Genetics - Theses
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ItemCharacterisation of putative fatty acyl-CoA synthetases and genes involved in �-oxidation in Aspergillus nidulans(University of Melbourne, 2009)Aspergillus nidulans is able to use short and long chain fatty acids as sole carbon and energy sources via the 13-oxidation pathway. This pathway occurs in both peroxisomes and mitochondria of A. nidulans. While various genes encoding the mitochondrial 13- oxidation enzymes are known, the genes encoding the peroxisomal ?-oxidation proteins have not been fully described. To investigate the first step of peroxisomal 13-oxidation two putative fatty acyl-CoA dehydrogenases, AcdA and AcdB, and two putative fatty acyl-CoA oxidases, AoxA and AoxB, were identified in the genome by blast search using the Saccharomyces cerevisiae fatty acyl-CoA oxidase FoxA and a Neurospora crassa fatty acyl-CoA dehydrogenase. The localisation of these proteins, the induction of the genes and the gene deletion phenotypes have been characterised to assess their possible involvement in ?-oxidation. The results have shown that AoxA is the major fatty acyl-CoA oxidase for peroxisomal long chain fatty acid utilisation, however, the leaky loss-of-growth phenotype of the aoxA? strain implies that there are additional peroxisomal ?-oxidation pathways. The induction of the four putative fatty acyl-CoA dehydrogenases and fatty acyl-CoA oxidases in response to fatty acids was shown to be dependent on the transcriptional regulators FarA required for the induction by short and long chain fatty acids and FarB and ScfA required for short chain fatty acid induction. This pattern of induction was observed for aoxA, while acdA, acdB and aoxB show a previously undescribed induction pattern with FarB and ScfA being required for short chain and long chain fatty acid induction and FarA for short chain fatty acid induction only. The occurrence of two induction patterns implies a more complex regulation by the three regulatory proteins. Six putative fatty acyl-CoA synthetases, FatA, FatB, FatC, FatD, FaaA and FaaB, have been identified using the S. cerevisiae fatty acyl-CoA transporters and synthetases Fat1, Fat2, Faa1, Faa2, Faa3 and Faa4. Investigation of the localisation of these proteins and phenotypes associated with the deletion of these genes showed that FaaB is likely to be the major peroxisomal fatty acyl-CoA synthetase and activates fatty acids with a wide range of chain lengths. The effect of deletion of the genes encoding these putative fatty acyl-CoA synthetases on the regulation of aoxA and acuJ was investigated to determine whether any of the putative fatty acyl-CoA synthetases were required to generate activated fatty acids for induction. It is clear that there are multiple genes contributing to fatty acid utilisation and that there is considerable redundancy. Additional genome and phylogenetics analyses have identified a variety of additional proteins, which may be involved in peroxisomal ?-oxidation pathways, as well as a potential mitochondrial fatty acyl-CoA synthetase (AN4659.3). This protein is predicted to localise to mitochondria, contains a short chain fatty acyl-CoA synthetase motif and is likely to be a synthetase activating short chain fatty acids, which are utilised in mitochondria.
