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Odorant receptor expressed sequence tags demonstrate olfactory expression of over 400 genes, extensive alternate splicing and unequal expression levels.

Young JM, Shykind BM, Lane RP, Tonnes-Priddy L, Ross JA, Walker M, Williams EM, Trask BJ - Genome Biol. (2003)

Bottom Line: Most of these genes were previously annotated as olfactory receptors based solely on sequence similarity.Our finding that different olfactory receptors have different expression levels is intriguing given the one-neuron, one-gene expression regime of olfactory receptors.We provide 5' untranslated region sequences and candidate promoter regions for more than 300 olfactory receptors, valuable resources for computational regulatory motif searches and for designing olfactory receptor microarrays and other experimental probes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA 98109, USA. jayoung@fhcrc.org

ABSTRACT

Background: The olfactory receptor gene family is one of the largest in the mammalian genome. Previous computational analyses have identified approximately 1,500 mouse olfactory receptors, but experimental evidence confirming olfactory function is available for very few olfactory receptors. We therefore screened a mouse olfactory epithelium cDNA library to obtain olfactory receptor expressed sequence tags, providing evidence of olfactory function for many additional olfactory receptors, as well as identifying gene structure and putative promoter regions.

Results: We identified more than 1,200 odorant receptor cDNAs representing more than 400 genes. Using real-time PCR to confirm expression level differences suggested by our screen, we find that transcript levels in the olfactory epithelium can differ between olfactory receptors by up to 300-fold. Differences for one gene pair are apparently due to both unequal numbers of expressing cells and unequal transcript levels per expressing cell. At least two-thirds of olfactory receptors exhibit multiple transcriptional variants, with alternative isoforms of both 5' and 3' untranslated regions. Some transcripts (5%) utilize splice sites within the coding region, contrary to the stereotyped olfactory receptor gene structure. Most atypical transcripts encode nonfunctional olfactory receptors, but can occasionally increase receptor diversity.

Conclusions: Our cDNA collection confirms olfactory function of over one-third of the intact mouse olfactory receptors. Most of these genes were previously annotated as olfactory receptors based solely on sequence similarity. Our finding that different olfactory receptors have different expression levels is intriguing given the one-neuron, one-gene expression regime of olfactory receptors. We provide 5' untranslated region sequences and candidate promoter regions for more than 300 olfactory receptors, valuable resources for computational regulatory motif searches and for designing olfactory receptor microarrays and other experimental probes.

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Sixty-two olfactory receptor cDNAs use splice sites within the coding region. The bar at the top represents an alignment of all olfactory receptor proteins, with transmembrane (TM) regions shaded gray and intracellular (IC) and extracellular (EC) loops in white. Above the bar, the jagged line plots information content [51] for each alignment position, with higher values representing residues conserved across more olfactory receptors. cDNAs with atypical splicing are plotted below, aligned appropriately to the consensus representation. Genbank accessions for each cDNA are shown on the right, and where more than one clone represents the same isoform, both names are given, but a composite sequence is drawn. Multiple isoforms from the same gene are grouped by gray background shading. Thick black lines represent cDNA sequence, and thin lines represent intronic sequence (with diagonal slash marks if not drawn to scale). The uppermost two cDNAs encode potentially functional olfactory receptors. A single cDNA drawn as white boxes (CB173065) is cloned into the vector in the reverse orientation. Introns that result in a frameshift relative to the olfactory receptor consensus are drawn as single dashed lines. The first in-frame methionine in the cDNA is marked with an 'M', and the first stop codon 5' to this methionine (if any) is marked with *. Most sequences are incomplete at the 3' end, as represented by paired dotted lines, although two sequences (CB174400 and CB174364), marked with '(A)n', contain the cDNA's poly(A) tail. The 'X' on sequence CB173500 marks an exon that does not align with genomic sequence near the rest of the gene or anywhere else in Celera's mouse genome sequence, and 'TM4' on sequence CB172879 notes an exon that matches to the reverse-complement of the fourth transmembrane domain of the next downstream olfactory receptor gene. For the two lowermost cDNAs, exon order in the cDNA clone is inconsistent with the corresponding genomic sequence, as represented by the curved intron lines.
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Figure 6: Sixty-two olfactory receptor cDNAs use splice sites within the coding region. The bar at the top represents an alignment of all olfactory receptor proteins, with transmembrane (TM) regions shaded gray and intracellular (IC) and extracellular (EC) loops in white. Above the bar, the jagged line plots information content [51] for each alignment position, with higher values representing residues conserved across more olfactory receptors. cDNAs with atypical splicing are plotted below, aligned appropriately to the consensus representation. Genbank accessions for each cDNA are shown on the right, and where more than one clone represents the same isoform, both names are given, but a composite sequence is drawn. Multiple isoforms from the same gene are grouped by gray background shading. Thick black lines represent cDNA sequence, and thin lines represent intronic sequence (with diagonal slash marks if not drawn to scale). The uppermost two cDNAs encode potentially functional olfactory receptors. A single cDNA drawn as white boxes (CB173065) is cloned into the vector in the reverse orientation. Introns that result in a frameshift relative to the olfactory receptor consensus are drawn as single dashed lines. The first in-frame methionine in the cDNA is marked with an 'M', and the first stop codon 5' to this methionine (if any) is marked with *. Most sequences are incomplete at the 3' end, as represented by paired dotted lines, although two sequences (CB174400 and CB174364), marked with '(A)n', contain the cDNA's poly(A) tail. The 'X' on sequence CB173500 marks an exon that does not align with genomic sequence near the rest of the gene or anywhere else in Celera's mouse genome sequence, and 'TM4' on sequence CB172879 notes an exon that matches to the reverse-complement of the fourth transmembrane domain of the next downstream olfactory receptor gene. For the two lowermost cDNAs, exon order in the cDNA clone is inconsistent with the corresponding genomic sequence, as represented by the curved intron lines.

