A novel olfactory receptor gene family in teleost fish

Abstract

While for two of three mammalian olfactory receptor families (OR and V2R) ortholog teleost families have been identified, the third family (V1R) has been thought to be represented by a single, closely linked gene pair. We identified four further V1R-like genes in every teleost species analyzed (Danio rerio, Gasterosteus aculeatus, Oryzias latipes, Tetraodon nigroviridis, Takifugu rubripes). In the phylogenetic analysis these ora genes (olfactory receptor class A-related) form a single clade, which includes the entire mammalian V1R superfamily. Homologies are much lower in paralogs than in orthologs, indicating that all six family members are evolutionarily much older than the speciation events in the teleost lineage analyzed here. These ora genes are under strong negative selection, as evidenced by very small d(N)/d(S) values in comparisons between orthologs. A pairwise configuration in the phylogenetic tree suggests the existence of three ancestral Ora subclades, one of which has been lost in amphibia, and a further one in mammals. Unexpectedly, two ora genes exhibit a highly conserved multi-exonic structure and four ora genes are organized in closely linked gene pairs across all fish species studied. All ora genes are expressed specifically in the olfactory epithelium of zebrafish, in sparse cells within the sensory surface, consistent with the expectation for olfactory receptors. The ora gene repertoire is highly conserved across teleosts, in striking contrast to the frequent species-specific expansions observed in tetrapod, especially mammalian V1Rs, possibly reflecting a major shift in gene regulation as well as gene function upon the transition to tetrapods.

Figure 1.

Phylogenetic tree of the fish Ora family. (A) Twenty-eight fish Ora (red), 15 frog Ora (green) with some mammalian V1R (light blue) representatives and T2R (orange) as the closest relatives. (B) Twenty-eight fish ora genes. Trees were constructed using the NJ method. Bootstrap support (total 1000 replications) is indicated at the major nodes. Scale bar indicates the number of amino acid substitutions per site. *, see Pfister and Rodriguez 2005; #, see Shi and Zhang 2007. Ora, olfactory receptors of class A; T2R, putative taste receptors of type 2 (Ishimaru et al. 2005); V1R, vomeronasal type 1 receptors (Grus et al. 2005). The V1R receptors are a subset of V1Rs from all mammalian organisms annotated in the NCBI database (mouse, rat, human) and described in publications (opossum, cow, dog; Grus et al. 2005). The phylogenetic position of the full mammalian V1R set of annotated and published genes is identical (cf. Supplemental Fig. 1).

Figure 2.

Conserved sequence motifs of the Ora family. Conservation of predicted amino acid sequence for the fish Ora repertoire is displayed as a sequence logo. In this representation, the relative frequency with which an amino acid appears at a given position is reflected by the height of its one-letter amino acid code in the logo, with the total height at a given position proportional to the level of sequence conservation. The regions corresponding to the transmembrane (TM) domains and the intracellular and intracellular domains (EC and IC) are numbered and indicated. Sequence alignments were manually edited (for details, see Methods). Of 14 motifs conserved in V1Rs (all of them single amino acids, identified by Rodriguez et al. 2002) eight are not conserved in ORs (cf. Niimura and Nei 2005) and consequently were chosen as analytical criterion here (*). (+) Residues conserved in ORs, some also in other GPCR families; (○) residues conserved in fish ora genes, but not in mammalian V1R genes.

Figure 3.

Identity, similarity, and conservation level of the ora genes. (A) d_N/d_S ratios of the six ora genes. For each gene, the d_N/d_S ratio was determined for all possible pairwise comparisons between orthologs and the mean value was plotted. (B) Amino acid percent identity is calculated for each gene comparisons between orthologs or species (paralog comparisons) by averaging the values of all possible pairwise comparisons inside the described group. (C) Amino acid % similarity is calculated for each gene or species by averaging the values of all possible pairwise comparisons inside the described group. (A–C) The bars correspond to the associated standard deviation as a measure of the variance within each group. (B,C) Differences between values for ortholog and paralog comparisons are highly significant.

Figure 4.

Sites under positive and negative selection in ora coding sequences. A schematic representation of site-by-site selective pressure is shown on ora receptor sequences drawn based on the edited nucleotide alignment, which was generated from the corresponding amino acid alignment used for Fig. 3. SLAC analysis shows the probability of sites being under selective pressure (negative selection in light blue (P < 0.2) or blue (P < 0.1), neutral selection in gray, positive selection not observed even at P < 0.2 level. All orthologs of each gene were used for this analysis; the results for three genes are shown: (A) ora1, (B) ora2, (C) ora4.

Figure 5.

Genomic structure of the 28 fish ora genes. Predicted exon/intron structure is drawn to scale for all the ora genes: six zebrafish oras (Dr, Danio rerio), six medaka (Ol, Oryzias latipes), six stickleback (Ga, Gasterosteus aculeatus), five fugu (Tr, Takifugu rubripes), and five tetraodon (Tn, Tetraodon nigroviridis) oras. Exons are represented by the black filled rectangles and introns are represented by the black line connecting the exons.

Figure 6.

Genomic arrangement of the ora1–ora2 and ora3–ora4 gene pairs. Exons are represented by the black filled rectangles, introns are represented by the gray filled rectangles, and the thick line represents the intergenic distance between the two members of a gene pair. All elements are drawn to scale.

Figure 7.

Expression of ora transcripts in the zebrafish olfactory system. (A) Expression of ora mRNA detected by RT-PCR. PCR amplifications were performed by using gene-specific primers (arrows above the gene structure scheme). BL, barbels + lips; OE, olfactory epithelium; OB, olfactory bulb; B, brain; G, gills; H, heart; L, liver; Gen, genomic DNA. Actin, both plus and minus RT, and genomic DNA as template for all oras and actin were used as controls. The single actin band as well as the absence of actin amplification products in the “minus RT” condition confirm the absence of genomic DNA contamination. That genomic DNA, if present, would have generated a visible amplification product is shown in the lane labeled “Gen.” Gel sections shown all correspond to the 400–900 bp range, with exception of the much larger genomic product of ora4. The weak band with ora3 in liver cDNA might be due to minor ectopic expression, as has been reported for several olfactory receptor genes (e.g., Vanderhaeghen et al. 1993). Arrows in the actin rows point to the expected position for the cDNA product. (B) Schematic representation of the localization of the OE followed by a drawing of a horizontal section of OE (lamellae are cut perpendicular to their flat face) and finally an enlargement of two lamellae. The central rose-colored area in the lamellae indicates the location of the sensory neuroepithelium (cf. Weth et al. 1996), gray lines, basal lamina, gray jagged spots, lumen. (C) In situ hybridizations with ora1, ora2, ora3, ora4, ora5, and ora6 in horizontal sections of the OE, with antisense RNA probes. The area shown corresponds roughly to one half of the schematical representation in the right panel of B. The black asterisks indicate the lumen. Each half-lamella is enclosed by dashed lines, thicker in the apical region and thinner in the basal region adjoining the basal lamina. Red arrowheads point to the labeled neurons.