Olfactory Genes Research Articles

Copy number variation analysis of a Chinese Han family with autism spectrum disorder

To study the copy number variation (CNV) in a Chinese Han autistic spectrum disorder (ASD) pedigree. The pedigree involved six siblings, and three of them were autistic. B lymphocytes of the pedigree were immortalized with EBV and used as studying materials. Karyotyping and Affymatrix 500k SNP chip assay were performed to assess the genetic defects among the members of the pedigree. Karyotyping indicated that the chromosomes were normal. However, the 15q11 locus was located as de novo CNV region in all autistic siblings of the pedigree. In this locus, the fragment in 19827281-19998230 illustrated "loss" of CNV, while other three fragments with 37 kb, 1316 kb and 37 kb indicated "gain" of CNV. In this study, olfactory genes OR11K1P, OR4Q1P, OR4H6P, OR4M2, etc. in the sites with loss and gain of CNV may provide a new clue for genetic research of autism spectrum disorder.

Effects of aggressive interaction on downstream movement and olfactory function in masu salmon, Oncorhynchus masou

In the present study, behavioral, electrophysiological, and molecular biological experiments were developed to examine the role of aggressive interaction (“fighting experience”) on downstream movement and olfactory function in relation to smoltification in hatchery-reared 1-year-old masu salmon (Oncorhynchus masou). Individual salmon that lost the fighting contest in April and May moved downstream earlier than the winners. The olfactory response to salinity sensitivity of the losers, measured by electro-olfactogram to 10−1M sodium chloride, was significantly higher than that of the winners during the period from April to June. In May, the olfactory response to imprinting activity of the losers, previously exposed for 1week to 10−4M l-glutamic acid was significantly high, and the salmon olfactory imprinting-related gene was also highly expressed. During the smoltification period, losing the aggressive interaction might exert strong influences on downstream movement and olfactory function in masu salmon. The present newly developed experiments provide new evidence for evaluating the relation between the aggressive interaction and the smoltification in masu salmon.

Sensory sea slugs

Molluscs are a large and diverse group of aquatic and terrestrial animals that rely heavily on chemical communication. Aplysia is an excellent model in which to investigate and develop breakthrough principles into the molecular aspects of chemoreception in molluscs. We recently identified a large family of rhodopsin-like G-protein coupled receptors expressed in the chemosensory rhinophore of Aplysia that may be key components of sensory detection. Here, we summarize these findings and provide further insight into the molecular olfactory toolkit used by Aplysia, by taking advantage of our knowledge of their attraction pheromones. Our characterization of rhinophore genes upregulated following pheromone stimulation helps explain the dynamics of olfactory gene expression following chemical stimulation.

Dietary restriction mitigates cocaine‐induced alterations of olfactory bulb cellular plasticity and gene expression, and behavior

Because the olfactory system plays a major role in food consumption, and because 'food addiction' and associated morbidities have reached epidemic proportions, we tested the hypothesis that dietary energy restriction can modify adverse effects of cocaine on behavior and olfactory cellular and molecular plasticity. Mice maintained on an alternate day fasting (ADF) diet exhibited increased baseline locomotion and increased cocaine-sensitized locomotion during cocaine conditioning, despite no change in cocaine conditioned place preference, compared with mice fed ad libitum. Levels of dopamine and its metabolites in the olfactory bulb (OB) were suppressed in mice on the ADF diet compared with mice on the control diet, independent of acute or chronic cocaine treatment. The expression of several enzymes involved in dopamine metabolism including tyrosine hydroxylase, monoamine oxidases A and B, and catechol-O-methyltransferase were significantly reduced in OBs of mice on the ADF diet. Both acute and chronic administration of cocaine suppressed the production of new OB cells, and this effect of cocaine was attenuated in mice on the ADF diet. Cocaine administration to mice on the control diet resulted in up-regulation of OB genes involved in mitochondrial energy metabolism, synaptic plasticity, cellular stress responses, and calcium- and cAMP-mediated signaling, whereas multiple olfactory receptor genes were down-regulated by cocaine treatment. ADF abolished many of the effects of cocaine on OB gene expression. Our findings reveal that dietary energy intake modifies the neural substrates underlying some of the behavioral and physiological responses to repeated cocaine treatment, and also suggest novel roles for the olfactory system in addiction. The data further suggest that modification of dietary energy intake could provide a novel potential approach to addiction treatments.

