Genetics - Theses
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ItemCharacterization of the tamA gene of Aspergillus nidulans(University of Melbourne, 2000)In Aspergillus nidulans, the GATA zinc finger protein AreA activates the expression of enzymes that metabolize less favoured nitrogen sources in the absence of preferred nitrogen sources, such as ammonium or glutamine. The amount and activity of AreA are modulated by a number of mechanisms, including an interaction with the NmrA protein through the GATA zinc finger and C-terminal regions of AreA to inhibit DNA binding under nitrogen-sufficient conditions. The TamA protein has also been implicated in nitrogen regulation, with mutants described as having reduced levels of a number of nitrogen metabolic enzymes. This thesis describes the characterization of the tamA gene and investigates its role in nitrogen regulation. tamA encodes a 739 amino acid protein that contains features common to DNA-binding transcription factors, including a potential Zn(II)2Cys6 DNA-binding domain. Uga35p of S. cerevisiae shows some similarity in both structure and function to TamA, and remarkably the Zn(II)2Cys6-like domains of both proteins are not required for function. To define important regions of TamA, sequence changes in a number of tamA mutants were determined and constructs containing deletions of various regions were tested for function. While the most N-terminal and C-terminal regions of TamA were dispensable for function, changes affecting even small parts of other regions of the protein abolished function. This suggests that the overall protein conformation is critical. Constructs encoding the TamA protein fused to DNA-binding domains were shown to activate gene expression in A. nidulans by recruitment of AreA. The Aspergillus oryzae AreA and Neurospora crassa NIT2 proteins were able to substitute for A. nidulans AreA in this interaction. Although the GATA zinc finger did not seem to be involved, the 12 amino acids at the AreA C-terminus were essential for interaction with TamA. This region is also involved in the interaction with NmrA, suggesting that competition for binding to the AreA C-terminus may be a part of the function of TamA. Uga35p was not able to interact with AreA and also could not complement a tamA? mutation, demonstrating differences in the coevolution of nitrogen regulatory mechanisms between different species.
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ItemNo Preview AvailableComparison of amidase genes in Aspergillus species(University of Melbourne, 1998)A study was undertaken to identify evolutionally conserved sequences within the acetamidase gene regulatory region across Aspergillus species which may define binding sites for transcriptional control regulatory proteins. Six amdS genes were cloned and characterised from five Aspergillus species and compared to A. nidulans and A. oryzae amdS genes. The amdS gene from A. echinulatus was predicted to be non functional due to a termination codon after amino acid 23. A proposed amdS pseudogene was identified in A. ustus. In this species two genes with amdS homology were identified, one with acetamidase activity and one lacking detectable amidase activity. A comparison of the Aspergillus spp. amdS coding region sequences revealed a very high level of conservation. The hydrolysis of amides by the Aspergillus spp. amdS genes was studied by heterologous gene expression in A. nidulans. Acetamide, propionamide, valeramide and butyramide were found to be substrates for A. ustus and A. unguis AmdS. A comparison of the transcriptional regulatory controls governing expression of Aspergillus spp. amdS and A. nidulans amdS genes was performed by heterologous expression of amdS genes fused to a reporter gene. Only some of the transcriptional control pathways regulating A. nidulans amdS were shown to regulate the different Aspergillus spp. amdS genes. During the course of this study, an amidase gene family was discovered in Aspergillus. The identification, cloning, functional characterisation, regulation and evolution of two members of the amidase gene family, gmdB (encoding general amidase B) and gmdC (encoding general amidase C) are described. Four gmdB genes were studied from four Aspergillus species and one gmdC gene was studied in A. nidulans. Substrates for the GmdB enzymes are valeramide, hexanamide and tyrosine amide, however substrate preferences for amide hydrolysis by the different GmdB enzymes differ between Aspergillus species. Transcriptional regulation of gmdB, determined by heterologous gene expression of reporter constructs in A. nidulans is not conserved between the species. The amidase family of genes comprising amdS, gmdB and gmdC is highly conserved in sequence and gene structure and suggests evolution from a common ancestor.
