Human Molecular Genetics, 2001, Vol. 10, No. 21 2373-2383
© 2001 Oxford University Press
Transcriptional activity of multiple copies of a subtelomerically located olfactory receptor gene that is polymorphic in number and location
1Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA, 2Department of Bioengineering, 3Department of Genetics, 4Department of Molecular Biotechnology, University of Washington, Seattle, WA 98195, USA, 5Institute for Systems Biology, Seattle, WA 98105, USA and 6Department of Otolaryngology, University of Washington, Seattle, WA 98195, USA
Received June 5, 2001; Revised and Accepted August 13, 2001.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
We report here on the transcriptional activity of multiple copies of a subtelomerically located olfactory receptor (OR) gene, OR-A. Due to recent duplication events, both the copy number and chromosomal location of OR-A vary among humans. Sequence analyses of 180 copies of this gene, derived from 12 chromosome ends in 22 individuals, show that the main coding exon of all but one copy is an intact open reading frame with 0–5 predicted amino acid differences. We detected transcription of OR-A in both olfactory epithelium and testis tissue using RT–PCR amplification with primers designed on the basis of a computationally predicted gene structure. Two alternatively spliced forms of transcripts, one encoding an isoform with an extended N-terminus, were found in both tissues. A third transcript, derived from a second promoter, was also observed in testes. The start methionine is predicted in all transcripts to lie in an upstream exon rather than the main coding exon, as is typical for most other OR genes. By examining sequence variants among transcripts, we show that transcription of this gene occurs at multiple chromosomal locations. Our results lend credence to the idea that OR diversity could be generated in rearrangement-prone subtelomeric regions and show that polymorphism in subtelomeric regions could lead to individual-to-individual variation in the expressed repertoire of OR genes.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The subtelomeric regions of human chromosomes are unusually dynamic and structurally complex regions of the human genome (1–7). These regions are composed of sequences, some of which encode genes, that have been distributed to multiple chromosomal ends. The presence of members of the olfactory receptor (OR) gene family in subtelomeric regions is of particular interest to us. The subtelomeric regions appear to have been shaped by duplications, rearrangements and transfers of material from one chromosome to another (1,6,8). Some of these rearrangements are quite recent, such that individuals are polymorphic for the presence or absence of large blocks of sequence on some chromosomes (1,4,7).
The evolutionary plasticity of the subtelomeric regions is illustrated well by the f7501 subtelomeric block. This block is single-copy in non-human primates, but is extremely polymorphic in copy number and chromosomal location in humans (7). In 44 unrelated individuals analyzed by fluorescence in situ hybridization (FISH), the number of copies ranged from 7 to 11 per diploid genome. The block was observed to be polymorphically present on 11 different chromosomal ends, in addition to common sites on 3qter, 15qter and 19qter (7). This block is also flanked by other subtelomeric sequences that are present on different sets of chromosome ends (7).
The function of these rearrangement-prone subtelomeric regions is largely unknown. The region may simply be a repository of accumulated junk, genomic material that is no longer in use. Alternatively, the subtelomeric regions may be transcriptionally active regions of the genome and could even serve as a nursery for new genes. The subtelomeres appear to be less constrained than other genomic regions for recombination, duplication, conversion and mutation (9,10), and these processes could facilitate the generation of new genes. Humans may have benefited from the dynamic processes that act on genes in these regions. Evidence exists in support of both of these views.
The ‘repository’ model is supported by the presence in subtelomeric regions of many repeats and pseudogenes, as well as partial regions of homology to genes whose functional copies are located elsewhere in the genome. The ‘nursery’ model is supported by observations of genes that are transcribed from subtelomeric regions. For example, the interleukin-9 receptor gene (IL9R) is transcribed from the pseudoautosomal regions of the active and inactive X and the Y (11). A small GTPase RABL2 is transcribed in multiple tissues from its locations on 22qter and the ancestral telomere–telomere fusion band 2q13 (12). These two examples demonstrate that functional genes reside in subtelomeric zones and that copies of these genes, produced by duplication processes, can have transcriptional activity. There is also precedence for the involvement of subtelomeric domains in generating functional diversity in other organisms. Frequent subtelomeric gene conversion provides diversity for surface antigens in trypanosomes (13), and expression of Plasmodium falciparum var genes from subtelomeric domains has important implications for the generation of novel antigenic and adhesive phenotypes (14).
The occurrence of members of the OR gene family in subtelomeric regions may shed light on the functional significance of these regions. We have so far identified eight OR-like sequences, at least four of which are present in multiple subtelomeric regions (one in up to 19 copies). Together, they comprise a subtelomeric collection of at least 30 paralogous sequences (7 and unpublished data). The f7501 subtelomeric block, referred to earlier, contains three regions of homology to known OR genes (7). At least one of these genes, OR-A, appears to be potentially functional from sequence analysis of the prototypic copy on chromosome 19. [The prototypic chromosome 19 copy of this gene is called OR4F19 and hOR19.06.01 by other groups (15,16).] This gene, like the rest of the block, is present on multiple chromosomes and is polymorphic in both copy number and chromosomal location (7).
The association of a subset of OR genes and subtelomeric regions is intriguing given the size and diversity of this gene family (15,16). OR genes encode a family of proteins that recognize an enormous variety of odorants (
10 000 odors) (17). This large gene family is distributed among >40 sites in the human genome, with a bias for terminal bands (18). Functional studies in rodents have demonstrated that expression of OR genes in sensory neurons is very selective—only a single allele of a single gene is expressed per cell (19), yet the total repertoire in the olfactory epithelium comprises 1000 receptor types (20). Although these genes were originally discovered expressed in olfactory epithelium (17), their expression has also been demonstrated in testis and other tissues (21–23). These findings suggest that the proteins encoded by these genes may have additional functions related to recognition of an even larger ligand collection. The diversity of function coupled with the large repertoire of different receptors implies the need to expand and modify the repertoire over time, especially while adapting to new environments. The association of the OR genes with subtelomeres and the dynamic processes acting on them could therefore be significant.
