Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (27)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Rouquier, S.
Right arrow Articles by Giorgi, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rouquier, S.
Right arrow Articles by Giorgi, D.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics Pages 1337-1345  

A gene recently inactivated in human defines a new olfactory receptor family in mammals
Introduction
Results
   Isolation of a chromosome 11-specific OR sequence
   Cloning and sequencing of the 912-93 full-length sequence
   Characterization of the 912-93 gene from non-human primates
   Sub-chromosomal localization of the 912-93 gene in hominoids
Discussion
Materials And Methods
   Chromosome flow sorting and isolation of the 9120-93 partial sequence
   Chromosome assignment
   Chromosome 11 cosmid library screening
   Cosmid clone analysis and subcloning
   Fluorescence in situ hybridization (FISH)
   Sequence analyses of the 5[prime]-region of unrelated individuals
   Cloning of the 912-93 gene in non-human primates
   Multiple alignments and phylogenetic trees
Acknowledgements
References

A gene recently inactivated in human defines a new olfactory receptor family in mammals

A gene recently inactivated in human defines a new olfactory receptor family in mammals

Sylvie Rouquier, Cynthia Friedman1, Cécile Delettre, Ger van den Engh1, Antoine Blancher2, Brigitte Crouau-Roy3, Barbara J. Trask1 and Dominique Giorgi*

IGH, CNRS UPR 1142, 141 rue de la Cardonille, 34396 Montpellier Cédex 5, France, 1Department of Molecular Biotechnology, Box 357730, University of Washington, Seattle, WA 98195, USA, 2Laboratoire d'Immunologie, Hôpital Purpan, 31059 Toulouse Cédex, France and 3CIGH, CNRS UPR 8291, Hôpital Purpan, 31300 Toulouse, France

Received April 7, 1998; Revised and Accepted June 13, 1998

DDBJ/EMBL/GenBank accession nos AF045576-AF45580

The olfactory receptor (OR) gene family constitutes one of the largest multigene families and is distributed among many chromosomal sites in the human genome. Four OR families have been defined in mammals. We previously demonstrated that a high fraction of human OR sequences have incurred deleterious mutations, thus reducing the repertoire of functional OR genes. In this study, we have characterized a new OR gene, 912-93, in primates. This gene is unique and it defines a new OR family. It localizes to human chromosome 11q11-12 and at syntenical sites in other hominoids. The sequence marks a previously unrecognized rearrangement of pericentromeric material from chromosome 11 to the centromeric region of gibbon chromosome 5. The human gene contains a nonsense point mutation in the region corresponding to the extracellular N-terminus of the receptor. This mutation is present in humans of various ethnic groups, but is absent in apes, suggesting that it probably appeared during the divergence of humans from other apes, <4 000 000-5 000 000 years ago. A second mutation, a frameshift at a different location, has occurred in the gorilla copy of this gene. These observations suggest that OR 912-93 has been recently silenced in human and gorilla, adding to a pool of OR pseudogenes whose growth may parallel a reduction in the sense of smell in primates.

INTRODUCTION

The olfactory system has the remarkable capacity to discriminate a wide range of odour molecules. The identification of a large multigene family encoding olfactory receptors (OR) belonging to the G protein-coupled receptors (GPCR) gene superfamily has provided insight into the mechanism of olfaction (1,2). Families of odourant receptor genes related to those initially described in the rat (3) have now been found in a variety of species, including human (4-7), mouse (8), dog (4,9,10), catfish (11), Xenopus (12) and honeybee (13). All these odourant receptors share features of sequence and structure, including seven hydrophobic transmembrane domains ([alpha]-helices TM1-TM7), which are common to all members of the GPCR superfamily. The OR genes cloned so far show a high degree of homology in several TM regions, allowing identification of new OR genes. Study of the family is facilitated by the fact that OR genes are usually intronless in their coding region and are ~1000 bp long (for a review see ref. 14). A widely accepted current classification defines sequences that share >40% identity as a family and those with >60% identity as a sub-family. Accordingly, eight OR gene families have been identified, four containing only mammalian sequences and four containing only fish sequences.

The human sense of smell is classified as microsmatic, i.e. it is greatly reduced relative to that of other mammals such as dog or rodents. Part of this difference can be explained by differences in the anatomical structures devoted to olfaction (10). Variations in the size and diversity of the OR gene family could also explain differences in sensory perception among organisms. The size of the OR gene family in the human genome has been estimated to contain 200-1000 members, on the basis of indirect arguments (15). We have recently shown that OR genes are located in the human genome at >25 sites, many of which contain clusters of OR genes (16). Our survey of 87 OR sequences revealed that a remarkably high fraction of human OR genes appear to be pseudogenes; 72% of the OR sequences isolated from flow-sorted chromosomes using degenerate primers for the conserved TM2 and TM7 regions showed frameshifts and/or in-frame stop codons (16). This finding led us to hypothesize that the reduction in the sense of smell in humans could be correlated to a reduction, through mutation, in the repertoire of functional OR genes.

Here we present the characterization of a particular new OR gene, 912-93. This gene is highly conserved in hominidae, but it has mutated into a pseudogene very recently in humans and gorillas. It is also sufficiently diverged from other known OR genes that it defines a new mammalian OR gene family. Because of its unique sequence, the 912-93 OR gene can be followed easily during evolution and constitutes an excellent model for expression studies.

RESULTS

Isolation of a chromosome 11-specific OR sequence

As part of a study of OR sequences from different human chromosomes, we previously characterized OR-related sequences derived from the chromosome 9-12 group (16) (chromosomes 9-12 were analyzed as a group because they cannot be separated by flow sorting). OR sequences from these chromosomes were obtained by first randomly amplifying flow-sorted material by DOP-PCR and then subjecting this product to OR-specific amplification using degenerate consensus primers in the TM2 and TM7 coding domains of OR sequences. These PCR products were cloned and sequenced. Fourteen of these sequences were reported elsewhere (16). All 14 were pseudogenes, showing one or more frameshifts and/or in-frame stop codons.

