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

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.

Figure 1

Olfactory receptor genes whose expression in the mouse olfactory epithelium was confirmed in this study. Genes whose expression has been confirmed by our cDNA screen are colored blue on a phylogenetic tree of 1,107 intact mouse olfactory receptors. Genes whose expression was confirmed by PCR methods are colored red (genes listed in Additional data file 1 were confirmed by specific PCR of the cDNA library or reverse-transcribed RNA, and genes confirmed using the class I degenerate primer for RT-PCR are AY317681, AY317698, AY317700, AY317767, AY317773, AY317774, AY317797 and AY317923). Other olfactory receptors are colored gray, and a chemokine outgroup is colored black. Class I olfactory receptors are bracketed, and the remaining olfactory receptors are class II.

Figure 2

The cDNA screen suggests different expression levels for different olfactory receptors. Distribution of number of cDNAs observed (dots) and expected (triangles, line) per olfactory receptor gene among 1,176 olfactory receptor cDNAs identified, based on a Poisson distribution.

Figure 3

Differential expression levels among six olfactory receptor genes determined by quantitative PCR. (a) Expression levels of olfactory receptor genes can vary by almost 300-fold (for example, genes A and D). Relative expression levels of six selected olfactory receptor genes (A, AY318555; B, AY318107; C, AY318644; D, AY317365; E, AY317773; and F, AY317797) were determined in olfactory epithelium RNA samples from three mice. Expression levels for each gene were first determined relative to a standard curve made using mouse genomic DNA templates, and then values for each mouse were normalized so that a housekeeping gene, ribosomal S16, had a value of 1 (arbitrary units) (not shown). Error bars show one standard deviation (six replicate reactions). Genes E and F (AY317773 and AY317797) are class I olfactory receptors. Numbers of cDNAs observed in our screen are shown under each gene name. (b) Expression levels of each gene are similar, with some variation, among the three mice sampled. Graphs show pairwise comparisons between the three mice sampled, with relative expression levels (arbitrary units) in one mouse plotted along the x-axis and in a second on the y-axis.

Figure 4

Olfactory receptors showing different expression levels. Different expression levels of one pair of olfactory receptors is due to different numbers of expressing cells and different transcript levels per cell. RNA in situ hybridization with digoxigenin-labeled probes for (a) gene A (AY318555) and (b) gene D (AY317365) on coronal sections of the olfactory turbinates of an adult mouse, shown at low magnification and inset (boxed) at high magnification. Endoturbinates II and III and ectoturbinate 3 are labeled in (b).

Figure 5

Many olfactory receptor genes show alternate splicing. Distribution of the number of transcriptional isoforms observed for the 82 olfactory receptors for which we have identified at least four cDNAs.

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.