Genetics - Theses
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ItemMolecular and genetic analysis of Bt and spinosad resistance in diamondback moth, Plutella xylostella(University of Melbourne, 2005)Diamondback moth, Plutella xylostella, is a destructive global pest of nutritional and economically important cruciferous plants. Over the last 50 years, field strains have developed resistance to all major classes of insecticides used against them, including Bacillus thuringiensis (Bt) toxins and spinosyns. Molecular and genetic analysis of Bt resistance Diamondback moth is the only insect to develop open field resistance to Bt toxins. Many other insect species have been selected for Bt resistance under laboratory conditions, and in three lepidopteran pests, resistance has been genetically associated with mutations in a CrylA-binding midgut cadherin protein. Here, diamondback moth strains SCI and NO-QA were analysed. Both are resistant to CrylAa, CrylAb, CrylAc, CryIF and CrylJ however SCI was also resistant to Cry1C. AFLP linkage mapping was used to identify twenty-nine of the expected 31 linkage groups in SCI. CrylA resistance was significantly associated with a single linkage group (LG22 ?2=15.6, dfi=1, P<0.0001) and Cry 1C resistance was associated with two others (?2 =6.3 and 4.8, df=1). This data confirms different genetic mechanisms are responsible for CrylAc and CrylC resistance in this strain. Genes for lepidopteran Bt binding proteins were identified and mapped in SCI, including the 12-domain midgut cadherin, aminopeptidase N, alkaline phosphatase, glycosyl transferase, and P252-like genes, however none mapped to CrylAc or CrylC resistance associated linkage groups. Molecular analysis and interstrain complementation tests for allelism show mutations in the same genes are responsible for CrylAc resistance in SCl and the second resistant strain, NO-QA, Comparative genomics identified seven ribosomal genes that mapped to the CrylAc resistance linkage. Where possible, these single copy anchor loci were used to construct linkage maps for NO-QA and SCI. In NO-QA, 9 AFLP markers clustered around the Cry 1 Ac resistance locus were identified using bulked segregant analysis. Sequencing and inverse PCR were carried out on two of these AFLP markers and a PCR based resistance detection assay designed from one of these markers. Molecular and genetic analysis of spinosad resistance Spinosad is a novel class of insecticides that primarily targets nACh Receptors. The mechanism for field evolved spinosad resistance in diamondback moth strain Pearl-Sel (18,600 fold resistant) is hypothesised to be a single, recessive, autosomal gene. AFLP linkage mapping identified 28 linkage groups including one autosome responsible for spinosad resistance (?2=21.1, df=1, P<0.0001). Degenerate PCR identified five nAChR genes, two of which mapped to the resistance linkage group (nAChR a7-1 and a7-2). A linkage map of the resistance linkage group positioned nAChR ?7-1 more than 30 Haldane centimorgans (cM) and nAChR ?7-2 only 4.2 cM from the theoretical resistance locus, SpR- 1.
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ItemNo Preview AvailableCopper homeostasis and the Alzheimer's disease amyloid precursor protein(University of Melbourne, 2005)The transition metal copper is both essential for normal cellular function and potentially highly toxic, thus it is critical that all living organisms, from bacteria to humans, have developed copper homeostasis mechanisms. Alzheimer's disease is characterised by the accumulation of amyloid-? peptide, which is cleaved from the copper-binding Amyloid Precursor Protein. The normal biological function of the Amyloid Precursor Protein is poorly understood. Amyloid Precursor Protein is a member of a multi-gene family, including Amyloid Precursor like Proteins-1 and -2. The copper-binding domain is similar among Amyloid Precursor Protein family members, suggesting an overall conservation in its function or activity. This study investigated the hypothesis that the Amyloid Precursor Protein functions in copper homeostasis and that copper levels may regulate Amyloid precursor protein gene expression. To investigate the hypothesis that Amyloid Precursor Protein has a functional role in cellular copper homeostasis, mammalian cell culture systems in both neuronal and non-neuronal cells with varied Amyloid Precursor Protein expression were characterised for copper transport utilising a radio-copper assay. Over-expression of Amyloid Precursor Protein increased intracellular copper accumulation in non-neuronal and neuronal mammalian cells. Studies utilising mouse primary neuronal cortical cultures, demonstrated a gene dosage-dependent effect of Amyloid Precursor Protein expression on increasing cellular copper levels in cells that have a genetic ablation of Amyloid Precursor-like Protein-2 expression. In contrast, overexpression of the Swedish mutant of Amyloid Precursor Protein in primary cortical neurons reduces cellular copper levels. These findings provide evidence for the role of Amyloid Precursor Protein in neuronal copper homeostasis as a copper detoxification and/or efflux protein. To investigate the hypothesis that copper may regulate