We identified DNA sequences of Tas1r3 and Tas1r2 from the domestic cat by screening a feline BAC library and using a PCR strategy on cat genomic DNA with degenerate primers. The feline sequences were compared with those of other species, and gene structures were determined. The expression of these two receptors was then evaluated by in situ hybridization and immunohistochemistry.

Molecular Cloning of Cat Tas1r3 and Tas1r2: Sequence and Gene Structure

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/).

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 1. Alignment of Deduced Amino Acid Sequences of T1R3 and T1R2 from Five Species This figure shows the alignment of the deduced sequences of the taste receptor proteins, T1R3 and T1R2, from domestic cat, domestic dog, human, mouse, and rat. Amino acids that are identical among species are shaded in black; conservative amino acid substitutions are shaded in gray. The cat T1R3 sequence shows high similarity with that of human and rodents, with especially high similarity with that of dog. The predicted cat T1R2 sequence is truncated at amino acid 355 due to a premature stop codon at bp 57–59 in exon 4, which results from a 247-bp deletion in exon 3. The underlined amino acids from 316 to 355 of the cat T1R2 result from the frame shift brought by the 247-bp deletion in exon 3. Note that the deduced amino acid sequence of dog T1R2 predicts an apparently normal protein showing high similarity with that of rat, mouse, and human. https://doi.org/10.1371/journal.pgen.0010003.g001

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).

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 2. Gene Structures of Cat Tas1r3, Human TAS1R3, and Cat Tas1r2, Human TAS1R2 The exons are shown in black (size in bp of each exon is in parentheses). Boundaries of gene sequences used to produce probes for in situ hybridization studies (Figure 3) are shown by the horizontal lines labeled “P1” and “P2” under the sketch of the cat Tas1r3 and cat Tas1r2. Boundaries of sequence used to generate peptide antigens for immunohistochemical studies (Figure 4) are shown by the horizontal lines labeled “A” under the sketch of the cat Tas1r3 and cat Tas1r2. The locations referred to in the vertical explanation text above the asterisks and the spade symbol indicate the position in bp within each exon. Intron sizes shown in the figure are not proportionally scaled on both (A) and (B) because of the large size of Tas1r2 introns. Under each human exon is the percent similarity between each human exon and its cat counterpart at the nucleotide level (Figure 2B). The exons for cat Tas1r2 refer to parts corresponding to human exons. The spade symbol (♠) indicates the position of microdeletion in exon 3 of cat Tas1r2. Asterisks (*) indicate the stop codon positions in exon 4 and 6 of cat Tas1r2. Note that nucleotide numbers of the exon 3 in human TAS1R2 and cat Tas1r2 are not identical. https://doi.org/10.1371/journal.pgen.0010003.g002

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 3. RNA Expression of Cat Tas1r2 and Tas1r3 from Circumvallate Papillae Digoxigenin-labeled sense and antisense cRNA probes corresponding to exons 3 and 6 of cat Tas1r2 and Tas1r3 were synthesized using DIG RNA labeling kit (Roche Applied Science, Indianapolis, Indiana, United States) (See Figure 2 for the locations of in situ probes, and Table 3 for identity of primers.) Hybridizations were carried as described [39]. Panel (A) shows result of antisense probes for Tas1r3, whereas panel (B) shows the result of the sense probes for Tas1r3. Panel (C) shows results of the antisense probes for Tas1r2 whereas panel (D) shows results of the sense probes. Scale bar, shown only in panel (A), = 60 μm for (A), (B), (C), and (D). https://doi.org/10.1371/journal.pgen.0010003.g003

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 4. Protein Expression of Cat T1R2 and T1R3 Cat T1R3 expression is detected in taste buds of circumvallate papilla (CV) (A) and a fungiform papilla (Fun) just anterior to the intermolar eminence (B) by labeling with anti-mouse T1R3 antibody. Cat T1R2 expression is not detectable in either circumvallate (C) or fungiform (D) using an anti-cat T1R2 antibody. Control studies demonstrated that the anti-cat T1R2 antibody labeled a subset of taste bud cells in rat circumvallate (data not shown). Scale bar, shown only in panel (A) and (B), = 60 μm for (A) and = 45 μm for (B). Scale for panel (C) is the same as that of panel (A); scale for panel (D) is the same as that of panel (B). https://doi.org/10.1371/journal.pgen.0010003.g004

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.