Genetics - Theses
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ItemCharacterisation of lipase genes in Helicoverpa armigera( 2006)Helicoverpa armigera (cotton bollworm) is a major agricultural pest in Australia, Asia and Africa. Analysis of a midgut cDNA library revealed a surprising diversity of genes encoding lipases, enzymes that metabolise lipids. Prior to and during this project, 13 neural lipases and three acidic ones were discovered. These were classified and compared to other insect lipases using sequence alignments and phylogenetic analysis. Novel non-catalytic subfamilies were discovered, including one containing an H. armigera lipase. At least three other subfamilies were found to contain H. armigera lipases. Lipidopteran lipases were found to be almost entirely distinct from Dipteran ones.
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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)
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ItemDNA methylation at the neocentromere( 2006-01)The Centromere is a vital chromosomal structure that ensures faithful segregation of replicated chromosomes to their respective daughter cells. With such an important structure, one would expect the underlying centromeric DNA sequence would be highly conserved across all species. It turns out that the underlying centromeric DNA sequences between species ranging from the yeast, fly, mouse to humans are in fact highly diverged suggesting a DNA sequence independent or an epigenetic mechanism of centromere formation. Neocentromeres are centromeres that form de-novo at genomic locations that are devoid of highly repetitive a-satellite DNA sequences of which normal centromeres are usually comprised from. To date, the 10q25 neocentromere is the most well-characterised, fully functional human centromere that has been used previously to characterise the extent of a number of centromeric protein binding domains and characterise the properties of the underlying DNA sequence. Along with other factors, the existence of neocentromeres has given rise to a hypothesis where centromeres are defined by epigenetic or DNA sequence independent mechanisms. The putative 10q25 neocentromere domain was recently redefined by high resolution mapping of Centromeric protein A (CENP-A) binding through a chromatin immunoprecipitation and array (CIA) analysis. The underlying DNA sequence was investigated to determine and confirm that the formation of the 10q25 neocentromere was through an epigenetic mechanism. Through a high-density restriction fragment length polymorphism (RFLP) analysis using overlapping PCR amplified DNA derived from genomic DNA representing the 10q25 region before and after neocentromere activation. No sequence polymorphisms, large insertions or deletions were detected and confirmed the epigenetic hypothesis of centromere formation. DNA methylation is one of many epigenetic factors that are important for cellular differentiation, gene regulation and genomic imprinting. As the mechanisms and functions of DNA methylation have been well characterised, its role at the 10q25 neocentromere was investigated to try and identify the candidate epigenetic mechanism involved in the formation of centromeres. DNA methylation across the neocentromere was assessed using sodium bisulfite PCR and sequencing of selected CpG islands located across the 10q25 neocentromere. Overall, the methylation level of the selected CpG islands demonstrated no difference in DNA methylation before and after neocentromere activation. However, significant hypomethylation upon neocentromere formation was detected close to the protein-binding domain boundaries mapped previously suggesting that this may have a role in demarcating protein binding domains at the neocentromere. Further analysis of DNA methylation investigated non-CpG island methylation at sites defined as CpG islets and CpG orphans. Interestingly, the DNA methylation level measured at selected CpG islets and CpG orphans across the 10q25 neocentromere were not completely hypermethylated as previously thought, but demonstrated variable methylation that became fully hypermethylated upon neocentromere activation in most sites investigated. These results suggested that a role for DNA methylation existed at the 10q25 neocentromere and that it occurred at sites devoid of CpG islands. This study has found that DNA methylation at non-CpG island sites was variable contrary to popular belief and, was linked with neocentromere formation through the observation of increased DNA methylation at the 10q25 neocentromere. Inhibition of DNA methylation demonstrated increased neocentromere instability and a decrease in methylation of these CpG islets and CpG orphans confirming the importance of DNA methylation at neocentromeres. This study has characterised a new class of sequences that are involved in the maintenance of chromatin structure through DNA methylation at the 10q25 neocentromere.