A new sequence from the 9-12 group, clone 912-93, stands out from these others in several respects. First, it shows an uninterrupted open reading frame between TM2 and TM7. Second, the sequence of 912-93 is significantly divergent from the other sequences from the 9-12 group. The previous sequences derived from chromosomes 9-12 fell into two groups, G1 and G2. Sequences within each group were [ge]60-70% similar, but the two groups showed only ~50% nucleotide sequence identity (NSI) with each other. 912-93 does not fit well in either group; it shows only 55% NSI with its closest relative among the 87 sequences we have characterized (912-92 in G1).

At the predicted amino acid sequence level, 912-93 shows <40% amino acid identity (ASI) in the TM2-TM7 region with any of the previously defined OR families (Fig. 1). Among mammalian OR sequences in the public databases, 912-93 is most similar, albeit distantly related, to dog CfOLF1 and CfOLF2 (DDBJ/EMBL/GenBank accession nos U53679 and U53680; 50 and 48% ASI respectively) and human HsOLF1 (51% ASI) (9,10). Thus 912-93 and these three OR sequences define a new mammalian OR family, designated number 9. Four sub-families (A-D) were defined in family 9 on the basis of their divergence, i.e. >60% ASI. 912-93 is the sole member in subfamily A. Database sequence comparisons also revealed 44-47% ASI with chicken OR (COR1-3 and COR5-6) and 55% ASI with COR4 (DDBJ/EMBL/GenBank accession no. X94744)">http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=n&form=6&uid=X94744&dopt=g">X94744) (Fig. 1). To our surprise, 912-93 shows a very high degree of sequence identity (96 and 97% NSI, 89 and 93% ASI) with two olfactory receptor sequences (DDBJ/EMBL/GenBank accession nos S76956 and S76957) derived from the honeybee (Apis mellifera) (13; see Discussion).


Figure 1. Phylogenetic tree placing the predicted protein encoded by OR 912-93 (in a square) relative to selected human predicted OR proteins representative of OR families (defined as having [ge]40% ASI) and sub-families (having [ge]60% ASI). Data are from Rouquier et al. (16), Ben-Arie et al. (7) (shadowed sequences), Issel-Tarver and Rine (CfOLF1/2 and HsOLF1; 10) and Leibovici et al. (COR4; 40). Families are represented by circled numbers and sub-families by letters in squares. Not shown in the figure are families 4 and 5-8, which are defined by rat and fish olfactory receptors respectively (7). Sequences were aligned using CLUSTAL W (26). The numbers on internal branches represent the frequency of occurrence among 1000 trees (bootstrap method). Each sequence number refers to the chromosome and clone number (e.g. 912-93 for clone 93 derived from chromosomes 9-12). Note that all protein sequences are predicted from genomic sequences. It is not known whether any of these putative ORs are expressed at either the mRNA or protein level. Hs, Homo sapiens; Cf, Canis familiaris; C, Gallus gallus.

To determine the chromosomal location of 912-93, we screened a human-rodent somatic cell hybrid panel by PCR with 912-93-specific primers. Primers 9F and 9R were designed from the TM3 and TM4 coding regions of 912-93. These regions were chosen because they are highly divergent from rodent OR sequences in the databases and were, therefore, unlikely to amplify sequences in the rodent background of the hybrid lines in the human monochromosomal panel. This primer pair unambiguously assigned clone 912-93 to chromosome 11 (Fig. 2).

Cloning and sequencing of the 912-93 full-length sequence

To isolate the full-length sequence of 912-93, we used the 700 bp PCR product as a probe to screen the ICRF chromosome 11-specific cosmid library. The screening was performed at high stringency to minimize isolation of related OR sequences. Of the 18 816 clones screened (a 5.8-fold coverage of chromosome 11), only one clone (ICRF c107E0141D1, referred to henceforth as cos 1-93) was strongly positive in duplicate. The screening was then confirmed by Southern blot analysis of restriction enzyme-digested cosmid DNA (Fig. 3). We subcloned the 3.2 kb EcoRI fragment containing the 912-93 sequence into Bluescript KS- and sequenced the 912-93 gene using vector or specific primers (primer walking). The sequence is shown in Figure 4. The sequence revealed an in-frame stop codon in the extracellular N-terminal portion at position 11. Alignment of the sequence with the predicted amino acid sequences of other OR genes indicated that Glu is most frequently found at this position. Thus, the nonsense mutation is most likely due to a G->T transversion (GAA Glu->TAA stop).


Figure 2. Chromosomal assignment of human sequence 912-93 by PCR amplification (primers 9F and 9R, see Fig. 4) using a human-rodent somatic cell hybrid panel. The human chromosome present in each of the 24 somatic cell hybrids is shown above the lanes (chromosomes 1-22, X and Y). Lanes M, CH and H contain mouse, Chinese hamster and human DNA respectively. Lane H2O is a negative control with no DNA.


Figure 3. Analysis of cosmid 1-93 containing the human 912-93 OR gene. (Left) Restriction analysis of cos 1-93 by different restriction endonucleases is indicated at the top. DNAs were run on a 0.4% agarose gel. A molecular weight marker (1 kb ladder; Life Technologies) was run in parallel. The gel was then alkali blotted on nylon membrane and hybridized with the 912-93 probe. (Right) Autoradiogram of the membrane. The positive 3.2 kbp EcoRI fragment indicated by an arrow was then subcloned in pBluescript KS- (Stratagene) and sequenced.


