NIEHS Scientist Serves at White House

Background: Olfactory receptor (OR) genes were discovered more than a decade ago, when Buck and Axel observed that, in rats, certain G-protein coupled receptors are expressed exclusively in the olfactory epithelium. Subsequently, protein sequence similarity was used to identify entire OR gene repertoires of a number of mammalian species, but only in mouse were these predictions followed up by expression studies in olfactory epithelium. To rectify this, we have developed a DNA microarray that contains probes for most predicted human OR loci and used that array to examine OR gene expression profiles in olfactory epithelium tissues from three individuals. Results: We detected expression of 437 (76%) human OR genes in these olfactory epithelia. Interestingly, we detected widespread expression of OR pseudogenes, an observation that may shed light on the mechanism of OR gene choice in the olfactory sensory neurons. To address the hypothesis that OR genes may carry out additional functions, we also characterized the expression of OR genes in a number of non-olfactory tissues. Conclusion: While our results corroborate the functional annotation of the majority of predicted human odorant receptors, we find that a large number of putative human OR genes are expressed in non-olfactory tissues, sometimes exclusively so. Our evolutionary analysis of ectopically expressed human OR genes does not lend support to the hypothesis that these genes have alternative functions. Background Buck and Axel [1] identified the odorant receptor (OR) gene family based partly on the observation that OR genes were expressed in olfactory epithelium, but were not detected in lung, liver, spleen, kidney, retina, and brain. Subsequently, additional OR genes were recognized in genomic sequences by their similarity to the first set of identified OR genes [2,3], and by the presence of certain predicted protein motifs [1,4]. Recently, the complete genomic sequence of a number of mammalian species became available, permitting Published: 17 May 2007 Genome Biology 2007, 8:R86 (doi:10.1186/gb-2007-8-5-r86) Received: 17 January 2007 Revised: 10 April 2007 Accepted: 17 May 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/5/R86 Genome Biology 2007, 8:R86 R86.2 Genome Biology 2007, Volume 8, Issue 5, Article R86 Zhang et al. http://genomebiology.com/2007/8/5/R86 interspecies comparisons of complete OR gene repertoires. The analyses of published mammalian genomes suggested that the original estimate of the size of the mammalian OR gene repertoire, approximately 100 genes, was a severe underestimate. Indeed, it is now thought that mammalian genomes carry 800-1,400 OR genes [5-10], which are typically organized in gene clusters and are found on many chromosomes. With roughly 3% of all genes coding for odorant receptors, OR genes are by far the largest gene family in mammalian genomes. To date, however, mammalian OR genes have remained largely orphan receptors. In fact, until recently, there was no systematic study of putative mammalian OR gene expression in olfactory epithelium [7,11], such that the functional annotation of OR genes remained unclear. Moreover, expression of several predicted mammalian OR genes was detected only in non-olfactory tissues, notably in testis [12-14]. These observations raised the possibility that a subset of predicted OR genes may not be odorant receptors at all but have other functions, with important implications for functional studies in olfaction and comparisons of mammalian OR gene repertoires. Alternatively, OR genes may have a function beyond odor recognition, for example, in sperm chemotaxis [15]. Recently, Zhang et al. [16] studied the expression of nearly all predicted OR genes in mouse using a newly developed DNA microarray [16]. Most (approximately 80%) predicted mouse OR genes were confirmed to be expressed in olfactory epithelium, but a subset were found to be expressed only in nonolfactory tissues and, consequently, their functional annotation is now in question [16]. In humans, it is not known how many of the predicted OR genes are expressed in the olfactory epithelium, and hence how many are likely to participate in odorant binding. Moreover, the predicted human OR gene repertoire includes nearly 600 pseudogenes [5] and it remains unknown how often they are expressed. Since olfactory sensory neurons are believed to express only a single functional OR gene, if these pseudogenes are routinely expressed in the olfactory epithelium, a large proportion of neurons may either express a single non-functional gene, or co-express a functional and non-functional OR genes [17]. A recent study [14] used expressed sequence tag (EST) data and results of genome-wide microarrays to survey human OR gene expression in olfactory epithelium and several nonolfactory tissues. However, that analysis was limited by shortcomings of the available data, including biases and inaccuracies in the EST databases and incomplete sampling of OR genes on the human genome-wide microarray (which includes probe-sets for only 356 predicted OR genes and pseudogenes). Moreover, the genome-wide microarray was not optimized specifically to measure OR gene expression, so many of the probes may be susceptible to cross-hybridization by other OR genes [14]. Indeed, the authors' analysis of the probe-set sequences suggested that the expression of only 217 human OR genes and pseudogenes could be estimated with confidence using the genome-wide microarray data [14]. To comprehensively and reliably assess expression of predicted human OR genes, we designed a new microarray with probes for nearly all human OR genes. We used this microarray to characterize the expression of human OR genes in olfactory epithelium as well as in a number of other tissues. Results and discussion To measure the expression of human OR genes, we extracted total RNA from three samples of human olfactory epithelium tissues collected by the National Disease Research Interchange)[18] within eight hours of the donor's death. We confirmed that RNA was extracted from olfactory epithelium tissues by amplification of the odorant binding protein 2B (OBP2B) gene (Figure 1), which is expressed exclusively in olfactory epithelium [19]. In addition, we tested for the presence of olfactory sensory neurons in each sample by amplifying the olfactory sensory neuron marker gene, the olfactory marker protein (OMP) [20]. Once we confirmed the source of the RNA, we proceeded by labeling and hybridizing each olfactory epithelium RNA sample, in two independent technical replicates, to a custom human OR gene microarray (see Materials and methods). Similarly, we hybridized RNA from human liver, lung, kidney, heart, and testis (purchased from Ambion (Austin, TX, USA)) to the microarray in two technical replicates each. Expression of OR genes in human olfactory epithelium Our first goal was to detect which of the predicted human OR genes are expressed in olfactory epithelium, thereby lending support to their functional annotation as odorant receptors. One way to examine this is to rely on the absent/present calls that the Affymetrix software provides for each probe-set. However, microarrays were not designed to detect expression but rather to compare levels of expression between samples or treatments and, as a result, existing algorithms are not well suited for our application [21,22]. In particular, as probes vary in their specificity and sensitivity, using cut-offs for absolute hybridization intensity as a detection tool is unreliable [23-25]. An alternative is to use a comparison of gene expression levels across the studied RNA samples in order to detect genes that are expressed [16]. The rationale of this approach is that genes with significantly higher expression in sample A compared to sample B are clearly expressed in A. Thus, OR genes with significantly elevated expression in olfactory epithelium compared to other tissues can be considered 'detected'. Accordingly, we compared expression of OR genes in the olfactory epithelium samples with their expression levels in the five non-olfactory tissues (see Materials and methods). Genome Biology 2007, 8:R86 http://genomebiology.com/2007/8/5/R86 Genome Biology 2007, Volume 8, Issue 5, Article R86 Zhang et al. R86.3