In this paper, we demonstrate that the subtelomerically located OR-A gene is transcribed in both olfactory epithelium and testis tissue. In addition, we show that two alternative 5' splice variants of OR-A are transcribed in both tissues. A third transcript derived from an alternative promoter is found in testes. Sequence analyses of the main coding exon of the OR-A gene from multiple chromosomal locations demonstrate that most copies have an intact open reading frame (ORF) and are therefore potentially functional. The proteins encoded by these genes are predicted to differ by up to five amino acid substitutions in addition to N-terminal differences resulting from alternative splicing. By analyzing subtle sequence variants among transcripts, we show that transcription of the gene can occur from multiple chromosomal locations. Thus, subtelomeric duplications have spawned a multiplicity of OR-A copies that are still intact and active. These results support the idea that the subtelomeric regions could be a nursery for generating diversity in the expressed OR repertoire and a source of phenotypic variation among humans.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
In order to examine the potential function of the multicopy odorant receptor gene OR-A, we analyzed its structure using various gene prediction programs. The coding regions of eukaryotic OR genes are typically intronless and are preceded by an intron and one or two upstream non-coding exons (24). Thus, identification of a putative 5' untranslated exon would permit us to place primers in separate exons and thereby discriminate PCR products from cDNA from possible genomic contamination. Using sequence from cosmid f7501 (accession no. L78442), we analyzed the 10 kb region surrounding the OR-A gene to identify potential promoters and 5' untranslated exons. Two potential transcription start sites (TSS) with TATA promoters were predicted by linear discriminant function (LDF) using the TSSW program at positions 9241 (LDF – 5.35) and 8234 (LDF – 4.44). Only the first of these sites was also predicted by the TSSG (LDF – 4.16) and NNPP/Eukaryotic (score 0.87) programs. Three donor and two acceptor splice sites were predicted using SpliceView and Splice Site programs (Table 1). The program Genie predicted a transcript including two upstream exons and the full-length main coding exon.
|
Figure 1A shows the structure of the gene resulting from a combination of these sequence-analysis programs. The gene is predicted to have two alternative TATA-promoters, with splicing of 5' exons predicted in both cases. Two alternative splice variants can be transcribed from the more 5' promoter (TATA1). These transcripts are referred to here as the short transcript I and long transcript II. The long transcript is predicted to include exon 1, exon 2 and the main coding exon 3. Exon 2 is skipped in the short transcript I. A third two-exon transcript (III) is predicted using the second TATA promoter, TATA2.
|
Conceptual translation of all three potential transcripts suggests that the starting methionine lies in an upstream exon, not in the main coding exon as is typical for OR genes (24). Proteins translated starting with the next available methionine, which is located in the main coding exon, would lack a conserved N-terminal glycosylation site (25). Transcript II is predicted to encode a long isoform with an extended N-terminus (Fig. 1B).
We confirmed the conservation of the promoter region and the splice sites using copies of the OR-A gene residing on several human chromosomes. The regions spanning TATA1, exon 1, exon 2 and the main coding exon were amplified from six different somatic cell hybrids containing single copies of human chromosomes 3, 15 or 19 (two cell lines for each of those chromosomes). The sequences of the TATA-box, all predicted splice sites, exon 1 and exon 2 were conserved (data not shown), such that all copies were capable of producing transcripts I and II (exon 1a of transcript III was not analyzed). Several nucleotide and three predicted amino acid differences (more details below) were found in the main coding region among these copies. Analyses of two sequenced cosmids (AC005603 and AC005604) revealed that some versions of the OR-A gene have a complex rearrangement involving deletion of exons 1 and 2 (Fig. 1A), which co-segregates with a characteristic Alu insertion (8). These versions are therefore not capable of producing transcripts I and II.
The same regions were sequenced from chimpanzee, gorilla and orangutan DNA. These species carry only two allelic copies of the OR-A gene (7). The nucleotide divergences of the main coding exons of chimpanzee, gorilla and orangutan from human genes average 0.6, 1.8 and 3.0%, respectively (data not shown). Several sequence variations were detected between the alleles in each species (data not shown). Although the TATA1-box and all splicing sites are conserved in great apes, only the orangutan gene appears functional. The main coding regions of the gorilla and chimpanzee genes contain a premature stop codon and frameshift, respectively. In addition, all non-human primates have a 35 bp insertion in the predicted exon 1 sequence, which disrupts the predicted ORF of the long transcript II (Fig. 2). This 35 bp sequence was most likely deleted from the human genome after the evolutionary split of humans from the other great apes. As a consequence, only the human genes appear capable of generating a long transcript II encoding a protein with an extended N-terminus.
|
RT–PCR was used to test for transcription of the OR-A gene in human olfactory epithelium, testis, prostate and seminal vesicle tissues. Using the predicted gene structure, we designed primers specific to the predicted exons and flanking the 5'-intron, such that PCR products from transcripts could be discriminated by size from those derived from genomic DNA contamination. If the genes are transcribed as predicted, primer pair P1/P2 should amplify transcripts I and II from TATA1, and primer pair L/P2 should amplify transcript III from TATA2 (Fig. 1A).