Figure 4. Sequence of the human OR gene 912-93. The nucleotide sequence is represented by lower case (untranslated regions) and upper case (coding region) letters. The predicted translated protein sequence is indicated below. The deleterious mutation generating an in-frame stop codon (TAA) is indicated in bold and by an asterisk (residue 11). The indel frameshift (Go550[Delta]T) found in gorilla is indicated by a vertical arrow. The different primer pairs used in this study are indicated by horizontal arrows above the nucleotide sequence. Positions of consensus OR primers OR5B and OR3B are indicated by thick shaded arrows. Protein motifs discussed in the text are underlined by dashed lines.

Hydrophobicity plots based on the sequence are consistent with a structure containing seven transmembrane segments (data not shown). The gene contains the PMY(F/L)FL motif common to most olfactory receptors, but has an MAYDCYVAIC motif, already found in another example (7,16), instead of the classical MAYDRYVAIC feature. Cys98 and Cys179, which may form a disulfide bridge, Ser231, which has been described as a potential phosphorylation site, and Ser218 and Tyr219 are also conserved (Fig. 4).

Multiple individuals show the same nonsense mutation in the N-terminal extracellular domain. To verify that the stop codon found in the 5[prime]-portion of the gene was not a cosmid cloning artefact, a PCR-induced sequencing error or a polymorphism, we designed a pair of primers (93-FP1 and 93RP1) to amplify 181 bp around this site (Fig. 4) from genomic DNAs. These primers were first tested on total human and rodent genomic DNAs and on a monochromosomal somatic cell hybrid panel. As expected, a 181 bp product was amplified only from human DNA and the chromosome 11 hybrid in the panel. Genomic DNAs of seven unrelated Caucasian individuals were PCR amplified and the products directly sequenced. The nonsense mutation was present in all samples. To test whether this mutation predated the emergence of different human populations or was more recent, we then PCR amplified and sequenced genomic DNAs of five non-Caucasian individuals, three Africans (Anaclet, Bissangou and N'Dong) and two Asians (Chinese). The same nonsense mutation was detected in all individuals.

Characterization of the 912-93 gene from non-human primates

The PCR primer pair 93-FP1 and 93R4 surrounding the coding region of 912-93 was used on genomic DNAs of different non-human primates. A single band of the expected size was amplified from the non-human hominoid samples (chimpanzee, gorilla, orangutan and gibbon), but not from lower species (baboon and macaque). PCR products from the four species were subcloned and ~12 clones/species were confirmed by Southern blot using the human 912-93 probe. Two independent clones from each species were then sequenced. In each case, the sequences of the two clones were identical. The human and non-human primate sequences are highly identical (96-98% NSI, 94-97% ASI) (Fig. 5A). However, none of the ape sequences presents the N-terminal nonsense mutation, all coding for a glutamic acid (GAA). The chimpanzee, orangutan and gibbon sequences all exhibit an intact open reading frame. However, a deletion of one base (550[Delta]T) was observed in gorilla (one individual from each of two species were sequenced), leading to a frameshift (Figs 4 and 5). In addition, amino acid 34 in intracellular loop 1 is missing (deletion of an ACT codon) in gorilla, gibbon and orangutan, whereas a threonine is present in human and chimpanzee sequences (Fig. 5). The phylogenetic tree based on 912-93 sequences is consistent with the generally accepted hominoid structure (Fig. 5A).


Figure 5. Predicted sequences of OR 912-93 proteins from hominoids. (A) The sequence alignment was performed using the methods described in Figure 1. Amino acid identity is indicated by a dash (-) and amino acid deletion by a delta ([Delta]). The frameshift observed in gorilla (between residues 183 and 184) is indicated by a vertical bar. The putative seven transmembrane domains (TM1-TM7) are indicated by horizontal lines. Conserved positions are indicated by asterisks (Cons). Hum, human; Ch, chimpanzee; Go, gorilla; Oo, orangutan; Gib, gibbon. (B) Unrooted phylogenetic tree of hominoid OR 912-93 proteins generated as described in Figure 1.

Sub-chromosomal localization of the 912-93 gene in hominoids

The cos 1-93 DNA was labeled by nick-translation and hybridized onto metaphase chromosomes of various hominoids to determine its location. As shown in Figure 6A, this sequence maps to a single location, 11q11-12, in human chromosomes. The sequence maps to the locations syntenic to 11q11-12 on chimpanzee PPA 9, gorilla GGO 9 and orangutan PPY 8 (Fig. 6B and C). The orangutan chromosome 8 has undergone several rearrangements, resulting in positioning of the 11q11-12 region on the distal short arm of this chromosome (Fig. 6C). The 912-93 sequence maps near the centromere of Hylobates lar (gibbon) chromosome 5 (HLA 5). This region was not previously known to contain orthologs of sequences on HSA 11 (28). However, dual hybridization of the 912-93 sequence and a human chromosome 11 paint clearly demonstrate the presence of a significant segment of chromosome 11 material at this location. We established, using additional chromosome paints, that HLA5 is otherwise composed of material homologous to human chromosomes 18 and 1, consistent with earlier studies (28).


Figure 6. FISH localization of the 912-93 sequence in hominoids. (A) Cosmid 1-93 containing the 912-93 gene maps to a single site in the human genome corresponding to 11q11-12. (B and C) The 1-93 sequence maps to the syntenic locations in hominoid chromosomes. (B) The only labeled chromosomes from representative metaphases of each species. (C) The homology of HSA11, PPA9, GGO9 and PPY8 as described by Yunis and Prakash (34). Although the chimpanzee and gorilla chromosomes have a different number, they represent unrearranged versions of human 11 and, as expected, the 912-93 sequence maps to the regions corresponding to 11q11-12 on these chromosomes. In orangutan, chromosome 8 is the result of three rearrangements of chromosome 11 material as indicated by the arrows connecting PPY8 and HSA11 in (C). The location of the 912-93 sequence on the p arm of this chromosome is consistent with conservation of synteny of the 11q11-12 region. The 912-93 sequence maps to H.lar 5 near the centromere (B and D). A human chromosome 11 painting probe (green) demonstrates that this region contains chromosome 11 material (D). The diagram in (C) summarizes the results of hybridization of the gibbon chromosomes with a variety of human chromosome paints in combination with the 912-93 probe.

