Klaus Raming

A candidate olfactory receptor subtype highly conserved across different insect orders

Candidate olfactory receptors of the moth Heliothis virescens were found to be extremely diverse from receptors of the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae, but there is one exception. The moth receptor type HR2 shares a rather high degree of sequence identity with one olfactory receptor type both from Drosophila (Dor83b) and from Anopheles (AgamGPRor7); moreover, in contrast to all other receptors, this unique receptor type is expressed in numerous antennal neurons. Here we describe the identification of HR2 homologues in two further lepidopteran species, the moths Antheraea pernyi and Bombyx mori, which share 86–88% of their amino acids. In addition, based on RT-PCR experiments HR2 homologues were discovered in antennal cDNA of the honey bee (Apis mellifera; Hymenoptera), the blowfly (Calliphora erythrocephala; Diptera) and the mealworm (Tenebrio molitor; Coleoptera). Comparison of all HR2-related receptors revealed a high degree of sequence conservation across insect orders. In situ hybridization of antennal sections from the bee and the blowfly support the notion that HR2-related receptors are generally expressed in a very large number of antennal cells. This, together with the high degree of conservation suggests that this unique receptor subtype may fulfill a special function in chemosensory neurons of insects.

A divergent gene family encoding candidate olfactory receptors of the moth Heliothis virescens

The antennae of moths have been an invaluable model for studying the principles of odour perception. In spite of the enormous progress in understanding olfaction on the molecular level, for the moth one of the key elements in olfactory signalling, the odourant receptors, are still elusive. We have assessed a genome database of a heliothine moth (Heliothis virescens, Noctuidae) and employed exon‐specific probes to screen an antennal cDNA library of this species. Analysis of isolated cDNA‐clones led to the discovery of a divergent gene family encoding putative seven‐transmembrane domain proteins. The notion that they may encode candidate olfactory receptors of the moth, was supported by a tissue‐specific expression; several of the subtypes were exclusively expressed in antennae. By means of double‐labelling in situ hybridization studies it was demonstrated that the receptors are indeed expressed in antennal sensory neurons; moreover, each receptor subtype appears to be expressed in a distinct population of sensory cells. The results strongly suggest that the newly discovered gene family indeed encodes olfactory receptors of moth.

Expression of odorant receptors in spatially restricted subsets of chemosensory neurones

From a rat olfactory library a cDNA clone (OR37) which is supposed to encode an odorant receptor protein has been isolated and characterized. Specific antisense RNA and in situ hybridization techniques have been employed to monitor the olfactory epithelium for the distribution of olfactory neurones expressing the OR37-gene. The OR37-transcripts were detected only in a subset of receptor cells segregated in two restricted areas of the olfactory epithelium. The clusters of reactive cells appear symmetrically in both nasal cavities. Within a reactive region only a subset of the cells expressed the receptor. The segregation of neurones expressing a distinct receptor supports the notion that a spatial component may be involved in coding odour quality.

Odorant binding proteins of Heliothis virescens

cDNA clones coding for three different binding proteins were isolated from an antennal library of Heliothis virescens. The deduced amino acid sequences showed only moderate homology to each other but shared several common structural features. Based on a comparison with the predicted primary structures of antennal binding proteins from different moth species, one of the clones (Hel-1) was found to encode a pheromone binding protein, whereas the two others (Hel-10 and -11) encode general odorant binding proteins.

Cloning and functional expression of GABAB receptors from Drosophila

The neurotransmitter GABA (γ‐aminobutyric acid) functions as the major inhibitory neurotransmitter in the central nervous system of vertebrates and invertebrates. In vertebrates GABA signals both through ionotropic receptors (GABAA, GABAC), which induce fast synaptic inhibitory responses, and through metabotropic receptors (GABAB), which play a fundamental role in the reduction of presynaptic transmitter release and postsynaptic inhibitory potentials. Whilst GABAA and GABAC receptors have been cloned from vertebrates as well as invertebrates, GABAB receptors have only been identified in vertebrate species to date, although indirect evidence suggests their existence in arthropods, too. Here we report the cloning of three putative invertebrate GABAB receptor subtypes (D‐GABABR1, R2 and R3) isolated from Drosophila melanogaster. Whilst D‐GABABR1 and R2 show high sequence identity to mammalian GABABR1 and R2, respectively, the receptor D‐GABABR3 seems to be an insect‐specific subtype with no known mammalian counterpart so far. All three D‐GABABR subtypes are expressed in the embryonic central nervous system. In situ hybridization of Drosophila melanogaster embryos shows that two of the D‐GABABRs (D‐GABABR1 and R2) are expressed in similar regions, suggesting a coexpression of the two receptors, whilst the third D‐GABABR (D‐GABABR3) displays a unique expression pattern. In agreement with these results we have only been able to functionally characterize D‐GABABR1 and R2 when the two subtypes are coexpressed either in Xenopus laevis oocytes or mammalian cell lines, whilst D‐GABABR3 was inactive in any combination. The pharmacology of the coexpressed D‐GABABR1/2 receptor was different from the mammalian GABABRs: e.g. baclofen, an agonist of mammalian GABABRs, showed no effect.

Molecular cloning of an insect pheromone-binding protein

Pheromone binding protein; cDNA cloning; Nucleotide sequence; Primary structure; RNA blot hybridization

Cloning of genomic and complementary DNA encoding insect pheromone binding proteins: evidence for microdiversity

Genomic DNA from the silk moth Antheraea pernyi bearing the gene of a pheromone binding protein has been isolated from a partial genomic library using specific cDNA probes. The DNA spans 3.5 kilobases, contains three exons and two intervening sequences that interrupt the protein coding region of the gene. A DNA fragment of a second gene was isolated and the complete primary structure of a corresponding cDNA clone was unravelled. The expression of two different genes, giving rise to different pheromone binding proteins, implies a more specific function of these proteins than was hitherto assumed.

A novel class of binding proteins in the antennae of the silk moth Antheraea pernyi

Abstract Using recombinant DNA approaches a cDNA clone was isolated from antennal libraries of Antheraea pernyi which encodes the complete sequence of a putative pheromone or odorant binding protein. The deduced protein sequence consist of a signal peptide of 19 amino acids and a mature protein of 141 amino acid residues. It shares a number of structural features with previously identified pheromone binding proteins although the predicted primary structures show only moderate homologies; Furthermore, the identified clone is preferentially expressed in female antennae.

Primary structure of a pheromone-binding protein from Antheraea pernyi: homologies with other ligand-carrying proteins

SummaryAn antennal cDNA clone encoding the complete sequence (163 amino acids) of a pheromone-binding protein precursor from the male silk moth, Antheraea pernyi, was isolated using oligonucleotide probes. The cloned cDNA was expressed and the translation product detected by specific antibodies. The deduced protein sequence consists of a signal peptide of 21 amino acids and a mature binding protein of 142 amino acid residues. The predicted structure of this protein is homologous to binding-proteins from different insect species which have previously been identified, but shows no similarities to odorant-binding proteins from vertebrates, suggesting that soluble odorant-binding proteins in insects and vertebrates represent an evolutionary convergence.

Odorant-sensitive phospholipase C in insect antennae.

Exogenous tritiated phosphatidylinositol bisphosphate added to antennal preparations from locust and cockroach was hydrolysed releasing inositol trisphosphate. High activity of phospholipase C was detected in the soluble as well as in the membrane fraction. At low free calcium concentrations hydrolysis of the labelled lipid was stimulated by odorants and pheromones in a GTP-dependent manner. Consequently the level of inositol trisphosphate in antennal preparations increased upon odorant stimulation.