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ItemTaxonomy, biogeography and population genetic structure of the southern Australian intertidal barnacle fauna( 2008)Barnacles are a unique organism in that they have both a planktonic larval stage followed by an irreversibly sessile adult stage. Widely distributed throughout the world, they have been studied by many prominent scientists, with much of the work undertaken focusing on ecology and taxonomy. However, most of the taxonomic work had been undertaken based on morphological characteristics, with phylogenetic studies only undertaken more recently. Many of these studies have failed to include Australian species, most of which are endemic to the continent. Newly produced and Genbank records of mitochondrial DNA sequence were used to confirm the taxonomic status of Australian species. The status of most species was confirmed, with a few notable exceptions. In particular, data analysis suggested the existence of cryptic species within Elminius modestus. In addition, the divergence between these three species and Elminius kingii was great enough to warrant the introduction of a new genus, Austrominius. This genus now contains three species, A. modestus, A. adelaidae and A. covertus. The two-phase life history of barnacles also made them the ideal organism for the study of the dynamics of the southern Australian marine environment. Three marine biogeographic provinces are recognised in the region, with both historical and contemporary ecological factors predicted to be responsible. In order to investigate the biogeography and hydrography of the region, both mitochondrial sequence data and microsatellite data were used to investigate the phylogeography and population genetic structure of Catomerus polymerus. The mitochondrial data showed a deep phylogeographical split, dividing the species into eastern and western lineages. Dating this split using a molecular clock indicated that the repeated emergence of the Bassian Isthmus during glacial periods was most likely responsible, having provided a barrier to gene flow between the two lineages. However, subsequent gene flow during interglacial periods prevented the lineages from diverging into two separate species. Analysis of the microsatellite data indicated that the species comprised four groups or subregions; one in South Australia, one in New South Wales and eastern Victoria, one in central Victoria, and one in western Victoria and Tasmania. Further analysis of the data indicated that these subregions could be due to the influence of the major ocean currents (Leeuwin, East Australian and Zeehan currents), and the reduction in gene flow across two biogeographic breaks (Ninety Mile Beach, The Coorong). This correlates reasonably well with the previously recognised biogeographic provinces. Finally, mitochondrial data were used to examine the phylogeography of two species of barnacle, Chthamalus antennatus and Chamaesipho tasmanica. In contrast with the study of C. polymerus, these species did not show any significant structure across their entire distribution. There are a number of possible explanations for this, with most relating to the longevity and durability of the larvae. However, it is also possible that these species could colonise southern Tasmania during the glacial periods, thereby being unaffected by the present of the Bassian Isthmus, and maintaining a single panmictic population.
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ItemSelection and characterisation of Arabidopsis mutants resistant to a glutathione biosynthesis inhibitor( 2003)This thesis describes the investigation of GSH regulation and signaling in Arabidopsis thaliana by a genetic approach. A procedure is described to select for mutants resistant to a chemical inhibitor of glutathione (GSH) biosynthesis, L-buthionine-SR-sulfoximine (BSO). Fifteen mutants were selected and preliminary genetic examinations indicated that BSO-resistance phenotypes are often highly variable. Consequently five mutants were chosen for further genetic, molecular and biochemical characterisation. All five of these mutants were shown to have altered GSH levels. Moreover, root growth among these mutants varied indicating that different mechanisms may be conferring BSO-resistance. Molecular mapping techniques were used to assign the BSO-resistance loci of four of the mutants to chromosome regions. Two mutants (a T-DNA insertion mutant and an EMS-derived mutant) were found to map to the same position between the markers CHS1 and R89998 on chromosome 5. Subsequently, complementation tests confirmed that these mutants define a single locus. The mutated locus in the T-DNA insertion mutant was determined using molecular techniques. The gene that contained the insert encodes a putative transport protein and was designated the BSO Resistance Transporter1 (BRT1). No transcript was detected in the T-DNA insertion mutant (brt1-1) which suggests that brt1-1 is likely to be a null allele. The EMS-derived mutant (brt1-2) contained an intron splice-site consensus sequence mutation which resulted in transcripts that included an unspliced intron. The function of the BRT1 gene was investigated by phenotype testing and despite the brt1 mutants having 2-fold GSH levels they were not resistant to known oxidising agents. Northern analysis of GCS and the use of a GCS-GUS reporter line indicated that the increase in GSH levels in these mutants was not accompanied by increases in GCS transcription and translation. This suggests that BRT1 is a novel component influencing GSH metabolism. BRT1 defines a novel family of three genes in Arabidopsis. Preliminary experiments indicate that these three genes are expressed in the same tissues providing for possible a level of redundancy between them. Supporting this is a newly identified insertion mutant in a second BRT gene (brt2-1) which is also resistant to BS0. Moreover, the double mutant, brt1-1;brt2-1, exhibits increased BSO-resistance suggesting an additive effect of the mutations. Database searches revealed that there were homologues in other organisms including one from the malaria parasite (PfCRT). Mutations in PfCRT confer resistance to a widely used antimalarial, chloroquine (CQ). However the function of PfCRT is not known. The BRT1 mutant was shown to have a level of functional conservation with a malaria parasite homologue, PfCRT, and therefore further characterisation of these proteins may provide insights into GSH metabolism and perhaps CQ-resistance in the malaria parasite.
