Genetics - Theses
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ItemStudies on minor non-metrical skeletal variants in the mouse and man(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.
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ItemCharacterisation of genes involved in L-Arabinose metabolism in Arabidopsis(University of Melbourne, 2003)Glycosyl hydrolases are important mediators of plant cell wall modification during plant development. These enzymes catalyse the hydrolytic release of specific sugars, such as L-arabinose, from the polysaccharide-rich cell wall matrix. Here, the cloning and expression analysis of two genes, AtASD1 and AtASD1, encoding putative ?-L-arabinofuranosidases, is reported in Arabidopsis thaliana. AtASD1 and AtASD2 identities were assigned on the basis of homology to plant and microbial family 51 glycoside hydrolases. Despite numerous attempts, no enzyme activity was demonstrated for either protein. Over-expression of AtASD1 in the cell wall mutant, mur1, resulted in a genetic interaction that caused extreme dwarfism in plants. An AtASD2 T-DNA knockout mutant presented no visible phenotypes. Using the experimental approaches of RT-PCR, RNA gel blot analysis and GUS reporter gene expression analysis, AtASD1 and AtASD2 were shown to have different developmental expression profiles. High levels of AtASD1 promoter activity are present in multiple tissues during vegetative and reproductive growth. In comparison, AtASD2 expression is limited to the vasculature of older root tissue and to some floral organs and floral abscission zones. These findings implicate multiple and complex developmental roles for L-arabinosidases in addition to a simple role in carbon homeostasis. A previously described Arabidopsis mutant, ara1, exhibits a visible sensitivity to exogenous L-arabinose and encodes a defective L-arabinose kinase gene allele. The molecular characteristics of an ARA1 homologue, denoted ARA2, are reported. Expression studies revealed ARA1 is expressed during early vegetative growth, in floral organs and in conjunction with ARA2, developing pollen. Attempts to identify an ARA2 T-DNA mutant were made. Thus, a total of four genes that are involved in L-arabinose metabolism in Arabidopsis, are described. In summary, these findings have contributed significant insights into an intricate and highly regulated mechanism, whereby the concerted action of many enzymes mediate development-related cell wall remodelling that ultimately influences plant form and function.
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ItemFunctional analysis of conserved domains in the Drosophila TRPL channel(University of Melbourne, 2003)The Drosophila Transient Receptor Potential (TRP) and Transient Receptor Potential Like (TRPL) proteins form non selective cation channels whose activity depolarize the photoreceptor neurons in response to light. The initial steps that trigger the photoreceptor response are well characterized. However, the molecular mechanisms that directly activate and regulate the channels remain unknown. The elucidation of critical domains and their contribution to channel activation might constitute a starting point to understand the activating mechanism of TRPL and other TRP channels. The function of conserved regions amongst TRP channels was explored by creating mutations in the TRPL channel (truncations and point mutations) in three regions of the protein with high conservation through human and Drosophila TRP channels. The first conserved domains studied were the Ankyrin Repeats (AR) motifs that are present in the Amino-terminal region of the TRPC and TRPV subfamilies of TRP channels. These motifs were deleted in the TRPL channel, expressed in HEK cells and studied by patch-clamp whole cell recordings. Two truncated channels lacking all or only the 3rd and 4th AR showed activation by GTP-y-S included in the patch pipette, indicating that these conserved motifs do not play a key role in TRPL channel activation. Within the transmembrane domains region, the loop linking the S4 and S5 transmembrane domains, which shows significant degree of conservation, was studied. The substitution of Leucine 542 and Serine 545 for Alanines (mutant LS) did not affect TRPL activation, whereas changing Glycine 540 and Glutamine 543 for Alanines (mutant GO) eliminated the channel response to GTP-?-S. The possible role of this region was also investigated by dialysis of a peptide derived from this S4-S5 loop into HEK cells expressing wild type TRPL. This treatment elicited currents in cells expressing TRPL as well as in cells expressing the human homologue TRPC6. In overlay assays this peptide bound a Drosophila head protein of approximately 200 KDa. In conjunction these results indicate that the region is necessary for normal channel activation and that it probably regulates TRPL channel activity by interacting with other proteins. The C-terminal region was the third region investigated. The deletion of aminoacids 682-1025 (mutant ?C1), located immediately C-terminal to the last transmembrane domain, abolished TRPL channel responses to both GTP-?-S and Linolenic acid (LNA). The deletion of the highly conserved region 682-698, which is present in all TRPC channels, significantly reduced the response to GTP-?-S and prevented LNA activation of the channel. These results provide evidence of the regions within the channel that are required for its normal activation by GTP-?-S or by lipids. Although, the actual role that these identified regions is playing in channel functioning can not be established, different possible scenarios are discussed.
