By sequencing positive BAC clones retrieved from a feline genomic BAC library (Felis silvestris catus; BACPAC Resources, Oakland, California, United States), we obtained more than 3 kb of genomic sequences containing the open reading frame for cat Tas1r3, and approximately 10 kb of genomic sequences containing the open reading frame for cat Tas1r2. Because exons 1 and 2 of Tas1r2 were not found in the positive BAC clones, we employed a PCR strategy using degenerate primers to amplify these regions from cat genomic DNA (Novagen, San Diego, California, United States) (See Materials and Methods). We aligned the cDNA sequences and the deduced amino acid sequences from cat Tas1r3 and Tas1r2 with their dog, human, mouse, and rat orthologs (Figure 1). (We obtained the sequences of domestic dog genes, Tas1r3 and Tas1r2, by screening a dog genomic library using the same overgo probes and methods as for the feline genomic BAC library and by taking advantage of the limited data available at that time from the public dog genome database at http://www.ncbi.nlm.nih.gov/genome/guide/dog/).

Table 1 presents the percent similarity of the Tas1r3 and Tas1r2 genes at both the cDNA and the protein levels between all possible pairs of five species: cat, dog, human, mouse, and rat. The cat Tas1r3 gene shows high similarity at the cDNA level with that of dog (87%), human (79%), rat (75%), and mouse (74%) (Table 1). The cat Tas1r3 gene predicts a protein of 865 amino acids (Figure 1) showing 85% similarity with deduced protein of dog, and 73%, 72%, and 72% with that of human, mouse, and rat, respectively (Table 1). Initially we predicted the exon–intron boundaries of cat Tas1r3 by comparison with the known boundaries of human TAS1R3. To confirm these exon–intron boundaries for cat Tas1r3, we performed both RT-PCR on cDNA from cat taste bud–containing circumvallate and fungiform papillae, and PCR on cat genomic DNA using intron-spanning primers. By comparing the cDNA sequence with the genomic sequence, we confirmed the boundaries predicted from human TAS1R3 (Figure 2A). Both the cat Tas1r3 and human TAS1R3 genes are composed of six similarly sized exons and five introns (Figure 2A).

We identified the exon–intron boundaries of cat Tas1r2 by comparison with known human boundaries (Figure 2B). Examining the sequence of cat Tas1r2, we discovered a microdeletion of 247 base pairs (bp) within exon 3. This deletion is responsible for a frame shift that results in a premature stop codon at bp 57–59 of exon 4 (Figure 2B). Assuming, for the moment, that a protein is translated from cat Tas1r2, then, because of the deletion and premature stop codon, the gene sequence predicts a peptide of 355 amino acids, the first 315 of which show high similarity with their rat, mouse, human, and dog counterparts (see Figure 1). Because of the frame shift introduced by the 247-bp deletion, the remaining deduced 40 amino acids show no similarity with their rat, mouse, human, or dog counterparts (underlined sequence of cat T1R2; Figure 1). The predicted similarity of this hypothetical 355–amino acid protein was compared with its truncated counterparts from dog, human, mouse, and rat. It ranges from 55% to 69% (Table 1). In contrast, the percent similarity of the full-length T1R2 protein within pairs of other species is between 91% (mouse–rat) and 69% (mouse–human).

By aligning cat Tas1r2 DNA sequences of exons 4, 5, and 6 with their human counterparts, we found four additional stop codons: one in exon 4 due to a deletion at bp 123, and three in exon 6 due to a substitution at bp 95 and a deletion at bp 247 (Figure 2B). The multiple stop codons indicate that the cat Tas1r2 is a pseudogene.

In an attempt to confirm the cat Tas1r2 exon–intron boundaries, we performed RT-PCR on cDNA from cat circumvallate and fungiform taste papillae. Despite using numerous (> 70) primers corresponding to deduced message from the Tas1r2 gene, we were unable to detect it.