Two products representing transcripts I and II were obtained using cDNA from both olfactory epithelium and testis tissue (Fig. 3A). This pair of products was also observed using testicular cDNA from three additional different sources (data not shown). Sequencing of obtained PCR products validated the computer-predicted gene structure. PCR products were subcloned and used for subsequent analysis of transcribed variants (see below). No products were obtained using primers P1 and P2 from prostate or seminal vesicle cDNA.
|
A product representing spliced transcript III was obtained in a PCR reaction from testicular cDNA (Fig. 3B). This result was confirmed using testicular cDNA from a different source (data not shown). Sequencing verified the predicted splice junction. No products were obtained using the L/P2 primer pair on cDNA from olfactory epithelium, prostate or seminal vesicle (data not shown).
We have previously shown using FISH that the number of copies of the subtelomerically located OR-A gene can vary from seven to 11 per diploid genome (7). We were interested in testing whether all copies of OR-A are potentially functional and in assessing the variability of OR-A gene repertoires among individuals. In order to address these questions, we determined the sequence of the main coding exon of the OR-A gene for each chromosomal copy found by FISH analysis in 22 individuals. The main coding exon accounts for 
88% of the amino acids in the proteins predicted by the three transcripts. Because the gene is present on multiple chromosomes, each chromosome carrying the OR-A gene was isolated from copies elsewhere in the genome by flow sorting, before PCR amplification and sequence analysis (Materials and Methods). A total of 180 copies from 12 different chromosomal ends were sequenced in these individuals.
Twenty variant sites were observed among these copies within the 972 bp main coding exon. Rare variants were verified by sequencing products amplified in an independent PCR reaction to rule out PCR error. Eight of the changes are silent, 11 cause amino acid alterations and one results in a premature stop codon. The non-synonymous changes occur throughout the OR-A protein (Fig. 4). Combinations of the 20 polymorphic sites define 21 different OR-A gene (DNA) haplotypes (see below) that can be conceptually translated into 14 intact proteins differing by 1–5 amino acid substitutions and one prematurely truncated protein. The complex deletion of exons 1 and 2 was observed in 24 of 105 chromosomes tested (8); these versions cannot encode isoforms PI and PII but are capable of forming isoform III.
|
The frequency of protein haplotypes encoded by gene copies found at different chromosome ends is shown in Figure 5A. Six of the protein haplotypes (P1, P2, P3, P5, P6, P7) are present on multiple chromosome ends, probably as a result of recent exchanges between non-homologous chromosomes (8), and three of these are relatively rare overall. The most common protein haplotype was observed on 10 different chromosome ends and has an overall frequency of 42%. Notably, the haplotype with a premature stop codon was observed on only one of the 180 copies sequenced (haplotype P15). Seven additional protein haplotypes were also observed only once.
|
The genomic repertoire of OR-A genes varies considerably as a consequence of variation in both sequence and copy number among individuals. Among individuals carrying 7–11 copies of the gene, the number of different gene haplotypes varies from 3 to 7. These gene haplotypes encode 3–7 different protein types per individual, in varying copy numbers (Fig. 5B). Some protein haplotypes are present in similar numbers, but in different chromosomal locations, in different individuals (e.g. protein type P1 in individuals 1 and 16 in Fig. 5B). Others, such as protein type P2, exhibit wide variation in copy number among individuals (e.g. one copy in individual 4 and four copies in individual 8 in Fig. 5B).
Our sequence analyses show that potentially functional copies of the OR-A gene reside at many chromosome ends. In order to determine whether more than one chromosomal location of this multicopy gene is transcribed, we took advantage of nucleotide polymorphisms among olfactory epithelial transcripts and genomic copies from the same individual. Two independent PCR amplifications using the P1/P2 primer pair were performed on the same olfactory epithelial cDNA template in order to be able to discriminate true variants from PCR-induced errors. PCR products were subcloned and inserts of multiple clones were sequenced. Sequences were aligned and analyzed using CONSED and PolyPhred programs (26,27). A haplotype was considered validated if it was encountered in both independent PCR reactions.
The results of haplotyping olfactory epithelial transcripts from one individual are summarized in Figure 6A. We sequenced 35 and 23 clones obtained from two independent PCR reactions, respectively. Two haplotypes, H1 and H2, were validated by their presence in both pools (a total of 47 validated clones). Both haplotypes were present as the short transcript I, but the H2 haplotype was observed five times more frequently among the clones with validated haplotypes. Haplotype H1 was also present as the long alternatively spliced transcript II.
|
We used chromosome sorting to analyze the genomic copies of the OR-A gene in the same individual. The H1 haplotype was assigned to chromosomes 3 and 19, and the H2 haplotype to chromosomes 15 and 16. Thus, at least two different chromosomal locations of the OR-A gene are transcribed in the olfactory epithelium of this individual. Additional haplotypes were present on chromosomes 9, 11 and 19, but transcripts of those haplotypes were not detected in our sample. Because the H1 haplotype is present on two different chromosomes, it is impossible to tell whether the short and the long transcript forms are transcribed from the same or different chromosomal locations.
Analyses of OR-A transcripts I and II from the testes of a different individual also show that this gene is transcribed from multiple subtelomeric locations (Fig. 6B). PCR products obtained in two independent reactions were cloned, and 32 and 20 clones were sequenced from each pool. Four haplotypes, H1, H3, H4 and H5, could be validated by their presence in both pools (a total of 38 clones with validated haplotypes). Three haplotypes (H1, H3 and H5) were expressed as the long transcript II, with H5 observed five times less frequently among the clones than either H3 or H1. Haplotype H4 was expressed as the short form. Although no genomic DNA was available for this individual with which to assign these haplotypes to chromosomal locations, the presence of four haplotypes implies that two or more chromosomal locations of the OR-A gene are transcribed in testis, as was also the case in the olfactory epithelial sample.
