At least 419 mouse olfactory receptor genes are expressed in the olfactory epithelium
We have isolated 1,264 olfactory receptor cDNA clones, which together confirm the olfactory epithelial expression of 419 annotated olfactory receptor genes. We used low-stringency hybridization with degenerate olfactory receptor DNA probes to screen around 4,100,000 plaque-forming units (pfu) of an adult mouse olfactory epithelium cDNA library and around 640,000 pfu of an embryonic olfactory epithelium library. We obtained sequences from 1,715 hybridization-positive cDNAs following secondary screens to isolate single clones. Of these clones, 1,264 yielded olfactory receptor-containing sequences. The 26% false-positive rate is a consequence of using low-stringency hybridization to obtain maximal sensitivity. Continuing the screen would have resulted in cDNAs from additional olfactory receptors, but we reached a point of limiting returns: our final batch of 45 olfactory receptor-positive sequences represented 33 different olfactory receptors, of which only four had not been encountered previously in our screen.
Sequence analysis shows that the libraries are of high quality. Firstly, directional cloning was successful: only eight out of 1,430 cDNA sequences with any protein homology matched that protein on the reverse strand. Secondly, genomic contamination is rare: when the 83 olfactory receptor-containing sequences that had a 5' UTR of 400 bp or longer were aligned to genomic sequence, 80 spliced across at least one intron, leaving a maximum of three clones (3.6%) that potentially represent genomic contamination of the libraries. Thirdly, most clones are of a reasonable length: although we did not determine whether clones are full-length, 881 of 1,264 (70%) olfactory receptor cDNAs contain the gene's start codon and at least some 5' UTR sequence.
In order to match cDNAs to their genomic counterparts, first we updated our catalog of mouse olfactory receptor genes [1] based on Celera's most recent genome assembly (Release 13) [29]. Previous reports of the mouse olfactory receptor repertoire [1,2] were based on the Release 12 assembly. Release 13 consists of fewer, longer scaffold sequences containing fewer, smaller gaps than Release 12. Using the BLAST-based methods detailed previously [1], we identified 1,490 olfactory receptor sequences in the new assembly, including 1,107 intact olfactory receptor genes (compared to 866 intact olfactory receptors in the old assembly) reflecting the reduced sequence error rate and increased coverage of the new assembly (Table 1). We created a local database of genomic sequences including all olfactory receptor loci and 0.5 Mb flanking sequences (if available) and compared each cDNA sequence to this 'olfactory subgenome' database using sim4 [30].
cDNAs were assigned to individual genes based on their best match to an olfactory receptor coding region or its upstream region (see Materials and methods). Of the 1,264 olfactory receptor cDNAs, 1,176 matched a total of 419 olfactory receptor genes; the remaining cDNAs either matched an olfactory receptor below our 96% nucleotide identity threshold or had ambiguous matches encompassing more than one olfactory receptor gene region (see below).