The olfactory receptor gene superfamily: data mining, classification, and nomenclature

The vertebrate olfactory receptor (OR) subgenome harbors the largest known gene family, which has been expanded by the need to provide recognition capacity for millions of potential odorants. We implemented an automated procedure to identify all OR coding regions from published sequences. This led us to the identification of 831 OR coding regions (including pseudogenes) from 24 vertebrate species. The resulting dataset was subjected to neighbor-joining phylogenetic analysis and classified into 32 distinct families, 14 of which include only genes from tetrapodan species (Class II ORs). We also report here the first identification of OR sequences from a marsupial (koala) and a monotreme (platypus). Analysis of these OR sequences suggests that the ancestral mammal had a small OR repertoire, which expanded independently in all three mammalian subclasses. Classification of ``fish-like' (Class I) ORs indicates that some of these ancient ORs were maintained and even expanded in mammals. A nomenclature system for the OR gene superfamily is proposed, based on a divergence evolutionary model. The nomenclature consists of the root symbol `OR', followed by a family numeral, subfamily letter(s), and a numeral representing the individual gene within the subfamily. For example, OR3A1 is an OR gene of family 3, subfamily A, and OR7E12P is an OR pseudogene of family 7, subfamily E. The symbol is to be preceded by a species indicator. We have assigned the proposed nomenclature symbols for all 330 human OR genes in the database. A WWW tool for automated name assignment is provided. Received: / Accepted:     

Similar numbers but different repertoires of olfactory receptor genes in humans and chimpanzees

Animals recognize their external world through the detection of tens of thousands of chemical odorants. Olfactory receptor (OR) genes encode proteins for detecting odorant molecules and form the largest multigene family in mammals. It is known that humans have fewer OR genes and a higher fraction of OR pseudogenes than mice or dogs. To investigate whether these features are human specific or common to all higher primates, we identified nearly complete sets of OR genes from the chimpanzee and macaque genomes and compared them with the human OR genes. In contrast to previous studies, here we show that the number of OR genes ( approximately 810) and the fraction of pseudogenes (51%) in chimpanzees are very similar to those in humans, though macaques have considerably fewer OR genes. The pseudogenization rates and the numbers of genes affected by positive selection are also similar between humans and chimpanzees. Moreover, the most recent common ancestor between humans and chimpanzees had a larger number of functional OR genes (>500) and a lower fraction of pseudogenes (41%) than its descendents, suggesting that the OR gene repertoires are in a phase of deterioration in both lineages. Interestingly, despite the close evolutionary relationship between the 2 species, approximately 25% of their functional gene repertoires are species specific due to massive gene losses. These findings suggest that the tempo of evolution of OR genes is similar between humans and chimpanzees, but the OR gene repertoires are quite different between them. This difference might be responsible for the species-specific ability of odor perception.     

Local and cis effects of the H element on expression of odorant receptor genes in mouse

From the approximately 1,200 odorant receptor (OR) genes in the mouse genome, an olfactory sensory neuron is thought to express only one gene. The mechanisms of OR gene choice are not understood. A 2.1 kilobase region (the H element) adjacent to a cluster of seven OR genes has been proposed as a trans- and pan-enhancer for OR gene expression. Here, we deleted the H element by gene targeting in mice. The deletion abolishes expression of a family of three OR genes proximal to H, and H operates in cis on these genes. Deletion of H has a graded effect on expression of a distal group of four OR genes, commensurate with genomic distance. There is no demonstrable effect on expression of OR genes located outside the cluster. Our findings are not consistent with the hypothesis of H as an essential trans-acting enhancer for genome-wide regulation of OR gene expression.     

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

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.     