Comparison of the transcript haplotypes from testis to the 21 genomic DNA haplotypes observed among the 180 sequenced copies of the main coding exon allows us to speculate about the chromosomal origin of the transcripts. Figure 6C tabulates the DNA haplotype frequency observed at each chromosome location in 22 individuals and over all 180 chromosomes analyzed. Because we examined transcripts only in the initial 814 bp of the main coding exon, we cannot discriminate among transcripts that might differ in the terminal 158 bp of the main coding exon. Therefore, several full-length exon haplotypes may be represented by one partial transcript haplotype, as indicated in Figure 6C. Transcript haplotype H1, which could correspond to genomic haplotypes A1a, A9 and/or A15, is the most frequently observed haplotype at the genomic level. It could have derived from any of nine different chromosome ends (Fig. 6C). Transcript haplotype H4 corresponds to genomic haplotype A5a, which has been observed on chromosomes 9 and 19; haplotype H3 could represent genomic haplotype A3c or A3d and has only been seen on chromosome 19. Haplotype H5 was not observed in our sample of 180 genomic copies.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
We have demonstrated here that a subtelomerically located OR gene, which varies in number, sequence and locations among individuals, is transcriptionally active in olfactory neuroepithelium and testis. For low-abundance transcripts from multicopy genes, such as the OR-A gene studied here, possible genomic contamination in cDNA is a very important concern when testing for transcription using RT–PCR. Because most eukaryotic OR genes do not have introns in their coding region, it was necessary to infer the 5'-untranslated region (5'-UTR) structure of the OR-A gene using a combination of computational tools in order to design primers that flank 5' introns. In this way, PCR amplification products from cDNA and genomic DNA could be distinguished by size. We confirmed the predicted gene structure by sequencing PCR amplification products obtained from cDNA using primers designed according to our predictions.
Our results demonstrate that transcripts of the OR-A gene undergo 5'-splicing. Through the work of several groups who have employed 5'-rapid amplification of cDNA ends (5'-RACE) to analyze OR transcripts in rodents, 5'-splicing is emerging as a general phenomenon for this family of genes (22,23,28). In addition, alternative splicing of human OR genes was inferred recently from RT–PCR products generated from mRNA pooled from five individuals (24) and by analyses of expressed sequence tag (EST) sequences from non-olfactory tissue (29). Our results extend these observations by providing the first demonstration of alternative splicing (exon skipping) of olfactory gene transcripts within a given tissue of one individual. We observed alternative splicing, which resulted in two transcript forms, in both olfactory epithelium and testis tissue. In addition, our data indicate that two alternative promoters are used in testes. Alternative splicing of OR genes could play an important role in regulating mRNA stability, translation and/or membrane targeting.
The OR-A gene is an exception to the dogma that the coding regions of higher eukaryotic OR genes are intronless. Based on conceptual translation, all three 5' splice variants of the OR-A gene are predicted to start at methionines situated upstream of an intron in a 5' exon. Translation from these upstream methionines—but not from the first methionine in the main coding exon—results in inclusion of a conserved N-terminal glycosylation site (25) in the protein. In addition, one alternatively spliced variant of the OR-A gene is predicted to encode an isoform with an extended N-terminus, which could confer additional functions to the protein. Such a possibility was also suggested for the rat SCR D-8 gene (22).
The OR-A gene is also unusual in that it is multicopy, and its copy number and chromosomal location vary among individuals due to recent duplication events involving subtelomeric regions. By taking advantage of slight sequence differences among the transcribed haplotypes and the many genomic copies of the OR-A gene, we conclude that multiple chromosomal locations are transcriptionally active in both olfactory epithelium and testis tissue. We detected two haplotypes, one as both long and short forms, in one individual. Using flow-sorted chromosomes from the same individual, we could map these haplotypes to four chromosomal locations (H1 to chromosomes 3 and 19, and H2 to chromosomes 15 and 16). Three additional haplotypes were detected on other chromosomes, but were not picked up in our limited sample of olfactory transcripts. We do not yet know whether these recently duplicated genes are regulated like other members of the OR family, such that only a single gene is expressed per sensory neuron, or whether multiple chromosomal copies are expressed in the same cell.
We found a different, larger collection of transcripts expressed in the testes tissue of a second individual. Four haplotypes, three as the long alternatively spliced form and one as the short form, were observed. Cells were not available for this individual to determine the chromosomal origin of these transcripts. Our analysis of 180 genomic sequences from 22 individuals suggests likely chromosomes of origin for some haplotypes. However, subtelomeric regions of non-homologous chromosomes undergo exchanges sufficiently frequently that few, if any, subtelomeric haplotypes are chromosome-specific (8). Therefore, the chromosomal origin of a given transcript can only be known exactly if both transcripts and flow-sorted chromosomes are haplotyped from the same individual. Our results further indicate that different individuals may express different combinations of OR-A gene haplotypes and/or that more haplotypes are expressed in testis than in olfactory epithelium. Conceptual translation of the different transcribed haplotypes shows at least one non-conservative amino acid change (with 1–5 revealed in comparison of the entire main coding exon), suggesting the possibility of slight functional differences among the proteins.
Our sequence analysis of 180 copies of the OR-A gene from 12 chromosome ends in 22 individuals indicates that individuals can vary greatly in the genomic sets of OR-A genes. First, the number of copies of the gene ranges from 7 to 11 per individual. Secondly, the number of different gene (DNA) haplotypes present per genome ranges from 3 to 7, and the number of different protein types that may potentially be expressed also ranges from 3 to 7. Finally, the chromosome location, as well as the overall gene copy number, for each potential protein may vary among individuals. Such variation in sequence, copy number and chromosomal context of the OR-A gene could result in subtle phenotypic differences among individuals.
