Number Of Olfactory Receptors Research Articles

A machine learning-based electronic nose system using numerous low-cost gas sensors for real-time alcoholic beverage classification.

This study introduces numerous low-cost gas sensors and a real-time alcoholic beverage classification system based on machine learning. Dogs possess a superior sense of smell compared to humans due to having 30 times more olfactory receptors and three times more olfactory receptor types than humans. Thus, in odor classification, the number of olfactory receptors is a more influential factor than the number of receptor types. From this perspective, this study proposes a system that utilizes distinctive data patterns resulting from heterogeneous responses among numerous low-cost homogeneous MOS-based sensors with poor gas selectivity. To evaluate the performance of the proposed system, learning data were gathered using three alcoholic beverage groups including different aged whiskeys, Korean soju with 99% same compositions, and white wines made from the Sauvignon blanc variety, sourced from various countries. The electronic nose system was developed to classify alcoholic samples measured using 30 gas sensors in real time. The samples were injected into a gas chamber for 60 seconds, followed by a 60-second injection of clean air. After preprocessing the time-series data into four distinct datasets, the data were analyzed using a machine learning algorithm, and the classification results were compared. The results showed a high classification accuracy of over 99%, and it was observed that classification performance varied depending on data preprocessing. As the number of gas sensors increased, the prediction accuracy improved, reaching up to 99.83 ± 0.21%. These experimental results indicated that the proposed electronic nose system's classification performance was comparable to that of commercial electronic nose systems. Additionally, the implementation of an alcoholic beverage classification system based on a pretrained LDA model demonstrated the feasibility of real-time classification using the proposed system.

Olfactory system-inspired electronic nose system using numerous low-cost homogenous and hetrogenous sensors

This paper presents an electronic nose system inspired by the biological olfactory system. When comparing the human olfactory system to that of a dog, it’s worth noting that dogs have 30 times more olfactory receptors and three times as many types of olfactory receptors. This implies that the number of olfactory receptors could be a more important parameter for classifying chemical compounds than the number of receptor types. Instead of using expensive precision sensors, the proposed electronic nose system relies on numerous low-cost homogeneous and heterogeneous sensors with poor cross-interference characteristics due to their low gas selectivity. Even if the same type of sensor shows a slightly different output for the same chemical compound, this variation becomes a unique signal for the target gas being measured. The electronic nose system comprises 30 sensors, the e-nose had 6 differing sensors with 5 replicates of each type. The characteristics of the electronic nose system are evaluated using three different volatile alcoholic compounds, more than 99% of which are the same. Liquid samples are supplied to the sensor chamber for 60 seconds using an air bubbler, followed by a 60-second cleaning of the chamber. Sensor signals are acquired at a sampling rate of 100 Hz. In this experimental study, the effects of data preprocessing methods and the number of sensors of the same type are investigated. By increasing the number of sensors of the same type, classification accuracy exceeds 99%, regardless of the deep learning model. The proposed electronic nose system, based on low-cost sensors, demonstrates similar results to commercial expensive electronic nose systems.

General Chemical Reaction Network Theory for Olfactory Sensing Based on G-Protein-Coupled Receptors: Elucidation of Odorant Mixture Effects and Agonist-Synergist Threshold.

This work presents a general chemical reaction network theory for olfactory sensing processes that employ G-protein-coupled receptors as olfactory receptors (ORs). The theory can be applied to general mixtures of odorants and an arbitrary number of ORs. Reactions of ORs with G-proteins, in both the presence and absence of odorants, are explicitly considered. A unique feature of the theory is the definition of an odor activity vector consisting of strengths of odorant-induced signals from ORs relative to those due to background G-protein activity in the absence of odorants. It is demonstrated that each component of the odor activity defined this way reduces to a Michaelis-Menten form capable of accounting for cooperation or competition effects between different odorants. The main features of the theory are illustrated for a two-odorant mixture. Known and potential mixture effects, such as suppression, shadowing, inhibition, and synergy, are quantitatively described. Effects of relative values of rate constants, basal activity, and G-protein concentration are also demonstrated.