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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.
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ItemNo Preview AvailableCloning and analysis of the acuG, idpA and maeA genes involved in carbon metabolism in felamentous fungus Aspergillus nidulans(University of Melbourne, 2002)This thesis describes work on cloning and analysis of the three genes encoding enzymes of carbon catabolism from the filamentous fungus Aspergillus nidulans. NADP-dependent isocitrate dehydrogenase enzymes catalyze the decarboxylation of isocitrate to 2-oxoglutarate accompanied by the production of NADPH. In mammals two different genes encode mitochondrial and cytoplasmic/peroxisomal located enzymes, while in Saccharomyces cerevisiae three separate genes specify compartment specific enzymes. In A. nidulans a single gene, idpA, is shown to specify a protein with a high degree of identity to mammalian and S. cerevisiae enzymes. Two idpA transcripts were identified and two transcription start points were determined by sequencing cDNA clones and by 5'RACE. The shorter transcript was found to be inducible by acetate and by fatty acids while the longer transcript was present in higher amounts during growth in glucose containing media. The longer transcript is predicted to encode a polypeptide containing an N-terminal mitochondrial targetting sequence as well as a C-terminal tripeptide (ARL). The shorter transcript is predicted to encode a polypeptide lacking the mitochondrial targetting signal but retaining the C-terminal sequence. Immunoblotting using antibody raised against S. cerevisiae Idp1p detected two polypeptides consistent with these predictions. The functions of the predicted targetting sequences were confirmed by analysis of transformants containing fluorescent protein fusion constructs. Using anti-Idp1p antibodies, protein localisation to mitochondria and peroxisomes was observed during growth on glucose while cytoplasmic and peroxisomal localisation was found upon acetate or fatty acid induction. Therefore it has been established that by the use of two transcription start points a single gene is sufficient to specify localisation of NADP-dependent isocitrate dehydrogenase to three different cellular compartments in A. nidulans. A deletion of the idpA gene was generated and the phenotype indicates a possible role in providing NADPH for protection against oxidative stress. The single acuG gene encodes fructose-1,6-bisphosphatase, a crucial enzyme in gluconeogenesis. It has been shown to be under strong control by CreA mediated carbon catabolite repression. A significant effect is also exerted by endogenous induction in carbon starvation conditions. This pattern of relatively weak regulation of acuG in A. nidulans is very different to the strong regulation of FBP1 gene expression and FBP activity in S. cerevisiae which acts on the level of transcription, mRNA stability and glucose inactivation of the protein. The putative novel regulators AcuK and AcuM were found to play a key role in fructose-1,6-bisphosphatase regulation. These novel proteins may be global gluconeogenic regulators in A. nidulans as they have been found to affect the regulation of other gluconeogenic enzymes: acuF encoding PEPCK, acuN encoding enolase (M.J. Hynes, personal communication) and maeA encoding malic enzyme (see below). This pattern of regulation allows gluconeogenesis to occur during growth on any carbon source metabolised via TCA cycle intermediates in A. nidulans. The malic enzyme plays an intermediary role between the TCA cycle and gluconeogenesis. Two genes with different cofactor specificity have been identified in A. nidulans and work was concentrated on the maeA gene encoding a conserved NADP-dependent malic enzyme. Two closely located starts of transcription were detected by 5'RACE and transcripts showed strong regulation by carbon sources with strong gdhB101 dependent induction by sources of glutamate. A deletion of the maeA gene was generated and the phenotype indicates a possible role in NADPH generation and providing pyruvate for acetyl-CoA synthesis to maintain carbon flux and operation of the TCA cycle during growth on compounds metabolised via the cycle.