Having detected message from cat Tas1r3, but not from cat Tas1r2, by RT-PCR, we used the more tissue-specific approaches of in situ hybridization and immunohistochemistry to refine the search for cat Tas1r2 gene expression, using the cat Tas1r3 gene for comparison. Probes for in situ hybridization were constructed from the gene sequences corresponding to the lines marked “P” in Figures 2A and 2B. (See Materials and Methods for details.) Figure 3 shows that message from Tas1r3 is expressed in taste buds of cat circumvallate papillae whereas Tas1r2 expression is not detectable by in situ hybridization. Antisense probes for Tas1r3 result in positive labeling (Figure 3A); the arrows indicate three of the many labeled taste buds. The control sense probes show no labeling (Figure 3B). In contrast, antisense probes for cat Tas1r2 show no detectable labeling (Figure 3C) as is the case for the sense control (Figure 3D).

To detect the presence of taste receptor proteins from Tas1r2 and from Tas1r3, we exposed 10-μm sections of cat circumvallate and fungiform papillae to polyclonal antibodies developed against deduced amino acid peptide antigens marked by the line labeled “A” in Figure 2A and 2B. T1R3-like immunoreactivity was present in the taste buds of every circumvallate (10) and fungiform (4) papilla used in this study (Figure 4A and 4B) whereas immunoreactivity to T1R2 was not detected in these same tissues (Figure 4C and 4D). (Each circumvallate papilla of the cat contains approximately 400 taste buds, whereas the large fungiform papillae used in this study, located in the area of the eminence, contain from 1 to about 15 taste buds each.) The antibody to cat T1R2 did, however, label a subset of taste buds in rat circumvallate papillae (results not shown).

Because the feline BAC genomic library was constructed from a single individual cat, we confirmed the sequence of Tas1r2 in six additional, unrelated, healthy adult domestic cats. Genomic DNA was obtained by cheek swabs from five of the six cats and through a blood sample from the remaining cat, amplified by PCR using primers that flanked the deletion and stop codons of the known cat Tas1r2, and sequenced. In addition, to test whether other species of Felidae display similar sequence anomalies in their Tas1r2 gene, we performed PCR on genomic DNA of one tiger (Therion International, Saratoga Springs, New York, United States) and one cheetah (a gift from the San Diego Zoo). We found that Tas1r2 in all six cats, the tiger, and the cheetah show the identical 247-bp deletion in exon 3, and all have stop codons at the same positions in exon 4 (Table 2). In exon 6, we found evidence for two alleles at position 93–95 in domestic cat, wherein two cats show the stop codon, TGA (homozygotes TGA/TGA); one cat shows TGR (heterozygote TGA/TGG); and three of the domestic cats, the one tiger, and the single cheetah show TGG (homozygotes TGG/TGG) (Table 2). The second exon 6 stop codon is also common to all three species (TGA for domestic cat, TAG for tiger and cheetah). Although the third stop codon of exon 6 at bp 697–699 was found in all six domestic cats, the corresponding region in tiger and cheetah could not be amplified by PCR.

Collectively, these data indicate that cat Tas1r3 is an expressed and likely functional receptor, whereas cat Tas1r2 is an unexpressed pseudogene.

By comparison with other known T1R3 proteins and other proteins of Class C, family 3, the sequence and gene structure of cat Tas1r3 predict a functional receptor of 865 amino acids (see Figure 1). Cat Tas1r3 is assumed to be located on cat Chromosome C1, syntenic with human 1p36, where human TAS1R3 is located [19,20]. As with other Tas1r3 genes, the cat Tas1r3 is composed of six exons, each approximately the same size as those of human (see Figure 2A). The sequence of cat Tas1r3 predicts a seven-transmembrane GPCR with extended N-terminal domain (first transmembrane region spanning amino acids 572–595), features common to other T1R3 receptors. Important Class C, family 3, structural motifs can also be located in cat T1R3 including the xPKxY motif at amino acids 814–818, and the FHSCCY motif at amino acids 517–522. Additionally, although most members of Class C, family 3, GPCRs show a highly conserved arginine residue at the extreme 3′ end of transmembrane segment 3, an exception is found with human, mouse, and rat T1R3, which substitute glutamic acid (E) for arginine (R) [21]. This substitution is also found in cat T1R3 at amino acid 660 (see Figure 1; the deduced dog T1R3 substitutes glutamine (Q) for arginine at the end of TM3).