Expressed Sequence Tags from Cephalic Chemosensory Organs of the Northern Walnut Husk Fly,Rhagoletis suavis, Including a Putative Canonical Odorant Receptor

Rhagoletis fruit flies are important both as major agricultural pests and as model organisms for the study of adaptation to new host plants and host race formation. Response to fruit odor plays a critical role in such adaptation. To better understand olfaction in Rhagoletis, an expressed sequence tag (EST) study was carried out on the antennae and maxillary palps of Rhagoletis suavis (Loew) (Diptera: Tephritidae), a common pest of walnuts in eastern United States. After cDNA cloning and sequencing, 544 ESTs were annotated. Of these, 66% had an open reading frame and could be matched to a previously sequenced gene. Based on BLAST sequence homology, 9% (49 of 544 sequences) were nuclear genes potentially involved in olfaction. The most significant finding is a putative odorant receptor (OR), RSOr1, that is homologous to Drosophila melanogaster Or49a and Or85f. This is the first tephritid OR discovered that might recognize a specific odorant. Other olfactory genes recovered included odorant binding proteins, chemosensory proteins, and putative odorant degrading enzymes.

Expression of Trace Amine-Associated Receptors in Human Nasal Mucosa

Trace amine-associated receptors (TAARs) have recently been identified in mouse olfactory epithelium (OE) and may be implicated in the detection of volatile amines, including pheromones. We investigated TAAR expression in human nasal mucosa, evaluating 32 nasal mucosa biopsies obtained from 16 patients with normal olfactory function undergoing routine nasal surgery. OE was identified on the basis of olfactory marker protein gene expression, and it was included in 11 samples out of 32. With the exception of TAAR1, which was detected at trace levels in a few samples of both groups, TAAR expression was limited to OE. TAAR5 was expressed in all OE samples at levels ranging from 15 to 1,480 copies/50 ng cDNA; TAAR8 was detected in 5 OE samples at trace levels and in another 5 OE samples at levels ranging from 11 to 33 cDNA copies/50 ng RNA; TAAR9, TAAR2, and TAAR5 were expressed at trace levels in 5, 3, and 3 OE samples, respectively. In conclusion, most TAARs, and particularly TAAR5, are selectively expressed in human OE. TAAR5 might play a significant functional role since it is known to be activated by trimethylamine, which is present in human secretions and has been implicated in behavioral responses.

Olfactory imprinting of amino acids in lacustrine sockeye salmon.

Juvenile salmon have an olfactory ability to imprint their natal stream odors, but neither the odor properties of natal stream water nor the imprinting timing and duration have been clarified as yet. Here we show, using electrophysiological and behavioral experiments, that one-year-old lacustrine sockeye salmon (Oncorhynchus nerka) can be imprinted around the stage of parr-smolt transformation (PST) by a single amino acid, 1 µM L-proline (Pro), or L-glutamic acid (Glu). We also show by real-time PCR that changes occur in mRNA levels of the salmon olfactory imprinting-related gene (SOIG) around PST. The electro-olfactogram (EOG) responses of test fish exposed to Pro in March (before PST) and April–June (during PST) for 2 weeks were significantly (1.7-fold) greater than those of non-exposed control fish, but not those of test fish exposed in July (after PST). When Pro and control water were added to the water inlets of a two-choice test tank during the spawning season 2 years after the test water exposure, 80% of maturing and matured test fish exposed before and during PST showed a preference for Pro, whereas those exposed after PST did not. The EOG response of test fish exposed to Pro or Glu for 1 hour, 6 hours, 1 day, 7 days, or 14 days in May revealed that only the response after 14 days of exposure was significantly (1.8-fold) greater than the control. The expression levels of SOIG mRNA increased before and during PST, and decreased after PST. We conclude that one-year-old lacustrine sockeye salmon can be imprinted by a single amino acid before and during PST, and that imprinting requires exposure for at least 14 days.