Comparative Analysis of Olfactory Receptor Repertoires Sheds Light on the Diet Adaptation of the Bamboo-Eating Giant Panda Based on the Chromosome-Level Genome

The giant panda (Ailuropoda melanoleuca) is the epitome of a flagship species for wildlife conservation and also an ideal model of adaptive evolution. As an obligate bamboo feeder, the giant panda relies on the olfaction for food recognition. The number of olfactory receptor (OR) genes and the rate of pseudogenes are the main factors affecting the olfactory ability of animals. In this study, we used the chromosome-level genome of the giant panda to identify OR genes and compared the genome sequences of OR genes with five other Ursidae species (spectacled bear (Tremarctos ornatus), American black bear (Ursus americanus), brown bear (Ursus arctos), polar bear (Ursus maritimus) and Asian black bear (Ursus thibetanus)). The giant panda had 639 OR genes, including 408 functional genes, 94 partial OR genes and 137 pseudogenes. Among them, 222 OR genes were detected and distributed on 18 chromosomes, and chromosome 8 had the most OR genes. A total of 448, 617, 582, 521 and 792 OR genes were identified in the spectacled bear, American black bear, brown bear, polar bear and Asian black bear, respectively. Clustering analysis based on the OR protein sequences of the six species showed that the OR genes distributed in 69 families and 438 subfamilies based on sequence similarity, and the six mammals shared 72 OR gene subfamilies, while the giant panda had 31 unique OR gene subfamilies (containing 35 genes). Among the 35 genes, there are 10 genes clustered into 8 clusters with 10 known human OR genes (OR8J3, OR51I1, OR10AC1, OR1S2, OR1S1, OR51S1, OR4M1, OR4M2, OR51T1 and OR5W2). However, the kind of odor molecules can be recognized by the 10 known human OR genes separately, which needs further research. The phylogenetic tree showed that 345 (about 84.56%) functional OR genes were clustered as Class-II, while only 63 (about 15.44%) functional OR genes were clustered as Class-I, which required further and more in-depth research. The potential odor specificity of some giant panda OR genes was identified through the similarity to human protein sequences. Sequences similar to OR2B1, OR10G3, OR11H6 and OR11H7P were giant panda-specific lacking, which may be related to the transformation and specialization from carnivore to herbivore of the giant panda. Since our reference to flavoring agents comes from human research, the possible flavoring agents from giant panda-specific OR genes need further investigation. Moreover, the conserved motifs of OR genes were highly conserved in Ursidae species. This systematic study of OR genes in the giant panda will provide a solid foundation for further research on the olfactory function and variation of the giant panda.

Dog olfactory receptor gene expression profiling using samples derived from nasal epithelium brushing

Dogs have an exquisite sense of olfaction. In many instances this ability has been utilized by humans for a wide range of important situations including detecting explosives and illegal drugs. It is accepted that some breeds have better senses of smell than others. Dogs can detect many volatile compounds at extremely low concentrations in air. To achieve such high levels of detection, the canine olfactory system is both complex and highly developed requiring a high density of olfactory receptors capable of detecting volatiles. Consequently the dog genome encodes a large number of olfactory receptor (OR) genes. However, it remains unclear as to what extent are all of these OR genes expressed on the cell surface. To facilitate such studies, a nasal brushing method was developed to recover dog nasal epithelial cell samples from which total RNA could be extracted and used to prepare high quality cDNA libraries. After capture by hybridization with an extensive set of oligonucleotides, the level of expression of each transcript was measured following next generation sequencing (NGS). The reproducibility of this sampling approach was checked by analyzing replicate samples from the same animal (up to 6 per each naris). The quality of the hybridization capture was also checked by analyzing two DNA libraries; this offered an advantage over RNA libraries by having an equal presence for each gene. Finally, we compared this brushing method performed on living dogs to a nasal epithelium biopsy approach applied to two euthanized terminally ill dogs, following consent from their owners.Comparison the expression levels of each transcript indicate that the ratios of expression between the highest and the least expressed OR in each sample are greater than 10,000 (paralog variation). Furthermore, it was clear that a number of OR genes are not expressed.The method developed and described here will allow researchers to further address whether variations observed in the OR transcriptome relate to dog ‘life experiences’ and whether any differences observed between samples are dog-specific or breed-specific.