Available evidence indicates that the products of cat Tas1r3 are expressed in taste buds. RT-PCR readily detected the message from Tas1r3 in lingual taste bud–containing tissues (results not shown). In situ hybridization studies confirmed the presence of message and localized it to taste buds (see Figure 3A). Polyclonal antibodies developed against T1R3 labeled taste buds in both cat circumvallate (Figure 4A) and fungiform papillae (Figure 4B). While only a few cells showed evidence of T1R3-like immunoreactivity, nearly every taste bud was labeled by both in situ hybridization and immunohistochemistry.

These commonalities in gene structure and sequence, together with evidence that the cat Tas1r3 gene is expressed, are consistent with the assumption that cat Tas1r3 codes for a functional receptor.

Cat Tas1r2, on the other hand, while retaining structure similar with that of the human TAS1R2 gene (see Figure 2B), is an unexpressed pseudogene. The likely important molecular event that resulted in cat Tas1r2 becoming a pseudogene is the 247-bp deletion in exon 3. This deletion results in a frame shift that brings about a premature stop codon in exon 4 (Figure 2B). An additional stop codon can be found in exon 4, with three more in exon 6 (Figure 2B). This apparent accumulation of mutations suggests that there is no pressure from natural selection on the cat Tas1r2 gene. To determine if this gene is expressed, we performed studies to detect message from cat Tas1r2 by RT-PCR of taste bud–containing lingual papillae and by in situ hybridization. For RT-PCR, numerous (>70) primers were constructed based on sequences from exons 1–6. For in situ hybridization, probes were designed from exon 3 and from exon 6 (Figure 2B; Table 3). Both techniques failed to detect message from cat Tas1r2 (see Figure 3C and 3D). Consistent with these attempts to detect message from cat Tas1r2, immunohistochemistry using an antibody developed from a deduced amino acid sequence spanning exons 2 and 3 revealed no labeling of taste buds in circumvallate or fungiform papillae (Figure 4C and 4D).

These results suggest that the cat Tas1r2 pseudogene is not transcribed, or if it is transcribed, it rapidly degrades, perhaps through a nonsense-mediated mRNA decay pathway [22].

The generality of the pseudogene nature of cat Tas1r2 was confirmed by sequencing the deletion and stop codon areas from six individual healthy adult cats. All showed the deletion and similar stop codons with some polymorphism (see Table 2). To assess the generality of the pseudogene nature of Tas1r2 in Felidae, we sequenced the stop codon areas and the area including the exon 3 microdeletion from genomic DNA of tiger and cheetah. These too displayed microdeletion and stop codons at the same location as the domestic cat. These observations, suggesting that in at least three species of Felidae Tas1r2 is not expressed, are consistent with behavioral evidence showing that, not only domestic cats, but also tigers and cheetahs do not prefer sweetened water over plain water [1].

According to morphological and molecular evidence, the available phylogeny of the order Carnivora consists of two groups, the Feliformia (cats, mongooses, civets, and hyenas) and the Caniformia (wolves, bears, raccoons, mustelids, and pinnipeds) [23,24]. It is difficult to determine when the alteration of Tas1r2 occurred and whether it preceded or followed the cat ancestor's change in diet to exclude plants. Clearly, because dogs have a human-like T1R2 structure (see Figure 1) and an avidity for sweet carbohydrates [25], the changes in the cat Tas1r2 must have occurred after the divergence of the Feliformia and the Caniformia.

