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Vertebrate ORs have a roughly 1-kb intronless coding region that codes for a polypeptide with a little more than 300 amino acids. As a group, ORs can be distinguished from other seven transmembrane receptors (7TMRs) by a few conserved amino acid motifs and some conserved single residues. Seventeen hypervariable amino acid residues in transmembrane domains 3-5 are thought to define an odorant binding pocket; these domains have high diversity and little conservation among OR subfamilies (Mombaerts 2001). OR genes have variable noncoding exons and splice sites, suggesting that they are alternatively spliced (e.g., Sosinsky, Glusman et al. 2000).

The range of odorants detectable by this variety of receptors includes molecules with very different chemical structures, implying that OR ligand-binding domains must be diverse as well, and there is corresponding variability in OR structure. An OR seems to respond optimally to a particular molecular structure—for example, only one of a series of specific odorants used in experimental tests. It is often assumed that the OR has been "tuned" for this specificity by evolution (Dreyer 1998), but the use of this adjective by sensory biologists suggests an implicitly strong darwinian view of precise selection. Whether, when, or how the OR-specific response pattern was shaped by (odorant-specific?) natural selection or whether it is simply the empirical property of any given binding pocket is not clear. As a rule, odorants are not detected in nature by single receptors (a feature shared with antigen recognition by antibodies), and, as noted earlier, odorants do not have a clear or cohesive chemical "spectrum" as do light and sound.

Transduction of an olfactory signal is initiated by the binding of an odorant molecule to the receptor on the cilia of an olfactory neuron dendrite. This triggers the intracellular G protein cascade and ultimately results in the delivery of the signal to the olfactory centers in the brain where the signal is decoded. G protein linked ORs activate signal transduction in one of two ways. Olfaction-specific G proteins convert abundant ATP into cAMP to generate an action potential in the ON. Some ORs increase intracellular cAMP concentration, opening cAMP-gated cation channels and allowing an influx of sodium ions, which depolarizes the cell and initiates a nerve impulse that travels along the axon to the brain. Others activate the inositol phospholipid pathway, IP3-gated Ca2+ channels, in the plasma membrane, to activate second-messenger signal transduction molecules. ONs typically produce multiple OR copies and a given cell's response may be thought of as the effect of the binding of odorants to a sufficient number of receptors on its surface over a suitably short time period, which, in aggregate, generates a threshold action potential.

ONs are the only sensory neurons whose axons connect directly to the brain (see Figure 13-3 for a schematic drawing of basic olfactory wiring to the brain). The mapping between specific odorant receptors and specific locations in the brain seems to be quite precise within an individual, as will be discussed in Chapters 15 and 16. The essential "wiring" characteristic may be that different ONs that express the same OR gene send axons to the same glomerulus (neuronal cluster), in the olfactory bulb (e.g., Kauer 2002; Mombaerts 2001; Zou et al. 2001). Replacement ONs are generated mitotically during life from cells at the base of the olfactory epithelium and express the same OR gene and send their axons to the same glomerulus as their predecessor, which seems to be an important means by which odor perception remains more or less constant throughout an organism's life. How ON-glomerular conservation occurs is not yet completely clear, but it appears to be ON-guided; an ON could either switch which OR gene it expresses or use OR-specific axonal redirection to the glomerulus appropriate for the OR gene it is already expressing (P. Mombaerts, personal communication).

DNA sequence phylogenies suggest that there are two main classes of vertebrate OR genes (Kratz et al. 2002; Mombaerts 2001) (see Figure 13-2B). Class I appears

Piriform cortex

Figure 13-3. Basic olfactory wiring diagram showing pathways of two different odorant receptors. In the olfactory epithelium, sensory neurons expressing a single OR gene are located in the same zone. In the olfactory bulb their axons synapse with mitral cells (small circles) in the same few glomeruli (large circles). The mitral cells carrying signal of specific ORs synapse with clusters of neurons (differently shaded dots in large ovals) at stereotypical sites in olfactory areas of the cortex, creating a sensory map. Figure redrawn from (Zou, Horowitz et al. 2001) with permission. Original figure copyright 2001 by Nature Publishing.

