Diversification of olfactory receptor genes in the Japanese medaka fish, Oryzias latipes

Abstract

Vertebrate olfactory receptors (OR) exists as the largest multigene family, scattered throughout the genome in clusters. Studies have shown that different animals possess remarkably diverse set of OR genes to recognize diverse odor molecules. In order to examine the evolutionary process of OR diversification, we examined three OR gene subfamilies from Japanese medaka fish (seven lines sampled from four populations). For each subfamily, the sequences of ancestral genes were inferred based on distance method. Examination of d_N/d_S ratios for each branch of phylogenetic trees suggested that purifying selection is the major force of evolution in medaka OR genes. However, for the mfOR1 and mfOR2 paralogous gene pairs, a nonrandom distribution of fixed amino acid changes and the d_N>d_S in a branch suggested that diversifying selection occurred after gene duplication. The fixed amino acid changes were observed in the third, fifth and sixth transmembrane domains, which has been predicted to serve as a ligand-binding pocket in a structural model. Compatibility test suggested that interlocus recombinations involving the fourth transmembrane domain occurred between the mfOR1 and mfOR2 gene pairs. The pattern of nucleotide substitutions in other OR genes agrees with the hypothesis that a limited number of amino acid residues are involved in odorant binding. Such comparative analyses of paralogous OR genes should provide bases for understanding the evolution, the structure, and the functional specificity of OR genes.

Introduction

Olfaction is, for most animals, a crucial sensory modality for controlling many aspects of behavior; seeking for specific food and mate, and escaping from danger. Odor recognition begins with the binding of odorants to olfactory receptor proteins located in the membrane of the olfactory neuron cilia. This activates an inner cellular signal transduction pathway, transmitting neuronal excitation to the central nervous system leading to odorant recognition. OR has been characterized in Fish, amphibia, birds, mammals, as well as in Drosophila and Caenorhabditis elegans (reviewed in Dryer and Berghard, 1999). It comprise the largest family of G-protein coupled receptors and is encoded by multigene family of unparalleled size and diversity. The size of the OR gene family is estimated to be about 1000 in mouse and about 100 in catfish (Buck and Axel, 1991, Ngai et al., 1993). Each OR gene is expressed in different olfactory neuron, and presumably bind different sets of odorant molecules (Malnic et al., 1999). OR genes are disposed in clusters on several chromosomes, which suggests that the OR gene family was built after complex multistep duplication of individual genes (Glusman et al., 1996) and of gene clusters (Lancet and Ben-Arie, 1993, Trask et al., 1998).

Overall amino acid identity within vertebrate species ranges from 20% to >90%. The genes with high amino acid identity (i.e. above 60%) are conventionally called subfamilies. There are no similarities in the amino acid sequences among vertebrates, Drosophila and C. elegans olfactory receptors, and a remarkable diversity of OR genes is present both within and across species. Phylogenetic analysis suggests that OR gene families explosively emerged several times in an independent manner in the early stages of evolution (Dryer and Berghard, 1999). What caused the emergence of OR genes and how duplicated OR gene pairs acquired new function is not known, however, it is likely that ORs evolved in response to the variety of odors in the surrounding. Recent study in human suggests impact of gene conversion and weak positive selection on the evolution of the olfactory repertoire (Gilad et al., 2000). However, studies from other organisms are necessary to elucidate the diversification process of olfactory receptor gene family.

Medaka is a freshwater fish native to East Asia with a small genome size of 680–850 M (approximately one half of zebra fish and twice as large as pufferfish) and a detailed linkage map (Naruse et al., 2000). The Japanese medaka is divided into four genetically different groups, the Northern population (i.e. the northern coast of the Sea of Japan), the Southern population (i.e. eastern, western and southern Japan), the East Korean population (i.e. East and South Korea) and the China-Korean population (i.e. China and West Korea). The genetic diversity estimated between these populations is known to be as large as interspecific comparisons (Sakaizumi et al., 1983). We have previously identified four OR genes, mfOR1–mfOR4, from a Japanese medaka (Oryzias latipes; Sun et al., 1999). Recently, Yasuoka et al. (1999) independently cloned seven Japanese medaka ORs, E1–E4 and Y1–Y3, which belonged to the same subfamily as mfOR3 and mfOR4, respectively. High sequence similarity and the organization of the genes belonging to each subfamily indicate that they are apparently paralogous genes evolved from one another through gene duplication (Yasuoka et al., 1999).

In this study, we have newly determined nucleotide sequences of the four OR genes (mfOR1–mfOR4) from five medaka strains. We examined the characteristics of intraspecific variation within each locus and genetic differences between the paralogous OR genes. The evolutionary process underlying the diversification of OR genes are examined.