Challenges and possible solutions for decoding extranasal olfactory receptors.

Olfactory receptors are primarily known to be expressed in the olfactory epithelium of the nasal cavity and therefore assist in odor perception. With the advent of high-throughput omics technologies such as tissue microarray or RNA sequencing, a large number of olfactory receptors have been reported to be expressed in the nonolfactory tissues. Although these technologies uncovered the expression of these olfactory receptors in the nonchemosensory tissues, unfortunately, they failed to reveal the information about their cell type of origin. Accurate characterization of the cell types should be the first step towards devising cell type-specific assays for their functional evaluation. Single-cell RNA-sequencing technology resolved some of these apparent limitations and opened new means to interrogate the expression of these extranasal olfactory receptors at the single-cell resolution. Moreover, the availability of large-scale, multi-organ/species single-cell expression atlases offer ample resources for the systematic reannotation of these receptors in a cell type-specific manner. In this Viewpoint article, we discuss some of the technical limitations that impede the in-depth understanding of these extranasal olfactory receptors, with a special focus on odorant receptors. Moreover, we also propose a list of single cell-based omics technologies that could further promulgate the opportunity to decipher the regulatory network that drives the odorant receptors expression at atypical locations.

Comparative Evaluation of Sensitivity to Hexanal Between Human and Canine Olfactory Receptors

It is known that a dog possesses much better sense of smell than a human. It has been reported that a dog has advantages (compared to a human) in the number of olfactory receptor (OR) genes, proteins and cells, and also nasal structure. However, a definitive reason for superior sensitivity of the canine olfactory system remains controversial. In this study, we compared sensitivity of human and canine olfactory receptors relative to the same condition. Human OR (hOR2W1) and canine OR (cfOR0312), previously identified to recognize hexanal, were inserted to the pcDNA3 vector. This vector was transfected to HEK293 cells. Hexanal-discriminating ability of ORs was confirmed using Fura-2 AM, a dye which illuminates when binding to calcium ions flowing into cells once olfactory signaling occurs. Consequently, cfOR0312 was more sensitive to hexanal than hOR2W1. Comparison of OR sensitivity would be one of the major factors in clarifying difference of sensitivity between human and canine olfactory systems.

Olfactory receptor repertoire size in dinosaurs.

The olfactory bulb (OB) ratio is the size of the OB relative to the cerebral hemisphere, and is used to estimate the proportion of the forebrain devoted to smell. In birds, OB ratio correlates with the number of olfactory receptor (OR) genes and therefore has been used as a proxy for olfactory acuity. By coupling OB ratios with known OR gene repertoires in birds, we infer minimum repertoire sizes for extinct taxa, including non-avian dinosaurs, using phylogenetic modelling, ancestral state reconstruction and comparative genomics. We highlight a shift in the scaling of OB ratio to body size along the lineage leading to modern birds, demonstrating variable OR repertoires present in different dinosaur and crown-bird lineages, with varying factors potentially influencing sensory evolution in theropods. We investigate the ancestral sensory space available to extinct taxa, highlighting potential adaptations to ecological niches. Through combining morphological and genomic data, we show that, while genetic information for extinct taxa is forever lost, it is potentially feasible to investigate evolutionary trajectories in extinct genomes.

Molecular Basis of Mammalian Odor Discrimination: A Status Report.