Mentions: We identified 62 cDNAs (5% of all olfactory receptor clones) from 38 intact olfactory receptors and one olfactory receptor pseudogene where a splice site within the protein-coding region is used. For two genes (top two cDNAs, Figure 6), the predicted protein appears to be an intact olfactory receptor with three or ten amino acids, including the initiating methionine, contributed by an upstream exon. A similar gene structure was described previously for a human olfactory receptor [25]. One of these two mouse genes has no start codon in its otherwise intact main coding exon. The unusual splicing thus rescues what would otherwise be a dysfunctional gene. In most cases (60 out of 62 cDNAs), the unusual transcript appears to be an aberrant splice form - the transcript would probably not encode a functional protein because the splice introduces a frameshift or removes conserved functional residues (Figure 6). For two clones (bottom two cDNAs, Figure 6), exon order in the cDNA clone is inconsistent with the corresponding genomic sequence. It is difficult to imagine what kind of cloning artefact resulted in these severely scrambled cDNAs: we suggest that they derive from real but rare transcripts. However, their low frequency in our cDNA collection suggests that splicing contrary to genomic organization does not contribute significantly to the olfactory receptor transcript repertoire. For 21 of the 26 genes for which unusually spliced cDNAs were found, we also observe an alternative ('normal') isoform that does not use splice sites within the coding region. (For the remaining 13 of the 3' genes showing odd splicing, we have identified only one cDNA so have not determined whether normal isoforms are present.)


Odorant receptor expressed sequence tags demonstrate olfactory expression of over 400 genes, extensive alternate splicing and unequal expression levels.

Young JM, Shykind BM, Lane RP, Tonnes-Priddy L, Ross JA, Walker M, Williams EM, Trask BJ - Genome Biol. (2003)

