Genetics - Theses

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Studies on minor non-metrical skeletal variants in the mouse and man

Kellock, Wendy Lorraine. (University of Melbourne, 1970)

This thesis consists of papers presenting the results of studies on the genetical, developmental and anthropological aspects of minor non-metric al variants in man and the house mouse. The work is mainly on variants of the skeleton, particularly the cranium, but includes a limited discussion of published data on minor non-metrical variants of the muscular and vascular systems. Each study is based on a number of variants, and, where applicable, single measures have been obtained to express the overall difference in skeletal variability between populations or the overall effect on skeletal variability of certain environmental factors. Investigations into the role of genotype and environment in the determination of minor skeletal variants in mice and man indicate that most of them are under some genetic control but that maternal physiology and other non-genetic factors may influence the frequency of individual variants. Data presented here (Publication 1) on 25 minor skeletal variants in inbred strains of mice and their hybrids suggest that genotype is more important than environment in determining skeletal variability. Although the frequency of a few individual variants was found to be significantly affected by certain non-genetic factors, when many variants were considered together the environment had no overall significant effect. In contrast, large differences, due mainly to genetic factors, were observed between inbred strains and hybrids. Further studies on inbred strains of mice and hybrids (Publication 2) indicate that stabilizing mechanisms operate during the formation of the skeleton. For most of the 29 bilateral minor non-metrical variants studied , the frequency of asymmetrical mice (i.e., those with the variant present on only one side) was less than expected on the assumption that the number of mice with the variant present on both, one or neither sides depends solely on the frequency of the variant on each side. This tendency for the development of the skeleton to be canalized against asymmetry has been described as a form of morphogenetic homeostasis. The same phenomenon has been observed for bilateral minor non-metrical variants in man (Publication 3) for the skeletal, muscular and vascular systems (based on data published by Danforth in 1924) and for the skeletal system of Australian Aborigines. Studies on inbred strains of mice (e.g., Publication l) indicate that genotype plays the major role in determining the frequency of minor non-metrical variants. If these findings can be extrapolated to man, minor non-metrical variants may be of use in anthropological work. A general survey of skeletal variation, based on 30 such variants, was carried out on Aboriginal crania from many parts of Australia (Publication 4). Regional differences in the pattern of cranial morphology were observed which appear to culminate in two extreme populations: one in the north and north-west of the continent, the other in south-eastern Australia. These results were considered in relation to some current theories on the origin and ethnic composition of the Australian Aborigines.
Nitrogen utilization and its regulation in Aspergillus nidulans

Askin, Marion C. (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
Nitrogen utilization and its regulation in Aspergillus nidulans

Askin, Marion C. (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
Investigating the structure and function of lymphocyte perforin

Thia, Marie-Claude. (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.
Insecticide resistance in the Australian sheep blowfly, Lucilia cuprina : selection, chance and response

Scott, Megan. (University of Melbourne, 2000)

The influences of random genetic drift and selection by the insecticides dieldrin and diazinon on the initial establishment of the resistant allele (Rdl or Rop1) and subsequent allelic frequency changes within populations of L. cuprina were investigated. Discrete generation population cages were initiated with Rdl and Rop1 frequencies of 1 per cent or 5 per cent and maintained for 17 or 10 generations respectively on media with a concentration range that kills between 0 and 100 per cent of susceptible individuals. The resistant allele frequency was recorded at each generation. The probability of the initial establishment of the Rdl allele in a population was consistently greater at the 5 per cent frequency and dependent on the concentration of dieldrin in the medium for both starting frequencies. Once the resistant allele was established, responses to selection were concentration-dependent. In the absence of dieldrin the susceptible allele was selectively favoured, at concentration 0.00005 per cent (w/v) selection was approximately neutral and at concentrations above this the Rdl allele was at a selective advantage. Fixation of Rdl occurred at higher concentrations. The probability of the initial establishment of the Rop1 allele in a population was found to be independent of both founding allele frequency and the concentration of diazinon on which the population was maintained. Once the resistant allele was established, responses to selection were concentration-dependent. In the non-modifier populations, in the absence of diazinon, the susceptible allele was selectively favoured, at concentration 0.00002 per cent (w/v) selection was approximately neutral and at concentrations above this the Rop1 allele was at a selective advantage, with fixation occurring at higher concentrations. In populations where the modifier gene was present the resistant allele was maintained in all populations with selection generally favouring resistant phenotypes. In populations maintained on insecticide, that fixed for the susceptible allele neither resistance system provided evidence of a polygenic response. Computer simulations were conducted to estimate the relative genotypic fitness values that could give rise to the allele frequencies produced by the population cage experiments and then to extrapolate the results to field situations. Results support the hypothesis that in the absence of the chemical the susceptible allele is at an advantage and in the presence of the chemical the resistant allele is selected for in both the dieldrin and diazinon resistance systems. Population size and genetic drift size were found to have little effect on the development of insecticide resistance when founding allele frequencies were at 1 and 5 per cent. When founding allele frequencies representative of field situations were simulated population size and drift dramatically influenced the initial establishment, and thus the development of resistance, in both the dieldrin and diazinon resistance systems. The resistant allele failed to establish in the population on all experimental concentrations when a finite population size of 100 was analysed. Furthermore, the resistant allele failed to establish in an infinite population in the absence of the chemical and when selection coefficients were low. When selection coefficients were high, and population size was infinite, results obtained were basically the same as those achieved when the simulation was run using 1 and 5 per cent founding allele frequencies. The resistance system-specific impact of genetic drift and selection on the evolution of insecticide resistance is discussed in the context of the applicability of the results to resistance management strategies or for resistance systems as a general model of microevolution.
Insecticide resistance in the Australian sheep blowfly, Lucilia cuprina : selection, chance and response

