Genetics - Theses
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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.
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ItemCloning and characterisation of the regulatory gene, amdA, of Aspergillus nidulans(University of Melbourne, 1993)Semi-dominant mutations in the amdA gene lead to elevated expression of the gene encoding acetamidase, amdS. These mutations also cause constitutive expression of the acetate-inducible gene, aciA. In the amdS 5' regulatory region, two cis-acting mutations, amdI66 and amdI666, have been isolated which specifically affect amdA activation of amdS. These mutations affect an 18 bp GA-rich sequence, thought to define the amdA site of action within the amdS promoter region. Similar GA-rich sequences have also been found in the 5' region of aciA. The work presented in this thesis describes the cloning and initial functional characterisation of the amdA gene and three of its mutant alleles. The wild-type amdA gene has been cloned by undertaking a chromosome walk from genes gatA and alcC on linkage group VII and localised by complementation of an amdA loss-of-function mutation. Transcriptional analysis reveals that the gene is expressed at low levels and constitutively under growth conditions which affect expression of amdS and aciA. The gene is predicted to encode an 880 amino acid protein which contains two C2H2 zinc fingers, similar to those found in Xenopus laevis transcription factor TFIIIA, a potential nuclear localisation sequence and two potential transcriptional activation domains. Functional analysis of in vitro-generated deletion mutations and of the semi-dominant mutant allele, amdA7, indicate that the acidic activation domain identified in the protein sequence is functionally significant. Interestingly, the C2H2 zinc finger motifs identified in the protein are similar to those found in the carbon catabolite repressor protein, CreA, which also regulates amdS and recognises sequences which coincide with the proposed site of action for AmdA within the promoter of this structural gene.
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ItemStructural and functional analysis of the amdR positively-acting regulatory gene in Aspergillus oryzae(University of Melbourne, 1992)The mechanism of the amdR regulation has been well characterized in Aspergillus nidulans. The corresponding A.oryzae gene was cloned and characterized in this study, which provides an insight into the conservation of the regulatory proteins during evolution. The A.oryzae homologue was isolated by cross hybridization with the A.nidulans amdR gene, and shown to complement the A.nidulans amdR44- mutant. Induction studies indicated that the A.oryzae gene product binds similar sequences and responds to inducer in a similar manner to the A.nidulans protein. Inactivation of the A.oryzae amdR gene further confirmed its regulatory role in GABA utilization. Transformation of the A.nidulans amdR gene restored the amdR activity in the gene inactivation mutant of A.oryzae, indicating again that these two amdR genes are functionally interchangeable. Sequence comparison of the two amdR genes showed that the overall homology of DNA sequence throughout the coding region was 64%; and that the conservation of the two polypeptides was 85%, with 76% identity. The conserved amino acid sequences are concentrated in the putative functional domains, such as DNA binding, nuclear localization, transcription activation, and inducer interaction. Two stretches of amino acid sequences which are outside the known functional domains are significantly divergent between two species, suggesting that these represent regions of little functional importance. The A.nidulans amdR gene contains three introns, and the first and third introns have been found at identical positions within the A.oryzae gene. Intron II region is probably a coding region in A.oryzae due to its lack of consensus 5' splice site and a high degree of homology with the corresponding A.nidulans sequence. The transcriptional initiation, termination and splice signals are similar between these two amdR genes, although the overall DNA sequences in these regions have diverged. The extent of sequence divergence between these two amdR genes indicates that the species have evolved separately for a period of time. However, the sequence divergence has been restrained to those changes which have little effect on the functional activity of the amdR gene products.