Random expression of main and vomeronasal olfactory receptor genes in immature and mature olfactory epithelia of Fugu rubripes

Main olfactory receptor genes were isolated from a seawater fish, Fugu rubripes (pufferfish), and characterized. Two subfamilies of genes encoding seven transmembrane receptors were identified; one consists of five or more members, termed FOR1-1 to 5 of FOR1 subfamily, and the other appears to be a single copy gene, termed the FOR2 subfamily. FOR1 members show extremely high amino acid sequence similarities of about 95% to one another, and are distantly related to catfish-1 with the highest similarity of 37%. FOR2 shows 43% similarity to goldfish-A28. Phylogenically, both FOR members are categorized among pedigrees of the fish main olfactory receptor family outside the mammalian receptor family, although similarities between Fugu receptors and those of fresh-water fishes are lower than those among fresh-water fishes. In situ hybridization shows that both subfamilies of receptor genes are expressed randomly over the olfactory epithelium throughout all developmental stages, and no segregation of the signals was found. On the other hand, when three members of a vomeronasal olfactory receptor gene family, related to the Ca(2+)-sensing receptor, were used as probes, they were also randomly expressed over the same epithelium as the main olfactory receptors. This is in contrast to the expression profiles observed for zebrafish and goldfish, where the main or vomeronasal olfactory receptors are expressed in segregated patterns. It is thus suggested that the expression pattern of fish olfactory receptors varies depending on the species, although fish olfactory receptors are highly related to one another in their primary structures, and are phylogenically distinct from those of mammals.     

Evolution of the “OR37” Subfamily of Olfactory Receptors: A Cross-Species Comparison

Genes encoding the olfactory receptors of the “OR37” subfamily of the mouse are characterized by special features including a clustered expression pattern, assembly in two distinct gene clusters, and highly conserved putative promoter motifs. Mining the rat and dog databases revealed that these two species possess highly conserved clusters of OR37 genes at two syntenic genomic loci. In a prototherian mammal, the platypus (Ornithorhynchus anatinus), none of the characteristic OR37 genes were found. Examination of a metatherian mammal, the gray short-tailed opossum (Monodelphis domestica) revealed seven canonical OR37 genes, all phylogenetically related to cluster II genes and also organized similar to cluster II of eutherian species. In addition, their 5′ upstream regions comprised sequence motifs related to the putative regulatory sequences of cluster II genes. Typical cluster I OR37 genes were identified only in the eutherian mammals examined, including the evolutionary ancient anteater, wherein OR37 genes related to both clusters were present. Together, these results reveal novel information concerning the phylogenetic origin and important evolutionary steps of the mammalian-specific OR37 olfactory receptor family. [Reviewing Editor: Dr. Lauren Ancel Meyers] Reiner Hoppe and Thomas D. Lambert are contributed equally to this work.     

Homozygous deletion of six olfactory receptor genes in a subset of individuals with Beta-thalassemia

Progress in the functional studies of human olfactory receptors has been largely hampered by the lack of a reliable experimental model system. Although transgenic approaches in mice could characterize the function of individual olfactory receptors, the presence of over 300 functional genes in the human genome becomes a daunting task. Thus, the characterization of individuals with a genetic susceptibility to altered olfaction coupled with the absence of particular olfactory receptor genes will allow phenotype/genotype correlations and vindicate the function of specific olfactory receptors with their cognate ligands. We characterized a 118 kb β-globin deletion and found that its 3' end breakpoint extends to the neighboring olfactory receptor region downstream of the β-globin gene cluster. This deletion encompasses six contiguous olfactory receptor genes (OR51V1, OR52Z1, OR51A1P, OR52A1, OR52A5, and OR52A4) all of which are expressed in the brain. Topology analysis of the encoded proteins from these olfactory receptor genes revealed that OR52Z1, OR52A1, OR52A5, and OR52A4 are predicted to be functional receptors as they display integral characteristics of G-proteins coupled receptors. Individuals homozygous for the 118 kb β-globin deletion are afflicted with β-thalassemia due to a homozygous deletion of the β-globin gene and have no alleles for the above mentioned olfactory receptors genes. This is the first example of a homozygous deletion of olfactory receptor genes in human. Although altered olfaction remains to be ascertained in these individuals, such a study can be carried out in β-thalassemia patients from Malaysia, Indonesia and the Philippines where this mutation is common. Furthermore, OR52A1 contains a γ-globin enhancer, which was previously shown to confer continuous expression of the fetal γ-globin genes. Thus, the hypothesis that β-thalassemia individuals, who are homozygous for the 118 kb deletion, may also have an exacerbation of their anemia due to the deletion of two copies of the γ-globin enhancer element is worthy of consideration.     