Identification of an atypical insect olfactory receptor subtype highly conserved within noctuids

Olfaction is primarily mediated by the large family of olfactory receptors. Although all insect olfactory receptors share the same structure with seven transmembrane domains, they present poor sequence homologies within and between species. As the only exception, Drosophila melanogaster OR83b and its orthologues define a receptor subtype singularly conserved between insect species. In this article, we report the identification of a new subtype of putative olfactory receptors exceptionally conserved within noctuids, a taxonomic group that includes crop pest insects. Through homology-based molecular cloning, homologues of the previously identified OR18 from Heliothis virescens were identified in the antennae of six noctuid species from various genera, presenting an average of 88% sequence identity. No orthologues were found in genomes available from diverse insect orders and selection pressure analysis revealed that the noctuid OR18s are under purifying selection. The OR18 gene was studied in details in the cotton leafworm, Spodoptera littoralis, where it presented all the characteristic features of an olfactory receptor encoding gene: its expression was restricted to the antennae, with expression in both sexes; its developmental expression pattern was reminiscent of that from other olfactory genes; and in situ hybridization experiments within the antennae revealed that the receptor-expressing cells were closely associated with the olfactory structures, including pheromone- and non-pheromone-sensitive structures. Taken together, our data suggest that we have identified a new original subtype of olfactory receptors that are extremely conserved within noctuids and that might fulfil a critical function in male and female noctuid chemosensory neurones.

Olfactory Proteins Mediating Chemical Communication in the Navel Orangeworm Moth, Amyelois transitella

BackgroundThe navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control — like pheromone-based approaches — are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins.MethodologyBy analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components.ConclusionWe have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.

YY1's DNA-Binding motifs in mammalian olfactory receptor genes

BackgroundYY1 is an epigenetic regulator for a large number of mammalian genes. While performing genome-wide YY1 binding motif searches, we discovered that the olfactory receptor (OLFR) genes have an unusual cluster of YY1 binding sites within their coding regions. The statistical significance of this observation was further analyzed.ResultsAbout 45% of the olfactory genes in the mouse have a range of 4-8 YY1 binding sites within their respective 1 kb coding regions. Statistical analyses indicate that this enrichment of YY1 motifs has likely been driven by unknown selection pressures at the DNA level, but not serendipitously by some peptides enriched within the OLFR genes. Similar patterns are also detected in the OLFR genes of all mammals analyzed, but not in the OLFR genes of the fish lineage, suggesting a mammal-specific phenomenon.ConclusionYY1, or YY1-related transcription factors, may help regulate olfactory receptor genes. Furthermore, the protein-coding regions of vertebrate genes can contain cis-regulatory elements for transcription factor binding as well as codons.

Highly conserved regions in the 5’ region of human olfactory receptor genes

Regulation of human olfactory receptor (hOR) genes is a complex process of control and signalization with various structures and functions that are not clearly understood. To date, nearly 390 functional hOR genes and 462 pseudogenes have been discovered in the human genome. Enhancer models and trans-acting elements for the regulation of different hOR genes are among the few examples of our knowledge concerning regulation of these genes. We looked for upstream control elements that might help explain these complex control mechanisms. To analyze the human olfactory gene family, we looked for functional genes and pseudogenes common to all hOR genes obtained from public databases. Subsequently, we analyzed sequences upstream of the transcription start sites with data mining and bioinformatics tools. We found two highly conserved regions, which we called HCR I and HCR II, upstream of the transcription start sites in 77 hOR genes and 87 pseudogenes. These regions showed possible enhancer functions common to both genes and pseudogenes, an intriguing feature that may be associated with the expression of pseudogenes. Based on these HCRs, we propose a structural model of gene regulation for the olfactory gene family.