We hypothesized that if the subtelomeric regions are a nursery for generating diversity in the OR gene family, different chromosomal copies of the OR-A gene might be accumulating variants in regions of the gene thought to be involved in odorant binding. Alternatively, if these regions of the genome are repositories for ‘junk’ DNA, multiple copies would contain variants that abolish gene function, including nonsense and frameshift mutations. Our analyses show that the main coding exons of 179 of 180 OR-A genes analyzed have ORFs and are therefore potentially functional; only a single copy of the gene harbors an obviously deleterious mutation in this region.
The promoter and exons 1 and 2 were shown to be intact in the six copies analyzed from somatic cell hybrid lines (two each of chromosomes 3, 15 and 19). These copies represent two of the three major phylogenetic clades identified in the accompanying paper (8). Given the high linkage disequilibrium and low mutation rate observed in that analysis for the OR-A and non-coding loci (which flank the upstream region), it is likely that the upstream regions are similarly intact in copies that share the same OR-A coding haplotypes as these six representatives, although additional sequence could reveal rare variants. The OR-A sequence haplotypes represented by these six chromosomes (A1a, A3c, A4a) were observed on a total of 96 of the 180 chromosomes analyzed.
The upstream regions of 41 copies are predicted to carry a deletion involving exons 1 and 2. This deletion co-segregates with DNA haplotypes A2, A7 and A14 (8). While this deletion precludes transcription of transcripts I and II, transcript III may still be expressed from the TATA2 promoter. As none of the genomic copies of the individual whose transcripts were analyzed contains this deletion, it remains to be tested whether transcript III is expressed from chromosomes carrying the deletion.
Variation in OR-A sequences is comparable to variation observed among copies of nearby non-coding sequence (overall nucleotide diversity of 0.28% in the OR-A exon and 0.36% in non-coding DNA) (8), but we see no striking pattern that would suggest that positive selection for diversity is operating on these paralogous OR-A genes. Of the 20 variable sites in the OR-A main coding exon, eight changes are synonymous, 11 cause amino acid alterations and one results in a premature stop codon. The 11 amino acid sites that vary among the 14 potential proteins identified in this study are distributed throughout the protein (Fig. 4). Although the most variable regions of OR proteins are transmembrane (TM) domains 3, 4 and 5 (17), and Pilpel and Lancet (30) have proposed 17 hypervariable residues within these domains that may constitute the odorant-complementarity-determining regions, only four of the 11 changes observed in this study occur in TM3 and TM4, while the remaining seven changes are in the N-terminus, extracellular domain 2, intracellular domain 3, TM7 and the C-terminus (1, 2, 2, 1 and 1 changes, respectively). Some of the amino acid changes are non-conservative and may therefore result in slight functional differences among the proteins, although this remains to be tested.
The demonstration of transcriptional activity of a multicopy OR gene suggests that the subtelomeric association of OR genes could be significant, and that subtelomeres could be a place where new genes are created by duplication and subsequent modification processes. These processes, which have resulted in considerable variability in the subtelomeric OR gene repertoires, could therefore be important in allowing organisms to adapt to their environments over evolutionary time.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Gene structure prediction
Potential promoters and corresponding TSS were identified using TSSG and TSSW (31) and by NNPP (32) via the BCM Search Launcher Gene Feature Searches (http://dot.imgen.bcm.tmc.edu:9331/seq-search/gene-search.html). Acceptor and donor splicing sites were detected by the programs Splice Site Prediction by Neural Network (http://www.fruitfly.org/seq_tools/splice.html) and SpliceView – Splice Prediction by using Consensus Sequences (http://l25.itba.mi.cnr.it/~webgene/wwwspliceview.html). We employed several exon-prediction programs including GRAIL-1.3, FGENEH, FEXH, HEXON, GENESCAN and Genie via the BCM Search Launcher Gene Feature Searches (http://dot.imgen.bcm.tmc.edu:9331/seq-search/gene-search.html).
RT–PCR
Total cellular RNA was extracted from normal adult male tissues by homogenization in TRIZOL reagent (Gibco, Rockville, MD) following the manufacturer’s instructions. First-strand cDNA was synthesized using reagents from Perkin Elmer (Gaithersburg, MD) RNA PCR kit. One microgram of total RNA was incubated at 42°C for 15 min in a 20 µl volume containing 5 mM MgCl2, 1 mM dNTPs, 1 U/µl Rnase inhibitor and 2.5 µM random hexamers (Perkin Elmer) and reverse transcribed with 2.5 U/ µl MuLV reverse transcriptase and then boiled for 5 min following incubation. Reagent volumes were scaled accordingly for reverse transcription of more than 1 µg of RNA. PCR amplification was carried out using 5 µl of the reverse transcription mix in a 25 µl reaction buffer 1 µM of each primer, 0.2 mM of each dNTP and 1 U of Taq DNA polymerase (Perkin Elmer) and amplified using touchdown PCR with the following parameters: 95°C for 1 min initial denature, 94°C for 20 s, 67°C for 30 s, 72°C for 2 min x 4 cycles, annealing temperature 65°C for 30 cycles. P1/P2 and L/P2 primer pairs were used for PCR amplification (P1, 5'-AAATTGCTGTAGTCTCTTCCAG-3'; P2, 5'-GGCCAGGCATAAATAAAGACAC-3'; L, 5'-GGCTCTTCATGGTTGCTACA-3'). Five microlitres of PCR products were analyzed on a 1.5% ethidium bromide-stained agarose gel. Products showing two transcripts were reloaded with 15–20 µl of PCR product on a 1% LMP agarose gel, excised and purified using Qiaquick Gel Extraction kit (Qiagen, Valencia, CA). Bulk PCR products were sequenced to verify splice junctions. For transcript analysis, RT–PCR products were TA-subcloned into pCRII 9 (Invitrogen, Carlsbad, CA) and inserts were sequenced (see below) after PCR amplification, using the primer pairs used for the PCR.