DISCUSSION

The olfactory receptor genes constitute one of the largest multigene families in mammals, but relatively few of the hundreds of predicted genes have been characterized (29). We previously isolated almost 90 chromosome-specific OR sequences starting from flow-sorted chromosomes (16). Here, we describe a new chromosome 11-specific sequence, 912-93, which is highly diverged from all the other OR sequences. The protein sequence of OR 912-93 defines a new mammalian OR family. The uniqueness of the 912-93 sequence with respect to other mammalian OR sequences allowed us to isolate its genomic counterpart without the usual problems of cross-hybridization with other OR sequences due to their high sequence similarity (24; V. Brand-Apron, S. Rouquier, H. Massa, P. de Jong, C. Ferraz, P. Ioannou, J.G. Demaille, B.J. Trask and D. Giorgi, submitted for publication). A single clone out of almost 19 000 was isolated, suggesting that 912-93 is not duplicated on chromosome 11. No cross-hybridization to other chromosomes was observed by FISH or by PCR of the hybrid panel, suggesting that 912-93 has no close relatives in family 9. The closest mammalian relatives detected through database comparisons are CfOLF1 and HsOLF1, which at ~50% ASI are quite divergent.

Sequence comparisons in databases revealed a high NSI (>96%) and ASI (>90%) with insect OR sequences isolated from honeybee (Apis mellifera) (13). Given the genetic divergence in the evolutionary process and speciation events, such conservation of sequence between human and honeybee is surprising. For example, the nucleotide sequence identity among human and rodent orthologs of OR genes is typically 60-70% (S. Rouquier et al., unpublished data). To date, all attempts to clone insect OR sequences using strategies based on sequence conservation with mammalian OR sequences have failed (30), suggesting that odour detection in insects involves different receptors or that the divergence of insects and mammals is too great to cross species using PCR or hybridization strategies. Therefore, we suspect that the OR sequences identified from honeybee represent mammalian DNA contamination.

The 912-93 OR sequence displays all the features of G protein-coupled receptors, except that the classical DRY motif is replaced by DCY. The DCY motif has already been documented in several other examples (7,16). These observations suggest either that the DRY motif is not a critical site for G protein coupling in OR, as it is for vasopressin receptors (31,32), or that this motif has also incurred a deleterious mutation, because an intact DRY is observed in other species. The complete sequence of the human gene revealed an in-frame stop codon in the first N-terminal part, demonstrating that 912-93 is a pseudogene. In this respect, 912-93 is like many other OR sequences in the human genome: 72% of the 87 OR sequences we previously characterized from the human genome, including all six from chromosome 11, were pseudogenes (16). This nonsense mutation was found in 12 unrelated individuals from several ethnic groups. Thus, the mutation is not a PCR-induced error or a polymorphism. The presence of only a single deleterious mutation in all 12 individuals suggests that this mutation appeared recently during evolution but in an ancestor common to these human populations (African, Caucasian and Chinese). It has been observed that once a gene is silenced by one mutation, it tends to accumulate further mutations in the absence of selective constraints, thereby lowering the chance of recovering its function (33), as observed previously (16). Accordingly, the human sequence contains nine additional species-specific amino acid changes relative to the non-human primate sequences.

Primers complementary to human sequences flanking the 912-93 gene successfully amplified the orthologous genes in the hominidae superfamily (chimpanzee, gorilla, orangutan and gibbon), but not in the cercopithecidae (macaque and baboon). Because Old World monkeys diverged from hominoids ~25 000 000 years ago, we conclude that the primer sequences were too diverged to allow PCR amplification. The other primate sequences were highly similar to human 912-93. The NSI varied from 98.3% between human and chimpanzee to ~96% between human and gibbon or orangutan. All non-human primate sequences presented a glutamic acid (GAA) at N-terminal position 11 instead of the nonsense (TAA) mutation seen in humans. Chimpanzee, orangutan and gibbon presented a complete ORF, suggesting that the gene may be functional in these species. In gorilla, however, 912-93 is also a pseudogene, due to a frameshift mutation. These two deleterious mutations, one human-specific and one gorilla-specific, occurred independently. The primate sequence comparisons show that the nonsense mutation found in humans appeared after divergence of human from gorilla and chimpanzee, 4 000 000-5 000 000 years ago. The gorilla mutation is also relatively recent, since it was detected in two gorilla species, but not in chimpanzee or humans. Notably, 912-93 sequence comparisons place chimpanzee closer to humans on the phylogenetic tree, consistent with many, but not all, earlier findings on the human-chimpanzee-gorilla trichotomy (34).

FISH analysis of the location of OR 912-93 in hominoid genomes adds to the picture of chromosome evolution in primates. The 912-93 sequence maps to a single location, 11q11-12 in human chromosomes and to the syntenic location in chimpanzee, gorilla, orangutan and gibbon chromosomes. A previously unrecognized rearrangement of chromosome 11 material encompassing this gene has occurred along the Hylobatidae branch. The sequence maps near the centromere of chromosome 5 in H.lar. Previous analysis of the H.lar karyotype with human chromosome-specific painting probes did not detect chromosome 11 material at this site (28). However, dual hybridization of a chromosome 11 painting probe and the 912-93 probe clearly demonstrate that a portion of chromosome 11, including 912-93, has been inserted, perhaps in inverted orientation, near the centromere of HLA 5. Chromosome 5 in H.lar is otherwise composed of material homologous to chromosomes 18 and 1. Interestingly, a small portion of chromosome 11 material, presumably containing the 912-93 sequence, is present near the centromere of chromosome 4 in a different gibbon species, H. concolor (35). This material is associated on this chromosome with material from human 18 (as in H.lar 5), but with chromosome 3 instead of chromosome 1 material. The genesis of these different chromosomes remains to be explained and will require analysis of region-specific markers for human chromosomes 3 and 1 of these two gibbon species.

