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Functional characterisation of Epiphyas postvittana odorant receptor 1 36 Neuhaus et al., 2004; Sakurai et al., 2004; Nakagawa et al., 2005; Kiely et al., 2007; Sato et al., 2008; Anderson et al., 2009; Jordan et al., 2009). The empty-neuron system, a mutant fly strain that does not express the OR22a and OR22b receptors was originally developed for characterising Drosophila ORs (Dobritsa et al., 2003).This approach has been successfully applied to the expression and characterisation of ORs from other insects such as mosquitoes and moths (Syed et al., 2006; Kurtovic et al., 2007; Lu et al., 2007; Syed et al., 2010). Syed et al. (2006) successfully co-expressed functional B. mori OR1 with B. mori PBP in the Drosophila empty-neuron system and showed that it bound the B. mori sex pheromone, bombykol, albeit with low signal onset that lasted for an unusually long time, implying the empty-neuron expressing the BmOR1 is devoid of pheromone degrading enzymes that would otherwise degrade bombykol (Syed et al., 2006). Functional studies of moth ORs have mainly been carried out using heterologous expression systems. Several assays for characterising moth ORs have been carried out in X. laevis oocytes by two-electrode voltage clamping (Sakurai et al., 2004; Mitsuno et al., 2008; Miura et al., 2009). Briefly, the OR of interest is co-expressed with the species-specific Drosophila OR83b homologue in X. laevis oocytes and the cells are left to grow for 2–3 days at 20°C. Two-electrode voltage clamps are used for recording whole-cell currents which measures response upon stimulation with odorants as a variation from the holding voltage. Sakurai et al. (2004) expressed the B. mori pheromone receptor, BmOR1 together with BmGαq in X. laevis oocytes and showed that 10 to 15% of the transfected cells were responsive to the B. mori sex pheromone bombykol with an EC50 value of 3.4 x 10 -5 M (Sakurai et al., 2004). In a similar experiment, the B. mori homologue of OR83b, BmOR2 was co-expressed with BmOR1 and BmGαq in X. laevis oocytes (Nakagawa et al., 2005). Stimulation of the transfected cells with bombykol rendered ~95% of the transfected cells responsive, with an EC50 value of 1.5 x 10 -6 M and the lowest threshold concentration for response of 1 x 10 -7 M, indicating a role of OR83b in enhancing the sensitivity of ORs.
Functional characterisation of Epiphyas postvittana odorant receptor 1 37 Miura et al. (2009) co-expressed O. scapulalis OR1 (OscaOR1) together with its species-specific OR83b homologue in X. laevis oocytes and showed that it responded highly to E11-14:OH and a small response was shown to Z11-16:OAc (Miura et al., 2009). These compounds have been identified as pheromone components of other Ostrinia species, while the sex pheromone components of O. scapulalis (E11- and Z11- 14:OAc) did not confer a response from OscaOR1, indicating that even though OscaOR1 is homologous to the pheromone receptors of Ostrinia species, it is not specific to O. scapulalis sex pheromone. Wanner et al. (2010) expressed Ostrinia nubilalis OR1, and ORs 3–6 in X. laevis oocytes and showed that they bound all the components of the Ostrinia sex pheromone, as shown in Table 1.1 (Wanner et al., 2010). Mitsuno et al. (2008) expressed the pheromone receptor together with their species specific OR83b homologue from three different moth species (P. xylostella, M. separata and D. indica) in X. laevis oocytes and showed via electrophysiological recordings that they all bound their respective sex pheromone components with an EC50 in the micromolar range and the lowest binding threshold of 10 -7 M, values which are comparable with B. mori PR assays conducted by Nakagawa et al. (2005). Modified versions of HEK293 cells, containing a mouse Gα15 gene have also been used for successfully expressing and characterising moth ORs in fluorometric assays. Stable cell lines of H. virescens HvOR13, HvOR14 and HvOR16 have been generated and characterised to bind H. virescens sex pheromone components in HEK cells with high affinity. HvOR13 can recognise Z11-16:Al in DMSO with an EC50 of 1.2 x 10 -9 M (Große-Wilde et al., 2007). These results are comparable to an earlier study in which B. mori OR1 and OR3 were expressed and characterised in HEK293 cells. BmOR1 bound the sex pheromone bombykol with an EC50 of 10 -10 M (Große-Wilde et al., 2006). Together these results show that expression of moth ORs in HEK293 cells yield functional ORs, that are highly responsive and sensitive to their ligands. Kiely et al. (2006) developed an insect cell system for assaying insect ORs. Drosophila OR22a was transiently expressed in Sf9 cells, and after a 48 hour incubation period, the cells were loaded with the calcium sensitive dye Fluo-4 and change in fluorescence of cells in response to stimulation by odorants was measured
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Page 3 and 4: Abstract Abstract Olfaction or the
Page 5 and 6: Acknowledgements Acknowledgements I
Page 7 and 8: Contents v Contents Abstract……
Page 9 and 10: Contents 4.2.5 Degenerate PCR .....
Page 11 and 12: List of Figures List of Figures Fig
Page 13 and 14: List of Tables List of Tables Table
Page 15 and 16: List of Abbreviations gDNA Genomic
Page 17 and 18: 1.1 General overview 1 General Intr
Page 19 and 20: General Introduction 3 moth in loca
Page 21 and 22: General Introduction 5 Figure 1.1:
Page 23 and 24: General Introduction 7 1.5 Componen
Page 25 and 26: General Introduction 9 OBPs have al
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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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Identification of putative odorant
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Microarray OR 454 Transcriptome OR
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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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