Small subfamily of olfactory receptor genes: structural features, expression pattern and genomic organization

doi:10.1016/S0378-1119(99)00275-9

Gene

Volume 236, Issue 2, 20 August 1999, Pages 281-291

https://doi.org/10.1016/S0378-1119(99)00275-9 Get rights and content

Abstract

Olfactory receptors of the OR37 subfamily are characterized by distinct sequence features and are expressed in neurons segregated in a restricted area of the olfactory epithelium. In the present study, we have characterized the complement of OR37-like genes in the mouse. Five OR37-like genes were identified. They reside within only 60 kb of DNA on chromosome 4. About 70 kb distant from this cluster, two additional olfactory receptor genes are located, which are members of distinct receptor subfamilies. Phylogenetic analysis demonstrated that the two physically linked receptors are closely related to the OR37 subfamily. Studies of gene expression showed that both genes are also expressed in clustered neuron populations located in the typical OR37 region of the epithelium. These data suggest the involvement of locus-dependent mechanisms for the spatial control of OR gene expression.

Introduction

Olfactory receptors (ORs) are G-protein coupled seven-transmembrane proteins responsible for the recognition of odorous molecules; they are encoded by a large multigene family originally identified in rat (Buck and Axel, 1991). OR genes have been cloned from a variety of other vertebrate species (Freitag et al., 1999, Freitag et al., 1995, Nef et al., 1996, Ngai et al., 1993, Parmentier et al., 1992, Ressler et al., 1993, Selbie et al., 1992; for review see Mombaerts, 1999). In rodents, olfactory sensory neurons expressing a given OR gene are restricted to one of three or four zones of the nasal neuroepithelium extending along the anterior–posterior axis of the nose (Ressler et al., 1993, Strotmann et al., 1994a, Strotmann et al., 1994b, Vassar et al., 1993). In situ hybridization experiments revealed that each OR probe hybridizes to a small subpopulation of neurons, suggesting that individual cells express a small number of genes. The mechanisms controlling the selective expression of OR genes in distinct topographic zones are still elusive. For other multigene families, it has been shown that the genomic organization may provide a basis for regulatory principles (Krumlauf, 1992). Exploring the chromosomal distribution of the OR multigene family has demonstrated that OR genes are arranged in clusters, which are widely distributed in the genome (Ben-Arie et al., 1994, Rouquier et al., 1998, Trask et al., 1998); in mouse, 11 loci on seven different chromosomes have been mapped (Sullivan et al., 1996). It is still unclear whether a correlation exists between the clustering of OR genes and their expression zone. Not all the OR genes expressed in a particular zone are arranged in one cluster (Sullivan et al., 1996); however, whether all genes of a genomic cluster are expressed in the same zone, remains unanswered.

Recently, subtypes of ORs (OR37) that are characterized by distinct structural features have been identified in mammals. The OR37-like proteins comprise an extended third extracellular loop with numerous charged amino acids. Neurons expressing OR-37-like genes are selectively found in small groups on the tip of central turbinates, but not on the septum (Kubick et al., 1997, Strotmann et al., 1994a, Strotmann et al., 1994b, Strotmann et al., 1992). This patch-like distribution remains thus far the only exception to the zonal expression rule. In this study, we have characterized the repertoire of OR37-like OR genes in the mouse. The five OR37-like genes that have been identified are arranged in a distinct gene cluster. The OR37 locus contains two additional, non-OR37 OR genes which are also expressed in clustered populations of neurons in the epithelium.