The vestigial olfactory receptor subgenome of odontocete whales: phylogenetic congruence between gene-tree reconciliation and supermatrix methods

The macroevolutionary transition of whales (cetaceans) from a terrestrial quadruped to an obligate aquatic form involved major changes in sensory abilities. Compared to terrestrial mammals, the olfactory system of baleen whales is dramatically reduced, and in toothed whales is completely absent. We sampled the olfactory receptor (OR) subgenomes of eight cetacean species from four families. A multigene tree of 115 newly characterized OR sequences from these eight species and published data for Bos taurus revealed a diverse array of class II OR paralogues in Cetacea. Evolution of the OR gene superfamily in toothed whales (Odontoceti) featured a multitude of independent pseudogenization events, supporting anatomical evidence that odontocetes have lost their olfactory sense. We explored the phylogenetic utility of OR pseudogenes in Cetacea, concentrating on delphinids (oceanic dolphins), the product of a rapid evolutionary radiation that has been difficult to resolve in previous studies of mitochondrial DNA sequences. Phylogenetic analyses of OR pseudogenes using both gene-tree reconciliation and supermatrix methods yielded fully resolved, consistently supported relationships among members of four delphinid subfamilies. Alternative minimizations of gene duplications, gene duplications plus gene losses, deep coalescence events, and nucleotide substitutions plus indels returned highly congruent phylogenetic hypotheses. Novel DNA sequence data for six single-copy nuclear loci and three mitochondrial genes (> 5000 aligned nucleotides) provided an independent test of the OR trees. Nucleotide substitutions and indels in OR pseudogenes showed a very low degree of homoplasy in comparison to mitochondrial DNA and, on average, provided more variation than single-copy nuclear DNA. Our results suggest that phylogenetic analysis of the large OR superfamily will be effective for resolving relationships within Cetacea whether supermatrix or gene-tree reconciliation procedures are used.     

Interchromosomal interactions and olfactory receptor choice

The expression of a single odorant receptor (OR) gene from a large gene family in individual sensory neurons is an essential feature of the organization and function of the olfactory system. We have used chromosome conformation capture to demonstrate the specific association of an enhancer element, H, on chromosome 14 with multiple OR gene promoters on different chromosomes. DNA and RNA fluorescence in situ hybridization (FISH) experiments allow us to visualize the colocalization of the H enhancer with the single OR allele that is transcribed in a sensory neuron. In transgenic mice bearing additional H elements, sensory neurons that express OR pseudogenes also express a second functional receptor. These data suggest a model of receptor choice in which a single trans-acting enhancer element may allow the stochastic activation of only one OR allele in an olfactory sensory neuron.     

Extensive gains and losses of olfactory receptor genes in mammalian evolution

Odor perception in mammals is mediated by a large multigene family of olfactory receptor (OR) genes. The number of OR genes varies extensively among different species of mammals, and most species have a substantial number of pseudogenes. To gain some insight into the evolutionary dynamics of mammalian OR genes, we identified the entire set of OR genes in platypuses, opossums, cows, dogs, rats, and macaques and studied the evolutionary change of the genes together with those of humans and mice. We found that platypuses and primates have <400 functional OR genes while the other species have 800-1,200 functional OR genes. We then estimated the numbers of gains and losses of OR genes for each branch of the phylogenetic tree of mammals. This analysis showed that (i) gene expansion occurred in the placental lineage each time after it diverged from monotremes and from marsupials and (ii) hundreds of gains and losses of OR genes have occurred in an order-specific manner, making the gene repertoires highly variable among different orders. It appears that the number of OR genes is determined primarily by the functional requirement for each species, but once the number reaches the required level, it fluctuates by random duplication and deletion of genes. This fluctuation seems to have been aided by the stochastic nature of OR gene expression.     

基因组水平蝙蝠嗅觉受体基因的分子进化

翼手目动物（俗称蝙蝠）的食性分化显著,不同食性的蝙蝠具有显著不同的嗅球大小。为了研究嗅觉是否影响了蝙蝠食性的进化,我们利用网上已公布的10种蝙蝠基因组的数据,通过同源比对的方法鉴定出所有的嗅觉受体基因,并进行嗅觉受体基因亚家族的分类,进而比较嗅觉受体基因亚家族的数目差异。结果显示,蝙蝠的嗅觉受体基因与其它哺乳动物一样,都可以分为13个单系起源的亚家族;在Yinpterochiroptera亚目中,OR1/3/7、OR2/13、OR5/8/9等3个嗅觉受体亚家族在食果蝙蝠中均发生了不同程度的扩张,基因数目显著地多于食虫蝙蝠,提示嗅觉在食果蝙蝠取食过程中具有重要的作用。因此,本研究在基因组水平上重现了蝙蝠嗅觉受体基因的进化历史,揭示了3个嗅觉受体基因亚家族的功能可能与食果蝙蝠的食性相关,突出了嗅觉对动物食性的重要作用.     