Genome-wide analysis of chimpanzee genes with premature termination codons

BackgroundPremature termination codons (PTCs) cause mRNA degradation or a truncated protein and thereby contribute to the transcriptome and proteome divergence between species. Here we present the first genome-wide study of PTCs in the chimpanzee. By comparing the human and chimpanzee genome sequences we identify and characterize genes with PTCs, in order to understand the contribution of these mutations to the transcriptome diversity between the species.ResultsWe have studied a total of 13,487 human-chimpanzee gene pairs and found that ~8% were affected by PTCs in the chimpanzee. A majority (764/1,109) of PTCs were caused by insertions or deletions and the remaining part was caused by substitutions. The distribution of PTC genes varied between chromosomes, with Y having the highest proportion. Furthermore, the density of PTC genes varied on a megabasepair scale within chromosomes and we found the density to be correlated both with indel divergence and proximity to the telomere. Within genes, PTCs were more common close to the 5' and 3' ends of the amino acid sequence. Gene Ontology classification revealed that olfactory receptor genes were over represented among the PTC genes.ConclusionOur results showed that the density of PTC genes fluctuated across the genome depending on the local genomic context. PTCs were preferentially located in the terminal parts of the transcript, which generally have a lower frequency of functional domains, indicating that selection was operating against PTCs at sites central to protein function. The enrichment of GO terms associated with olfaction suggests that PTCs may have influenced the difference in the repertoire of olfactory genes between humans and chimpanzees. In summary, 8% of the chimpanzee genes were affected by PTCs and this type of variation is likely to have an important effect on the transcript and proteomic divergence between humans and chimpanzees.

Olfaction, psychiatric disorders and autoimmunity: Is there a common genetic association?

Psychiatric diseases are often associated with mild alterations in immune functions (e.g., schizophrenia) as well as autoimmune features. Recent evidence suggests that autoimmune diseases (AD) demonstrate a higher prevalence of psychiatric disorders, such as depression and psychosis, than in the normal population. Patients with AD often have an olfactory impairment as well, based on smell studies, accompanied by olfactory regional alterations in neuroimaging. Some evidence suggests that olfactory gene receptors have additional functions in the brain, related to their direct anatomical connection to the limbic system. For example, odor sensing may explain HLA-dissimilar mate selection in humans and animals. Recently, a large cluster of the olfactory receptor (OR) genes was mapped in proximity to the HLA locus on chromosome 6. The HLA and linked OR genes are clustered in haplotypes and are highly polymorphic. This finding may constitute an association among autoimmunity, psychiatric disorders and smell impairment. In this review, we examine the anatomic, genetic and clinical clues that may support an association among these conditions.

Initiation of olfactory placode development and neurogenesis is blocked in mice lacking both Six1 and Six4

Mouse olfactory epithelium (OE) originates from ectodermally derived placode, the olfactory placode that arises at the anterior end of the neural plate. Tissue grafting and recombination experiments suggest that the placode is derived from a common preplacodal domain around the neural plate and its development is directed by signals arising from the underlying mesoderm and adjacent neuroectoderm. In mice, loss of Six1 affects OE morphogenesis but not placode formation. We show here that embryos lacking both Six1 and Six4 failed to form the olfactory placode but the preplacodal region appeared to be specified as judged by the expression of Eya2, which marks the common preplacodal domain, suggesting a synergistic requirement of Six1 and Six4 in patterning the preplacodal ectoderm to a morphologic placode. Our results show that Six1 and Six4 are coexpressed in the preplacodal ectoderm from E8.0. In the olfactory pit, Six4 expression was observed in the peripheral precursors that overlap with Mash1-expressing cells, the early committed neuronal lineage. In contrast, Six1 is highly distributed in the peripheral regions where stem cells reside at E10.5 and it overlaps with Sox2 expression. Both genes are expressed in the basal and apical neuronal progenitors in the OE. Analyses of Six1;Six4 double mutant embryos demonstrated that the slightly thickened epithelium observed in the mutant was not induced for neuronal development. In contrast, in Six1−/− embryos, all neuronal lineage markers were initially expressed but the pattern of their expression was altered. Although very few, the pioneer neurons were initially present in the Six1 mutant OE. However, neurogenesis ceased by E12.5 due to markedly increased cell apoptosis and reduced proliferation, thus defining the cellular defects occurring in Six1−/− OE that have not been previously observed. Our findings demonstrate that Six1/4 function at the top of early events controlling olfactory placode formation and neuronal development. Our analyses show that the threshold of Six1/4 may be crucial for the expression of olfactory specific genes and that Six1 and Six4 may act synergistically to mediate olfactory placode specification and patterning through Fgf and Bmp signaling pathways.

