Genetics - Theses

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Type: PhD thesis × Subject: Aspergillus nidulans -- Genetics × Author: Szewczyk, Edyta. ×

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    Cloning and analysis of the acuG, idpA and maeA genes involved in carbon metabolism in felamentous fungus Aspergillus nidulans
    Szewczyk, Edyta. (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.