Characterizing the expression of the human olfactory receptor gene family using a novel DNA microarray

Background  

Olfactory receptor (OR) genes were discovered more than a decade ago, when Buck and Axel observed that, in rats, certain G-protein coupled receptors are expressed exclusively in the olfactory epithelium. Subsequently, protein sequence similarity was used to identify entire OR gene repertoires of a number of mammalian species, but only in mouse were these predictions followed up by expression studies in olfactory epithelium. To rectify this, we have developed a DNA microarray that contains probes for most predicted human OR loci and used that array to examine OR gene expression profiles in olfactory epithelium tissues from three individuals.     

Subfamily of Olfactory Receptors Characterized by Unique Structural Features and Expression Patterns

Abstract: The complex chemospecificity of the olfactory system is probably due to the large family of short-looped, heptahelical receptor proteins expressed in neurons widely distributed throughout one of the several zones within the nasal neuroepithelium. In this study, a subfamily of olfactory receptors has been identified that is characterized by distinct structural features as well as a unique expression pattern. Members of this receptor family are found in mammals, such as rodents and opossum, but not in lower vertebrates. All identified subtypes comprise an extended third extracellular loop that exhibits amphiphilic properties and contains numerous charged amino acids in conserved positions. Olfactory sensory neurons expressing these receptor types are segregated in small clusters on the tip of central turbinates, thus representing a novel pattern of expression for olfactory receptors. In mouse, genes encoding the new subfamily of receptors were found to be harbored within a small contiguous segment of genomic DNA. Based on species specificity as well as the unique structural properties and expression pattern, it is conceivable that the novel receptor subfamily may serve a special function in the olfactory system of mammals.     

A novel family of ancient vertebrate odorant receptors

Vertebrate odorant receptor (OR) genes have been isolated and characterized in several taxa, including bony fish and mammals. However, the search for more ancient vertebrate OR genes has been unsuccessful to date, indicating that these ancient genes share little sequence identity with previously isolated ORs. The lamprey (Lampetra fluviatilis) olfactory epithelium does not appear to express any of the modern vertebrate ORs previously identified in bony fish and mammals. We have isolated and characterized an ancient family of vertebrate membrane receptors from the olfactory epithelium of the lamprey. Sequence analysis reveals similarities with other Class A (rhodopsin-like) G protein-coupled receptors such as serotonin, dopamine, and histamine receptors, but the expression patterns of members of the new family, as well as certain conserved motifs, strongly suggest that the sequences encode ORs. Sequence similarity within the lamprey OR family is low, and Southern blot analysis suggests reduced-sized subfamilies. This novel vertebrate OR gene family, the most ancient isolated to date, is proposed to be involved in the detection of water-borne molecules in jawless fishes. Lamprey OR genes therefore represent a new level of diversity within the vertebrate OR gene family, but also provide clues as to how vertebrate ORs might have emerged. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 383–392, 1998     

Expression of odorant receptor family, type 2 OR in the aquatic olfactory cavity of amphibian frog Xenopus tropicalis