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ItemNo Preview AvailableComparison of amidase genes in Aspergillus species(University of Melbourne, 1998)A study was undertaken to identify evolutionally conserved sequences within the acetamidase gene regulatory region across Aspergillus species which may define binding sites for transcriptional control regulatory proteins. Six amdS genes were cloned and characterised from five Aspergillus species and compared to A. nidulans and A. oryzae amdS genes. The amdS gene from A. echinulatus was predicted to be non functional due to a termination codon after amino acid 23. A proposed amdS pseudogene was identified in A. ustus. In this species two genes with amdS homology were identified, one with acetamidase activity and one lacking detectable amidase activity. A comparison of the Aspergillus spp. amdS coding region sequences revealed a very high level of conservation. The hydrolysis of amides by the Aspergillus spp. amdS genes was studied by heterologous gene expression in A. nidulans. Acetamide, propionamide, valeramide and butyramide were found to be substrates for A. ustus and A. unguis AmdS. A comparison of the transcriptional regulatory controls governing expression of Aspergillus spp. amdS and A. nidulans amdS genes was performed by heterologous expression of amdS genes fused to a reporter gene. Only some of the transcriptional control pathways regulating A. nidulans amdS were shown to regulate the different Aspergillus spp. amdS genes. During the course of this study, an amidase gene family was discovered in Aspergillus. The identification, cloning, functional characterisation, regulation and evolution of two members of the amidase gene family, gmdB (encoding general amidase B) and gmdC (encoding general amidase C) are described. Four gmdB genes were studied from four Aspergillus species and one gmdC gene was studied in A. nidulans. Substrates for the GmdB enzymes are valeramide, hexanamide and tyrosine amide, however substrate preferences for amide hydrolysis by the different GmdB enzymes differ between Aspergillus species. Transcriptional regulation of gmdB, determined by heterologous gene expression of reporter constructs in A. nidulans is not conserved between the species. The amidase family of genes comprising amdS, gmdB and gmdC is highly conserved in sequence and gene structure and suggests evolution from a common ancestor.
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ItemAnalysis of the facB gene of Aspergillus nidulans(University of Melbourne, 1995)The facB gene of Aspergillus nidulans encodes a transcriptional activator which contains a Zn(II)2Cys6 DNA binding cluster, a putative leucine zipper-like dimerization motif and potential acidic activation domains. facB mediates acetate induction of amdS (acetamidase) and the genes of acetate utilisation (facA, acuD, and acuE). This thesis describes an analysis of the facB gene product using a mutational approach and in vitro DNA binding studies. A facB null mutant was constructed by gene replacement This mutant is unable to grow when acetate is the sole carbon source, indicating that facB is required for utilisation of acetate. Important functional regions of FacB were identified by the localisation of lesions in facB mutant alleles. Regions of FacB likely to be involved in DNA binding, dimerization, transactivation, and a possible structural function in acetate metabolism were identified. The Zn(II)2Cys6 motif was shown to be necessary for facB function by in vitro mutagenesis of specific cysteine residues. A facB allele harbouring this Zn(H)2Cys6 mutation failed to complement for growth on acetate upon transformation into a facB null mutant Transformation of this facB allele into a wildtype strain resulted in decreased growth on acetate and acetamide media. The DNA binding function of FacB was examined in detail. A FacB fusion protein was expressed in Escherichia coli and was shown to bind DNA fragments from facB-regulated promoters in vitro in a sequence-specific manner. Abolition of in vitro binding by an expressed FacB fusion protein containing mutated cysteines in the Zn(II)2Cys6 cluster confirmed that this motif was required for DNA binding activity. Expressed FacB proteins with mutations flanking the Zn(II)2Cys6 motif showed altered DNA binding specificity, implicating these regions in determination of DNA binding site specificity. FacB binding sites were defined by Gel Mobility Shift Assay (GMSA), DNase I and Missing Contact Interference Footprinting analyses. FacB binding sites fall into two dissimilar classes. Mutant binding sites from the amdS promoter, known to modulate expression, were shown to alter FacB binding in vitro.