Pattern of the divergence of olfactory receptor genes during tetrapod evolution

The olfactory receptor (OR) multigene family is responsible for the sense of smell in vertebrate species. OR genes are scattered widely in our chromosomes and constitute one of the largest gene families in eutherian genomes. Some previous studies revealed that eutherian OR genes diverged mainly during early mammalian evolution. However, the exact period when, and the ecological reason why eutherian ORs strongly diverged has remained unclear. In this study, I performed a strict data mining effort for marsupial opossum OR sequences and bootstrap analyses to estimate the periods of chromosomal migrations and gene duplications of OR genes during tetrapod evolution. The results indicate that chromosomal migrations occurred mainly during early vertebrate evolution before the monotreme-placental split, and that gene duplications occurred mainly during early mammalian evolution between the bird-mammal split and marsupial-placental split, coinciding with the reduction of opsin genes in primitive mammals. It could be thought that the previous chromosomal dispersal allowed the OR genes to subsequently expand easily, and the nocturnal adaptation of early mammals might have triggered the OR gene expansion.     

One neuron-one receptor rule in the mouse olfactory system

Similar to the expression of antigen receptor genes in lymphocytes, the mammalian odorant receptor (OR) genes are expressed in a mutually exclusive and monoallelic manner in olfactory sensory neurons (OSNs). DNA rearrangement has long been regarded as a possible mechanism for the allelic exclusion of the OR genes. However, mice cloned from mature OSN nuclei expressed the full repertoire of ORs, and the possibility of irreversible gene translocation was excluded as a mechanism to activate a single OR gene in each OSN. How is allelic exclusion achieved in the olfactory system? Recent transgenic experiments indicated an inhibitory role of the OR protein in preventing further activation of other OR genes. Stochastic activation of an OR gene and negative-feedback regulation by the OR gene product might ensure the maintenance of the one neuron-one receptor rule in the mammalian olfactory system.     

嗅觉受体基因的研究进展

嗅觉在动物的生命活动中起着重要的作用, 与嗅觉相关的基因主要是嗅觉受体(Olfactory receptor, OR)基因。文章介绍了嗅觉受体基因的结构、表达调控、分布、分子进化及其多态性研究进展, 并讨论了该基因与嗅觉功能和嗅觉障碍的关系。     

Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 adrenergic receptor

Odorant receptors (ORs) provide the core determinant of identity for axons of olfactory sensory neurons (OSNs) to coalesce into glomeruli in the olfactory bulb. Here, using gene targeting in mice, we examine how the OR protein determines axonal identity. An OR::GFP fusion protein is present in axons, consistent with a direct function of ORs in axon guidance. When the OR coding region is deleted, we observe OSNs that coexpress other ORs that function in odorant reception and axonal identity. It remains unclear if such coexpression is normally prevented by negative feedback on OR gene choice. A drastic reduction in OR protein level produces axonal coalescence into novel, remote glomeruli. By contrast, chimeric ORs and ORs with minor mutations perturb axon outgrowth. Strikingly, the beta2 adrenergic receptor can substitute for an OR in glomerular formation when expressed from an OR locus. Thus, ORs have not evolved a unique function in axon guidance.     

Characterization of a cluster comprising ∼100 odorant receptor genes in mouse

With ∼1000 genes, the odorant receptor (OR) gene repertoire is the largest gene family in the mouse genome. Here we have established a 129/Sv BAC contig for mouse OR gene cluster 7 (Olfr7) on Chromosome (Chr) 9. The assembled ∼2-Mb contig consists of 75 BACs and may contain as many as 100 OR genes, or ∼10% of the mouse repertoire. Facilitated by the lack of introns in the coding region, we have determined the nucleotide sequence of 37 full-length, 2 partial, and 3 pseudo coding regions. These 42 OR genes and 3 additional OR genes previously mapped to the mouse Olfr7 cluster can be organized into 13 classes based on OR probe cross-hybridizations with 129/Sv mouse genomic DNA. OR genes belonging to the same class tend to be located next to each other within the cluster. Comparison of published full-length mouse and rat OR coding sequences with those identified here shows that the Olfr7 OR genes are highly related to each other, clustering on two major branches of an unrooted phylogenetic tree. Eight ORs contain an unusual NXC sequon at the amino-terminal extracellular domain that may represent a novel N-linked glycosylation site. The BAC contig presented here provides the substrate for sequencing of the cluster. Received: 27 June 2000 / Accepted: 17 August 2000     

Primate evolution of an olfactory receptor cluster: diversification by gene conversion and recent emergence of pseudogenes.