Humans have 396 unique, intact olfactory receptors (ORs), G-protein coupled receptors (GPCRs) containing receptor-specific binding sites; other mammals have more. Activation of these transmembrane proteins by an odorant initiates a signaling cascade, evoking an action potential leading to perception of a smell. Because the number of distinguishable odorants vastly exceeds the number of ORs, research has focused on mechanisms of recognition and signaling processes for classes of odorants. In this review, selected recent examples will be presented of "deorphaned" mammalian receptors, where the OR ligands (odorants) as well as key aspects of receptor-odorant interactions were identified using odorant-mediated receptor activation data together with site-directed mutagenesis and molecular modeling. Based on cumulative evidence from OR deorphaning and olfactory receptor neuron activation studies, a receptor-ligand docking model rather than an alternative bond vibration model is suggested to best explain the molecular basis of the exquisitely sensitive odor discrimination in mammals.

Olfactory Receptors as Biomarkers in Human Breast Carcinoma Tissues.

Olfactory receptors (ORs) are known to be expressed in a variety of human tissues and act on different physiological processes, such as cell migration, proliferation, or secretion and have been found to function as biomarkers for carcinoma tissues of prostate, lung, and small intestine. In this study, we analyzed the OR expression profiles of several different carcinoma tissues, with a focus on breast cancer. The expression of OR2B6 was detectable in breast carcinoma tissues; here, transcripts of OR2B6 were detected in 73% of all breast carcinoma cell lines and in over 80% of all of the breast carcinoma tissues analyzed. Interestingly, there was no expression of OR2B6 observed in healthy tissues. Immunohistochemical staining of OR2B6 in breast carcinoma tissues revealed a distinct staining pattern of carcinoma cells. Furthermore, we detected a fusion transcript containing part of the coding exon of OR2B6 as a part of a splice variant of the histone HIST1H2BO transcript. In addition, in cancer tissues and cell lines derived from lung, pancreas, and brain, OR expression patterns were compared to that of corresponding healthy tissues. The number of ORs detected in lung carcinoma tissues was significantly reduced in comparison to the surrounding healthy tissues. In pancreatic carcinoma tissues, OR4C6 was considerably more highly expressed in comparison to the respective healthy tissues. We detected OR2B6 as a potential biomarker for breast carcinoma tissues.

Identification of Complete Repertoire of Apis florea Odorant Receptors Reveals Complex Orthologous Relationships with Apis mellifera.

We developed a computational pipeline for homology based identification of the complete repertoire of olfactory receptor (OR) genes in the Asian honey bee species, Apis florea. Apis florea is phylogenetically the most basal honey bee species and also the most distant sister species to the Western honey bee Apis mellifera, for which all OR genes had been identified before. Using our pipeline, we identified 180 OR genes in A. florea, which is very similar to the number of ORs identified in A. mellifera (177 ORs). Many characteristics of the ORs including gene structure, synteny of tandemly repeated ORs and basic phylogenetic clustering are highly conserved. The composite phylogenetic tree of A. florea and A. mellifera ORs could be divided into 21 clades which are in harmony with the existing Hymenopteran tree. However, we found a few nonorthologous OR relationships between both species as well as independent pseudogenization of ORs suggesting separate evolutionary changes. Particularly, a subgroup of the OR gene clade XI, which had been hypothesized to code cuticular hydrocarbon receptors showed a high number of species-specific ORs. RNAseq analysis detected a total number of 145 OR transcripts in male and 162 in female antennae. Most of the OR genes were highly expressed on the female antennae. However, we detected five distinct male-biased OR genes, out of which three genes (AfOr11, AfOr18, AfOr170P) were shown to be male-biased in A. mellifera, too, thus corroborating a behavioral function in sex-pheromone communication.

Expression and identification of olfactory receptors in sciatic nerve and dorsal root ganglia of rats

The olfactory receptor (OR) genes are expressed mainly in the cell membrane of olfactory sensory neurons of the nasal epithelium, and the binding of specific odorant ligands to OR proteins leads to odor detection. ORs are also expressed in non-olfactory tissues and cells, but their functions are often elusive. In this study, microarray analysis was used to detect the presence of ORs in peripheral nerves. We found that a number of ORs were differentially expressed in sciatic nerve and dorsal root ganglia (DRGs) following sciatic nerve injury. The expression and expression profile of several ORs in sciatic nerve were verified by in situ hybridization and real time quantitative RT-PCR. We also observed that the expression of some ORs in primary culture of Schwann cells was up-regulated under H2O2 stimulation. Overall, all the results suggest that there may be a possible relationship between the differential expression of ORs in injured peripheral nerves and peripheral nerve regeneration.