Study of a Synthetic Human Olfactory Receptor 17-4: Expression and Purification from an Inducible Mammalian Cell Line

In order to begin to study the structural and functional mechanisms of olfactory receptors, methods for milligram-scale purification are required. Here we demonstrate the production and expression of a synthetically engineered human olfactory receptor hOR17-4 gene in a stable tetracycline-inducible mammalian cell line (HEK293S). The olfactory receptor gene was fabricated from scratch using PCR-based gene-assembly, which facilitated codon optimization and attachment of a 9-residue bovine rhodopsin affinity tag for detection and purification. Induction of adherent cultures with tetracycline together with sodium butyrate led to hOR17-4 expression levels of ∼30 µg per 150 mm tissue culture plate. Fos-choline-based detergents proved highly capable of extracting the receptors, and fos-choline-14 (N-tetradecylphosphocholine) was selected for optimal solubilization and subsequent purification. Analysis by SDS-PAGE revealed both monomeric and dimeric receptor forms, as well as higher MW oligomeric species. A two-step purification method of immunoaffinity and size exclusion chromatography was optimized which enabled 0.13 milligrams of hOR17-4 monomer to be obtained at >90% purity. This high purity of hOR17-4 is not only suitable for secondary structural and functional analyses but also for subsequent crystallization trials. Thus, this system demonstrates the feasibility of purifying milligram quantities of the GPCR membrane protein hOR17-4 for fabrication of olfactory receptor-based bionic sensing device.

Zebrafish orthologue of mental retardation protein IL1RAPL1 regulates presynaptic differentiation

IL1-receptor accessory protein-like 1 (IL1RAPL1), a member of interleukin-1/toll receptor (TIR) family, is responsible for a nonsyndromic form of mental retardation (MR). The zebrafish orthologue of mammalian IL1RAPL1, designated as Il1rapl1b, was expressed widely in the brain and in the olfactory placode. We employed an olfactory sensory neuron-specific gene manipulation system in combination with in vivo imaging of transparent zebrafish embryos to examine the functional role of Il1rapl1b in synaptic vesicle accumulation and subsequent morphological remodeling of axon terminals, the characteristic features of presynaptic differentiation of zebrafish olfactory sensory neurons during synapse formation. Antisense morpholino oligonucleotide against il1rapl1b suppressed both the synaptic vesicle accumulation and axon terminal remodeling. Consistently, the overexpression of Il1rapl1b stimulated synaptic vesicle accumulation. Swapping the carboxyl-terminal domain of Il1rapl1b with that of mouse IL-1 receptor accessory protein abolished the stimulatory effect. On the other hand, a substitution mutation in the TIR domain suppressed the morphological remodeling of axon terminals. Thus, the regulation of synaptic vesicle accumulation and subsequent morphological remodeling by Il1rapl1b appeared to be mediated by distinct domains. These results suggest that Il1rapl1b plays an important role in presynaptic differentiation during synapse formation.

