Genetics - Theses
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ItemThe genetics of DDT resistance in Drosophila melanogaster: novel insights into an old debate( 2014)The genetics of DDT resistance in Drosophila melanogaster has been a topic of debate for over 60 years. Beginning with James Crow’s observation that his lab selected flies exhibited polygenic inheritance for DDT resistance, the old debate has concerned itself with what mode of inheritance – polygenic or monogenic – best explains DDT resistance in D. melanogaster. The field has had many important contributions, and both the polygenic and monogenic “schools” can muster substantial evidence in their favour. However a reading of the relevant literature, and an appraisal of developments in the field of population genetics should suggest that these seemingly dichotomous views are not mutually exclusive. We may expect to find that DDT resistance factors span a range of effect sizes, some large perhaps many more of smaller effect. The problem has been that very little effort has been deployed in a quantitative analysis of DDT resistance. Instead of asking do x genes confer resistance or not, we should perhaps ask how much of the variation in DDT resistance does each gene explain. While DDT resistance in the majority of resistant D. melanogaster strains can be said to not be monogenic, very few if any resistance genes have been identified with them. In contrast the monogenic school has provided an extensively validated resistance gene, the Cytochrome P450 Cyp6g1. However the old debate about genetic architectures is reflected in a newer debate about Cyp6g1’s contribution to DDT resistance. Can it explain the resistance seen in lab selected strains? Is it only selected in field populations? It would appear that some of this confusion stems from previously undiscovered functional variation at Cyp6g1. The second chapter of this thesis dissects an adaptive allelic series at Cyp6g1, and determines that it contributes greatly to variation in DDT resistance in field populations. However it can only explain half the genetic variation in DDT resistance at most. The third chapter of this thesis reports on a Genome Wide Association Study for DDT resistance in another D. melanogaster population that identifies novel DDT resistance genes, but perhaps paradoxically finds that Cyp6g1 does not contribute to DDT resistance in this population. Previously such an observation would provide grist for the mill in minimising Cyp6g1’s role in DDT resistance. However a quantitative genetics approach suggests that this discrepancy merely reflects that fact that resistance alleles of Cyp6g1 have already swept through most populations of D. melanogaster – it was but is no longer a DDT resistance factor in some populations. In contrast the newly identified DDT resistance loci do not bear the footprints of recent positive selection. This result neatly demonstrates the difference between the genetic basis of standing variation versus the adaptive response, but also points to the challenge in identifying a polygenic signal of adaptation in the background of a strong single gene sweep.
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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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ItemIdentification and characterisation of copper homeostasis genes( 2010)Copper is essential for life, yet also potentially toxic in excess. Copper homeostasis is therefore regulated at the cellular, tissue and organismal levels. Studies with eukaryotic model systems, primarily yeast and mammals, have identified conserved mechanisms for copper uptake, distribution, sequestration and efflux. Nevertheless, there is much we do not know about how copper levels are sensed and regulated. Additional proteins involved in copper homeostasis under both ‘normal’ and ‘diseased’ conditions remain to be identified. The emergence of Drosophila melanogaster as a bona fide model system for the study of copper homeostasis has coincided with research aiming to identify and characterise novel copper regulatory genes in this organism. Whereas others have focused on in vivo studies, the studies reported in this thesis are focussed on cultured D. melanogaster S2 cells. The first aim of this project was to use cDNA microarrays to identify genes transcriptionally regulated by copper levels in S2 cells. The second and third aims were to characterise the function and localisation of novel copper homeostasis genes in vitro and in vivo respectively. Initial characterisation of D. melanogaster S2 cells found these cells express orthologues of key mammalian copper regulatory genes. Copper uptake primarily occurs via Ctr1A and Ctr1B, the orthologues of human Ctr1. Copper efflux occurs via DmATP7, the sole D. melanogaster orthologue of the mammalian P-type ATPases, ATP7A and ATP7B. S2 cells are highly copper tolerant and primarily rely on metallothionein-mediated copper sequestration and copper efflux to maintain homeostasis. Whereas ATP7A and ATP7B undergo copper-induced trafficking between the trans-Golgi network and plasma membrane of mammalian cells, this does not appear to occur with endogenous DmATP7 in cultured D. melanogaster cells. Interestingly, when expressed in mammalian cells DmATP7 does undergo copper-induced trafficking to the plasma membrane and can facilitate copper efflux, demonstrating functional conservation of localisation and trafficking motifs in these P-type ATPases. Malvolio, the orthologue of Divalent Metal ion Transporter 1, also contributes to copper uptake in S2 cells and D. melanogaster, with impaired function associated with sensitivity to copper limitation. D. melanogaster therefore utilises this general metal transporter in addition to the copper-specific Ctr1 pathway. cDNA microarrays were used to identify genes transcriptionally regulated by copper in S2 cells, with RNA interference used to determine whether candidate genes could affect copper homeostasis. Several components of the COPI vesicle trafficking pathway, including ADP-ribosylation factor 1 (Arf1), were found to affect copper levels in S2 cells. Arf1 was found to have a conserved role in regulating copper uptake in cultured mammalian and D. melanogaster cells, and this is likely to be mediated via the localisation of Ctr1 at the plasma membrane. Taken together these studies demonstrate the value of D. melanogaster S2 cells for the study of copper homeostasis, thereby complementing the D. melanogaster model system. These novel findings should stimulate additional research in both D. melanogaster and mammalian systems and facilitate a greater understanding of copper homeostasis.