The localization of 912-93 to 11q11-12 in humans places it at or near one of the three locations on this chromosome that have been found to contain OR genes (11p15, 11q12-14 and 11p23-24) (16,24,36). Since many OR genes are known to be clustered in the genome (7,37), this gene may be part of the 11q12-14 array. Interestingly, HsOLF1, its closest known relative in the human genome, also maps to 11q11 (10). However, no other OR genes were found in the cosmid by hybridization using the OR consensus probe and no other clone was isolated during screening of the 5.8-fold redundant chromosome 11-specific cosmid library with the 912-93 sequence.

Our observations are pertinent to the question of the evolution of the olfactory sense. Our sense of smell is known to be reduced with respect to rodents, probably because smelling is not as essential for primate survival as it is for rodents (seeking food, predator detection, etc.). We previously demonstrated that a large fraction of human OR sequences are pseudogenes (16). Here we have shown that the pool of pseudogenes is still growing. One possibility is that, in the absence of selective pressure, the OR gene repertoire in primates is progressively being restricted towards a minimal set of genes reflecting a loss of function. The mutated state of the interphotoreceptor retinoid binding protein in the blind marsupial mole is another such example (38). Alternatively, the OR family may be very dynamic, regularly spawning receptors with new odour specificities as well as pseudogenes. Sequence analyses of a large subset of OR genes in a variety of species should differentiate between these two hypotheses in the future.

The 912-93 gene constitutes an excellent model to study the evolution and expression of OR genes. Its uniqueness makes it easy to analyse in isolation from the hundreds of other OR genes. Because it is a unique member of a new OR family, we hypothesize that 912-93 may bind a well-defined class of odour molecules that humans may no longer be able to recognize. Using the adenovirus system discussed by Firestein et al. (39) and the corrected gene (with Glu11), it may be possible to identify odour ligands and study ligand-receptor interactions.

MATERIALS AND METHODS

Chromosome flow sorting and isolation of the 9120-93 partial sequence

The isolation of OR-related sequences from flow-sorted chromosomes 9-12 has been described elsewhere (16). Briefly, 2000 chromosomes from the unresolved 9-12 group were flow sorted and subjected to DOP-PCR amplification with primer 6MW (17,18). This material was then PCR amplified using the OR consensus degenerate oligonucleotides OR5B (in TM2) and OR3B (in TM7) (7). PCR products were subcloned in the TA vector (InVitrogen) and recombinant clones were identified by PCR. The partial sequence of OR gene 912-93 was obtained from one of these clones. Sequencing of this portion of 912-93 was performed and sequences were assembled and analysed as detailed elsewhere (16).

Chromosome assignment

The PCR primer pair 9F and 9R was chosen between TM3 and TM4 (9F, 5[prime]-GTGCTTTGCAGCAGTGGTTC-3[prime]; 9R, 5[prime]-ATTCATGAGTCCAATGACATAG-3[prime]) to be sufficiently specific that it could be used to determine the chromosomal location of 912-93, using a human-rodent somatic cell hybrid panel (mapping panel 2; NIGMS, Coriell). The PCR conditions used to type the panel were an initial denaturation step at 94°C for 2 min followed by 30 cycles of 94°C for 15 s, 55°C for 30 s and 72°C for 45 s, and a final extension at 72°C for 10 min. PCR products were analysed on an ethidium bromide stained 1% agarose gel.

Chromosome 11 cosmid library screening

A chromosome 11-enriched cosmid library (reference library ICRF 107 L4/FS11) was prepared from flow-sorted chromosome 11 DNA isolated from human cell line FC11 (19). The FC11 cell line contains a deletion of the 11q22-q23 chromosomal region (5% of the chromosome length) that allows chromosome 11 to be separated from chromosomes 9, 10 and 12 (19). This library was gridded on high density filters and screened with a 912-93 probe. To generate the probe, the 912-93 clone was subjected to PCR amplification using vector primers. This PCR product was radiolabelled to a specific activity of 108-109 c.p.m./µg by random hexamer priming (20) using [[alpha]-32P]dCTP. Filter hybridization was performed in duplicate and carried out overnight at 65°C in 6× SSC, 5× Denhardt's, 0.5% SDS, 100 µg/ml sonicated herring sperm DNA with a labelled probe concentration of 106 c.p.m./ml. Following hybridization, filters were washed once in 2× SSC, 0.1% SDS at 65°C and twice at 0.1× SSC, 0.1% SDS at 65°C. Filters were then exposed overnight to X-ray film at -80°C.

Cosmid clone analysis and subcloning

DNA was purified from the 912-93-positive cosmid ICRF c107E0141D1 (referred to here as 1-93) on Qiagen columns and digested with various restriction enzymes in buffers supplied by the manufacturer (Life Technologies). The restriction digests were analysed by electrophoresis on 0.4% agarose gels. The gels were alkali blotted as described (21) onto nylon membranes (Hybond N+; Amersham) and used for hybridization as described above. Because of its extreme instability, the cosmid DNA was rescued by in vitro packaging and transfected in the bacterial host DH5[alpha]MCR (22) using the Gigapack III Gold Packaging extract from Stratagene and following the instructions of the manufacturer.

