Genetics - Theses

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Type: PhD thesis × Subject: Genetic regulation × Author: Littlejohn, Timothy Graham. ×

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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.