Section snippets

BAC-library screen

A filter library of mouse genomic ES-cell DNA (129 SvJ) in a BAC vector (Release II; Genome Systems, St. Louis, MO) was screened with 50–100 ng of mOR37A-DNA (Kubick et al., 1997) labeled with [α-³²P]-ATP (NEN, Boston, MA) by random priming with Klenow enzyme (Life Technologies, Eggenstein, Germany) according to standard protocols (Sambrook et al., 1989). The probe was hybridized at 65°C in 5×SSPE (20×SSPE is 3.6 M NaCl, 200 mM NaH₂PO₄, 20 mM EDTA, pH 7.4), 5×Denhardt's, 0.1% SDS and 10 μg/ml salmon

Results

A genomic library with high molecular weight inserts of 129/SvJ-mouse DNA in a BAC vector was screened with a ³²P-labelled probe covering the coding region of mOR37A (Kubick et al., 1997). Using high-stringency conditions, nine positive clones were obtained and subjected to further analysis. To characterize the repertoire of OR37-like receptor genes located within these genomic fragments, each BAC clone was used as a template in PCR reactions with primers, which have previously been employed to

Discussion

It was the aim of the present study to characterize the complement of OR37-like olfactory receptor types in mouse. Using Southern blot and PCR approaches, five genes were identified in the mouse genome encoding proteins with an extended third extracellular loop of six additional amino acids, typical for OR37-like receptors. By this criterion, they are members of a distinct family of OR types present in different mammals, but not in non-mammalian species (Kubick et al., 1997, Strotmann et al.,

Acknowledgements

This research was supported by the Deutsche Forschungsgemeinschaft. Grant support to P.M. came from the National Institutes of Health.