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ItemRegulation of amidase genes in Aspergillus nidulans : mechanisms of nitrogen metabolite repression(University of Melbourne, 2002)The filamentous fungus Aspergillus nidulans can utilise a wide variety of amides as the sole nitrogen source. The expression of the required catabolic enzymes is tightly regulated in response to nitrogen availability. The global mechanism responsible for this response is termed nitrogen metabolite repression, and is mediated by the GATA zinc finger activator protein AreA. During nitrogen limitation, AreA activates the expression of genes required for the utilisation of alternative nitrogen sources. If a more easily assimilated source such as ammonium is present changes in areA mRNA stability and interaction with the negatively acting NmrA protein prevent AreA activation of catabolic gene expression. This thesis describes the isolation of three genes involved in amide utilisation in A. nidulans and an analysis of their regulation. The fmdS gene encodes a formamidase unlike the characterised acetamidase of A. nidulans, and is required for the hydrolysis of formamide. The gmdA and bzuA genes are required for the utilisation of benzamide as a nitrogen source, and encode general amidase and benzoate para-hydroxylase, respectively. Like the acetamidase, the general amidase belongs to the amidase signature family of enzymes. In addition to benzamide, the general amidase also mediates the hydrolysis of a variety of long chain amides. BzuA is required to prevent benzoate toxicity following the hydrolysis of benzamide by GmdA. The transcription of both fmdS and gmdA is highly regulated by AreA-mediated nitrogen metabolite repression, with a low level of expression during growth on preferred nitrogen sources such as ammonium and increased levels during growth on an alternative source such as alanine. In addition, this activation was shown to be enhanced during nitrogen starvation, defining an additional level of AreA function. The transcription of these genes is affected by carbon availability, with transcription halted upon carbon starvation irrespective of nitrogen availability. Detailed deletion analysis of the fmdS promoter was performed, and the only changes in fmdS transcription revealed were though deletion of putative AreA recognition sequences. This suggested the mechanism behind the carbon starvation response is probably inactivation of AreA. Expression of fmdS was also found to be affected by transcriptional interference from an upstream gene (usgS), whose transcript overlaps the fmdS coding region. Beyond its affect on reducing fmdS transcription when in cis, gene inactivation revealed no indication of the role of the usgS gene. The nitrogen starvation and carbon starvation responses of nitrogen metabolite repression are independent of the two mechanisms already characterised in this global regulatory phenomenon - NmrA interaction and areA transcript stability. In an attempt to identify factors which may play a role in the starvation mechanisms, a possible A. nidulans homologue of the S. cerevisiae URE2 gene was cloned. In yeast, Ure2 is a negatively acting factor that interacts with and represses the AreA homologue Gln3. Several ESTs with similarity to URE2 were identified, allowing the gstA gene to be cloned. Gene inactivation showed gstA encodes a functional glutathione S-transferase involved in resistance to xenobiotics and heavy metals. The evidence presented here strongly suggests the A. nidulans genome does not contain a true URE2 homologue that is involved in nitrogen metabolite repression. A gene replacement strategy was developed to allow epitope tagging of AreA, yielding a variety of strains with which this regulatory factor could be studied at the protein level. Western blot analysis has indicated that AreA is differentially phosphorylated under different conditions of nitrogen and carbon availability. The starvation responses in AreA regulated structural gene expression correspond to the post-translational modifications observed suggesting that it is functionally significant.
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ItemSystematics, phylogeography and population genetics of the large bent-wing bat, Miniopterus schreibersii (Chiroptera)(University of Melbourne, 2001)This thesis is a multi-faceted approach to consider the population and evolutionary genetics of the widely distributed Miniopterus schreibersii complex. The first section focuses on phylogeography. It investigates the monophyly of the M. schreibersii complex and the number of species present, using the mitochondrial gene ND2. These data reveal separate lineages within the M. schreibersii complex. The second section involves the Australian forms of Miniopterus. This section investigates the recognised taxonomy within Australia using molecular and morphological characters. Three distinct lineages are found to exist on this continent with only two of these previously known. These lineages are described as subspecies. This situation is discussed with references made to historical processes including migration events and vicariance. The third section focuses on the south-eastern Australian populations which include two of the three subspecies. This section incorporates both molecular and ecological information and investigates the pattern of gene flow and dispersal along with the processes of vicariance and philopatry. The final section investigates the patterns of distribution and genetic variation present in the bat parasites living on the southern Australian bat populations. These patterns mirror those described in the bats.