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ItemRegulation of gluconeogenesis in aspergillus nidulans(University of Melbourne, 2009)
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ItemThe isolation and analysis of the hap genes of Aspergillus nidulans(University of Melbourne, 1996)The 5' regulatory region of the amdS gene of Aspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence required for setting the basal level of transcription. Mobility shift assays have identified a factor in A. nidulans nuclear extracts that binds specifically to this CCAAT sequence. In Saccharomyces cerevisiae, the HAP3 and HAP5 genes encode components of a highly conserved multi subunit complex which is able to bind CCAAT sequences. The identification, cloning and sequencing of genes from A. nidulans with homology to HAP3 and HAP5, known as hapC and hapE respectively, is described here. The predicted amino acid sequences of the proteins encoded by the hapC and hapE genes share extensive sequence identity to conserved regions in HAP3 and HAP5 respectively. Furthermore, they both show identity to the histone-fold motif, a motif used widely as a means for protein-protein and DNA- protein interactions. A haploid carrying a hapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially poorly on acetamide as a sole carbon and nitrogen source, indicating that hapC plays a role in amdS expression. In agreement with this notion, the hapC deletion results in reduced levels of amdS expression, particularly under conditions of carbon limitation. Nuclear extracts prepared from the hapC deletion mutant show no CCAAT specific binding to the amdS or gatA promoter, indicating that hapC encodes a component of the complex binding at this sequence. In the presence of the hapC deletion growth on acetamide and amdS
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ItemCharacterisation of the facB88 translocation of Aspergillus nidulans(University of Melbourne, 1996)The positively acting regulatory gene facB of Aspergillus nidulans mediates acetate induction of the amdS (acetamidase) gene and genes required for acetate metabolism (facA, acuD and acuE). facB encodes a gene product with a Zn(II)2Cys6 DNA binding cluster, heptad repeats and potential activation domains, and binds to sequences 5' of amdS, facA, acuD and acuE. facB orthologues from A. oryzae and A. niger have been compared to the A. nidulans facB gene at the level of DNA and predicted protein sequences. Highly conserved regions in the predicted translation products have been identified and discussed in terms of their relevance to protein function. Putative transcription factor binding sites were identified in the 5' non-coding regions of each orthologue. The facB88 reciprocal translocation results in high-level constitutive amdS expression (superactivation) and this is mediated by a chimeric gene formed by the translocation event. This chimeric gene was found to encode the N-terminal half of FacB, including the DNA binding domain, fused to a new gene encoding two C2H2 zinc finger DNA binding motifs. The new gene was designated amdX, the cloning, sequencing and transcriptional analysis of which is reported here. Inactivation of amdX and creation of multicopy strains by transformation revealed that amdX is a minor positive regulator of amdS expression. The DNA binding function of AmdX was examined, using an Escherichia coli-expressed AmdX fusion protein, by gel mobility shift assays and DNase I footprinting. AmdX was shown to bind to two sites in the amdS 5' region which overlap the binding sites for the AmdA and CreA regulatory proteins. The facB88 chimeric gene mediating amdS superactivation was designated facB-amdX. Molecular dissection of facB-amdX showed that both the FacB and AmdX DNA binding domains are required for amdS superactivation and that they contribute to amdS expression in a synergistic manner. Cooperative DNA binding of the FacB and AmdX DNA binding domains to the amdS 5' region is proposed to mediate the superactivation ability of the facB-amdX gene product.
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ItemMolecular studies of gene regulation in Aspergillus nidulans( 1985)The amdA regulatory gene of Aspergillus nidulans controls the expression of both the acetamidase (amdS) gene and a gene, designated aciA, which encodes a 42 000 dalton polypeptide. The cloning and analysis of the aciA gene is described in this thesis. The aciA gene is a single copy gene approximately 2kb in size and encodes two messenger RNA species of 1.45 and 1.55kb•in size. Both of these aciA transcripts are regulated in an identical fashion. The aciA gene is demonstrated to be induced in the presence of the carbon source acetate and evidence is presented which indicates that it may also be subject to carbon catabolite repression. None of the other regulatory circuits which are known to control amdS expression affect the expression of the aciA gene. The 5' non-coding region of the aciA gene was sequenced and both the primary and secondary structures within this region are compared with the corresponding regions of the amdS gene and a cis-acting mutant of the amdS gene which increases the amdA-mediated regulation of this gene. The aciA gene possesses a canonical TATA box and also contains a number of other sequences which are similar to sequences found in the 5' non-coding region of the amdS gene. The most interesting of these is a 23bp purine-rich region which is similar to a purine-rich region occurring in approximately the same position in the amdS gene. This sequence is found to be duplicated in the amdS mutant which is subject to increased regulation by the amdA gene. Experiments in which A. nidulans was transformed with multicopy plasmids containing DNA fragments from the aciA gene have indicated that the aciA sequence responsible for the titration of the amdA gene product is located in the 5' non-coding region of this gene. A model for the regulation of the aciA gene by the amdA gene, acetate and carbon catabolite repression is presented. The possible significance of small repeated sequences within the purine-rich regions of aciA and amdS is also discussed.