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The roles of Epiphyas postvittana GOBP2 in odour detection 58 Different methods have been used to examine the roles of PBPs both in vivo and in vitro. Electrophysiological recordings of single antennal branches in A. polyphemus upon stimulation with pheromone have shown that ApolPBP decreases the concentration of pheromone required to elicit ORN response by 100-fold, hence indicating a role of ApolPBP as a solubiliser of the pheromone (van den Berg and Ziegelberger, 1991). In in vitro characterisation assays using calcium imaging of HEK293 cells expressing BmOR1 and HvOR13, organic solvents were successfully replaced by the respective species‟ PBP as the solubiliser of the pheromone (Große- Wilde et al., 2006; Große-Wilde et al., 2007). A. polyphemus PBP has also been shown by in vitro binding assays to act as a protector of the pheromone, as measured by reduced activity of PDE on radio-labelled pheromone in the presence of PBP (Vogt et al., 1985). Several assays have provided evidence for interaction of the OBP/ligand complex with the dendritic bound ORs. The ab3A neuron of Drosophila has been used for expressing BmOR1 and sensitivity of the OR to its ligand bombykol was enhanced upon co-expression of BmPBP in the empty neuron (Syed et al., 2006). Similarly, when BmOR1 was expressed in HEK293 cells, replacement of the organic solubiliser DMSO with BmPBP selectively showed activity to BmPBP-bombykol complex and the DMSO solubilised activity of bombykal was lost, as measured by calcium imaging of the BmOR1 expressing HEK293 cells (Große-Wilde et al., 2006). In vitro fluorescence measurements of endogenous tryptophan residues of BmPBP in complex with bombykol at physiological pH of 7.0 and dendritic pH of 4.7 showed a decrease in fluorescence indicating a dissociation of the OBP/ligand complex at low pH (Leal et al., 2005). A similar study has been done in A. polyphemus to show conformational changes of ApolPBP at different pH values (Mohanty et al., 2003; Mohanty et al., 2004). Other characterisation assays have also been used to deorphan insect OBPs. These include the cold binding assay, a two-phase binding assay and a volatiles odorant binding assay (Briand et al., 2001; Leal et al., 2005; Zhou et al., 2009). The cold binding assay allows for testing binding of ligands to OBPs at different test conditions such as pH and temperature and is able to separate free ligand from protein bound ligand using a centrifugal filter device (Leal et al., 2005; Zhou et al., 2009). In this assay, the test odorants are added to the protein solution and the mixture left to incubate. The mixture is then centrifuged to remove unbound odorants in the filtrate
The roles of Epiphyas postvittana GOBP2 in odour detection 59 while the protein bound ligands are maintained in the retentate. The ligand is released from the protein by altering the pH of the mixture and eluted in a layer of hexane, which is then analysed for the presence of the ligands that were bound to the protein by gas chromatography (GC). Recently, Zhou et al. (2009) proposed the two-phase binding assay, due to the potential release of the ligand during the washing step in cold binding assay. In this assay, the ligands are dissolved in a layer of hexane that covers the protein solution, and the depletion of the odorants in the hexane layer is measured as the uptake of odorants by the proteins in the bottom layer. The identification of ligands for proteins, especially if the test odorants are hydrophobic volatiles becomes difficult due to the tendency for such odorants to adhere to surfaces such as test vial walls and if mixed directly to the solution, may form micelles. The technique developed in the volatiles odorant binding assay (VOBA) circumvents these problems by introducing the volatile slowly into the protein solution via diffusion through a gas phase intermediate (Briand et al., 2001). This technique was successfully developed and used by Briand et al. (2000) to characterise an OBP isolated from rat nasal mucus. It has also been shown to facilitate the uptake of a range of compounds by the honeybee Apis mellifera OBP (Briand et al., 2001). The VOBA technique introduces the volatile slowly, molecule by molecule so the distribution of odorants in the protein solution is uniform. An excess of the odorant is used and the reaction is allowed to reach equilibrium so any adherence of the odorant to the walls of the test vial becomes negligible. In E. postvittana, three PBPs and one GOBP were identified from native protein gel analysis and one PBP and another GOBP each identified from EST sequences (Newcomb et al., 2002; Jordan et al., 2008). All the E. postvittana PBPs and one GOBP have been shown on western blot and by qRT-PCR analysis to be expressed in both male and female antennae, albeit with varying levels. Two of the PBPs were shown to have higher expression in male than female antennae. Further sequence analysis revealed them to be allelic variants of the same PBP, consisting of six amino acid substitutions and differing in their electrophoretic properties, thus they were named PBP1-fast and PBP1-slow. Both the PBP1 proteins bind the major sex pheromone component of E. postvittana, (E)-11-tetradecenyl acetate in gel assays. The third PBP, called PBP2 was shown to have higher expression in female than male
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Abstract Abstract Olfaction or the
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Acknowledgements Acknowledgements I
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Contents v Contents Abstract……
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Contents 4.2.5 Degenerate PCR .....
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List of Figures List of Figures Fig
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List of Tables List of Tables Table
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List of Abbreviations gDNA Genomic
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1.1 General overview 1 General Intr
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General Introduction 3 moth in loca
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General Introduction 5 Figure 1.1:
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Page 31 and 32: General Introduction 15 Tanaka et a
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Page 37 and 38: General Introduction 21 et al., 200
Page 39 and 40: General Introduction 23 the attract
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Page 45 and 46: General Introduction 29 Expression
Page 47 and 48: General Introduction 31 BmOr2 HvOr2
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Page 51 and 52: Functional characterisation of Epip
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Identification of putative odorant
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5.1 Introduction 5 Concluding Discu
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Concluding Discussion 127 VOBA sett
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Concluding Discussion 129 redundant
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Concluding Discussion 131 against t
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Concluding Discussion 133 sequencin
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Appendix B 135 Buffered TB (Sambroo
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Appendix C 137 C. Appendix C - Clus
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Appendix D 139 D. Appendix D - Solu
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Appendix E 141 I II
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Appendix E 143 V
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Appendix E 145 VII continued Figure
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References 147 Ansebo, L., Ignell,
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References 149 Butenandt, A., Beckm
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References 151 Fedurco, M., Romieu,
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References 153 Große-Wilde, E., St
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References 155 Hrdy, I., F. Kuldova
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References 157 Kaissling, K. E. (19
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References 159 Krieger, J., Große-
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References 161 Lopez-Gomez, R. and
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References 163 Miura, N., Nakagawa,
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References 165 Pell, J. K., Macaula
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References 167 Røstelien, T., Stra
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References 169 Stowers, L. and Loga
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References 171 Turner, C. T., Davy,
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References 173 Vogt, R. G., Riddifo
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References 175 Widmayer, P., Heifet
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