Small subfamily of olfactory receptor genes: structural features, expression pattern and genomic organization☆
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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Olfactory receptor sequence data have been deposited under accession numbers AJ133424–AJ133430.