The olfactory receptor (OR) subgenome harbors the largest known gene family in mammals, disposed in clusters on numerous chromosomes. We have carried out a comparative evolutionary analysis of the best characterized genomic OR gene cluster, on human chromosome 17p13. Fifteen orthologs from chimpanzee (localized to chromosome 19p15), as well as key OR counterparts from other primates, have been identified and sequenced. Comparison among orthologs and paralogs revealed a multiplicity of gene conversion events, which occurred exclusively within OR subfamilies. These appear to lead to segment shuffling in the odorant binding site, an evolutionary process reminiscent of somatic combinatorial diversification in the immune system. We also demonstrate that the functional mammalian OR repertoire has undergone a rapid decline in the past 10 million years: while for the common ancestor of all great apes an intact OR cluster is inferred, in present-day humans and great apes the cluster includes nearly 40% pseudogenes.     

Activation of OR1A1 suppresses PPAR-γ expression by inducing HES-1 in cultured hepatocytes

Olfactory receptors (ORs) comprise the largest G protein-coupled receptor gene superfamily. Recent studies indicate that ORs are also expressed in non-olfactory organs, including metabolically active tissues, although their biological functions in these tissues are largely unknown. In this study, OR1A1 expression was detected in HepG2 liver cells. OR1A1 activation by (−)-carvone, a known OR1A1 ligand, increased the cyclic adenosine monophosphate (cAMP), but not intracellular Ca²⁺ concentration, thereby inducing protein kinase A (PKA) activity with subsequent phosphorylation of cAMP response element-binding protein (CREB) and upregulation of the CREB-responsive gene hairy and enhancer of split (HES)-1, a corepressor of peroxisome proliferator-activated receptor-γ (PPAR-γ) in hepatocytes. In (−)-carvone-stimulated cells, the repression of PPAR-γ reduced the expression of the target gene, mitochondrial glycerol-3-phosphate acyltransferase, which encodes a key enzyme involved in triglyceride synthesis. Intracellular triglyceride level and lipid accumulation were reduced in cells stimulated with (−)-carvone, effects that were diminished following the loss of OR1A1 function. These results indicate that OR1A1 may function as a non-redundant receptor in hepatocytes that regulates the PKA-CREB-HES-1 signaling axis and thereby modulates hepatic triglyceride metabolism.     

Identification of four gene variants associated with myocardial infarction

Family history is a major risk factor for myocardial infarction (MI). However, known gene variants associated with MI cannot fully explain the genetic component of MI risk. We hypothesized that a gene-centric association study that was not limited to candidate genes could identify novel genetic associations with MI. We studied 11,053 single-nucleotide polymorphisms (SNPs) in 6,891 genes, focusing on SNPs that could influence gene function to increase the likelihood of identifying disease-causing gene variants. To minimize false-positive associations generated by multiple testing, two studies were used to identify a limited number of nominally associated SNPs; a third study tested the hypotheses that these SNPs are associated with MI. In the initial study (of 340 cases and 346 controls), 637 SNPs were associated with MI (P<.05); these were evaluated in a second study (of 445 cases and 606 controls), and 31 of the 637 SNPs were associated with MI (P<.05) and had the same risk allele as in the first study. For each of these 31 SNPs, we tested the hypothesis that it is associated with MI, using a third study (of 560 cases and 891 controls). We found that four of these gene variants were associated with MI (P<.05; false-discovery rate <10%) and had the same risk allele as in the first two studies. These gene variants encode the cytoskeletal protein palladin (KIAA0992 [odds ratio (OR) 1.40]), a tyrosine kinase (ROS1 [OR 1.75]), and two G protein-coupled receptors (TAS2R50 [OR 1.58] and OR13G1 [OR 1.40]); all ORs are for carriers of two versus zero risk alleles. These findings could lead to a better understanding of MI pathophysiology and improved patient risk assessment.