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ItemNo Preview AvailableInsecticide resistance in Lucilla cuprina and Drosophila melanogaster(University of Melbourne, 2001)Insecticide resistance is a major challenge not only for agricultural production, but also in terms of environmental preservation and human health. Intensive pesticide use for the last half century has resulted in the rapid evolution of resistance not only in pest species, but also in beneficial and non-targeted insects. The options for resistance in insect species have been well investigated, and two major types of resistance - metabolic and target-site resistance have been identified. EMS mutagenesis and selection represent tools for generating resistant mutants in the laboratory, enabling us to predict the evolution of resistance to particular chemicals before it evolves in the field. This may prevent the use of ineffective chemicals and the evolution of resistance in other species that may have been targeted inadvertently. Laboratory studies in the Australian sheep blowfly, Lucilia cuprina have been shown to parallel the field situation. This study investigates the possibility of the existence of a resistance option for the organophosphorous compound, diazinon, other than the known Rop-1/E3-mediated resistance. Only one resistant mutant was generated after EMS-mutagenesis of the susceptible strain. Genetic mapping of the resistant mutation and molecular analysis showed that the mutation is Rop-1. Explanation for Rop-1/E3 bias in L. cuprina response to organophosphates is discussed. Furthermore, options for resistance to novel insecticides - Insect Growth Regulators (IGRs) were investigated, using Drosophila melanogaster as a model. IGRs were introduced in late 1970s as a group of highly selective insecticides with low toxicity for vertebrates and the environment. It was thought that resistance to chemicals with similarities to the insect hormones (juvenile hormone or ecdysone) would be unlikely to evolve. However, it did evolve and very rapidly to some of these chemicals. Resistance to lufenuron has been found in non-targeted species D. melanogaster, in strains isolated from the East coast of Australia. This resistance was mapped and its position within a chromosome determined. Another example of lufenuron resistance was found in EMS-induced cyromazine resistant strain, Cyr1b. Lufenuron resistance was mapped, to determine whether the same gene confers resistance to both chemicals. Phenotypic correlation of survivorship in Cyr1b cross-resistant strain was shown to be result of a high genotypic correlation, which suggests that single mechanism confers resistance to both, lufenuron and cyromazine. Cross-resistance studies were performed with dicyclanil, a recently introduced IGR, on all cyromazine-resistant mutants generated by EMS-mutagenesis. Resistance to dicyclanil in Cyr2 strain was mapped to the same region as Rst(2)Cyr. A field lufenuron-resistant strain was tested for cross-resistance to cyromazine, diflubenzuron and dicyclanil and a very unpredictable cross-resistance pattern among IGRs was found. This thesis highlights the importance of using the prediction of resistance, positional cloning and cross-resistance studies for insecticide resistance management in the field.