A 3.2 kb EcoRI fragment containing the complete OR 912-93 protein coding sequence was subcloned into pBluescript KS- (Stratagene) and sequenced using either -21M13 and M13rev Dye primers (Perkin Elmer) using the PRISM Ready Reaction AmpliTaq FS+ kit (Perkin Elmer) or specific primers (93F1, 5[prime]-AGTGCAACCTTCTCCCTG-3[prime]; 93R1, 5[prime]-GAACCACTGCTGCAAAGC-3[prime]; 93F2, 5[prime]-TATACTCAGTTGCTATG-3[prime]; 93R2, 5[prime]-ACAGTCTGCTGCCAC-3[prime]; 93F3, 5[prime]-GATCCGCTCTGCTGATGG-3[prime]; 93R3, 5[prime]-TTGCACTGGGATGTAC-3[prime]; 93R4, 5[prime]-GGAAGCCACACAATTACT-3[prime]) using the Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer). Sequences with the dye terminator cycle sequencing kit were also performed in parallel directly on cosmid DNA.

Fluorescence in situ hybridization (FISH)

Metaphase cell preparations were prepared from human phytohaemagglutinin-stimulated peripheral blood of a normal male donor, lymphoblastoid cell cultures of chimpanzee (CRL-1868, LENA), gorilla (CRL-1854, ROK) and orangutan (CRL-1850, Puti) and a lymphosarcoma-derived cell line from gibbon (H.lar, TIB-201), all from ATCC, according to published procedures (23,24). DNA from cos 1-93 was biotinylated by nick-translation and hybridized in the presence of human Cot1 DNA to metaphase cells fixed on slides. Methods for preparation of the slides and probe, hybridization, washing, detection with FITC, fluorescent banding and analysis have been described elsewhere (23,24). Chromosomes were banded (a QFH-like pattern) with DAPI staining. A human chromosome 11-specific paint was prepared by flow sorting ~500 copies of chromosome 11 from a somatic cell hybrid line, J1, which carries chromosome 11 as its only human material (25). This material was universally amplified by DOP-PCR using primer 6MW and then digoxigenin labelled by addition of digoxigenin-dUTP in additional PCR cycles as described elsewhere (23). The chromosome 11 paint was co-hybridized with cos 1-93 to gibbon metaphases. In this case, the paint was detected using mouse anti-digoxin and FITC-labelled anti-mouse IgG antibodies and the cosmid was detected with Texas Red-conjugated avidin (details of the procedure in 24ref. ). Additional human chromosome-specific paints were similarly generated from flow-sorted chromosomes as an aid to the identification of gibbon chromosomes. The FITC, Texas Red and DAPI fluorescence images were collected in register and displayed in false colours using a Princeton cooled CCD camera, ChromaTechnology spectral filters and image analysis software (IP Lab Spectrum). The DAPI fluorescence image was in some cases inverted to display a Giemsa-like banding pattern.

Sequence analyses of the 5[prime]-region of unrelated individuals

The 912-93 sequences of 12 unrelated individuals (seven Caucasian, three black African and two Chinese) were compared by amplifying this region from genomic DNA by PCR using primers 93-FP1 (5[prime]-TAGTAGAAAGCAGAACCAGTC-3[prime]) and 93-RP1 (5[prime]-ATCAATTCGGATGAGGACAATC-3[prime]). The PCR conditions were identical to those previously described for 9F and 9R. PCR products were purified by precipitation and directly sequenced using the same primers as described above.

Cloning of the 912-93 gene in non-human primates

Genomic DNA prepared from peripheral blood of chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), gibbon (H.lar), macaque (Macaca sylvanus), and baboon (Papio papio) was PCR amplified using primers 93-FP1 and 93R4 surrounding the putative coding region. PCR products were subcloned in the TA cloning vector (InVitrogen). Recombinant clones were verified by Southern blot using the human 912-93 probe and sequenced as described above.

Multiple alignments and phylogenetic trees

Multiple alignment was performed using the CLUSTAL W v.1.7 software package (26). Phylogenetic trees were constructed using the neighbour joining method (27), based on the number of nucleotide or amino acid substitutions. Bootstrap tests were also performed using the CLUSTAL W package.

ACKNOWLEDGEMENTS

We thank Dr Philippe Berta and Prof. Marie-Paule Lefranc for human DNA samples, Dr Jean Derancourt for helpful discussions and Prof. Jacques Demaille for his constant interest in this work. This work was supported by grants from the Programme Génome du CNRS, the Fondation pour la Recherche Médicale, NATO and the NIH (R01-GM 57070 to B.T.).

REFERENCES

1. Vassar, R., Ngai, J. and Axel, R. (1993) Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell, 74, 309-318. MEDLINE Abstract

2. Mombaerts, P., Wang, F., Dulac, C., Chao, S.K., Nemes, A., Mendelsohn, M., Edmonson, J. and Axel, R. (1996) Visualizing an olfactory sensory map. Cell, 87, 675-686. MEDLINE Abstract

3. Buck, L. and Axel, R. (1991) A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell, 65, 175-187. MEDLINE Abstract

4. Parmentier, M., Libert, F., Schurmans, S., Schiffmann, S., Lefort, A., Eggerickx, D., Ledent, C., Mollereau, C., Gerard, C. and Perret, J. (1992) Expression of members of the putative olfactory receptor gene family in mammalian germ cells. Nature, 355, 453-455. MEDLINE Abstract

5. Selbie, L.A., Townsend-Nicholson, A., Iismaa, T.I. and Shine, J. (1992). Novel G protein-coupled receptors: a gene family of putative human olfactory receptor sequences. Mol. Brain Res., 13, 159-163. MEDLINE Abstract

6. Schurmans, S., Muscatelli, F., Miot, F., Mattéi, M.-G., Vassart, G. and Parmentier, M. (1993) The OLFR1 gene encoding the HGMP07E putative olfactory receptor maps to the 17p13-p12 region of the human genome and reveals an MSPL restriction fragment length polymorphism. Cytogenet. Cell Genet., 6, 200-204.