Copy number variation in the speciation of pigs: a possible prominent role for olfactory receptors.

BackgroundUnraveling the genetic mechanisms associated with reduced gene flow between genetically differentiated populations is key to understand speciation. Different types of structural variations (SVs) have been found as a source of genetic diversity in a wide range of species. Previous studies provided detailed knowledge on the potential evolutionary role of SVs, especially copy number variations (CNVs), between well diverged species of e.g. primates. However, our understanding of their significance during ongoing speciation processes is limited due to the lack of CNV data from closely related species. The genus Sus (pig and its close relatives) which started to diverge ~4 Mya presents an excellent model for studying the role of CNVs during ongoing speciation.ResultsIn this study, we identified 1408 CNV regions (CNVRs) across the genus Sus. These CNVRs encompass 624 genes and were found to evolve ~2.5 times faster than single nucleotide polymorphisms (SNPs). The majority of these copy number variable genes are olfactory receptors (ORs) known to play a prominent role in food foraging and mate recognition in Sus. Phylogenetic analyses, including novel Bayesian analysis, based on CNVRs that overlap ORs retain the well-accepted topology of the genus Sus whereas CNVRs overlapping genes other than ORs show evidence for random drift and/or admixture.ConclusionWe hypothesize that inter-specific variation in copy number of ORs provided the means for rapid adaptation to different environments during the diversification of the genus Sus in the Pliocene. Furthermore, these regions might have acted as barriers preventing massive gene flow between these species during the multiple hybridization events that took place later in the Pleistocene suggesting a possible prominent role of ORs in the ongoing Sus speciation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1449-9) contains supplementary material, which is available to authorized users.

Activation of Olfactory Receptors on Mouse Pulmonary Macrophages Promotes Monocyte Chemotactic Protein-1 Production

BackgroundEmerging evidence suggests that non-olfactory tissues and cells can express olfactory receptors (ORs), however, the exact function of ectopic OR expression remains unknown. We have previously shown in mouse models that a unique cooperation between interferon-γ (IFN-γ) and lipopolysaccharide (LPS) drives the activation of pulmonary macrophages and leads to the induction of pathogenic responses in the respiratory tract. Further, through gene array studies, we have shown that activation of macrophages by these molecules results in the selective expression of a number of ORs. In this study, we validated the expression of these ORs in mouse airway and pulmonary macrophages in response to IFN-γ and LPS (γ/LPS) stimulation, and further explored the effect of odorant stimulation on macrophage function.Methodology/Principal FindingsOR expression in airway and pulmonary macrophages in response to IFN-γ, LPS or γ/LPS treatments was assessed by microarray and validated by q-PCR. OR expression (e.g. OR622) on macrophages was confirmed by visualization in immunofluoresence assays. Functional responses to odorants were assessed by quantifying inflammatory cytokine and chemokine expression using q-PCR and cell migration was assessed by a modified Boyden chamber migration assay. Our results demonstrate that eight ORs are expressed at basal levels in both airway and pulmonary macrophages, and that γ/LPS stimulation cooperatively increased this expression. Pulmonary macrophages exposed to the combined treatment of γ/LPS+octanal (an odorant) exhibited a 3-fold increase in MCP-1 protein production, compared to cells treated with γ/LPS alone. Supernatants from γ/LPS+octanal exposed macrophages also increased macrophage migration in vitro.Conclusions/SignificanceEight different ORs are expressed at basal levels in pulmonary macrophages and expression is upregulated by the synergistic action of γ/LPS. Octanal stimulation further increased MCP-1 production and the motility of macrophages. Our results suggest that ORs may mediate macrophage function by regulating MCP-1 production and cell migration.