Sixty-two olfactory receptor cDNAs use splice sites within the coding region. The bar at the top represents an alignment of all olfactory receptor proteins, with transmembrane (TM) regions shaded gray and intracellular (IC) and extracellular (EC) loops in white. Above the bar, the jagged line plots information content [51] for each alignment position, with higher values representing residues conserved across more olfactory receptors. cDNAs with atypical splicing are plotted below, aligned appropriately to the consensus representation. Genbank accessions for each cDNA are shown on the right, and where more than one clone represents the same isoform, both names are given, but a composite sequence is drawn. Multiple isoforms from the same gene are grouped by gray background shading. Thick black lines represent cDNA sequence, and thin lines represent intronic sequence (with diagonal slash marks if not drawn to scale). The uppermost two cDNAs encode potentially functional olfactory receptors. A single cDNA drawn as white boxes (CB173065) is cloned into the vector in the reverse orientation. Introns that result in a frameshift relative to the olfactory receptor consensus are drawn as single dashed lines. The first in-frame methionine in the cDNA is marked with an 'M', and the first stop codon 5' to this methionine (if any) is marked with *. Most sequences are incomplete at the 3' end, as represented by paired dotted lines, although two sequences (CB174400 and CB174364), marked with '(A)n', contain the cDNA's poly(A) tail. The 'X' on sequence CB173500 marks an exon that does not align with genomic sequence near the rest of the gene or anywhere else in Celera's mouse genome sequence, and 'TM4' on sequence CB172879 notes an exon that matches to the reverse-complement of the fourth transmembrane domain of the next downstream olfactory receptor gene. For the two lowermost cDNAs, exon order in the cDNA clone is inconsistent with the corresponding genomic sequence, as represented by the curved intron lines.
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Figure 6: Sixty-two olfactory receptor cDNAs use splice sites within the coding region. The bar at the top represents an alignment of all olfactory receptor proteins, with transmembrane (TM) regions shaded gray and intracellular (IC) and extracellular (EC) loops in white. Above the bar, the jagged line plots information content [51] for each alignment position, with higher values representing residues conserved across more olfactory receptors. cDNAs with atypical splicing are plotted below, aligned appropriately to the consensus representation. Genbank accessions for each cDNA are shown on the right, and where more than one clone represents the same isoform, both names are given, but a composite sequence is drawn. Multiple isoforms from the same gene are grouped by gray background shading. Thick black lines represent cDNA sequence, and thin lines represent intronic sequence (with diagonal slash marks if not drawn to scale). The uppermost two cDNAs encode potentially functional olfactory receptors. A single cDNA drawn as white boxes (CB173065) is cloned into the vector in the reverse orientation. Introns that result in a frameshift relative to the olfactory receptor consensus are drawn as single dashed lines. The first in-frame methionine in the cDNA is marked with an 'M', and the first stop codon 5' to this methionine (if any) is marked with *. Most sequences are incomplete at the 3' end, as represented by paired dotted lines, although two sequences (CB174400 and CB174364), marked with '(A)n', contain the cDNA's poly(A) tail. The 'X' on sequence CB173500 marks an exon that does not align with genomic sequence near the rest of the gene or anywhere else in Celera's mouse genome sequence, and 'TM4' on sequence CB172879 notes an exon that matches to the reverse-complement of the fourth transmembrane domain of the next downstream olfactory receptor gene. For the two lowermost cDNAs, exon order in the cDNA clone is inconsistent with the corresponding genomic sequence, as represented by the curved intron lines.
Mentions: We identified 62 cDNAs (5% of all olfactory receptor clones) from 38 intact olfactory receptors and one olfactory receptor pseudogene where a splice site within the protein-coding region is used. For two genes (top two cDNAs, Figure 6), the predicted protein appears to be an intact olfactory receptor with three or ten amino acids, including the initiating methionine, contributed by an upstream exon. A similar gene structure was described previously for a human olfactory receptor [25]. One of these two mouse genes has no start codon in its otherwise intact main coding exon. The unusual splicing thus rescues what would otherwise be a dysfunctional gene. In most cases (60 out of 62 cDNAs), the unusual transcript appears to be an aberrant splice form - the transcript would probably not encode a functional protein because the splice introduces a frameshift or removes conserved functional residues (Figure 6). For two clones (bottom two cDNAs, Figure 6), exon order in the cDNA clone is inconsistent with the corresponding genomic sequence. It is difficult to imagine what kind of cloning artefact resulted in these severely scrambled cDNAs: we suggest that they derive from real but rare transcripts. However, their low frequency in our cDNA collection suggests that splicing contrary to genomic organization does not contribute significantly to the olfactory receptor transcript repertoire. For 21 of the 26 genes for which unusually spliced cDNAs were found, we also observe an alternative ('normal') isoform that does not use splice sites within the coding region. (For the remaining 13 of the 3' genes showing odd splicing, we have identified only one cDNA so have not determined whether normal isoforms are present.)

Bottom Line: Most of these genes were previously annotated as olfactory receptors based solely on sequence similarity.Our finding that different olfactory receptors have different expression levels is intriguing given the one-neuron, one-gene expression regime of olfactory receptors.We provide 5' untranslated region sequences and candidate promoter regions for more than 300 olfactory receptors, valuable resources for computational regulatory motif searches and for designing olfactory receptor microarrays and other experimental probes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA 98109, USA. jayoung@fhcrc.org

ABSTRACT

Background: The olfactory receptor gene family is one of the largest in the mammalian genome. Previous computational analyses have identified approximately 1,500 mouse olfactory receptors, but experimental evidence confirming olfactory function is available for very few olfactory receptors. We therefore screened a mouse olfactory epithelium cDNA library to obtain olfactory receptor expressed sequence tags, providing evidence of olfactory function for many additional olfactory receptors, as well as identifying gene structure and putative promoter regions.

Results: We identified more than 1,200 odorant receptor cDNAs representing more than 400 genes. Using real-time PCR to confirm expression level differences suggested by our screen, we find that transcript levels in the olfactory epithelium can differ between olfactory receptors by up to 300-fold. Differences for one gene pair are apparently due to both unequal numbers of expressing cells and unequal transcript levels per expressing cell. At least two-thirds of olfactory receptors exhibit multiple transcriptional variants, with alternative isoforms of both 5' and 3' untranslated regions. Some transcripts (5%) utilize splice sites within the coding region, contrary to the stereotyped olfactory receptor gene structure. Most atypical transcripts encode nonfunctional olfactory receptors, but can occasionally increase receptor diversity.

Conclusions: Our cDNA collection confirms olfactory function of over one-third of the intact mouse olfactory receptors. Most of these genes were previously annotated as olfactory receptors based solely on sequence similarity. Our finding that different olfactory receptors have different expression levels is intriguing given the one-neuron, one-gene expression regime of olfactory receptors. We provide 5' untranslated region sequences and candidate promoter regions for more than 300 olfactory receptors, valuable resources for computational regulatory motif searches and for designing olfactory receptor microarrays and other experimental probes.

Show MeSH
Related in: MedlinePlus