FISH
In order to determine chromosomal distribution of the OR-A gene and to choose chromosomes for sorting and subsequent sequence analysis, FISH was performed using cosmid f7501 as a probe as described previously (7).
Cell lines
Somatic cell lines containing individual human chromosomes (3, NA10253 and NA11713; 15, NA11418 and NA11715; 19, NA10449 and NA10612) were obtained from NIGMS Human Genetic Mutant Cell Repository (Camden, NJ). The following lymphoblast cell lines were analyzed to determine genomic OR-A haplotypes. The lines are listed in order of presentation in Figure 5B: 1, GM10470; 2, GM10471; 3, GM10493; 4, GM10494; 5, GM10495; 6, GM10496; 7, GM10539; 8, GM10541; 9, GM10543; 10, GM10966; 11, GM10977; 12, GM10978; 13, GM11373; 14, GM11374; 15, GM11375; 16, GM11523; 17, GM11524; 18, GM11525; and 19, CGM1. Data for individuals 20 and 21 were obtained using peripheral blood lymphocyte cultures. Individual 22 is represented by primary fibroblasts cultured from the deceased donor of olfactory tissue, after appropriate informed consent was obtained. Lines with the GM prefix were obtained from the NIGMS repository.
PCR analyses of flow-sorted chromosomes
Chromosomes were isolated from lymphoblast cell lines or from phytohaemagglutinin-stimulated peripheral blood cell cultures into a polyamine buffer, stained with Hoechst 33258 and chromomycin A3, and sorted using a custom dual-laser flow cytometer, as described previously (33). The purity of flow-sorted chromosomes was periodically tested by using the flow-sorted chromosomes as template for generation of chromosome paints (34), then hybridized to cytogenetic preparations of metaphase spreads. There was little evidence for cross contamination by other chromosomes using this method or in sequence traces generated when bulk PCR products were used as sequencing templates (see below). In two situations, extra precautions were taken to prevent contamination. Because chromosomes 15 and 16 form neighboring peaks in a flow karyotype, we set especially narrow sort windows when the f7501 block was present on both of these chromosomes. When the block was present on both chromosomes 9 and 11, we resolved these chromosomes by measuring the fluorescence intensity of a fluorescein isothiocyanate (FITC)-labeled polyamide (Prolinx Inc., Bothell, WA), which targets a short sequence repeated in the heterochromatic region of chromosome 9 (M.Gygi et al., manuscript in preparation).
2000 copies of each chromosome carrying the f7501 block were sorted into a 0.5 µl PCR tube containing 10 µl sterile H2O and stored at –20°C before use. PCR ingredients [final concentrations: 1x high fidelity buffer 2, 200 µM each dNTP, 400 nM each primer, 0.7 U Expand high fidelity polymerase (Roche Molecular, Indianapolis, IN)] were added to the tube. After initial denaturation at 94°C for 2 min, the 25 µl reactions were subjected to 40 amplification cycles of 94°C 30 s, 60°C for 30 s and 72°C for 90 s using primers F4708 (5'-ATTGAGGCAATGTATGTGGAAG-3') and OLF-AR (5'-ACACTGAGAAGCCGAGATAACTGAA-3'), which encompass the main coding exon of OR-A. As necessary, 1 µl of product was re-amplified using primers OLA10 (5'-CCAACTTCACTATATTTTGTG-3') and OLA4 (5'-TCTGACTTCCTTCTCCTTCTC-3'), using the same PCR conditions for 35 additional cycles. The upstream region encompassing exons 1 and 2 was analyzed using primers U7984 (5'-CTGAATTTGTGCTGCTGAGG-3') or U8594 (5'-GCCTTAGCTTTCCTGTTTTT-3') and A9295 (5'-TGGCCAAGGGAAAACTTGTGA-3').
Sequencing
Excess dNTPs and primers from DNA produced by PCR amplification were removed with 2 U/µl shrimp alkaline phosphatase and 10 U/µl exonuclease I (Amersham, Piscataway, NJ) for each 5 µl of PCR product, or by purifying 20 µl of PCR product through Sephacryl 300 spin columns (Sigma-Aldrich, St Louis, MO). Bulk PCR products were sequenced with Ready Reaction Big-dye terminator PRISM kits with AmpliTaq FS (Perkin Elmer). Primers used for sequencing were the same as for PCR amplification. Analysis of individual clones was carried out only to determine the phase of variants detected in sequence traces generated from bulk PCR products. Results of sequence analysis were consistent with the presence of only one copy of the f7501 block on each chromosome (8).
|
ACKNOWLEDGEMENTS |
|---|
We thank Pete Nelson, Christopher Kuhr, Sylvie Rouquier and Dominique Giorgi for advice, David Coil for flow sorting assistance, and Melanie Gygi and Prolinx for polyamide probes. This work was supported in part by NIH grants R01 GM57070 and R01 DC04209 to B.J.T. H.C.M. was supported by T32 HG00035 and a Poncin fellowship.
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed at: 1100 Fairview Avenue N, Mailstop C3-168, P.O. Box 19024, Seattle, WA 98109, USA. Tel: +1 206 667 1470; Fax: +1 206 667 4023; Email: btrask@fhcrc.orgPresent address:Oanh Nguyen, Pacific Horizon Ventures, 1001 Fourth Avenue Plaza, Suite 4105, Seattle, WA 98154, USAThe authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Brown, W.R., MacKinnon, P.J., Villasante, A., Spurr, N., Buckle, V.J. and Dobson, M.J. (1990) Structure and polymorphism of human telomere-associated DNA. Cell, 63, 119–132.[ISI][Medline]
2 Cross, S., Lindsey, J., Fantes, J., McKay, S., McGill, N. and Cooke, H. (1990) The structure of a subterminal repeated sequence present on many human chromosomes. Nucleic Acids Res., 18, 6649–6657.
