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ItemMolecular evolution, structure and function of Glutathione S-Transferases in the genus Drosophila(University of Melbourne, 2009)GLUTATHIONE S-TRANSFERASES (GSTs) are a superfamily of ubiquitous enzymes that fulfill a variety of biological functions including detoxification. They have been implicated in insecticide resistance and may have evolved in response to toxins in the niche-defining feeding substrates of Drosophila species. In this thesis, a bioinformatic approach was used to identify GST genes of the twelve Drosophila species with recently sequenced genomes. This was followed by comprehensive analyses of their molecular evolution. Gene copy number variation is mainly attributable to unequal crossing over events in the large delta and epsilon clusters. Within these gene clusters there are also GST genes with slowly diverging orthologs. This implies that they have their own unique functions or have spatial/temporal expression patterns that impose significant selective constraints. Searches for positively selected sites within GSTs identified G171K in GSTD1, an enzyme with a promiscuous use of substrates. The same radical substitution (G171K) in GSTD1 has occurred at least three times in the Drosophila radiation. As D. melanogaster GSTD1 can metabolize DDT in a DDTase reaction, the hypothesis that the G171K mutation is associated with DDT selection was tested. Sequencing of historical alleles revealed that the G171K substitution was fixed prior to the use of DDT and so, this insecticide is not the selective agent. Both population genetic and biochemical analyses suggested that the DDTase activity in GSTD1 preceded the use of DDT in the field. In an effort towards probing GSTs binding potential against insecticides and substrates, a general strategy was tested. The strategy entails the following steps: 3D structure determination by X-ray crystallography, in silico molecular docking of candidate insecticide, and in vitro insecticide binding study using NMR spectroscopy. The utility of this approach is investigated with D. melanogaster GSTD1 interaction with DDT. The crystal structure of D. melanogaster GSTD1 has been determined to 1.1 � resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. After removing seven residues of the last helix, DDT was computationally docked into the active site in an orientation favoring catalysis. 2D 15N HSQC NMR experiments of GSTD1 suggest conformational changes occur in GSTD1 upon the binding of glutathione and DDT. Furthermore the 15NH resonances of residues 117 - 121 significantly broaden upon the addition of DDT and thereby support the predicted binding site. The overexpression of D. melanogaster GstD1 has been implicated in DDT resistance, oxidative stress protection and extension of lifespan. However, a study that controls for the genetic background so that the effect of the overexpression of this gene is the sole factor responsible for a phenotype has not been performed. The GAL4/UAS ?C31 integrase system was used to constitutively overexpress D. melanogaster GstD1 and GstD2 at 2 fold and 278 fold, respectively. The paralog, D. melanogaster GstD2 was used because it is not a DDTase and thus, it served as a control for DDTase activity. Probit mortality analysis revealed that the overexpression of D. melanogaster GstD1 did not lead to an increased detoxification of DDT. The overexpression of both D. melanogaster GstD1 and GstD2 resulted in lifespan extension with median survival age of 69 days whereas the control had a median survival age of 52 days. The overexpression of D. melanogaster GstD1 but not GstD2 can lead to an increased survival in the presence of paraquat, which is an oxidative stress generator. The 4-hydroxynonenal conjugating activity of D. melanogaster GstD1 and GstD2 may be the factor responsible for the paraquat resistance and lifespan extension results.
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ItemThe potential for evolution in the rainforest restricted species : drosophila bunnanda(University of Melbourne, 2008)Most quantitative genetic studies to date have focused on model and agricultural species and ecological traits unrelated to the distributional limits of species, leading to the evolutionary paradigm that species should not be limited in their ability to adapt to environmental changes by a lack of genetic variation. However whether this assumption can be extended to specialist species is unknown. The overall aim of this study was to investigate the adaptive potential of a rainforest restricted species of Drosophila. Using a large family experiment analyzed with a powerful animal model analysis I found low levels of additive genetic variation and narrow sense heritability (h2 < 0.10) for desiccation resistance in multiple populations of the rainforest restricted Drosophila bunnanda. Despite small standard errors, both additive genetic variation and narrow sense heritability estimates for desiccation resistance were not more than two standard errors from zero suggesting this rainforest restricted species will have difficulty adapting to lower humidity levels. However, additive genetic variation and narrow sense heritability estimates for wing size and wing aspect were significantly different from zero, suggesting genetic variation for quantitative traits is not generally low in this species. Furthermore, high variation and low population differentiation in microsatellite and a mitochondrial gene further indicate population processes, such as small population size or restricted dispersal, are probably not influencing low additive genetic variance for desiccation resistance in D. bunnanda. A comparison of neutral genetic population structure and phylogeographic history between rainforest restricted and generalist species of Drosophila found ecological and geographic distribution does not influence neutral genetic structure. To explore whether the apparent evolutionary limit towards increased desiccation could be overcome, I investigated whether a population bottleneck could increase VA for desiccation resistance in D. bunnanda by creating replicate single pair bottleneck lines. I found both epistatic and dominance components released by population bottleneck increased additive genetic variance and narrow sense heritability to a significant level in some lines, but no selection response was observed.