Recent genome wide in silico analyses discovered a new family (type 2 or family H) of odorant receptors (ORs) in teleost fish and frogs. However, since there is no evidence of the expression of these novel OR genes in olfactory sensory neurons (OSN), it remains unknown if type 2 ORs (OR2) function as odorant receptors. In this study, we examined expression of OR2 genes in the frog Xenopus tropicalis. The overall gene expression pattern is highly complex and differs depending on the gene and developmental stage. RT-PCR analysis in larvae showed that all of the OR2η genes we identified were expressed in the peripheral olfactory system and some were detected in the brain and skin. Whole mount in situ hybridization of the larval olfactory cavity confirmed that at least two OR2η genes so far tested are expressed in the OSN. Because tadpoles are aquatic animals, OR2η genes are probably involved in aquatic olfaction. In adults, OR2η genes are expressed in the nose, brain, and testes to different degrees depending on the genes. OR2η expression in the olfactory system is restricted to the medium cavity, which participates in the detection of water-soluble odorants, suggesting that OR2ηs function as receptors for water-soluble odorants. Moreover, the fact that several OR2ηs are significantly expressed in non-olfactory organs suggests unknown roles in a range of biological processes other than putative odorant receptor functions.     

Genomic organization and evolution of olfactory receptors and trace amine-associated receptors in channel catfish,Ictalurus punctatus

BackgroundChannel catfish (Ictalurus punctatus) live in turbid waters with limited visibility to chase prey within a certain distance. This can be compensated through detecting specific water-soluble substances by the olfactory receptors (ORs) and trace amine associated receptors (TAARs) expressed on the olfactory epithelium.MethodsWe identified the OR and TAAR repertoires in channel catfish, and characterized the genomic organizations of these two gene families by data mining available genomic resources.ResultsA total of 47 putative OR genes and 36 putative TAAR genes were identified in the channel catfish genome, including 27 functional OR genes and 28 functional TAAR genes. Phylogenetic and orthogroup analyses were conducted to illustrate the evolutionary dynamics of the vertebrate ORs and TAARs. Collinear analysis revealed the presence of two conserved orthologous blocks that contain OR genes between the catfish genome and zebrafish genome. The complete loss of a conserved motif in fish OR family H may contribute to the divergence of family H from other families. The dN/dS analysis indicated that the highest degree of selection pressure was imposed on TAAR subfamily 14 among all fish ORs and TAARs.ConclusionsThe present study provides understanding of the evolutionary dynamics of the two gene families (OR and TAAR) associated with olfaction in channel catfish.General significanceThis is the first systematic study of ORs and TAARs in catfish, which could provide valuable genomic resources for further investigation of olfactory mechanisms in teleost fish.     

Evolutionary analysis of putative olfactory receptor genes of medaka fish, Oryzias latipes

To obtain an understanding of the origin, diversification and genomic organization of vertebrate olfactory receptor genes, we have newly cloned and characterized putative olfactory receptor genes, mfOR1, mfOR2, mfOR3 and mfOR4 from the genomic DNA of medaka fish (Oryzias latipes). The four sequences contained features commonly seen in known olfactory receptor genes and were phylogenetically most closely related to those of catfish and zebrafish.Among them, mfOR1 and mfOR2 showed the highest amino acid (aa) similarity (93%) and defined a novel olfactory receptor gene family that is most divergent among all other vertebrate olfactory receptor genes. Southern hybridization analyses suggested that mfOR1 and mfOR2 are tightly linked to each other (within 24kb), although suitable marker genes were not available to locate their linkage group. Unlike observation in catfish olfactory receptor sequences, nucleotide (nt) substitutions between the two sequences did not show any evidence of positive natural selection. mfOR3 and mfOR4, however, showed a much lower aa similarity (26%) and were both mapped to a region in the medaka linkage group XX.After including these medaka fish sequences, olfactory receptors of terrestrial and aquatic animals formed significantly different clusters in the phylogenetic tree. Although the member genes of each olfactory receptor gene subfamily are less in fish than that in mammals, fish seem to have maintained more diverse olfactory receptor gene families. Our finding of a novel olfactory receptor gene family in medaka fish may provide a step towards understanding the emergence of the olfactory receptor gene in vertebrates.     

