level of AreA function. The transcription of these genes is affected by carbon availability, with transcription halted upon carbon starvation irrespective of nitrogen availability. Detailed deletion analysis of the fmdS promoter was performed, and the only changes in fmdS transcription revealed were though deletion of putative AreA recognition sequences. This suggested the mechanism behind the carbon starvation response is probably inactivation of AreA. Expression of fmdS was also found to be affected by transcriptional interference from an upstream gene (usgS), whose transcript overlaps the fmdS coding region. Beyond its affect on reducing fmdS transcription when in cis, gene inactivation revealed no indication of the role of the usgS gene. The nitrogen starvation and carbon starvation responses of nitrogen metabolite repression are independent of the two mechanisms already characterised in this global regulatory phenomenon - NmrA interaction and areA transcript stability. In an attempt to identify factors which may play a role in the starvation mechanisms, a possible A. nidulans homologue of the S. cerevisiae URE2 gene was cloned. In yeast, Ure2 is a negatively acting factor that interacts with and represses the AreA homologue Gln3. Several ESTs with similarity to URE2 were identified, allowing the gstA gene to be cloned. Gene inactivation showed gstA encodes a functional glutathione S-transferase involved in resistance to xenobiotics and heavy metals. The evidence presented here strongly suggests the A. nidulans genome does not contain a true URE2 homologue that is involved in nitrogen metabolite repression. A gene replacement strategy was developed to allow epitope tagging of AreA, yielding a variety of strains with which this regulatory factor could be studied at the protein level. Western blot analysis has indicated that AreA is differentially phosphorylated under different conditions of nitrogen and carbon availability. The starvation responses in AreA regulated structural gene expression correspond to the post-translational modifications observed suggesting that it is functionally significant.
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ItemCharacterization of the tamA gene of Aspergillus nidulans(University of Melbourne, 2000)In Aspergillus nidulans, the GATA zinc finger protein AreA activates the expression of enzymes that metabolize less favoured nitrogen sources in the absence of preferred nitrogen sources, such as ammonium or glutamine. The amount and activity of AreA are modulated by a number of mechanisms, including an interaction with the NmrA protein through the GATA zinc finger and C-terminal regions of AreA to inhibit DNA binding under nitrogen-sufficient conditions. The TamA protein has also been implicated in nitrogen regulation, with mutants described as having reduced levels of a number of nitrogen metabolic enzymes. This thesis describes the characterization of the tamA gene and investigates its role in nitrogen regulation. tamA encodes a 739 amino acid protein that contains features common to DNA-binding transcription factors, including a potential Zn(II)2Cys6 DNA-binding domain. Uga35p of S. cerevisiae shows some similarity in both structure and function to TamA, and remarkably the Zn(II)2Cys6-like domains of both proteins are not required for function. To define important regions of TamA, sequence changes in a number of tamA mutants were determined and constructs containing deletions of various regions were tested for function. While the most N-terminal and C-terminal regions of TamA were dispensable for function, changes affecting even small parts of other regions of the protein abolished function. This suggests that the overall protein conformation is critical. Constructs encoding the TamA protein fused to DNA-binding domains were shown to activate gene expression in A. nidulans by recruitment of AreA. The Aspergillus oryzae AreA and Neurospora crassa NIT2 proteins were able to substitute for A. nidulans AreA in this interaction. Although the GATA zinc finger did not seem to be involved, the 12 amino acids at the AreA C-terminus were essential for interaction with TamA. This region is also involved in the interaction with NmrA, suggesting that competition for binding to the AreA C-terminus may be a part of the function of TamA. Uga35p was not able to interact with AreA and also could not complement a tamA? mutation, demonstrating differences in the coevolution of nitrogen regulatory mechanisms between different species.