Scott, Megan. (University of Melbourne, 2000)

The influences of random genetic drift and selection by the insecticides dieldrin and diazinon on the initial establishment of the resistant allele (Rdl or Rop1) and subsequent allelic frequency changes within populations of L. cuprina were investigated. Discrete generation population cages were initiated with Rdl and Rop1 frequencies of 1 per cent or 5 per cent and maintained for 17 or 10 generations respectively on media with a concentration range that kills between 0 and 100 per cent of susceptible individuals. The resistant allele frequency was recorded at each generation. The probability of the initial establishment of the Rdl allele in a population was consistently greater at the 5 per cent frequency and dependent on the concentration of dieldrin in the medium for both starting frequencies. Once the resistant allele was established, responses to selection were concentration-dependent. In the absence of dieldrin the susceptible allele was selectively favoured, at concentration 0.00005 per cent (w/v) selection was approximately neutral and at concentrations above this the Rdl allele was at a selective advantage. Fixation of Rdl occurred at higher concentrations. The probability of the initial establishment of the Rop1 allele in a population was found to be independent of both founding allele frequency and the concentration of diazinon on which the population was maintained. Once the resistant allele was established, responses to selection were concentration-dependent. In the non-modifier populations, in the absence of diazinon, the susceptible allele was selectively favoured, at concentration 0.00002 per cent (w/v) selection was approximately neutral and at concentrations above this the Rop1 allele was at a selective advantage, with fixation occurring at higher concentrations. In populations where the modifier gene was present the resistant allele was maintained in all populations with selection generally favouring resistant phenotypes. In populations maintained on insecticide, that fixed for the susceptible allele neither resistance system provided evidence of a polygenic response. Computer simulations were conducted to estimate the relative genotypic fitness values that could give rise to the allele frequencies produced by the population cage experiments and then to extrapolate the results to field situations. Results support the hypothesis that in the absence of the chemical the susceptible allele is at an advantage and in the presence of the chemical the resistant allele is selected for in both the dieldrin and diazinon resistance systems. Population size and genetic drift size were found to have little effect on the development of insecticide resistance when founding allele frequencies were at 1 and 5 per cent. When founding allele frequencies representative of field situations were simulated population size and drift dramatically influenced the initial establishment, and thus the development of resistance, in both the dieldrin and diazinon resistance systems. The resistant allele failed to establish in the population on all experimental concentrations when a finite population size of 100 was analysed. Furthermore, the resistant allele failed to establish in an infinite population in the absence of the chemical and when selection coefficients were low. When selection coefficients were high, and population size was infinite, results obtained were basically the same as those achieved when the simulation was run using 1 and 5 per cent founding allele frequencies. The resistance system-specific impact of genetic drift and selection on the evolution of insecticide resistance is discussed in the context of the applicability of the results to resistance management strategies or for resistance systems as a general model of microevolution.
Molecular and cellular biological studies of the Drosophila melanogaster stoned gene

Smith, Michiko. (University of Melbourne, 1999)

