Comparative Genomics of Odorant- and Pheromone Receptor Genes in Rodents

Rat OR and V1Rs

In the rn3 (rat) assembly, 1525 OR genes and 181 V1R genes are mapped to specific genomic locations. 96.8% of the OR genes are mapped, as were a similar percentage of V1R genes. The number of OR/V1R genes on each chromosome is shown in Fig 1a. Chromosomes 1, 3, 7, 8 and 10 have the largest number of OR genes, whereas no OR genes are located on chromosomes 18 and Y. For V1R genes, the mapped genes are found on chromosome 1, 4, 7, 14 and 17. Only two isolated genes are located on chromosomes 14 and 17. Both OR and V1R genes tend to form tight clusters, but they are not intermingled with one another. In total, OR genes are distributed in 41 clusters, and V1R genes in 9 clusters. The distribution of OR and V1R genes is shown in Fig 1b, using chromosomes 1 and 4 as examples. Isolated single genes occur only rarely (1.5% for ORs, 2.7% for V1Rs) (shown in Table 1). Among these 23 isolated OR genes, 14 are pseudogenes, which is much higher than the average fraction of pseudogenes in the rat OR repertoire. For V1R genes, only 2 of the 5 single genes are intact.

As shown in Fig 1a, chromosomes 1 and 3 harbor the largest number of OR genes, where there are 344 ORs each. Characteristics, such as the number and density of genes and pseudogene frequency, vary among the clusters (see Table 2). The largest cluster, which is located on chromosome 3 and harbors about 1/6 of the entire rat OR repertoire, has the lowest fraction of pseudogenes, while in the third largest cluster on chromosome 7, containing 173 ORs, nearly 50% are pseudogenes. Thus there is no obvious correlation (Pearson r=0.25) between the fraction of pseudogenes and the density of clusters. For rat V1Rs, only clusters with more than 10 genes are shown in Table 2. In general, rat V1R clusters have a higher percentage of pseudogenes but a lower average density compared to rat OR clusters.

Mouse OR and V1Rs

In the mm5 assembly, 1303 ORs and 253 V1Rs are mapped to specific genomic locations, whereas 72 ORs and 53 V1Rs could not be mapped. In earlier reports based on the mm2 assembly (Zhang et al 2004), about one-third of V1R genes could not be mapped, but our updating using the mm5 assembly improves this to only 17% unmapped genes. The number of OR/V1R genes on each chromosome is shown in Fig 1c. Chromosomes 2, 7, 9, 10, 11 have the largest number of OR genes, whereas no OR genes are found on chromosomes 18 and Y. For V1R genes, the mapped genes are found on chromosomes 4, 6, 7, 13, 17, 18. Five isolated V1R genes are found on the following chromosomes: 1, 2, 5, 14, X. Similar to the rat OR and V1R genes, mouse ORs and V1Rs also tend to form tight clusters. Mouse OR genes are distributed in 43 clusters, and V1R genes in 10 clusters. The distribution of mouse OR and V1R genes is shown in Fig 1d, taking chromosomes 6 and 7 as examples. Isolated genes also occur rarely (1.5% for ORs, 2.6% for V1Rs), but have a higher fraction of pseudogenes. 11 of the 21 single ORs and 5 of the 8 isolated V1Rs do not appear to be functional.

As shown in Fig 1c, the largest OR clusters are localized on chromosomes 2 and 7, which harbor 344 and 267 ORs respectively. The characteristics of mouse clusters which are orthologous with rat OR clusters on the left are shown in Table 2. Note that Rat cluster 1_160 splits into two clusters (7_90 and 7_94) in mouse. The second large mouse cluster, which consists only of Class I ORs, is one of the densest clusters. For mouse V1Rs, clusters having more than 10 genes are listed. Unexpectedly, the largest mouse V1R cluster, 13_21, has the highest fraction of pseudogenes and shortest average inter-gene distance. Why some clusters have a much higher proportion of pseudogenes than others is unknown.

Phylogenetic Analysis of OR/V1R Repertoire

OR and V1R genes can be divided into families based on a phylogenetic analysis. Members from the same family are defined as sharing 40% or higher amino acid identity and more than 50% bootstrap support. Similar to the mouse, rat ORs also comprise two broad classes, the fish-like Class I and the mammalian Class II. Each of these broad classes can be further separated into families: 153 Class I ORs comprise 27 families, and 1423 Class II ORs can be organized into 133 families. The V1Rs comprise 12 families.