3 Ijdo, J.W., Lindsay, E.A., Wells, R.A. and Baldini, A. (1992) Multiple variants in subtelomeric regions of normal karyotypes. Genomics, 14, 1019–1025.[ISI][Medline]
4 Hoglund, M., Mitelman, F. and Mandahl, N. (1995) A human 12p-derived cosmid hybridizing to subsets of human and chimpanzee telomeres. Cytogenet. Cell Genet., 70, 88–91.[ISI][Medline]
5 Martin-Gallardo, A., Lamerdin, J., Sopapan, P., Friedman, C., Fertitta, A.L., Garcia, E., Carrano, A., Negorev, D., Macina, R.A., Trask, B.J. et al. (1995) Molecular analysis of a novel subtelomeric repeat with polymorphic chromosomal distribution. Cytogenet. Cell Genet., 71, 289–295.[ISI][Medline]
6 Wilkie, A.O., Higgs, D.R., Rack, K.A., Buckle, V.J., Spurr, N.K., Fischel-Ghodsian, N., Ceccherini, I., Brown, W.R. and Harris, P.C. (1991) Stable length polymorphism of up to 260 kb at the tip of short arm of human chromosome 16. Cell, 64, 595–606.[ISI][Medline]
7 Trask, B.J., Friedman, C., Martin-Gallardo, A., Rowen, L., Akinbami, C., Blankenship, J., Collins, C., Giorgi, D., Iadonato, S., Johnson, F. et al. (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.
8 Mefford, H.C., Linardopoulou, E., Coil, D., van den Engh, G. and Trask, B.J. (2001) Comparative sequencing of a multicopy subtelomeric region containing olfactory receptor genes reveals multiple interactions between non-homologous chromosomes. Hum. Mol. Genet., 10, 2363–2372.
9 Flint, J., Bates, G.P., Clark, K., Dorman, A., Willingham, D., Roe, B.A., Micklem, G., Higgs, D.R. and Louis, E.J. (1997) Sequence comparison of human and yeast telomeres identifies structurally distinct subtelomeric domains. Hum. Mol. Genet., 6, 1305–1313.
10 Baird, D.M., Coleman, J., Rosser, Z.H. and Royle, N.J. (2000) High levels of sequence polymorphism and linkage disequilibrium at the telomere of 12q: implications for telomere biology and human evolution. Am. J. Hum. Genet., 66, 235–250.[ISI][Medline]
11 Vermeesch, J.R., Petit, P., Kermouni, A., Renauld, J.-C., van den Berghe, H. and Marynen, P. (1997) The IL-9 receptor gene, located in the Xq/Yq pseudoautosomal region, has an autosomal origin, escapes X inactivation and is expressed from the Y. Hum. Mol. Genet., 6, 1–8.
12 Wong, A.C., Shkolny, D., Dorman, A., Willingham, D., Roe, B.A. and McDermid, H.E. (1999) Two novel human RAB genes with near identical sequence each map to a telomere-associated region: the subtelomeric region of 22q13.3 and the ancestral telomere band 2q13. Genomics, 59, 326–334.[ISI][Medline]
13 McCulloch, R., Rudenko, G. and Borst, P. (1997) Gene conversions mediating antigenic variation in Trypanosoma brucei can occur in variant surface glycoprotein expression sites lacking 70-base-pair repeat sequences. Mol. Cell. Biol., 17, 833–843.[Abstract]
14 Fischer, K., Horrocks, P., Preuss, M., Wiesner, J., Wunsch, S., Camargo, A.A. and Lanzer, M. (1997) Expression of var genes located within polymorphic subtelomeric domains of Plasmodium falciparum chromosomes. Mol. Cell. Biol., 17, 3679–3686.[Abstract]
15 Glusman, G., Yanai, I., Rubin, I. and Lancet, D. (2001) The complete human olfactory subgenome. Genome Res., 11, 685–702.
16 Zozulya, S., Echeverri, F. and Nguyen, T. (2001) The human olfactory receptor repertoire. Genome Biol., 2, RESEARCH0018.1–0018.12.
17 Buck, L. and Axel, R. (1991) A novel multigene family may encode odorant receptors: A molecular basis for odor recognition. Cell, 65, 175–187.[ISI][Medline]
18 Trask, B.J., Massa, H., Brand-Arpon, V., Chan, K., Friedman, C., Nguyen, O.T., Eichler, E., van den Engh, G., Rouquier, S., Shizuya, H. and Giorgi, D. (1998) Large multi-chromosomal duplications encompass many members of the olfactory receptor gene family in the human genome. Hum. Mol. Genet., 7, 2007–2020.
19 Chess, A., Simon, I., Cedar, H. and Axel, R. (1994) Allelic inactivation regulates olfactory receptor gene expression. Cell, 78, 823–834.[ISI][Medline]
20 Vassar, R., Ngai, J. and Axel, R. (1993) Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell, 74, 309–318.[ISI][Medline]
21 Vanderhaeghen, P., Schurmans, S., Vassart, G. and Parmentier M. (1997) Specific repertoire of olfactory receptor genes in the male germ cells of several mammalian species. Genomics, 39, 239–246.[ISI][Medline]
22 Walensky, L.D., Ruat, M., Bakin, R.E., Blackshaw, S., Ronnett, G.V. and Snyder, S.H. (1998) Two novel odorant receptor families expressed in spermatids undergo 5'-splicing. J. Biol. Chem., 273, 9378–9387.