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ItemThe role of cytochrome P450s in pyrethroid resistance in the AN02 strain of Helicoverpa armigera( 2003)Helicoverpa armigera is a lepidopteran moth species that is one of the largest agricultural pests in the world. In Australia, it is especially damaging to the cotton industry. Previous research on pyrethroid resistance in Helicoverpa armigera has suggested several resistance mechanisms present in the field. Research on a specific strain of H. armigera, AN02, suggested that resistance to pyrethroids in this particular strain is due to metabolism of the insecticide by cytochrome P450. This thesis describes work undertaken to gain further knowledge of the resistance mechanism present in this strain. Chapter 2 describes expression testing and elimination of the CYP6B cluster of P450s as candidates for resistance in the AN02, which were the most likely candidates available at the time. Chapter 3 describes construction, probing and sequence analysis of a midgut cDNA library of H.armigera to clone more P450 sequences and analyse the general transcriptome of one of the tissues where detoxification is likely to occur. Chapter 4 describes expression testing of new P450 sequences obtained from different sources to determine whether any of them have increased expression that is associated with the resistance genotype. Several new P450 genes were found to have increased expression and are candidates for further study.
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ItemCytochrome P450 gene expression in Drosophila melanogaster( 2008)Present in almost all living organisms, cytochrome P450s form one of the biggest enzyme superfamilies. They are versatile biocatalysts, capable of performing a range of biochemical reactions and are involved in a wide spectrum of biological functions. The vinegar fly, Drosophila melanogaster, has 85 P450s in its sequenced genome. Six of these have been found to catalyse the synthesis of the important insect molting hormone, 20-hydroxyecdysone and a handful have been implicated in insecticide resistance. The other P450s remained largely uncharacterised. In the first half of this thesis, the expression patterns of P450s in the D. melanogaster genome were characterised by in situ hybridisation at the third instar larval stage. Most P450s have defined expression patterns at this stage of development. A majority of P450s are expressed in the midgut, Malpighian tubules and fat body, tissues that are involved in the metabolism of xenobiotics. Other P450s are expressed in specific tissues, such as the prothoracic glands, the salivary glands and the gonads, where they might have roles in development or reproduction. In particular, Cyp6g2 is expressed in the corpus allatum (CA), where it could play a role in juvenile hormone synthesis. An RNAi lethality screen using lines that were available from the Vienna Drosophila RNAi Centre identified a number of P450s which are essential for development and viability. In the second half of the thesis, the transcriptional regulation of a P450 involved in insecticide resistance, Cyp6g1, was investigated. Cyp6g1 was regulated by two discrete cis-regulatory modules/enhancers, one controlling expression in the Malpighian tubules and one controlling expression in the midgut and fat body. Phenobarbital induction of Cyp6g1 is tissue-specific and is mediated by a fragment in the 5’ regulatory region that interacts with both enhancers. Characterisation of the long terminal repeat (LTR) of the Accord transposable element in the 5’ region of Cyp6g1, present in insecticide resistant populations, shows that the Accord LTR contains cis-regulatory elements which increase expression of Cyp6g1 in the fat body, midgut and Malpighian tubules, and contribute to insecticide resistance in these populations. This study shows that the diverse tissue distribution of different P450s in D. melanogaster is related to the diverse biological functions of the enzymes encoded. This is exemplified by the detailed examination of the regulation of the insecticide resistance-conferring P450, Cyp6g1. Its expression pattern reflects its detoxification function in the fly. The role of transposable element insertions in changing gene expression patterns and contributing to selectable variation in genomes is also demonstrated through the Cyp6g1 study.