Taste receptors are shaped by and reflect a species' food choices. The genes encoding taste receptors often show a good deal of variation both among species and among individuals. These variations, both subtle and obvious, can have a variety of effects on taste sensitivity and preference behavior. A textbook example of this effect is the individual variation seen in sensitivity to the bitter compound, phenylthiocarbamide (PTC). A gene of the human TAS2R family of bitter taste receptors, TAS2R38, associated with this individual variation, shows three coding single-nucleotide polymorphisms giving rise to five haplotypes worldwide, accounting for the 55% to 85% of the variance in PTC sensitivity [26]. Further, in Drosophila, the behavioral and electrophysiological responses to trehalose are diminished in two mutants that carry deletions in the trehalose recognition gene, Gr5a [27]. In the mouse, variation in preference for sweet-tasting stimuli maps to the gene for T1R3, located within the Sac locus [28,29]. This gene is allelic in mice, and several reports identify a missense mutation (I60T) as being the most likely mutation accounting for the phenotypic differences [13,14,16,30–33]. However, the same alleles are not involved in strain-dependent sweet-taste preference in rats [34].

In addition to the modulation of behavior that can be caused by point mutations, more profound behavioral changes can result from the abolishment of gene function through, for example, the generation of pseudogenes. An example of this effect in mammalian chemoreception lies within the large repertoire of olfactory receptor genes. More than 60% of the human olfactory receptor genes are pseudogenes [35], whereas, only 20% are classified as such in mouse [35,36]. Strikingly, the accumulation of these olfactory pseudogenes in primates reportedly occurred concomitant with the acquisition of trichromatic color vision, perhaps reflecting the overarching behavioral changes that such an acquisition engendered [37]. Similar generation of bitter-taste receptor pseudogenes, accompanied by a large number of coding region single-nucleotide polymorphism, can account for the broad diversity displayed by the bitter-taste receptor family. This diversity may possibly play an important role in both species-specific and individually manifested taste preference [38].

In the extreme case, where a species fails to respond to stimuli representative of an entire modality, such as the cat with sweet taste, the development of a unique food preference behavior, based on the remaining taste receptors, might be anticipated. Because, with the exception of the sweetness modality, the taste system of the cat is organized much like that of most other mammals, discovering the molecular basis for the cats' lack of response to sweet-tasting compounds gives us a window on the development of strict carnivorous behavior in Felidae.

It is known that Felidae do not detect sweetness of carbohydrates yet can taste amino acids. Our results indicate that the gene encoding one member of the sweet-taste receptor heteromer is an unexpressed pseudogene. Given this observation, we suggest that the most parsimonious explanation for the inability of Felidae to respond to sweeteners is the lack of a functional T1R2 protein.

We obtained cat taste tissue from healthy young-adult animals euthanized for reasons unrelated to this study. Animals were cared for under protocols 033400 and 057600 approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania to Dr. Mark Haskins of the School of Veterinary Medicine, University of Pennsylvania.

Overgo probes are comprised of two 22mers with a complementary eight-base overlap. They can be designed by a computer program (http://genome.wustl.edu/tools/?overgo=1) and are readily synthesized. To identify cat Tas1r2 and Tas1r3, overgo probes were designed by aligning conserved coding regions of Tas1r2 and Tas1r3 sequences from different species. The single-stranded overhangs (14 bases) were filled in with ³³P-labeled dATP and dCTP, and the overgo probes were used in hybridization procedures with the BAC libraries.

Tas1r2 and Tas1r3 overgo probes were radioactively labeled by the random hexa-nucleotide method, and hybridization and washing of membranes were as described [29]. We identified 47 positive BAC clones for cat Tas1r2 and cat Tas1r3, and sequenced all of the positive BAC ends. By aligning BAC ends sequences with human syntenic regions (human TAS1R2 and TAS1R3 are located on chromosome 1p36), we picked BAC clones positive for cat Tas1r2 and Tas1r3 for shotgun library preparation.

We prepared BAC DNAs from positive clones by using a Qiagen Large Construct Kit (Valencia, California, United States). The BAC DNAs were digested using Sau3A I and the digested BAC DNA fragments subcloned into pGEM-3Z (Promega) vector. After transformants were arrayed to a nylon membrane, two separate hybridizations were performed by using pooled Tas1r2 and Tas1r3 overgo probes. By sequencing positive clones from the shotgun libraries and by using a chromosome walking strategy, we obtained the full coding region of the cat Tas1r3 and exon 3 to exon 6 of cat Tas1r2.