We assigned families and names to rat ORs and V1Rs (see Supplementary table 1) based on earlier methods [4]. The nomenclature for rat ORs is in the format ROR[family number]-[index in the family]. Class I ORs have family numbers smaller than 100 and Class II ORs have family numbers higher than 100. The nomenclature for V1Rs is in the format RV1R[family]-[index in family]. For both ORs and V1Rs, the suffix ‘i’ was added to partial genes, and ‘p’ was added to pseudogenes (see Supplementary table 1).

In the mouse, the newly discovered ORs and V1Rs were analyzed for phylogenetic relatedness. Because sequences were updated in the new genome assembly, the classification of the OR and V1R repertoire was slightly different from the original ones based on mouse genome mm2 version[4; 10] (http://genome.ucsc.edu/). Our updated OR repertoire contains 1194 compared to 978 putative intact genes in the mm2 version. Our V1R repertoire contains 191 putative intact genes, in contrast to 134 in the mm2 version. This is because of the higher sequence quality and coverage in the new genome assembly, resulting in fewer errors in data mining. We classified the updated sequences into families, and mapped them to those in the old version (see Supplementary table 2). Since our main purpose in this paper is not to compare sequence differences in two assemblies, we will not discuss the details of them.

Species-specific Families of ORs/V1Rs

We sought to determine which families, if any, had evolved to be species specific in either rat or mouse. To highlight species-specific families, we performed a phylogenetic analysis with all ORs from mouse and rat pooled, and used the same criterion to define a family as applied to ORs from either species separately (see Methods). The total of all rodent ORs can be divided into 245 families, among which only 4 families include only rat ORs, and 12 families are specific for mouse. In total, 20 mouse ORs and 8 rat ORs are contained in these 16 species-specific families, but of these only 4 mouse ORs and 1 rat OR are intact genes. These analyses indicate that at the family level OR genes are closely related in mouse and rat since more than 99% of putatively functional ORs are clustered in families with counterparts from the other species.

Orthologous clusters were also identified, and presented in Fig 2a, where OR clusters and single genes from rat are displayed on the top, and from mouse along the bottom. As an example, rat cluster 3-68 is orthologous with mouse cluster 2-85, since the counterparts of the ORs from rat cluster 3-68 are always found in mouse cluster 2-85.

Additionally we found that many homologous regions were located around these orthologous clusters (data not shown). This could be an indication of segmental duplication across the two species, which might also account for the close relationship between the receptors. Alternatively, these regions could contain regulatory motifs as these non-coding regions appear not to have been subject to the expected random mutation.

V1R genes pooled from mouse and rat were subject to a similar phylogenetic analysis, but with quite different results, as shown in Fig 2b. Rat V1R clusters and single genes are displayed on the top, and mouse V1Rs on the bottom. The pooled rodent V1R genes can be divided into 53 families, among which 10 families are specific for rat, and 14 for mouse. In total, 79 mouse V1Rs and 35 rat V1Rs are contained in these 24 species-specific families. 42 mouse-specific V1Rs and 20 rat-specific V1Rs are putatively intact genes, which constitutes about 20 percent of the functional V1R repertoire. In particular, 68 V1Rs from mouse cluster 13-21 form 7 families in which only 2 rat V1Rs appear. Most of the rat-specific family members are from families c and g, and most of the mouse-specific family members are from families d and h [11].

The above analysis indicates that, in contrast to the OR genes, at the family level V1R genes tend to be specific to each species, though the mouse and rat diverged only a relatively short time ago. The higher species-specificity of V1R genes may be understandable in the context of the species-specific behaviors they are believed to mediate, while the ORs, with their primary role in detecting environmental odors, are common to both species.

Comparison of Orthologous OR/V1Rs

In Fig 3a, each OR gene (left panel) and V1R gene (right panel) were placed in the bin corresponding to their similarity level with their cross species counterparts. The colors represent the family membership (according to Fig 2) for each OR or V1R gene. In the top bars rat genes were compared with respect to the mouse genes, while in the bottom panel, rat genes served as the reference. The percentage of OR/V1R genes at different similarity levels is shown in Table 3. From the left panel, about 69% of rat ORs and 73% of mouse ORs have orthologs with over 80 percent identity, while only about 15% of rat and mouse V1Rs have orthologs at the same identity level. Most prominently, V1R genes do not have any ortholog with over 90 percent identity. Among ORs, Class I ORs are even more conserved between mouse and rat than Class II ORs (see Table 3). These analyses provide additional evidence, now at the individual gene level, that OR genes are more conserved between mouse and rat than V1R genes.

