Sequence analysis of mouse vomeronasal receptor gene clusters reveals common promoter motifs and a history of recent expansion

Abstract

We have analyzed the organization and sequence of 73 V1R genes encoding putative pheromone receptors to identify regulatory features and characterize the evolutionary history of the V1R family. The 73 V1Rs arose from seven ancestral genes around the time of mouse-rat speciation through large local duplications, and this expansion may contribute to speciation events. Orthologous V1R genes appear to have been lost during primate evolution. Exceptional noncoding homology is observed across four V1R subfamilies at one cluster and thus may be important for locus-specific transcriptional regulation.

Figure 1

pipmaker plots illustrating gene-block duplications and putative regulatory homologies at two mouse V1R loci. (A) The 788-kb sequence encompassing the mouse chromosome 6D V1R cluster is masked for repeats and compared with itself in both orientations. Homology is plotted according to position (horizontal axes) and percent identity (vertical axes, 50–100%). The positions of the V1R coding sequences with ORFs are indicated by black arrows above the plots (pseudogenes are indicated by gray arrows); the arrow direction indicates relative orientation. In contrast to a previous analysis of gene content and order by using PCR (19), we find V1ra1 and a novel pseudogene V1Rps8 located between V1Rb1 and V1Ra7. The 5′ UTRs, where determined by RACE-PCR, are indicated by medium-thick lines (exons) and thin lines (introns). The locus is bounded by two multiexon genes: the Tgr cDNA at 30–80 kb and the BC003332 cDNA between 761 and 773 kb (black arrows). Celera contigs are ordered in the scaffold by paired-end sequence assembly. Vertical bars within the plot indicate positions of unresolved gaps in the assembled sequence; hatched boxes indicate gaps of estimated size. Duplicated blocks are color-coded: red blocks are duplications of the A subfamily, green blocks are duplications of the B subfamily, and yellow blocks are duplications of the a7/a8 subfamily. The a9 gene block (not duplicated) is shaded gray. Repeat content is summarized along the top axis (light gray, low complexity; dark gray, simple repeats; brown, LTR content; yellow, all SINE repeats; red, L1_MM L1 repeats; blue, Lx L1 repeats; and green, all other Line repeats). Putative regulatory regions are shown with color-coded shading: purple patches are the common promoter homology, light blue patches are the common 5′ UTR homology, pink patches are the locus control region homology (see text). Regions marked with medium blue shading at 308 kb and 534 kb are homologous to each other and contain the common promoter homology (purple portion), but do not appear to be associated with V1R genes or any larger duplicated blocks. Unshaded pipmaker signals are local repetitive sequence. The portion of finished sequence contributed by us (RP23–27e23) lies between 467 kb and 666 kb. (B) The 708-kb scaffold encompassing a portion of a second mouse V1R cluster mapped by Celera to chromosome 6.56 (56 Mb from pter on chromosome 6) is similarly masked for repeats and compared with itself. The 20 V1R gene-block duplications are shaded blue. Regions shaded yellow share homology to the 6D locus. These regions are homologous to positions at ≈585 kb and ≈649 kb on the 6D map in A (regions marked 1 and 2). Regions shaded green are putative regulatory regions that share homology to each other, as well as other regions in the genome annotated for putative regulatory function (e.g., T cell receptor Vα gene promoter regions and DNase1 hypersensitivity sites). The region between 380.5 kb and 393.5 kb is a microsatellite-like repetitive sequence. All other features and labels are as described for A. See Fig. 8, which is published as supporting information on the PNAS web site, for an enlarged version of this figure.

Figure 2

Molecular tree illustrates local gene expansion. Unrooted paup (Sinauer Associates, Sunderland, MA) nucleotide distance tree of 44 V1R genes with ORFs from three chromosomal locations. The tree partitions into three monophyletic clades: one that contains all 16 V1R ORFs from the 6D locus, another that contains all 23 V1R ORFs from second region on chromosome 6 (6.56/6.67), and the third that contains all 5 V1R ORFs from the chromosome 13 BAC. Seven subfamilies (>25% nucleotide divergence in coding sequence) are color-coded: A subfamily, red; B subfamily, green; a7-a8 subfamily, yellow; a9 gene, unshaded; NC/BD/BC, blue (with BC subclade in light blue to reflect the distinct map location of this gene set); and the two chromosome 13 subfamilies, light and dark gray. Nomenclature is described in Methods.

Figure 3

Pairwise sequence divergence for noncoding and coding regions of duplicated V1R gene blocks. The duplicated blocks were compared within six of the seven V1R subfamilies (a9 is excluded, because it has not duplicated). The average size of the noncoding/nonrepeat portions of these blocks is 3.5 kb. All possible alignment combinations (293 pairs) of duplicated blocks containing V1R ORFs were analyzed for percent substitution. The resulting pairwise nucleotide divergence of the noncoding portions of the blocks is plotted versus the nucleotide divergence of the V1R coding sequences within the blocks. The right axis converts percent divergence into time, using a molecular clock rate of 0.5%/million years (Myr) for noncoding rodent sequences. The thicker black line between 20–29 Myr indicates the approximate date of mouse–rat speciation (36). The 252 possible comparisons of the NC/BD/BC subfamily (23 genes) are plotted as open blue circles, the 15 possible comparisons of the A-subfamily blocks (six genes) are plotted as red diamonds, the 21 possible comparisons of the B-subfamily blocks (seven genes) are plotted as green diamonds, the V1ra7-V1ra8 comparison (two genes) is plotted as a green triangle, and the three 13.3–13.4–13.8 (three genes) comparisons and the 13.6–13.9 comparison (two genes) are plotted as gray circles.

Figure 4

Common V1R promoter homology at the chromosome 6D locus. The common promoter regions of the 15 V1R genes with ORFs from the chromosome 6D locus are aligned (these regions correspond to the purple-shaded regions in Fig. 1A). The 15 V1R genes belong to four divergent subfamilies separated by lines. Identities are shaded black; nucleotide positions shared by at least three V1Rs are shaded gray. See Fig. 9, which is published as supporting information on the PNAS web site, for an enlarged version of this figure.