“Plus-C” odorant-binding protein genes in two Drosophila species and the malaria mosquito Anopheles gambiae
Introduction
Insect odorant-binding proteins (OBPs) are present at millimolar concentration in the olfactory sensillum lymph, where they receive and transport semiochemicals to the olfactory neurons and hence play an important role in the signal transduction of insect olfaction (Pelosi and Maida, 1995). It has been suggested that OBPs are not merely passive carriers of chemical signals but also show molecular recognition and are necessary for the binding of odorants to their receptors (ORs) (Pelosi and Maida, 1995). This view is clearly supported by more recent studies which have identified a diversity of OBPs within a species, for example in the tobacco hornworm Manduca sexta (Robertson et al., 1999) and the fruit fly Drosophila melanogaster Galindo and Smith, 2001, Hekmat-Scafe et al., 2002. In addition, some species, including D. melanogaster, have different OBPs associated with functionally distinct classes of olfactory sensilla Hekmat-Scafe et al., 1997, Park et al., 2000, Shanbhag et al., 2001 and each class of sensilla responds to a specific class of odours (Stensmyr et al., 2003) again suggesting a role in signal filtering. Since the first OBPs were reported in the Lepidoptera, many papers have been published describing the proteins and associated genes in a wide range of insect species in the orders Coleoptera (Nikonov et al., 2002), Hymenoptera (Briand et al., 2001), Diptera (Kim et al., 1998), Orthoptera (Picone et al., 2001), Dictyoptera (Riviere et al., 2003) and Heteroptera (Vogt et al., 1999). Although OBPs show little sequence similarity between species there is always conservation of six cysteines Pelosi and Maida, 1995, Pikielny et al., 1994, Field et al., 2000 and indeed this has become the hallmark by which genes are ascribed an OBP function even in the absence of functional data (Vogt et al., 1999). The best studied OBP is the Bombyx mori pheromone-binding protein 1 (BMPBP1) where the precise bridging between the cysteines has been established for the native protein (Scaloni et al., 1999) and recombinant protein has been used to study the PBP/pheromone complex by electrospray ionization mass spectrometry (Oldham et al., 2000) and X-ray crystallography Sandler et al., 2000, Lee et al., 2002.
In D. melanogaster, seven genes encoding putative OBPs have been cloned and sequenced. These are the OBP LUSH (Kim et al., 1998), the olfactory-specific proteins OS-E and OS-F (McKenna et al., 1994) and the pheromone-binding proteins PBPRP1, PBPRP2, PBPRP4 and PBPRP5 (Pikielny et al., 1994). Deletion of the LUSH gene results in abnormal attraction to food sources with high concentrations of ethanol, suggesting that LUSH plays a direct role in D. melanogaster olfaction Kim et al., 1998, Kim and Smith, 2001.
With the publication of the D. melanogaster genome in 2000, it became possible for the first time to look at how many OBP genes an insect might have. Galindo and Smith (2001) used the tBLASTn algorithm, along with comparisons with the accepted characteristics of OBPs, and estimated that the D. melanogaster genome had 35 putative OBP genes. This was later extended to 38 by Graham and Davies (2002), and Hekmat-Scafe et al. (2002) annotated 51 OBP genes of which, 32 encoded proteins with the typical six-cysteine motif. The other 19 were in two distinct subgroups, which Hekmat-Scafe et al. (2002) named the “minus-C” subgroup (seven proteins) with some cysteine residues missing, and the “Plus-C” subgroup (12 proteins), all with three extra cysteines and a conserved proline. The genome organisation of 23 D. melanogaster OBPs has also been analysed showing that they group at 12 loci with three groups with different exon boundaries (Vogt et al., 2002). The tissue-specific expression of many six-cysteine OBPs has also been determined indirectly, using OBP promoter-driven expression of the LacZ reporter gene (Galindo and Smith, 2001) and RT-PCR (Koganezawa and Shimada, 2002); however, in situ hybridisation has failed to demonstrate the expression of the Plus-C OBPs (Hekmat-Scafe et al., 2002), and indeed to date there is no evidence of an olfactory function for these putative OBPs. In this study, we have used semiquantitative RT-PCR to examine the tissue distribution of transcripts of each member of the putative Plus-C OBP genes and two dimer OBPs in D. melanogaster. We have also looked at the presence of such genes in other insects where the genome sequences are publicly available.
Section snippets
Insect material
The D. melanogaster, strain Canton S, were a gift from Dr. John Brookfield (University of Nottingham). Antennae, heads, legs and winged bodies of mixed sexes were separated on ice under a microscope and frozen immediately in liquid nitrogen.
RT-PCR
Each tissue was ground in liquid nitrogen, and total RNA extracted using the TRIZOL® reagent (Invitrogen). Messenger RNA was purified with the Dynalbeads purification kit (Dynal) and cDNA was synthesized using the SuperScript™ first-strand synthesis system
Expression of Drosophila Plus-C OBP and dimer OBP genes
Semiquantitative RT-PCR, using gene-specific primers along with primers for a control actin gene, was used to examine relative transcript levels in D. melanogaster tissues for all putative Plus-C OBP genes, the two dimer OBPs (OBP83CD and OBP83EF, see later) and two regular six-cysteine OBPs (LUSH and CG11218). The results for six of the genes are shown in Fig. 1 and a summary of the data for all genes is given in Fig. 2. One of the six-cysteine genes, LUSH, shows expression in antennae with
Conclusions
The challenge of annotating eukaryotic genomes has been comprehensively set out by Lewis et al. (2000). The use of algorithms and sequence similarity searches to identify putative gene functions can clearly play a role as applied by the present and previous studies for OBPs Galindo and Smith, 2001, Graham and Davies, 2002 and olfactory receptors (ORs) Kim and Carlson, 2002, Hill et al., 2002. Of course, the genes identified in this way are only “putative” OBPs and ORs. The value of such in
Acknowledgements
We thank the Drosophila genome project consortium, especially Prof. Michael Ashburner for making this work possible. WH and GZ were sponsored by the Chinese Government for overseas study. Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the UK.
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