Comparison of Paralogous OR/V1Rs

Using the same method, we analyzed the similarity between OR or V1R paralogs as shown in Fig 3b. On the top, rat OR/V1R genes are compared to their paralogs, and in the bottom, mouse OR/V1R genes are compared to their paralogs. The percentage of genes at each similarity level is shown in Table 3. On the left panel, 57% rat ORs and 52% mouse ORs have a paralog with over 80 percent identity. Note that these fractions are much lower than those found in the across species ortholog comparisons above. However, in the right panel, 41% rat V1Rs and 63% mouse V1Rs have a paralog at the same level, fractions that are much higher than the percentages found in across species ortholog comparisons. Class I ORs and Class II ORs do not show significant differences in terms of paralogous comparison, while mouse V1Rs are more conserved with their paralogs than rat V1Rs (see Table 3).

These similarity analyses show again that OR genes are more conserved across the two species than V1R genes. However, they also reveal that ORs in mouse and rat have a higher similarity with their orthologs than with their paralogs. As expected the opposite appears to be the case for the V1R genes. Measuring the number of nucleotide substitutions per codon for reciprocal mouse-rat orthologs we found that the rate was lower for the ORs than the V1Rs (ORs: 779 pairs, 0.241+-0.091; V1Rs: 45 pairs, 0.369+-0.078) further suggesting a difference in evolutionary rates between the two types of chemo-receptors.

Motifs of ORs

For OR genes, the 100 motifs discovered from the pool of 2478 mouse and rat intact ORs were schematically plotted in Fig4a. They tend to occur in Transmembrane domains (TM) 4, and 5 and Extracellular loop (EC) 3. Twelve of the generated motifs are highly conserved in the complete database (i.e., are present in more than 90% of ORs). The ‘MAYDRYVAIC[KN]P’ motif , which was previously found to be the most conserved motif in mouse, also stands out as the most conserved from our analysis with rat ORs added. For each motif we identified the species in which the support ORs are located. Somewhat surprisingly, we found no motif that is present only in mouse or rat ORs, although the number of support sequences from each species may vary considerably. For example, motif85 (see the supplementary table 3), exists in 16 rat ORs, but is shared by 26 mouse ORs.

As mentioned above, ORs can be classified into two classes, Class I (fish-like), and Class II (mammalian-like) and then further organized into phylogenetic families. We also studied the support sequences for each motif to indicate its specificity for the Class I/II subgroup and for the family subgroup. Among the 100 motifs, 31 are shared by both classes, whereas 10 motifs are specific to Class I ORs and 59 motifs are specific to Class II ORs. In the family subgroups, 75 motifs are carried by ORs from multiple families. Among the remaining 25 motifs that are specific for one family, there is only one motif specific for Class I, while the rest of the 24 are specific for Class II ORs.

Some of the most highly conserved and widely supported motifs are also well recognized in other GPCRs. For example, the four cysteines, occurring in EC3 and TM3, are thought to form two disulfide bonds [19], as they do in rhodopsins and amine receptors [20]. Aspartic acid, and asparagines, occurring in motif 4 and 2 respectively, are thought to interact with each other to stabilize the overall structure as they do in bovine rhodopsins [21].

Motifs of V1Rs

For V1Rs, we discovered 83 motifs from the pool of 306 intact V1R genes from mouse and rat, using the same method as we did for ORs. As shown in Fig. 4b and supplementary table 4 , and clearly distinct from ORs, the V1R motifs are distributed in a flat pattern, mainly because variable motifs are scattered from N-terminal to C-terminal, except in TM1, TM3 and TM5. Among the 83 motifs, 6 of them are highly conserved in the rodent V1R database (present in over 90% of V1Rs). These motifs display no sequence similarity with the OR motifs. Of the 83 motifs, 12 are specific for mouse and 3 are specific for rat. The remainder are shared by both species, although again the distribution of support sequences is different. For example, motif 14 is supported by 63 V1Rs, 55 of which are mouse V1Rs and 8 are rat V1Rs. There are fewer specific motifs in rat, probably because the rat genome only has half as many intact V1Rs as the mouse. As distinct from the ORs, the 15 motifs represented only by either mouse (12) or rat (3) again indicate that V1R genes are more species-specific than OR genes, now at this motif level of analysis.

We also investigated the family composition of the support sequences for each of the V1R motifs. 43 out of the 83 motifs are from multiple families, and the remaining 40 motifs are supported by one family without regard to the species. Compared to ORs, V1Rs have more motifs supported by single families. This indicates that V1Rs have more locally-conserved motifs than ORs.