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The roles of Epiphyas postvittana GOBP2 in odour detection 70 For the seven ligands identified in the scope of this study, octanol had the highest binding capacity for EpGOBP2, with 99 µM of octanol binding to 5 µM of EpGOBP2 while only 9 µM of pentyl acetate bound to the same concentration of EpGOBP2 (Figure 3.5). Ten compounds were identified as ligands for EpOR1 in Chapter 2, however, at lower concentrations, EpOR1 was able to elicit a response to only five of these (geranyl acetate, citral, methyl salicylate, geraniol and geranial) hence dose response curves were able to be obtained for only these five compounds. From these five ligands of EpOR1, geranyl acetate and methyl salicylate are able to bind to EpGOBP2, hence both these compounds were used for testing the response of EpOR1 expressing Sf9 cells by reconstituting the Sf9 cell assay system with EpGOBP2. 3.3.3 Reconstituted EpOR1 receptor activation assays In order to carry out reconstituted EpOR1 receptor activation assays in the Sf9 assay system with EpGOBP2, the common ligands of both EpGOBP2 and EpOR1 for which dose response curves have been determined in Chapter 2 are used. These ligands are methyl salicylate and geranyl acetate. The dose response of EpOR1 expressed in the Sf9 cell assay to methyl salicylate and geranyl acetate was measured under the following conditions: in the absence of both EpGOBP2 and DMSO (Figures 3.8A and 3.9A, no solubilising agent present hence the odorant does not elicit a response in the EpOR1 expressing cells); in the presence of EpGOBP2 only (Figures 3.8C and 3.9C, a dose response of the EpOR1 expressing cells is observed to both geranyl acetate and methyl salicylate indicating that the GOBP2 is able to solubilise these two odorants); or DMSO only (Figures 3.8B and 3.9B, DMSO acts as a solubilising agent for methyl salicylate and geranyl acetate as determined in Chapter 2); and in the presence of both DMSO and EpGOBP2 (Figures 3.8D and 3.9D, to determine whether the changes in the EC50 values of the various dose response curves are due to the presence of EpGOBP2 or DMSO). These sets of experiements showed that EpGOBP2 can act as a solubilising agent for methyl salicylate and geranyl acetate. To show that the dose response observed for both methyl salicylate and geranyl acetate in the presence of EpGOBP2 is not due to a concentration effect of the
The roles of Epiphyas postvittana GOBP2 in odour detection 71 EpGOBP2, a no-odorant dose response control assay of the EpOR1 expressing Sf9 cells was carried out. This showed that in the absence of a ligand, EpGOBP2 does not elicit a response in EpOR1 expressing Sf9 cells, as depicted in Figure 3.7. The concentrations at which half the maximal responses (EC50) were obtained for the different test concentrations is given in Table 3.1. Figure 3.7: Dose response of EpOR1 expressing Sf9 cells to EpGOBP2 in the concentration range of 10 -5 M to 10 -13 M. Error bars represent the standard error of six to eight cells.
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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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General Introduction 7 1.5 Componen
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General Introduction 9 OBPs have al
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General Introduction 11 A model has
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General Introduction 13 Figure 1.3:
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General Introduction 15 Tanaka et a
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General Introduction 17 Table 1.1:
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Page 37 and 38: General Introduction 21 et al., 200
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Page 45 and 46: General Introduction 29 Expression
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Page 51 and 52: Functional characterisation of Epip
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Identification of putative odorant
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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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