A Cleavable N-Terminal Signal Peptide Promotes Widespread Olfactory Receptor Surface Expression in HEK293T Cells

Olfactory receptors (ORs) are G protein-coupled receptors that detect odorants in the olfactory epithelium, and comprise the largest gene family in the genome. Identification of OR ligands typically requires OR surface expression in heterologous cells; however, ORs rarely traffic to the cell surface when exogenously expressed. Therefore, most ORs are orphan receptors with no known ligands. To date, studies have utilized non-cleavable rhodopsin (Rho) tags and/or chaperones (i.e. Receptor Transporting Protein, RTP1S, Ric8b and Gαolf) to improve surface expression. However, even with these tools, many ORs still fail to reach the cell surface. We used a test set of fifteen ORs to examine the effect of a cleavable leucine-rich signal peptide sequence (Lucy tag) on OR surface expression in HEK293T cells. We report here that the addition of the Lucy tag to the N-terminus increases the number of ORs reaching the cell surface to 7 of the 15 ORs (as compared to 3/15 without Rho or Lucy tags). Moreover, when ORs tagged with both Lucy and Rho were co-expressed with previously reported chaperones (RTP1S, Ric8b and Gαolf), we observed surface expression for all 15 receptors examined. In fact, two-thirds of Lucy-tagged ORs are able to reach the cell surface synergistically with chaperones even when the Rho tag is removed (10/15 ORs), allowing for the potential assessment of OR function with only an 8-amino acid Flag tag on the mature protein. As expected for a signal peptide, the Lucy tag was cleaved from the mature protein and did not alter OR-ligand binding and signaling. Our studies demonstrate that widespread surface expression of ORs can be achieved in HEK293T cells, providing promise for future large-scale deorphanization studies.

Odorant-binding proteins and olfactory coding in the solitary bee Osmia cornuta.

Solitary bees are major pollinators but their chemical communication system has been poorly studied. We investigated olfactory coding in Osmia cornuta from two perspectives, chemical and biochemical. We identified (E)-geranyl acetone and 2-hexyl-1,3-dioxolane, specifically secreted by females and males, respectively. A transcriptome analysis of antennae revealed 48 ORs (olfactory receptors), six OBPs (odorant-binding proteins), five CSPs (chemosensory proteins), and a single SNMP (sensory neuron membrane protein). The numbers of ORs and OBPs are much lower than in the honeybee, in particular, C-minus OBPs are lacking in the antennae of O. cornuta. We have expressed all six OBPs of O. cornuta and studied their binding specificities. The best ligands are common terpene plant odorants and both volatiles produced by the bee and identified in this work.

A Rapid Genotyping Assay for Segregating Human Olfactory Receptor Pseudogenes

Variation in odor perception between individuals is initiated by binding of "odorant" molecules to olfactory receptors (ORs) located in the nasal cavity. To determine the mechanism for variation in odor perception, identification of specific ligands for a large number of ORs is required. However, it has been difficult to identify specific ligands, and ligands have been identified for only 2-3% of the hundreds of mammalian ORs. One way to increase the number of identified ligands is to take advantage of >60 human OR genes that are segregating as a result of a single nucleotide polymorphism, between a functional intact allele and a nonfunctional pseudogene allele. Potential ligands for these ORs can be identified by correlating odor perception of an individual with their genotype [intact/intact (I/I) vs. pseudogene/pseudogene (P/P)] for an OR gene. For this type of study, genotypes must be determined for a large number of individuals. We have developed a PCR-based assay to distinguish between the intact and pseudogene alleles of 49 segregating human OR genes and to determine an individual's genotype for these genes. To facilitate rapid determination of genotypes for a large number of individuals, the assay uses a small number of simple steps and equipment commonly found in most molecular biology and biochemistry laboratories. Although this assay was developed to distinguish between polymorphisms in OR genes, it can easily be adapted for use in distinguishing single nucleotide polymorphisms in any gene or chromosomal locus.

