Genetics - Theses

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Start date: 1980 × End date: 1989 × Subject: Genetic regulation ×

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    Use of gene transfer to study amdS regulation in Aspergillus nidulans
    Littlejohn, Timothy Graham. (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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    The molecular characterization of genes involved in acetate metabolism in Aspergillus nidulans
    Sandeman, Ruth Ann. (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.