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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ItemMolecular characterisation of the structure and regulation of the gatA, lamA and lamB genes of Aspergillus nidulans(University of Melbourne, 1991)The amdR gene of Aspergillus nidulans controls the expression of structural genes needed for utilisation of omega amino acids and lactams. To understand this regulation the regulatory protein and the genes it controls need to be analysed. This thesis presents work on the structure and regulation of three genes of the amdR regulon, the gatA, lamA and lamB genes. The full sequence and structure of the gatA and lamB genes and the 5' sequence and structure of the lamA gene has been determined. Structural features of these are discussed in relation to other filamentous fungal genes. The gatA gene encodes a gamma-amino butyric acid transaminase and shows homology to the equivalent genes from Saccharomyces cerevisiae and Escherichia coli. The lamA promoter has been found to contain two unusual features: a potential Initiator element at the startpoint of transcription and a downstream TATA element. All three of these genes are under the control of the positive regulatory gene, amdR. Binding sites for the amdR protein in the 5' of gatA and between the divergently transcribed lamA and lamB genes have been identified by comparison with the known amdR protein binding region of the coregulated amdS gene. Analysis of the function of these sequences indicates that there is one major binding site in the gatA promoter and between the divergently transcribed lamA and lamB genes. Comparison of these functional sequences and the previously identified site in amdS with the non-functional sites indicates certain bases within the binding sequence which may be important for efficient binding. Three CCAAT factor binding sequences have been found in the gatA and lam promoters. Two of these sequences are closely associated with functional amdR protein binding sites as has also been shown by others to be the case for amdS.
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ItemNo Preview AvailableUse of gene transfer to study amdS regulation in Aspergillus nidulans(University of Melbourne, 1989)Expression of the amdS gene of Aspergillus nidulans is regulated by a number of different regulatory genes and coeffectors. In vivo generated cis mutants permitted initial identification of regions 5' to the amdS gene involved in regulation by some of these regulatory genes. In this study, an amdS-lacZ fusion gene was used to follow the regulatory consequences of in vitro generated mutants of the amdS controlling region. Numerous deletion, inversion, insertion and oligonucleotide based mutants were constructed and introduced into A. nidulans using a gene transfer (transformation) technique. Three approaches for the production of transformants suitable for regulatory analysis were assessed; cotransformation, single copy integrations at the argB locus, and gene replacements. A single region of the amdS controlling region was found to be responsible for amdR mediated regulation of amdS, The sequence of the 5' regions of three coregulated genes, gatA, lamA and lamB, revealed that these genes shared this sequence in common. A mutant amdR allele, amdR104c, regulated amdS expression from the same location as the wildtype product. Three regions 5' to amdS were found to be involved in facB mediated regulation of amdS; their action were seen to be synergistic under some circumstances. No homology was found between them, or with the 5' regions of other genes under facB control. A mutant facB allele, facB88, resulted in altered regulation of amdS. Insertion mutants indicated that the wild type products of the amdR and facB genes could regulate amdS when their site(s) of action were moved 5' by several hundred base pairs, and that the products of the mutant alleles showed different responses. In addition, a region 5' to amdS with homology to eukaryotic CCAAT boxes was shown to be required for establishing basal amdS expression. Titration analysis, an in vivo DNA-regulatory product binding assay, was used to show that the same sequences required for amdR mediated expression titrated the amdR product. Individual sites of action of the facB gene product were not seen to titrate the facB gene product, however.
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ItemThe molecular characterization of genes involved in acetate metabolism in Aspergillus nidulans(University of Melbourne, 1988)Two acetate-induced genes contained within lambda clones were identified by transformation into Aspergillus nidulans to complement existing mutations. In this way one clone was found to contain the acuE gene, encoding malate synthase, and the other clone contained the facA gene, encoding acetyl CoA synthase. These enzymes are involved in acetate metabolism in A.nidulans and have been shown to be coregulated, together with the acuD gene, isocitrate lyase, and amdS gene, acetamidase, by the facB gene product. The facA+ and acuE+ transformants were studied by Southern analysis, which helped to establish the extent of these genes within the lambda clones and provided a basis for further characterization of these genes. The identity of the facA gene was confirmed by Southern analysis of a facA translocation strain, FAD1. The transcripts of the facA and acuE genes were identified by Northern analysis, and found to be induced by acetate. Transcriptional mapping of both genes established the 5' startpoints of these mRNAs and localized two introns in this region of the facA transcript. The two genes were sequenced and their structures were compared with the gene structure of other sequenced fungal genes. The facA and acuE genes both contain introns, which conform to the expected size of fungal introns and contain recognisable splice site and signal sequences. Both genes also contain promoter and translation initiation termination sequences that conform to the consensus sequences established for other fungal genes. Preliminary Northern analysis of the facA and acuE genes established that the facB gene product is necessary for the induction of transcription by acetate and the 5' regions of these genes were examined for sequences that may be involved in binding the facB gene product. A comparison of the 5' regions of the facA and acuE genes, together with the acuD and amdS genes, failed to reveal sequences of strong homology. However, one sequence repeated in the 5' regions of the facA, acuE and acuD genes did show some homology to the amdl9 region of amdS, which has been shown to be necessary for facB-mediated induction of amdS. Finally, the facA gene sequence was compared to the acu5 gene sequence, which encodes acetyl CoA synthase in Neurospora crassa, and the acuE gene was compared to the aceB gene of Escherichia coli, encoding malate synthase. The facA and acu5 genes were found to be very similar, . although a number of structural differences were apparent at the 5' and N-terminal ends of these genes. These comparisons are discussed in relation to the molecular evolution of these genes.