Minigenes impart odorant receptor-specific axon guidance in the olfactory bulb

An olfactory sensory neuron (OSN) expresses selectively one member from a repertoire of approximately 1000 odorant receptor (OR) genes and projects its axon to a specific glomerulus in the olfactory bulb. Both processes are here recapitulated by MOR23 and M71 OR minigenes, introduced into mice. Minigenes of 9 kb and as short as 2.2 kb are selectively expressed by neurons that do not coexpress the endogenous gene but coproject their axons to the same glomeruli. Deletion of a 395 bp upstream region in the MOR23 minigene abolishes expression. In this region we recognize sequence motifs conserved in many OR genes. Transgenic lines expressing the OR in ectopic epithelial zones form ectopic glomeruli, which also receive input from OSNs expressing the cognate endogenous receptor. This suggests a recruitment through homotypic interactions between OSNs expressing the same OR.     

Untypical connectivity from olfactory sensory neurons expressing OR37 into higher brain centers visualized by genetic tracing

The OR37 subfamily of odorant receptors (ORs) exists exclusively in mammals. In contrast to ORs in general, they are highly conserved within and across species. These unique features raise the question, whether olfactory information gathered by the OR37 sensory cells is processed in specially designated brain areas. To elucidate the wiring of projection neurons from OR37 glomeruli into higher brain areas, tracing experiments were performed. The application of DiI onto the ventral area of the olfactory bulb, which harbors the OR37 glomeruli, led to the labeling of fibers not only in the typical olfactory cortical regions, but also in the medial amygdala and the hypothalamus. To visualize the projections from a defined OR37 glomerulus more precisely, transgenic mice were studied in which olfactory sensory neurons co-express the receptor subtype OR37C and the transsynaptic tracer wheat germ agglutinin (WGA). WGA became visible not only in the OR37C sensory neurons and the corresponding OR37C glomerulus, but also in cell somata located in the mitral/tufted cell layer adjacent to the OR37C glomerulus, indicating a transfer of WGA onto projection neurons. In the brain, WGA immunoreactivity was not detectable in typical olfactory cortical areas, but instead in distinct areas of the medial amygdala. Detailed mapping revealed that the WGA immunoreactivity was restricted to the posterior-dorsal subnucleus of the medial amygdala. In addition, WGA immunoreactivity was visible in some well-circumscribed areas of the hypothalamus. These results are indicative for a unique connectivity from OR37C sensory cells into higher brain centers.     

Taste perception and coding in Drosophila

BACKGROUND: Discrimination between edible and contaminated foods is crucial for the survival of animals. In Drosophila, a family of gustatory receptors (GRs) expressed in taste neurons is thought to mediate the recognition of sugars and bitter compounds, thereby controlling feeding behavior. RESULTS: We have characterized in detail the expression of eight Gr genes in the labial palps, the fly's main taste organ. These genes fall into two distinct groups: seven of them, including Gr66a, are expressed in 22 or fewer taste neurons in each labial palp. Additional experiments show that many of these genes are coexpressed in partially overlapping sets of neurons. In contrast, Gr5a, which encodes a receptor for trehalose, is expressed in a distinct and larger set of taste neurons associated with most chemosensory sensilla, including taste pegs. Mapping the axonal targets of cells expressing Gr66a and Gr5a reveals distinct projection patterns for these two groups of neurons in the brain. Moreover, tetanus toxin-mediated inactivation of Gr66a- or Gr5a-expressing cells shows that these two sets of neurons mediate distinct taste modalities-the perception of bitter (caffeine) and sweet (trehalose) taste, respectively. CONCLUSION: Discrimination between two taste modalities-sweet and bitter-requires specific sets of gustatory receptor neurons that express different Gr genes. Unlike the Drosophila olfactory system, where each neuron expresses a single olfactory receptor gene, taste neurons can express multiple receptors and do so in a complex Gr gene code that is unique for small sets of neurons.