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In the nose of mammals, odorous molecules are detected by olfactory sensory neurons residing in distinct nasal compartments, including the main olfactory epithelium, the septal organ, and the Grueneberg ganglion. In this chapter, the characteristics of these distinct chemosensory organs are reviewed with a particular focus on the functional implications and the molecular features of the respective olfactory neurons as well as their axonal connectivity with the olfactory bulb of the brain.
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Samples were cryoprotected in 15% and 30% sucrose in PBS at 4 °C, frozen in O.C.T. Compound (Tissue-Tek), sectioned at 12 μm with a Leica CM3500 cryostat, and collected onto glass slides. OR RNAs were detected with biotinylated riboprobes against Olfr156 (Strotmann et al., 1999); Olfr412 (forward 5′-ATGGACGGAGGCAACCAGA-3′, reverse 5′-GGCTTCCTTAGCAGTCTTCCC-3′), and Olfr552 (Addgene plasmid #15843; Hirota et al., 2007). Riboprobes were generated as described in Ishii et al. (2004).
In the mouse, odorant receptor proteins (ORs) are G-protein-coupled receptors expressed in mature olfactory sensory neurons (OSNs) of the main olfactory epithelium (MOE). ORs mediate odorant reception at the level of the OSN cilia. Most of the ∼1100 OR genes in the mouse genome are expressed, at the RNA level, in mature OSNs. The literature on antibodies against ORs is limited, and most reports are with antibodies that are not commercially available. Here we have screened 40 commercial antibodies against human and mouse ORs by immunofluorescence staining of coronal cryosections of the MOE of 21-day-old C57BL/6J mice. Various methods of antigen retrieval were tested. Of the 19 antibodies raised against human ORs, three yielded a consistent immunoreactive signal in the mouse MOE; of these three, two appeared to cross react against one or more, unknown, mouse ORs. Of the 21 antibodies raised against mouse ORs, six yielded a consistent immunoreactive signal in the mouse MOE; of these six, two also stained specific glomeruli in the olfactory bulb. Antibody specificity could be validated with gene-targeted mouse strains in the case of three ORs. The number of OSNs immunoreactive for the MOR28/Olfr1507 antibody is greater in C57BL/6J than in 129S6/SvEvTac wild-type mice. Taken together, our results are encouraging: 20–30% of these commercially available antibodies are informative in immunohistochemical analyses of the mouse MOE. The commercial availability of these antibodies should facilitate the study of OR proteins in the MOE and the olfactory bulb, and the replicability of results in the literature.
Structure, Expression, and Function of Olfactory Receptors
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Olfactory receptors (ORs) belong to the G protein-coupled receptor family and play a critical role in recognizing thousands of odorant molecules in the olfactory sensory system. Genome-wide searching for the OR family in various species has revealed the evolutional lineages that resulted in the dramatic changes in the numbers and sequences of the multigene family. Functional characterization of ORs has progressed rapidly due to the development of various expression systems. The detailed pharmacology of ORs has revealed agonist and antagonist specificity and has led to functional determination of the odorant-binding site. The three-dimensional configuration of the odorant-binding site, which is constructed by transmembrane helices, provides the molecular basis for odorant sensitivity and specificity. The one-neuron one-OR rule and OR-instructed axonal convergence, which have been explored by elegant genetic manipulation studies, produce neural circuits that ensure precise transmission of the combinatorial receptor codes for odorants from the olfactory epithelium to the olfactory bulb. The function of ORs has turned out to be much more diverse, including participation in the guidance of olfactory neuron axons, chemoreception in tissues other than the olfactory epithelium, and sensing of pheromones that affects innate behavior or neuroendocrine systems. Studies on the sense of smell have advanced to the point of elucidating mechanisms underlying integration of OR-mediated signals for odor perception related to survival and chemical communication.
Promotor elements governing the clustered expression pattern of odorant receptor genes
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Odorant receptor (OR) genes of family mOR262 are only expressed in olfactory sensory neurons (OSNs) segregated in a central patch of the nasal turbinates; they comprise conserved DNA elements upstream of their transcription start sites that are proposed to govern the distinct expression pattern. In mouse lines with a transgene containing the coding sequence and a short upstream region of the mOR262-12 gene, expression was restricted to OSNs that were segregated in the characteristic central patch, although the number of cells varied considerably. Only in one line, the transgene was also expressed in OSNs ectopically positioned outside the patch. The axons of transgene-expressing OSNs co-converged with those expressing the endogenous gene. The transgene was found to be expressed in a mutually exclusive manner and from only one allele indicating that the conserved upstream DNA elements play a critical role in controlling the specific expression pattern of these genes.
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Novel olfactory receptor-encoding genes that are expressed in olfactory sensory neurons arranged in a clustered pattern in the nasal epithelium, typical of the mOR262 (approved gene symbol Olfr) family, were identified. The genes share sequence motifs upstream of their transcription start sites that are highly related to those previously identified as characteristic of the mOR262 genes, suggesting that these regulatory elements may contribute to governing their unique expression pattern. Promoter analyses of genes encoding class I receptors that are expressed in the dorsal region of the epithelium revealed a different, but again common set of sequence motifs. A prominent feature of the class I gene promoters are multiple O/E-like binding sites, and O/E-type transcription factors that bind to the putative promoter region of class I OR genes were in fact identified. The findings support the concept that common elements in the promoter region of these OR genes may determine their congenic expression pattern in the epithelium.
A Multireceptor Genetic Approach Uncovers an Ordered Integration of VNO Sensory Inputs in the Accessory Olfactory Bulb
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^☆: Olfactory receptor sequence data have been deposited under accession numbers AJ133424–AJ133430.

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Small subfamily of olfactory receptor genes: structural features, expression pattern and genomic organization☆

Abstract

Introduction

Section snippets

BAC-library screen

Results

Discussion

Acknowledgements

Biochem. Biophys. Res. Commun.

Cell

Genomics

Gene

Genomics

Gene

Cell

Mech. Dev.

Cell

Mol. Brain Res.

Genomics

Cell

Olfactory receptor gene cluster on human chromosome 17: possible duplication of an ancestral receptor repertoire

Hum. Mol. Genet.

Location of mouse and human genes corresponding to conserved canine olfactory receptor gene subfamilies

Mammal. Genome

Two classes of olfactory receptors in Xenopus laevis

Neuron

Fast and sensitive multiple sequence alignments on a microcomputer

CABIOS

Evolution of the vertebrate hox homeobox genes

BioEssays