Because exon 1 and exon 2 of the cat Tas1r2 were not present in the positive BACs selected above, we designed degenerate primers based on Tas1r2 alignments from different species (human, rodents, and dog) and performed PCR using cat genomic DNA as a template. The PCR products were sequenced. The feline BAC library was then re-screened using PCR products as probes, and new positive BAC clones were retrieved. Using a chromosome walking strategy, we obtained the complete sequence of exon 1 and exon 2 of cat Tas1r2 from these newly retrieved BAC clones.

To examine the RNA expression and to determine the intron–exon boundaries of the cat Tas1r2 and Tas1r3 genes, we extracted total RNA using TRIZOl Reagent (Life Technologies Inc., Rockville, Maryland, United States) from cat taste bud–containing tissues, followed by reverse transcription (Superscript reverse transcriptase, Life Technologies). The cDNA samples were amplified using AmpliTaq DNA Polymerase with GeneAmp (Perkin Elmer Corporation, Branchburg, New Jersey, United States) and intron-spanning primers selected to distinguish genomic and cDNA. Single bands of expected sizes were excised from the gel, purified, and sequenced.

The probes corresponding to exons 3 and 6 of cat Tas1r2 and Tas1r3 were amplified by PCR using the primers described in Table 3. Digoxigenin-labeled cRNA probes were synthesized using a DIG RNA labeling kit (Roche). Taste bud–containing vallate tongue tissue was obtained as above. Fresh frozen sections (14 μm/section) of cat circumvallate papillae were attached to clean SuperFrost/Plus slides and prepared for in situ hybridization [39]. High-stringency hybridizations were carried out at 70 °C overnight in 50% formamide, 5X SSC, 5X Denhardt's, 250 μg/ml yeast RNA, and 500 μg/ml sperm DNA using the mixed cRNA probes. Sections were washed at 72 °C with 0.2X SSC three times. Signals were detected using alkaline phosphatase–conjugated antibodies to digoxigenin and standard chromogenic substrates and observed with a Nikon SA Microphot Microscope. Control hybridizations were performed with sense probes.

Polyclonal anti-cat T1R2 rabbit antisera directed against an N-terminal peptide of cat T1R2 (exons 2 and 3; see Figure 1) were generated by Zymed Laboratories, Inc. (South San Francisco, California, United States). Generation of antisera directed against N-terminal peptide of mouse T1R3 has been described previously [33]. Lingual tissue blocks containing cat circumvallate and fungiform papillae were fixed in 4% paraformaldehyde for 2–6 h, then processed [40]. The antibodies were incubated with the sections (10 μm/section) for 60 h at 4 °C. After washing, the sections were incubated with the secondary antibody (Cy3-conjugated goat anti-rabbit IgG; The Jackson Laboratory, Bar Harbor, Maine, United States) and observed with a Leica TCS SP2 Spectral Confocal Microscope (Leica Microsystems Inc., Mannheim, Germany). Single-channel fluorescence images (average projection of 20–25, 0.3-μm optical sections) were processed with Adobe Photoshop software and overlaid on their respective difference interference contrast images.

To confirm that Tas1r2 is a pseudogene in other cats, we obtained genomic DNA from cheek swabs or blood of six unrelated healthy adult cats. We sequenced the areas around the microdeletion and the stop codons by PCR using primers that flanked these areas of interest. In addition, to test whether other species of Felidae have a functional Tas1r2 gene, we performed PCR on genomic DNA of one tiger (Therion International, Saratoga Springs, New York, United States) and one cheetah (a gift from the San Diego Zoo) using the same primers above. All the PCR products are purified and sequenced.

The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the genomes discussed in this paper are cat Tas1r3 (AY819786), cat Tas1r2 (AY819787), dog Tas1r2 (AY916758), dog Tas1r3 (AY916759), human TAS1R3 (BK000152), and human TAS1R2 (NM_152232).