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|Some olfactory receptor genes are expressed at higher levels than others|
Our cDNA screen suggests that some olfactory receptor genes are expressed at higher levels than others. If all olfactory receptor genes were expressed at equal levels, and our screen and library were unbiased in their coverage of the class II olfactory receptors, the number of cDNAs detected per class II olfactory receptor should follow a Poisson distribution, calculated based on the assumption that all 983 intact class II olfactory receptors have an equal chance of being represented in the screen, but that class I olfactory receptors and pseudogenes cannot be found (Figure 2). We calculate a low probability (approximately one in 28) that we would observe any gene with at least eight matching cDNAs in the set of 1,176 cDNAs we assigned to single olfactory receptor sequences. However, for 17 olfactory receptors, we found ten or more matching cDNAs, suggesting that they might be expressed at higher levels than other olfactory receptor genes (Figure 2). The two genes for which we found most cDNAs (AY318726/MOR28 and AY318727/MOR10) are genomically adjacent and in the well-studied olfactory receptor cluster next to the T-cell receptor α/δ locus [18,31].
Quantitative RT-PCR of six olfactory receptors confirms that expression levels do indeed vary considerably between genes. We used quantitative (real-time) PCR to measure olfactory epithelium transcript levels of six olfactory receptor genes and the ribosomal S16 gene in three mice of the same inbred strain (Figure 3). These genes include two olfactory receptors with more than 20 matching cDNAs, two with one or two matching cDNAs and two class I olfactory receptors with no matching cDNAs. In these assays, we measure transcript level per genomic copy of the gene by comparing how well a gene-specific primer pair amplifies reverse-transcribed RNA, relative to a standard curve of amplification of mouse genomic DNA. We find that expression levels can vary by almost 300-fold between genes (for example, genes A and D, Figure 3). However, cDNA numbers are not a good indicator of expression level, a discrepancy that is likely to be due to bias in the screen (we used degenerate primers to make the probes, which will favor some genes over others) and in the libraries (oligo-dT priming will favor genes with shorter 3' UTRs). For example, we observe large expression differences in all three mice between two genes for which similar numbers of cDNAs were found (genes A and B, Figure 3), and conversely, similar expression levels between two genes with a ten-fold difference in number of cDNAs found (genes B and C, Figure 3). Expression levels are mostly consistent between different mice: we find similar expression-level differences between olfactory receptor genes in all three mice examined (that is, the rank order of the six genes is similar among the three mice), although there is variation in expression level of some genes between mice (for example, gene E, Figure 3).
In situ hybridization (Figure 4) shows that increased numbers of expressing cells account for some, but not all, of the difference in transcript levels between two of the genes tested by real-time PCR (genes A and D in Figure 3). We hybridized alternate coronal serial sections spanning an entire olfactory epithelium of a young mouse (P6) with probes for gene A and gene D. Southern blot and BLAST analyses show that both probes are likely to hybridize to their intended target genes and no others (not shown). Gene A is expressed in zone 4 of the epithelium according to the nomenclature of Sullivan et al.  (Figure 4a). The expression pattern of gene D does not correspond to any of the four 'classical' olfactory epithelial zones [14,15,32]: positive cells are found in regions of endoturbinates II and III and ectoturbinate 3, resembling the expression pattern seen previously for the OR37 subfamily and ORZ6 olfactory receptors [33,34] (Figure 4b). Counting the total number of positive cells in alternate sections across the entire epithelium, we find that gene A is expressed in 2,905 cells, about 12 times more cells than gene D, which is expressed in a total of 249 cells. This 12-fold difference in numbers of expressing cells does not account for the almost 300-fold difference in RNA levels observed by real-time PCR, implying that the transcript level per expressing cell for gene A is about 25 times higher than transcript level in each expressing cell for gene D. We note that hybridization intensities per positive neuron appear stronger for gene A than gene D after comparable exposure times, in accordance with the idea that transcript levels are higher per cell. Thus, we suggest that expression in more cells and in higher levels per cell together account for the almost 300-fold higher olfactory epithelial RNA levels of gene A relative to gene D (Figure 3).
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