odor

Olfactory Epithelium

Figure 13-3. Basic olfactory wiring diagram showing pathways of two different odorant receptors. In the olfactory epithelium, sensory neurons expressing a single OR gene are located in the same zone. In the olfactory bulb their axons synapse with mitral cells (small circles) in the same few glomeruli (large circles). The mitral cells carrying signal of specific ORs synapse with clusters of neurons (differently shaded dots in large ovals) at stereotypical sites in olfactory areas of the cortex, creating a sensory map. Figure redrawn from (Zou, Horowitz et al. 2001) with permission. Original figure copyright 2001 by Nature Publishing.

to be the oldest and is the only group found in fish. Amphibians have class I and class II genes; at least in frogs, the class I genes are only expressed in a water-sensitive chamber and class II genes in an air-sensitive chamber. The genomic cluster arrangement of mammalian ORs suggests that class I genes duplicated to form the ancestor of class II genes, followed by the dispersal of duplicate ORs, mainly in class

II, to other chromosomes. These facts suggest that class I genes specialize for waterborne and class II for volatile odorants. However, mammals retain many class I genes scattered in their genome, and a higher fraction of human class I genes are more functional than our class II genes; thus the former seem unlikely simply to be a relic of an early aquatic life.

As with other tandemly repeated genes, OR genes appear to have evolved by unequal crossing over, gene conversion, and occasional chromosomal translocations or duplications, followed by subsequent additional local duplication. In mammals, genes from different subclasses are dispersed within single chromosomal clusters. There are at least two OR gene clusters in zebra fish, 12 in mice, and more than 25 in humans (Kratz, Dugas et al. 2002; Mombaerts 2001), in whom ORs are found on all chromosomes except the short chromosome 20 and the Y (Glusman, Yanai et al. 2001) (Figures 13-4 and 13-5).These genes are distributed in clusters of various sizes. Each cluster largely comprises genes from the same OR subfamily. There are two major clusters of class I ORs on human chromosome 11, comprising over 40 percent of our entire repertoire, pointing to the importance of this cluster in the evolution of the vertebrate OR genes. Another cluster is linked to the major histocompati-bility complex (MHC) on chromosome 6, and it has been speculated that these ORs could be involved with mate identification, at least in some species like rodents (Younger et al. 2001).

There is at least some coherence in olfactory systems that may represent phy-logeny as well as development. Frequent duplication, mutation, and perhaps gene conversion means that homology between pairs of OR genes is highly variable; however, as might be expected, the cluster organization for identifiable orthologs generally appears to be retained among mammals. ORs closely related in sequence are closely related in chromosomal location and regulation and with a few exceptions tend to be expressed in similar regions in the olfactory epithelium (Lane et al. 2001; Mombaerts 1999); these in turn project to localized regions in the olfactory bulb (Kratz, Dugas et al. 2002; Tsuboi et al. 1999). Thus, each zone has its own expression mechanism, and its ONs project roughly to similarly distinct regions in the olfactory bulb.

Because these epithelial regions are similar among individual animals in the same species, they may reflect temporally coordinated aspects of gene expression and embryonic neural development. Interestingly, however, little regulatory sequence sharing has been detected among paralogous genes that are expressed in the same zone of the olfactory bulb (Lane, Cutforth et al. 2001), although some potential motif sharing has been suggested (Hoppe et al. 2000; Sosinsky, Glusman et al. 2000). One problem is that recently duplicated genes may share flanking sequence, which may or may not imply that that sequence is specifically regulatory; recall that the genome is nearly saturated with potential regulatory sequences; therefore, sequence analysis alone is usually not a definitive way to identify response elements. Experiments with different ORs have reported differing results regarding how near the gene the relevant regulatory regions are, whether a few kilobases or hundreds (e.g., Mombaerts 2001); this may mean either that the experiments have missed something important about OR regulation or that that regulation varies greatly from gene to gene.

Although these spatial and chromosomal clustering aspects of organization are relevant for developmental coherence and gene expression, they may be of little relevance for odorant detection itself. The zonal organization of the olfactory

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