Genetics - Theses
Permanent URI for this collection
Search Results
1 - 4 of 4
-
ItemNitrogen utilization and its regulation in Aspergillus nidulans(University of Melbourne, 2006)The filamentous fungus Aspergillus nidulans is able to utilize a wide range of compounds as sources of nitrogen. The presence of preferred nitrogen sources (ammonium and glutamine) signals nitrogen sufficiency, and genes required for the utilization of alternative sources are not expressed. In the absence of preferred nitrogen sources regulatory proteins activate expression of these genes. This constitutes nitrogen metabolite repression, and ensures the most efficient use of available nitrogen. In A.nidulans nitrogen metabolite repression is mediated by AreA, a positively acting GATA transcription factor. This thesis describes the investigation of two genes whose expression is subject to nitrogen metabolite repression and controlled by AreA, and the characterization of a fourth AMT/MEP gene in A. nidulans. areA102 is a specific mutation of the areA gene which results in a protein with altered promoter binding specificity, and areA102 mutants grow more strongly on a range of amino acids as nitrogen sources. Mutations at the sarA locus were first isolated as suppressors of the strong growth of an areA102 strain on histidine. In this study the sarA gene was characterized and confirmed to encode an L-amino acid oxidase (LAO) with broad substrate specificity. A sarA gene inactivation abolished LAO activity and suppressed the areA102 phenotype on histidine. An areA102 mutant was found to have increased utilization and stronger growth on amino acids which are LAO substrates. Investigation of sarA dependent and independent amino acid catabolism further defined the substrate specificity of LAO, and the contribution of other catabolic pathways was assessed. In A.nidulans the LAO was found to be the sole pathway for the catabolism of some amino acids, while for others this enzyme represents only a minor pathway. A.nidulans is known to possess four ammonium permeases differentially regulated by AreA. meaA encodes a low affinity ammonium transporter responsible for the majority of ammonium uptake. mepA has been shown to encode a high affinity permease which scavenges low concentrations of ammonium during nitrogen limitation, and mepB encodes a second high affinity permease only expressed during nitrogen starvation. To confirm the role of the fourth permease in ammonium acquisition, the mepC gene was cloned and its DNA and protein sequences analysed. The MepC ammonium transporter motifs differed somewhat from the consensus, and topology predictions indicated that mepC was more structurally divergent than the other A.nidulans ammonium transporters. The mepC gene was inactivated and the deletion strain was found to be indistinguishable from wildtype and from meaA, mepA, and mepB single, double and triple deletion backgrounds. However, strains over-expressing mepC from the highly inducible xylP promoter were able to partially complement the poor growth of an meaA?; mepA?; mepB? strain, indicating that MepC is capable of transporting ammonium. In other fungal systems certain ammonium permeases act as sensors of cellular nitrogen status. MeaA, MepA, and MepB do not act as nitrogen sensors. To determine whether MepC played a role in nitrogen sensing, amdS
-
ItemNitrogen utilization and its regulation in Aspergillus nidulans(University of Melbourne, 2006)The filamentous fungus Aspergillus nidulans is able to utilize a wide range of compounds as sources of nitrogen. The presence of preferred nitrogen sources (ammonium and glutamine) signals nitrogen sufficiency, and genes required for the utilization of alternative sources are not expressed. In the absence of preferred nitrogen sources regulatory proteins activate expression of these genes. This constitutes nitrogen metabolite repression, and ensures the most efficient use of available nitrogen. In A.nidulans nitrogen metabolite repression is mediated by AreA, a positively acting GATA transcription factor. This thesis describes the investigation of two genes whose expression is subject to nitrogen metabolite repression and controlled by AreA, and the characterization of a fourth AMT/MEP gene in A. nidulans. areA102 is a specific mutation of the areA gene which results in a protein with altered promoter binding specificity, and areA102 mutants grow more strongly on a range of amino acids as nitrogen sources. Mutations at the sarA locus were first isolated as suppressors of the strong growth of an areA102 strain on histidine. In this study the sarA gene was characterized and confirmed to encode an L-amino acid oxidase (LAO) with broad substrate specificity. A sarA gene inactivation abolished LAO activity and suppressed the areA102 phenotype on histidine. An areA102 mutant was found to have increased utilization and stronger growth on amino acids which are LAO substrates. Investigation of sarA dependent and independent amino acid catabolism further defined the substrate specificity of LAO, and the contribution of other catabolic pathways was assessed. In A.nidulans the LAO was found to be the sole pathway for the catabolism of some amino acids, while for others this enzyme represents only a minor pathway. A.nidulans is known to possess four ammonium permeases differentially regulated by AreA. meaA encodes a low affinity ammonium transporter responsible for the majority of ammonium uptake. mepA has been shown to encode a high affinity permease which scavenges low concentrations of ammonium during nitrogen limitation, and mepB encodes a second high affinity permease only expressed during nitrogen starvation. To confirm the role of the fourth permease in ammonium acquisition, the mepC gene was cloned and its DNA and protein sequences analysed. The MepC ammonium transporter motifs differed somewhat from the consensus, and topology predictions indicated that mepC was more structurally divergent than the other A.nidulans ammonium transporters. The mepC gene was inactivated and the deletion strain was found to be indistinguishable from wildtype and from meaA, mepA, and mepB single, double and triple deletion backgrounds. However, strains over-expressing mepC from the highly inducible xylP promoter were able to partially complement the poor growth of an meaA?; mepA?; mepB? strain, indicating that MepC is capable of transporting ammonium. In other fungal systems certain ammonium permeases act as sensors of cellular nitrogen status. MeaA, MepA, and MepB do not act as nitrogen sensors. To determine whether MepC played a role in nitrogen sensing, amdS