A “Microfluidic Nose”: Detection of Olfactory Sensory Neuron Responses to Odorants Across the Whole Olfactory Receptor Space

In vertebrates, odorant molecules are detected by olfactory sensory neurons (OSNs) present in the nasal epithelium. A large, G-protein coupled receptor gene family is responsible for transducing the detection of a cognate molecule for a given receptor. Each OSN appears to express only one of thousands of olfactory receptor (OR) genes in rodents. Odorants are perceived by combinatorial activation of a number of ORs (it is specific to a subset of odorants); each OR recognizes a range of odorants and odorants are typically recognized by a number of ORs. Given the approximately thousand OSN/OR types and the hundreds of thousands of potential ligands, measuring individual OSN activation with the usual in-vivo and in-vitro methods is a laborious task that is not suitable for interrogating the whole OR space. Hence a microfluidic and high-throughput system was developed to analyze these cells.Utilizing the techniques of soft-lithography, we developed a microwell array of ∼32,000 wells (20 um diameter, 10 um depth) to capture dissociated olfactory epithelia (OE) cells and sequentially exposing them to different odorants. Cell response was detected using the Fluo4AM calcium binding dye. By imaging the fluorescence change in each well, a response profile to each odorant can be constructed for thousands of individual OSNs simultaneously.

Common peptides shed light on evolution of Olfactory Receptors

BackgroundOlfactory Receptors (ORs) form the largest multigene family in vertebrates. Their evolution and their expansion in the vertebrate genomes was the subject of many studies. In this paper we apply a motif-based approach to this problem in order to uncover evolutionary characteristics.ResultsWe extract deterministic motifs from ORs belonging to ten species using the MEX (Motif Extraction) algorithm, thus defining Common Peptides (CPs) characteristic to ORs. We identify species-specific CPs and show that their relative abundance is high only in fish and frog, suggesting relevance to water-soluble odorants. We estimate the origins of CPs according to the tree of life and track the gains and losses of CPs through evolution. We identify major CP gain in tetrapods and major losses in reptiles. Although the number of human ORs is less than half of the number of ORs in other mammals, the fraction of lost CPs is only 11%.By examining the positions of CPs along the OR sequence, we find two regions that expanded only in tetrapods. Using CPs we are able to establish remote homology relations between ORs and non-OR GPCRs.Selecting CPs according to their evolutionary age, we bicluster ORs and CPs for each species. Clean biclustering emerges when using relatively novel CPs. Evolutionary age is used to track the history of CP acquisition in the collection of mammalian OR families within HORDE (Human Olfactory Receptor Data Explorer).ConclusionThe CP method provides a novel perspective that reveals interesting traits in the evolution of olfactory receptors. It is consistent with previous knowledge, and provides finer details. Using available phylogenetic trees, evolution can be rephrased in terms of CP origins.Supplementary information is also available at

Extensive Copy-Number Variation of the Human Olfactory Receptor Gene Family

As much as a quarter of the human genome has been reported to vary in copy number between individuals, including regions containing about half of the members of the olfactory receptor (OR) gene family. We have undertaken a detailed study of copy-number variation of ORs to elucidate the selective and mechanistic forces acting on this gene family and the true impact of copy-number variation on human OR repertoires. We argue that the properties of copy-number variants (CNVs) and other sets of large genomic regions violate the assumptions of statistical methods that are commonly used in the assessment of gene enrichment. Using more appropriate methods, we provide evidence that OR enrichment in CNVs is not due to positive selection but is because of OR preponderance in segmentally duplicated regions, which are known to be frequently copy-number variable, and because purifying selection against CNVs is lower in OR-containing regions than in regions containing essential genes. We also combine multiplex ligation-dependent probe amplification (MLPA) and PCR to assay the copy numbers of 37 candidate CNV ORs in a panel of approximately 50 human individuals. We confirm copy-number variation of 18 ORs but find no variation in this human-diversity panel for 16 other ORs, highlighting the caveat that reported intervals often overrepresent true CNVs. The copy-number variation we describe is likely to underpin significant variation in olfactory abilities among human individuals. Finally, we show that both homology-based and homology-independent processes have played a recent role in remodeling the OR family.