The Drosophila melanogaster stoned locus is an essential neurologically expressed gene, of which temperature-sensitive mutants exhibit a lethal interaction with the shibire locus. This, and subsequent evidence gathered from electrophysiological, localisation and sequence analyses of stoned have indicated a likely role critical to the synaptic vesicle cycle. Taking advantage of the genetic tools available for the dissection of stoned function, the role of stoned in the synaptic vesicle cycle was directly implicated in this study. The stoned transcript has a unique tandemly-arranged dicistronic structure, with both open reading frames (ORFs) separated by a fifty-five nucleotide sequence. Presented in this study, is firstly the identification of two distinct translatory products of the stoned ORFs, StonedA and StonedB. Although both novel proteins, the predicted amino acid sequences have allowed for the progressive identification of domains that have homology to a number of proteins implicated in the synaptic vesicle cycle. StonedA and StonedB were determined to have a direct association with synaptic vesicles by co-immunoprecipitation and sedimentation properties. However, rather than a ubiquitous association with synaptic vesicles, StonedA and StonedB are associated with separate subsets within the entire population. The biochemical localisation of StonedB repartitioned over D. melanogaster shibire background subcellular fractions. The data suggests that StonedA and StonedB are indeed part of the molecular mechanisms in the synaptic vesicle cycle. Perhaps StonedB-tagged synaptic vesicles are involved in the elusive kiss-and-run mechanism of the synaptic vesicle cycle.
Molecular and cellular biological studies of the Drosophila melanogaster stoned gene

Smith, Michiko. (University of Melbourne, 1999)

The Drosophila melanogaster stoned locus is an essential neurologically expressed gene, of which temperature-sensitive mutants exhibit a lethal interaction with the shibire locus. This, and subsequent evidence gathered from electrophysiological, localisation and sequence analyses of stoned have indicated a likely role critical to the synaptic vesicle cycle. Taking advantage of the genetic tools available for the dissection of stoned function, the role of stoned in the synaptic vesicle cycle was directly implicated in this study. The stoned transcript has a unique tandemly-arranged dicistronic structure, with both open reading frames (ORFs) separated by a fifty-five nucleotide sequence. Presented in this study, is firstly the identification of two distinct translatory products of the stoned ORFs, StonedA and StonedB. Although both novel proteins, the predicted amino acid sequences have allowed for the progressive identification of domains that have homology to a number of proteins implicated in the synaptic vesicle cycle. StonedA and StonedB were determined to have a direct association with synaptic vesicles by co-immunoprecipitation and sedimentation properties. However, rather than a ubiquitous association with synaptic vesicles, StonedA and StonedB are associated with separate subsets within the entire population. The biochemical localisation of StonedB repartitioned over D. melanogaster shibire background subcellular fractions. The data suggests that StonedA and StonedB are indeed part of the molecular mechanisms in the synaptic vesicle cycle. Perhaps StonedB-tagged synaptic vesicles are involved in the elusive kiss-and-run mechanism of the synaptic vesicle cycle.
The isolation and analysis of the hap genes of Aspergillus nidulans

Papagiannopoulos, Peter. (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
The inheritance of anthelmintic resistance in nematode parasites of sheep

Turney, Kerrin Linda. (University of Melbourne, 1993)

Studies on the inheritance of anthelmintic resistance were conducted. Three anthelmintics were considered; levamisole, ivermectin and closantel, and selection processes involved in conferring resistance were compared and contrasted. This study showed that sex-linked resistance was present in a field derived strain of Trichostrongylus colubriformis. Laboratory selection on another strain of T. colubriformis showing only a low level of levamisole resistance, led to the strain becoming highly resistant and the genetic basis of this resistance was sex-linked. The results indicated that high level levamisole resistance is inherited as a sex-linked recessive character and is probably monogenic. This confirms earlier work by Martin and McKenzie (1990b). The inheritance of resistance to ivermectin was found to be partially dominant in Haemonchus contortus and T. colubriformis and results were consistent with resistance in both species being controlled by a polygenic system. At the recommended dose rate of ivermectin the H. contortus heterozygotes showed a high level of survival and this has important consequences for the farmer as resistance will increase rapidly under selection (Georghiou and Taylor 1977). ' Results on the inheritance of resistance to closantel on resident worm burdens were consistent with resistance being conferred by many genes with a degree of recessiveness. Fitness estimates were made on susceptible, resistant and hybrid phenotypes. There were no significant differences in fitness between the F, and susceptible phenotypes at any time during the decay of closantel. However, both these phenotypes showed significantly reduced fitness compared with the resistant phenotype during the first 30 days after treatment with closantel. This suggests that there is no selection between the hybrids and the susceptibles at the concentrations of closantel used in this study, and implies that resistance is polygenic. The results provide information on the genetic basis of resistance and how selection for resistance may operate in the field. This information can be used to develop and modify worm control programs so that the onset of resistance is delayed or avoided.

Genetics - Theses

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