-
ItemInvestigating the structure and function of lymphocyte perforin(University of Melbourne, 2003)Cytotoxic T Lymphocytes (CTL) and Natural Killer (NK) cells are involved in the elimination of virus-infected and malignant cells. Perforin, a key cytotoxin secreted by cytotoxic lymphocytes acts synergistically with the co-secreted serine proteases (granzymes) to kill the target cell. The devastating effects of perforin deficiency are mirrored in perforin-deficient mice and children diagnosed with familial haemophagocytic lymphohistiocytosis (FHL), a lethal immune deficiency requiring bone marrow transplantation as the only successful therapy. Perforin�s pivotal role in killer cell function makes it an attractive target for therapeutic intervention. Currently however, a large gap exists in our understanding of how perform operates at the molecular level, principally due to a lack of expression systems capable of synthesising this cytotoxic protein. This thesis describes a novel retroviral expression system that was successfully used to express wild type perforin, allowing the first ever mutagenic analysis of the molecule. Using this technology, perforin was expressed in Rat Basophilic Leukemia (RBL) cells, which can synthesis and store the protein in secretory granules. Degranulation and perforin release were achieved through the use of an anti-trinitrophenyl (TNP) IgE antibody to crosslink the Fee receptor on RBL cells with TNP-labelled EL-4 target cells. This resulted in death of the EL-4 cells, however RBL cells transduced with empty viral vector did not induce cell death. Using the same methodology, two mutations identified in FHL (P5 mutation: G429E and P6 mutation: P345L) were expressed in the RBL cells and shown to be associated with complete loss of cytotoxic function. Both mutated perforin molecules were correctly targeted to the secretory granules, and released upon Fee receptor crosslinking. This suggested that in each case, the defect in perforin function mapped downstream of release from cytotoxic lymphocytes. Retroviral transduction was also used to investigate the role of putative calcium-binding aspartate residues located in perforin�s carboxy terminus C2-like domain. The negatively charged aspartate side chains have been predicted to cluster and bind calcium ions, which is an obligate requirement for perform function. Single and joint mutation of two of the five aspartate residues conserved in rat, mouse and human perforins caused a complete loss of perforin-mediated cytotoxicity, suggesting that aspartate residues 484 and 486 are both indispensable for perforin function.
-
ItemDissection of the genetic and physiological basis of desiccation resistance in Drosophila melanogaster using laboratory selection( 2011)Desiccation stress is a major factor underlying the distribution, abundance and evolution of insect species. This study utilises laboratory selection to dissect the physiological and genetic basis of desiccation resistance in a population of D. melanogaster recently collected from the field. The first part of this thesis assays selected and control flies for a series of physiological traits. Selected flies were found to lose water at a slower rate than their controls when exposed to desiccation stress, in keeping with previous investigations. Another major physiological mechanism underlying the selection response was increased water storage, possibly mediated via enlarged glycogen reserves. This study is the first to demonstrate that these traits can evolve in response to desiccation selection in fly lines recently derived from nature. Dehydration tolerance and whole-body lipid content however did not contribute towards the selection response, a finding consistent with most prior studies. The second part of this study investigates whether desiccation selection produced correlated responses in other environmental stress traits. In keeping with previous experiments, a strong correlated response was observed between desiccation and starvation resistance, suggesting that the two traits are shaped by at least partially overlapping physiological mechanisms. In contrast this investigation found no correlated response between desiccation resistance and cold tolerance, as measured by both cold mortality and chill coma recovery. The data surrounding heat tolerance were more complex; while higher heat knockdown resistance failed to evolve in response to desiccation selection, increased heat mortality and, to a lesser extent, Critical Thermal Maximum (CTMAX) did. This investigation also aimed to assess whether flies selected for increased desiccation resistance in the laboratory showed an increase in an aspect of field fitness under dry conditions. Female selected flies were better able than their controls to reach food resources following release into a natural environment but this was not the case for males. This result indicates that though laboratory selection for desiccation resistance can result in ecologically relevant evolutionary change, levels of field fitness are not always accurately predicted by laboratory-based assays. Finally, tiling microarrays were used to detect sequence variation between selected and control populations. Six hundred and seventy one Single Feature Polymorphisms (SFPs), located across all chromosomes, were found between the two treatments, none of which correspond to known desiccation resistance candidates. Sequence divergence was validated via sequencing for eight genes: CG7084, beat-VII, CG7638, omega, Dystrophin, SNF4Agamma, mol and CG14304. These genes may become solid desiccation resistance candidate genes if they are found to be associated with the trait in independent, natural populations.