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ItemNo Preview AvailableInsecticide resistance in Lucilla cuprina and Drosophila melanogaster(University of Melbourne, 2001)Insecticide resistance is a major challenge not only for agricultural production, but also in terms of environmental preservation and human health. Intensive pesticide use for the last half century has resulted in the rapid evolution of resistance not only in pest species, but also in beneficial and non-targeted insects. The options for resistance in insect species have been well investigated, and two major types of resistance - metabolic and target-site resistance have been identified. EMS mutagenesis and selection represent tools for generating resistant mutants in the laboratory, enabling us to predict the evolution of resistance to particular chemicals before it evolves in the field. This may prevent the use of ineffective chemicals and the evolution of resistance in other species that may have been targeted inadvertently. Laboratory studies in the Australian sheep blowfly, Lucilia cuprina have been shown to parallel the field situation. This study investigates the possibility of the existence of a resistance option for the organophosphorous compound, diazinon, other than the known Rop-1/E3-mediated resistance. Only one resistant mutant was generated after EMS-mutagenesis of the susceptible strain. Genetic mapping of the resistant mutation and molecular analysis showed that the mutation is Rop-1. Explanation for Rop-1/E3 bias in L. cuprina response to organophosphates is discussed. Furthermore, options for resistance to novel insecticides - Insect Growth Regulators (IGRs) were investigated, using Drosophila melanogaster as a model. IGRs were introduced in late 1970s as a group of highly selective insecticides with low toxicity for vertebrates and the environment. It was thought that resistance to chemicals with similarities to the insect hormones (juvenile hormone or ecdysone) would be unlikely to evolve. However, it did evolve and very rapidly to some of these chemicals. Resistance to lufenuron has been found in non-targeted species D. melanogaster, in strains isolated from the East coast of Australia. This resistance was mapped and its position within a chromosome determined. Another example of lufenuron resistance was found in EMS-induced cyromazine resistant strain, Cyr1b. Lufenuron resistance was mapped, to determine whether the same gene confers resistance to both chemicals. Phenotypic correlation of survivorship in Cyr1b cross-resistant strain was shown to be result of a high genotypic correlation, which suggests that single mechanism confers resistance to both, lufenuron and cyromazine. Cross-resistance studies were performed with dicyclanil, a recently introduced IGR, on all cyromazine-resistant mutants generated by EMS-mutagenesis. Resistance to dicyclanil in Cyr2 strain was mapped to the same region as Rst(2)Cyr. A field lufenuron-resistant strain was tested for cross-resistance to cyromazine, diflubenzuron and dicyclanil and a very unpredictable cross-resistance pattern among IGRs was found. This thesis highlights the importance of using the prediction of resistance, positional cloning and cross-resistance studies for insecticide resistance management in the field.
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ItemNo Preview AvailableRegulation of the G2(University of Melbourne, 2000)In order to survive DNA damage, cells respond with a range of mechanisms, including DNA repair, checkpoint responses, and survival mechanisms. The DNA damage checkpoint cannot be viewed as a simple signal transduction pathway. It is divided into several steps that are genetically separable. These observations have lead to the questions that are addressed in this project. Firstly, how does the checkpoint, interact with the core G2/M cell cycle machinery. I address this with a biochemical study of the affects of a DNA damage checkpoint on Y15 regulation of p34cdc2/cyclinB. This study allows the two pathways to be linked. Secondly, do we know all the proteins that are involved in the checkpoint. We do not know how the checkpoint detects DNA damage, nor how the checkpoint integrates with signals from DNA repair pathways, and so other, as yet unidentified, checkpoint genes may fill in these gaps. In this study I used a genetic screen to identify other members of the pathway. The mutants isolated in this screen allowed the posing of new questions about the DNA damage checkpoint pathway. It is becoming increasingly clear, through this and other studies, that essential genes will play roles in the checkpoint pathway. This allows us to link the checkpoint pathway with essential pathways such as DNA replication or chromatin organisation. In a screen for new genes involved in the DNA damage checkpoint, I isolated two essential genes that play roles in G2 checkpoint responses. cut5 provides a link between DNA replication and the G2 checkpoints responding to DNA damage and blocks to DNA replication. The isolation of a novel allele of rad18 in this project is the first evidence in S. pombe of a gene linking the DNA damage checkpoint and DNA repair. Several pieces of evidence point to rad18 playing an essential role in chromatin organisation and this then links chromatin regulation to the regulation of both repair and checkpoint responses to DNA. An allele-specific suppressor of this rad18 allele, brc1, suggested a link between checkpoint and repair pathways and independent regulators of genomic stability. The novel rad18 allele also allows us to address the issue of checkpoint maintenance and checkpoint initiation. The final gene isolated in the screen was a DNA repair gene that is not absolutely required for the ability to repair. As it is not clear what repair pathway this gene is involved in, it suggests a broad-range defect, which may also link the processes of chromatin organisation and DNA repair. The isolation of these genes links together multiple pathways of DNA damage responses. It suggests that genes with essential roles in processes such as DNA replication, chromatin organisation and maintenance of genomic stability may also play roles in checkpoint and other responses. Further screening, taking advantage of the types of phenotypes discovered in this screen, will be needed to identify these genes.