Amyloid Precursor Protein gene expression, a novel cell culture system was utilised. In this system, intracellular copper levels were genetically manipulated through altered expression of the Menkes protein, a major mammalian copper efflux protein. Cells lacking the Menkes protein show high intracellular copper levels due to reduced copper efflux, while restoration of Menkes protein function by over-expression restores copper efflux ability, resulting in dramatically decreased intracellular copper levels. Data presented in this study show that depletion of intracellular copper results in significantly reduced Amyloid Precursor Protein levels and a significant reduction in Amyloid Precursor Protein gene expression. In addition. Amyloid Precursor Protein promoter analysis suggests that putative metal regulatory elements, in the region -490 to +104 of the Amyloid Precursor Protein promoter, may be involved in mediating the response to copper depletion to regulate Amyloid Precursor Protein gene expression. This demonstrates a previously uncharacterised aspect of human Amyloid Precursor Protein gene regulation and supports the hypothesis that copper can regulate Amyloid Precursor Protein gene expression. Furthermore, these data support a role for the Amyloid Precursor Protein in copper homeostasis as a copper detoxification and/or efflux protein. Overall, this thesis presents strong evidence for the role of the Amyloid Precursor Protein in copper detoxification and/or efflux and the role of copper in the regulation of Amyloid Precursor Protein gene expression. Characterisation of copper homeostasis mechanisms of Amyloid Precursor Protein in copper detoxification and/or efflux and the elucidation of the copper-regulation mechanisms of the Amyloid Precursor Protein gene may provide potential therapeutic targets towards the treatment of Alzheimer's disease.
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ItemGenomics of insecticide resistance and xenobiotic metabolism in helicoverpa armigera(University of Melbourne, 2005)Helicoverpa armigera is a polyphagous lepidopteran that causes severe agricultural losses globally and it has a great capacity to develop resistance to insecticides. Previous research has shown that metabolic based resistance is the major cause of pyrethroid resistance in Australia and both cytochrome P450 monooxygenases and carboxylesterases have been implicated. AN02, a field strain of H. armigera exhibits a 50 fold resistance to fenvalerate in the larval stage. Resistance can be almost completely synergised by a P450 inhibitor, piperonyl butoxide, suggesting that resistance is metabolic based and likely to be mediated by P450 enzymes. The research presented in this thesis utilised two techniques, cDNA-AFLP and cDNA microarrays to identify differentially expressed genes between fenvalerate resistant and susceptible backcross progeny from an AN02 heterozygous resistant female and susceptible male. A novel cytochrome P450, CYP337B1 was found to be constitutively overexpressed in resistant individuals, and was mapped to within 1 cM of the resistance locus RFen1. Two other P450s which have previously been identified to be overexpressed in fenvalerate resistant H. armigera in Australia, CYP4G8 and CYP6B7 were also overexpressed in AN02, as were two glutathione S-transferases and one carboxylesterase. H. armigera larvae were also challenged with different xenobiotics; piperonyl butoxide, phenobarbital, esfenvalerate and jasmonic acid. Induction was studied in three separate tissues, integument, fatbody and midgut. Suites of inducible genes were identified, which may play a role in detoxifying these compounds. Notably, the three cytochrome P450s identified as constitutively upregulated in AN02 (CYP337B1, CYP4G8 and CYP6B7) were among the genes induced by esfenvalerate.
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ItemThe genetics of resistance to lufenuron in Drosophila melanogaster( 2005-02)The rise of large scale agriculture in the 20th century created the need for effective strategies to control insect pests. Treatment with chemical insecticides has been a weapon of choice, but the inevitable evolution of resistance has followed in many insect species. Resistance represents a major challenge, not only for agricultural production, but also for environmental preservation and human health. Two major options for resistance have been identified, and these are target-site based and metabolic-based resistance. Much insecticide resistance research focuses on identifying these mechanisms through genetic and molecular analysis. The insecticide lufenuron is the focus of this study. It belongs to a novel insecticidal group called the insect growth regulators, which were introduced in 1970s as highly selective insecticides with low vertebrate toxicity. Resistance to lufenuron in the non-pest species Drosophila melanogaster has been observed in field populations, despite the lack of field usage of lufenuron (Wilson & Cain, 1997; O’Keefe, 1997). This study has taken advantage of this phenomenon to investigate resistance mechanisms in natural populations. At least two detoxification mechanisms were identified. (For complete abstract open document)