7. Ben-Arie, N., Lancet, D., Taylor, C., Khen, M., Walker, N., Ledbetter, D.H., Carrozzo, R., Patel, K., Sheer, D., Lehrach, H. and North, M.A. (1994) Olfactory receptor gene cluster on human chromosome 17: possible duplication of an ancestral receptor repertoire. Hum. Mol. Genet., 3, 229-235. MEDLINE Abstract

8. Ressler, K.J., Sullivan, S.L. and Buck, L.B. (1993) A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell, 73, 597-609. MEDLINE Abstract

9. Issel-Tarver, L. and Rine, J. (1996) Organization and expression of canine olfactory genes. Proc. Natl Acad. Sci. USA, 93, 10897-10902. MEDLINE Abstract

10. Issel-Tarver, L. and Rine, J. (1997) The evolution of mammalian olfactory receptor genes. Genetics, 145, 185-195. MEDLINE Abstract

11. Ngai, J., Dowling, M.M., Buck, L., Axel, R. and Chess, A. (1993) The family of genes encoding odorant receptors in the channel catfish. Cell, 72, 657-666. MEDLINE Abstract

12. Freitag, J., Krieger, J., Strotman, J. and Breer, H. (1995) Two classes of olfactory receptors in Xenopus laevis. Neuron, 15, 1383-1392. MEDLINE Abstract

13. Danty, E., Cornuet, J.M. and Masson, C. (1994) Honeybees have putative olfactory receptor proteins similar to those of vertebrates. C.R. Acad. Sci. III, 317, 1073-1079.

14. Lancet, D. and Ben-Arie, N. (1993) Olfactory receptors. Curr. Biol., 3, 668-674.

15. Ressler, K.J., Sullivan, S.L. and Buck, L.B. (1994) A molecular dissection of spatial patterning in the olfactory system. Curr. Opin. Neurobiol., 4, 588-596. MEDLINE Abstract

16. Rouquier, S., Taviaux, S., Trask, B., Brand-Arpon, V., van den Engh, G., Demaille, J. and Giorgi, D. (1998) Distribution of olfactory receptor genes in the human genome. Nature Genet., 18, 1-10.

17. Telenius, H., Carter, N.P., Bebb, E.C., Nordenskjoeld, M., Ponder, B.A.J. and Tunnacliffe, A. (1992) Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics, 13, 718-725. MEDLINE Abstract

18. Rouquier, S., Trask, B.J., Taviaux, S., Diriong, S., van den Engh, G., Lennon, G.G. and Giorgi, D. (1995) Direct selection of cDNAs using whole chromosomes. Nucleic Acids Res., 23, 4415-4420. MEDLINE Abstract

19. Nizetic, D., Monard, S., Young, B., Cotter, F., Zehetner, G. and Lehrach, H. (1994). Construction of cosmid libraries from flow-sorted human chromosomes 1, 6, 7, 11, 13, and 18 for reference library resources. Mamm. Genome, 5, 801-802. MEDLINE Abstract

20. Feinberg, A. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., 132, 6-13. MEDLINE Abstract

21. Gemmill, R.M., Coyle-Morris, J.F., McPeek, F.D., Ware-Uribe, L.F. and Hecht, F. (1987) Construction of long-range restriction maps in human DNA using pulsed field gel electrophoresis. Gene Anal. Techniques, 4, 119-131.

22. Yokobata, K., Trenchak, B. and de Jong, P. (1990) Rescue of unstable cosmids by in vitro packaging. Nucleic Acids Res., 19, 403-404.

23. Trask, B.J. (1998) Fluorescence in situ hybridization. In Birren, B., Green, E., Hieter, P. and Myers, R. (eds), Genome Analysis: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

24. Trask, B., Friedman, C., Martin-Gallardo, A., Rowen, L., Akinbami, C., Blankenship, J., Collins, C., Giorgi, D., Iadonato, S., Johnson, F., Kuo, W.-L., Massa, H., Morrish, T., Naylor, S., Nguyen, O., Rouquier, S., Smith, T., Wong, D., Youngblom, J. and van den Engh, G. (1998) Members of the olfactory receptor gene family are contained in large blocks of DNA duplicated polymorphically near the ends of human chromosomes. Hum. Mol. Genet., 7, 13-26. MEDLINE Abstract

25. Trask, B.J., van den Engh, G., Christensen, M., Massa, H.F., Gray, J.W. and Van Dilla, M. (1991) Characterization of somatic cell hybrids by bivariate flow karyotyping by in situ hybridization. Somatic Cell Mol. Genet., 17, 117-136. MEDLINE Abstract

26. Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22, 4673-4780. MEDLINE Abstract

27. Saitou, N. and Nei, M. (1987) The Neighbor-Joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 4, 406-425. MEDLINE Abstract

28. Jauch, A., Wienberg, J., Stanyon, R., Arnold, N., Tofanelli, S., Ishida, T. and Cremer, T. (1992) Reconstruction of genomic rearrangements in great apes and gibbons by chromosome painting. Proc. Natl Acad. Sci. USA, 89, 8611-8615. MEDLINE Abstract

29. Buck, L.B. (1992) The olfactory multigene family. Curr. Opin. Genet. Dev., 2, 467-473. MEDLINE Abstract

30. Carlson, J.R. (1996) Olfaction in Drosophila: from odor to behavior. Trends Genet., 12, 175-180. MEDLINE Abstract

31. Rosenthal, W., Antaramian, A., Gilbert, S. and Birnbaumer, M. (1993) Nephrogenic diabetes insipidus. J. Biol. Chem., 268, 13030-13033. MEDLINE Abstract

