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Functional characterisation of Epiphyas postvittana odorant receptor 1 48 Table 2.1: Change in fluorescence at 10 -5 M of the compounds tested with EpOR1 in the Sf9 cell assay. Each odorant was tested in triplicate from a single transfection mix. Compound Group Compound Name ∆F a N c Monoterpene Limonene NR b 0 Myrcene NR 0 -pinene NR 0 Linalool NR 0 Citronellol NR 0 -terpineol NR 0 1,8 cineole 0.43 ± 0.25 6 Nerol 0.41 ± 0.14 8 Geraniol 0.31 ± 0.03 8 Citral 0.32 ± 0.02 7 Geranial 0.36 ± 0.02 6 Geranyl acetate 0.32 ± 0.02 6 Sesquiterpene -farnesene 0.41 ± 0.12 7 Caryophyllene NR 0 Humulene NR 0 Triterpene Squalene NR 0 Alcohol Hexanol NR 0 Nonanol NR 0 Pentanol NR 0 Octanol 0.25 ± 0.21 6 Aldehyde Hexanal NR 0 Ester Ethyl butyrate NR 0 Ethyl octanoate NR 0 Ethyl hexanoate NR 0 Propyl propanoate NR 0 Pentyl acetate 0.37 ± 0.31 8 Hexyl acetate NR 0 Dodecyl acetate NR 0 Octyl acetate NR 0 Propyl acetate NR 0 (E)-11-tetradecenyl acetate NR 0 Benzoate Eugenol NR 0 Methyl salicylate 0.31 ± 0.03 6 a. Average change in fluorescence ± SE of at least three cells. b. NR = no response as determined by no change in fluorescence of the cells upon stimulation with the odorant in triplicates at 10 -5 M. c. N = number of responding cells. A minimum of 300 cells were looked at to confirm that a compound did not elicit a calcium response in the cells.
Functional characterisation of Epiphyas postvittana odorant receptor 1 49 To examine the sensitivity of EpOR1 for the ten responding ligands, calcium assays were conducted over a range of ligand concentrations to enable the construction of dose response curves. Ligands were tested at ten-fold decreasing concentrations starting at 10 -5 M. Only five of the ten compounds (methyl salicylate, geranyl acetate, citral, geraniol and geranial) elicited a calcium response at concentrations lower than 10 -5 M and the compounds differed in their levels of sensitivity as shown in Figure 2.4. A relative measure of the difference between the binding affinities of EpOR1 to the different ligands can be represented by comparing estimated EC50 values, the concentration at which 50% of the maximal effect or activation of the ligand is observed (Motulsky and Christopoulos, 2003). The level of cooperativity of the ligands is given by the slope of the dose response curve and is calculated as the Hill slope. The lowest concentration of ligand to which a response of the cells was observed varied between the five best ligands and is given in Table 2.2, together with the EC50 and Hill slopes from the dose response curves.
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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
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:
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Page 25 and 26: General Introduction 9 OBPs have al
Page 27 and 28: General Introduction 11 A model has
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
Page 41 and 42: General Introduction 25 leaves and
Page 45 and 46: General Introduction 29 Expression
Page 47 and 48: General Introduction 31 BmOr2 HvOr2
Page 49 and 50: General Introduction 33 for lepidop
Page 51 and 52: Functional characterisation of Epip
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Page 73 and 74: 3.1 Introduction 3 The roles of Epi
Page 75 and 76: The roles of Epiphyas postvittana G
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Page 97 and 98: 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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