Evolution of major histocompatibility complex by “en bloc” duplication before mammalian radiation

Duplications are an important mechanism for the emergence of genetic novelties. Reports on duplicated genes are numerous, and mechanisms for polyploidization or local gene duplication are beginning to be understood. When a local duplication is studied, searches are usually done gene-by-gene, and the size of duplicated segments is not often investigated. Therefore, we do not know if the gene in question has duplicated alone or with other genes, implying that "en bloc" duplications are poorly studied. We propose a method for identification of "en bloc" duplication using mapping, phylogenetic and statistical analyses. We show that two segments present in the major histocompatibility complex (MHC) region of human chromosome 6 have resulted from an "en bloc" duplication that took place between divergence of amniotes and methaterian/eutherian separation. These segments contain members of the same multigenic families, namely olfactory receptors genes, genes encoding proteins containing B30.2 domain, genes encoding proteins containing immunoglobulin V domain and MHC class I genes. We will discuss the fact that olfactory receptors and MHC genes have undergone positive selection, which could have helped in fixation of the surrounding genes.

The distribution and conservation of retrotransposable elements in cattle

The objectives of this study were to investigate the distribution of retrotransposable elements in monoallelically-expressed genes including imprinted, X-linked and olfactory genes vs. biallelically-expressed genes and to analyze the conservation of these elements in imprinted and homeobox genes in human, mouse and cattle. The frequency of short interspersed nuclear elements (SINEs) was found to be significantly lower in imprinted genes compared to 2,748 nonimprinted genes from bovine chromosomes 4, 15, 29 and X. Consistent with human and mouse studies, long interspersed nuclear elements (LINEs) were enriched on bovine chromosome X compared with autosomal chromosomes. High densities of LINE-1s were found in 223 bovine and 402 mouse olfactory genes compared to imprinted genes. The low density of SINEs and LINEs found in imprinted genes compared to chromosome X implies that different mechanisms are involved in controlling genomic imprinting and X inactivation.

Molecular cytogenetic characterization of a unique and complex de novo 8p rearrangement

Human chromosome 8p is prone to recurrent rearrangements with inv dup del(8p) being most common. Each of these recurrent rearrangements is associated with different clinical manifestations. Some of these recurrent rearrangements at 8p are mediated by an 8p submicroscopic paracentric inversion between the olfactory gene clusters present in one of the parents. However, recent reports have shown that some of the rearrangements are unique and complex and are mediated by other repetitive elements within 8p. Here, we report on a unique and complex 8p rearrangement with seizures as the major presenting feature in the patient. Extensive fluorescence in situ hybridization and microarray analyses with tiling path 8p array showed that the rearrangement is unique in that the 8p duplication is a direct tandem duplication and, unlike the more common inv dup del(8p), is not derived from parental submicroscopic inversion. Also unlike the inv dup del(8p), the phenotype in our case is milder with no central nervous system malformations or cardiac defects.

Evolution of Gene Expression in the Drosophila Olfactory System

Host plant shifts by phytophagous insects play a key role in insect evolution and plant ecology. Such shifts often involve major behavioral changes as the insects must acquire an attraction and/or lose the repulsion to the new host plant's odor and taste. The evolution of chemotactic behavior may be due, in part, to gene expression changes in the peripheral sensory system. To test this hypothesis, we compared gene expression in the olfactory organs of Drosophila sechellia, a narrow ecological specialist that feeds on the fruit of Morinda citrifolia, with its close relatives Drosophila simulans and Drosophila melanogaster, which feed on a wide variety of decaying plant matter. Using whole-genome microarrays and quantitative polymerase chain reaction, we surveyed the entire repertoire of Drosophila odorant receptors (ORs) and odorant-binding proteins (OBPs) expressed in the antennae. We found that the evolution of OR and OBP expression was accelerated in D. sechellia compared both with the genome average in that species and with the rate of OR and OBP evolution in the other species. However, some of the gene expression changes that correlate with D. sechellia's increased sensitivity to Morinda odorants may predate its divergence from D. simulans. Interspecific divergence of olfactory gene expression cannot be fully explained by changes in the relative abundance of different sensilla as some ORs and OBPs have evolved independently of other genes expressed in the same sensilla. A number of OR and OBP genes are upregulated in D. sechellia compared with its generalist relatives. These genes include Or22a, which likely responds to a key odorant of M. citrifolia, and several genes that are yet to be characterized in detail. Increased expression of these genes in D. sechellia may have contributed to the evolution of its unique chemotactic behavior.