32. Marchese, A., Docherty, J.M., Nguyen, T., Heiber, M., Cheng, R., Heng, H.H.Q., Tsui, L.-C., Shi, X., George, S.R. and O'Dowd, B.F. (1994) Cloning of human genes encoding novel G protein-coupled receptors. Genomics, 23, 609-618. MEDLINE Abstract

33. Ohno, S. (1970) Evolution by Gene Duplication. Springer Verlag, Berlin, Germany.

34. Ruvolo, M. (1997) Molecular phylogeny of the hominoids: inferences from multiple independent DNA sequence data sets. Mol. Biol. Evol., 14, 248-265. MEDLINE Abstract

35. Koehler, U., Bigoni, F., Wienberg, J. and Stanyon, R. (1995) Genomic reorganization in the concolor gibbon (Hylobates concolor) revealed by chromosome painting. Genomics, 30, 287-292. MEDLINE Abstract

36. Buettner, J.A. and Evans, G.A. (1996) Organization and localization of olfactory receptor genes on human chromosome 11. Am. J. Hum. Genet., 59 (suppl.), 1737.

37. Sullivan, S.L., Adamson, M.C., Ressler, K.J., Kozak, C.A. and Buck, L.B. (1996) The chromosomal distribution of mouse odorant receptor genes. Proc. Natl Acad. Sci. USA, 93, 884-888. MEDLINE Abstract

38. Springer, M., Burk, A., Kavanagh, J., Waddell, V. and Stanhope, M. (1997) The interphotoreceptor retinoid binding protein gene in therian mammals: implications for higher level relationships and evidence for loss of function in the marsupial mole. Proc. Natl Acad. Sci. USA, 94, 13754-13759. MEDLINE Abstract

39. Zhao, H., Ivic, L., Otaki, J., Hashimoto, M., Mikoshiba, K. and Firestein, S. (1998) Functional expression of a mammalian odorant receptor. Science, 279, 237-242. MEDLINE Abstract

40. Leibovici, M., Lapointe, F., Aletta, P. and Ayer-Le Lièvre, C. (1996) Avian olfactory receptors: differentiation of olfactory neurons under normal and experimental conditions. Dev. Biol., 175, 118-131. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +33 4 99 61 99 35; Fax: +33 4 99 61 99 01; Email: giorgi@igh.cnrs.fr

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 12 Aug 1998
Copyright©Oxford University Press, 1998.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Gen Biol EvolHome page
D. Dong, G. He, S. Zhang, and Z. Zhang
Evolution of Olfactory Receptor Genes in Primates Dominated by Birth-and-Death Process
Gen Biol Evol, August 20, 2009; 2009(0): 258 - 264.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
F. Casals, A. Ferrer-Admetlla, M. Sikora, A. Ramirez-Soriano, T. Marques-Bonet, S. Despiau, F. Roubinet, F. Calafell, J. Bertranpetit, and A. Blancher
Human pseudogenes of the ABO family show a complex evolutionary dynamics and loss of function
Glycobiology, June 1, 2009; 19(6): 583 - 591.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
K. Hanada, C. Zou, M. D. Lehti-Shiu, K. Shinozaki, and S.-H. Shiu
Importance of Lineage-Specific Expansion of Plant Tandem Duplicates in the Adaptive Response to Environmental Stimuli
Plant Physiology, October 1, 2008; 148(2): 993 - 1003.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
I. Gaillard, S. Rouquier, A. Chavanieu, P. Mollard, and D. Giorgi
Amino-acid changes acquired during evolution by olfactory receptor 912-93 modify the specificity of odorant recognition
Hum. Mol. Genet., April 1, 2004; 13(7): 771 - 780.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Volz, A. Ehlers, R. Younger, S. Forbes, J. Trowsdale, D. Schnorr, S. Beck, and A. Ziegler
Complex Transcription and Splicing of Odorant Receptor Genes
J. Biol. Chem., May 23, 2003; 278(22): 19691 - 19701.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Y. Gilad, O. Man, S. Paabo, and D. Lancet
Human specific loss of olfactory receptor genes
PNAS, March 18, 2003; 100(6): 3324 - 3327.
[Abstract] [Full Text] [PDF]

Home page
 Chem SensesHome page
D. Giorgi and S. Rouquier
Identification of V1R-like Putative Pheromone Receptor Sequences in Non-human Primates. Characterization of V1R Pseudogenes in Marmoset, a Primate Species that Possesses an Intact Vomeronasal Organ
Chem Senses, July 1, 2002; 27(6): 529 - 537.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
J. M. Young and B. J. Trask
The sense of smell: genomics of vertebrate odorant receptors
Hum. Mol. Genet., May 15, 2002; 11(10): 1153 - 1160.
[Abstract] [Full Text] [PDF]

Home page
 Genome ResHome page
A. Ehlers, S. Beck, S. A. Forbes, J. Trowsdale, A. Volz, R. Younger, and A. Ziegler
MHC-Linked Olfactory Receptor Loci Exhibit Polymorphism and Contribute to Extended HLA/OR-Haplotypes
Genome Res., December 1, 2000; 10(12): 1968 - 1978.
[Abstract] [Full Text]

Home page
 Chem SensesHome page
D. Giorgi and S. Rouquier
LETTER TO THE EDITORS
Chem Senses, October 1, 2000; 25(5): 591 - 592.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Rouquier, A. Blancher, and D. Giorgi
The olfactory receptor gene repertoire in primates and mouse: Evidence for reduction of the functional fraction in primates
PNAS, March 14, 2000; 97(6): 2870 - 2874.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (27)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Rouquier, S.
Right arrow Articles by Giorgi, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rouquier, S.
Right arrow Articles by Giorgi, D.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?