23 Asai, H., Kasai, H., Matsuda, Y., Yamazaki, N., Nagawa, F., Sakano, H. and Tsuboi, A. (1996) Genomic structure and transcription of a murine odorant receptor gene: Differential initiation of transcription in the olfactory and testicular cells. Biochem. Biophys. Res. Commun., 221, 240–247.[ISI][Medline]
24 Sosinsky, A., Glusman, G. and Lancet, D. (2000) The genomic structure of human olfactory receptor genes. Genomics, 70, 49–61.[ISI][Medline]
25 Gat, U., Nekrasova, E., Lancet, D. and Natochin, M. (1994) Olfactory receptor proteins: Expression, characterization and partial purification. Eur. J. Biochem., 225, 1157–1168.[ISI][Medline]
26 Gordon, D., Abajian, C. and Green, P. (1998) Consed: a graphical tool for sequence finishing. Genome Res., 8, 195–202.
27 Nickerson, D., Tobe, V. and Taylor, S. (1997) PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res., 25, 2745–2751.
28 Bulger, M., Bender, M.A., van Doorninck, J.H., Wertman, B., Farrell, C.M., Felsenfeld, G., Groudine, M. and Hardison, R. (2000) Comparative structural and functional analysis of the olfactory receptor genes flanking the human and mouse ß-globin gene clusters. Proc. Natl Acad. Sci. USA, 97, 14560–14565.
29 Younger, R.M., Amadou, C., Bethel, G., Ehlers, A., Lindahl, K.F., Forbes, S., Horton, R., Milne, S., Mungall, A.J., Trowsdale, J., Volz, A., Ziegler, A. and Beck, S. (2001) Characterization of clustered mhc-linked olfactory receptor genes in human and mouse. Genome Res., 11, 519–530.
30 Pilpel, Y. and Lancet, D. (1999) The variable and conserved interfaces of modeled olfactory receptor proteins. Protein Sci., 8, 969–977.[Abstract]
31 Solovyev, V. and Salamov, A. (1997) The Gene-Finder computer tools for analysis of human and model organisms genome sequences. Proc. Int. Conf. Intell. Syst. Mol. Biol., 5, 294–302.[Medline]
32 Reese, M.G. and Eeckman, F.H. (1995) New neural network algorithms for improved eukaryotic promoter site recognition. In Venter, J.C. and Doyle, D. (eds) Genome Science and Technology. Proc. 7th Int. Genome Seq. Anal. Conf., 1, 45.
33 Mefford, H., van den Engh, G., Friedman, C. and Trask, B.J. (1997) Analysis of the variation in chromosome size among diverse human populations by bivariate flow karyotyping. Hum. Genet., 100, 138–144.[ISI][Medline]
34 Trask, B.J. (1999) Fluorescence in situ hybridization. In Birren, B., Green, E., Hieter, P., Klapholz, S., Myers, R., Riethman, H. and Roskams, J. (eds), Genome Analysis: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Vol. 4, pp. 303–413.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. H. De Maeyer, J. Aerssens, P. Verhasselt, and R. A. Lefebvre Alternative splicing and exon duplication generates 10 unique porcine 5-HT4 receptor splice variants including a functional homofusion variant Physiol Genomics, June 10, 2008; 34(1): 22 - 33. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Gilad, O. Man, and G. Glusman A comparison of the human and chimpanzee olfactory receptor gene repertoires Genome Res., February 1, 2005; 15(2): 224 - 230. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Young, M. Kambere, B. J. Trask, and R. P. Lane Divergent V1R repertoires in five species: Amplification in rodents, decimation in primates, and a surprisingly small repertoire in dogs Genome Res., February 1, 2005; 15(2): 231 - 240. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Amadou, R. M. Younger, S. Sims, L. H. Matthews, J. Rogers, A. Kumanovics, A. Ziegler, S. Beck, and K. Fischer Lindahl Co-duplication of olfactory receptor and MHC class I genes in the mouse major histocompatibility complex Hum. Mol. Genet., November 15, 2003; 12(22): 3025 - 3040. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Newman and B. J. Trask Complex Evolution of 7E Olfactory Receptor Genes in Segmental Duplications Genome Res., May 1, 2003; 13(5): 781 - 793. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Courseaux, F. Richard, J. Grosgeorge, C. Ortola, A. Viale, C. Turc-Carel, B. Dutrillaux, P. Gaudray, and J.-L. Nahon Segmental Duplications in Euchromatic Regions of Human Chromosome 5: A Source of Evolutionary Instability and Transcriptional Innovation Genome Res., March 1, 2003; 13(3): 369 - 381. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Fan, T. Newman, E. Linardopoulou, and B. J. Trask Gene Content and Function of the Ancestral Chromosome Fusion Site in Human Chromosome 2q13-2q14.1 and Paralogous Regions Genome Res., November 1, 2002; 12(11): 1663 - 1672. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
J. M. Young, C. Friedman, E. M. Williams, J. A. Ross, L. Tonnes-Priddy, and B. J. Trask Different evolutionary processes shaped the mouse and human olfactory receptor gene families Hum. Mol. Genet., March 1, 2002; 11(5): 535 - 546. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. C. Mefford, E. Linardopoulou, D. Coil, G. van den Engh, and B. J. Trask Comparative sequencing of a multicopy subtelomeric region containing olfactory receptor genes reveals multiple interactions between non-homologous chromosomes Hum. Mol. Genet., October 1, 2001; 10(21): 2363 - 2372. [Abstract] [Full Text] [PDF]
|
|
| |||||||||