Genetics - Theses

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End date: 1989 × Subject: Aspergillus -- Genetics. ×

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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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    Genetic and molecular analysis of a positive regulatory gene in Aspergillus nidulans
    Andrianopoulos, Alex. (University of Melbourne, 1989)
    In the ascomycete fungus Aspergillus nidulans, utilization of certain amides, omega-amino acids and cyclic amides (lactams) as carbon and/or nitrogen sources requires the structural genes of amdS, gatA, gabA, lamA and lamB, whose expression is coordinately controlled by the positively-acting regulatory gene amdR. Transcriptional activation by amdR is dependent on omega-amino acid inducers (ligands) such as GABA and ?-alanine. To understand the mechanisms by which amdR exerts its regulatory control over structural gene expression, a program was initiated to clone and characterize the amdR gene. Using DNA- mediated transformation of A.nidulans, the amdR regulatory gene was cloned from a genomic cosmid library. Transcript analysis showed that the 2.7kb amdR mRNA is constitutively transcribed at a very low level under all tested conditions. Sequence and transcriptional analysis of amdR showed that it contains three small introns, heterogeneous 5' and 3' transcription sites and multiple codons prior to the major AUG initiator. In addition, the semidominant amdR6c allele was cloned and its lesion identified. The predicted amdR protein sequence has a cysteine-rich "zinc-finger" DNA binding motif at the amino-terminal end, four putative acidic transcription activation motifs in the carboxyl- terminal half of the product and two sequences homologous to the SV40 large T antigen nuclear localization motif. A series of 5', 3� and internal deletions of amdR were examined in vivo for transcription activator function, showing that the amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator regions may function independently, but both are required for wildtype levels of transcription activation. A number of the amdR deletion products were also shown to compete with the wildtype amdR product in vivo. Dosage phenomena studies using amdR yielded transformants which exhibited stronger growth than the wildtype, indicating increased expression of the relevant structural genes. This suggests that the low constitutive level of amdR product sets the upper limits of basal and induced transcription of the structural genes. Further increases in amdR product concentrations in vivo, through overexpression of the amdR gene, yielded transformants with phenotypic abnormalities. From the molecular and genetic studies presented, a model for amdR-mediated regulation of structural gene expression was formulated.