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
with their dog, human, mouse, and rat orthologs (). (We obtained the sequences of domestic dog genes, Tas1r3
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/
Alignment of Deduced Amino Acid Sequences of T1R3 and T1R2 from Five Species
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%) (). The cat Tas1r3 gene predicts a protein of 865 amino acids () showing 85% similarity with deduced protein of dog, and 73%, 72%, and 72% with that of human, mouse, and rat, respectively (). 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 (A). Both the cat Tas1r3 and human TAS1R3 genes are composed of six similarly sized exons and five introns (A).
Similarity of Sweet Receptors between Species
Gene Structures of Cat Tas1r3, Human TAS1R3, and Cat Tas1r2, Human TAS1R2
We identified the exon–intron boundaries of cat Tas1r2 by comparison with known human boundaries (B). 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 (B). 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 ). 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; ). 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% (). 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 (B). 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.
RNA and Protein Expression
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 A and B. (See Materials and Methods
for details.) 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 (A); the arrows indicate three of the many labeled taste buds. The control sense probes show no labeling (B). In contrast, antisense probes for cat Tas1r2
show no detectable labeling (C) as is the case for the sense control (D).
RNA Expression of Cat Tas1r2 and Tas1r3 from Circumvallate Papillae
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 A and B. T1R3-like immunoreactivity was present in the taste buds of every circumvallate (10) and fungiform (4) papilla used in this study (A and B) whereas immunoreactivity to T1R2 was not detected in these same tissues (C and D). (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).
Protein Expression of Cat T1R2 and T1R3
Confirmation of Tas1r2 Sequence in Six Individual Cats, Tiger, and Cheetah
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 (). 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) (). 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.
Tas1r2 Stop Codons in Species of Felidae
Collectively, these data indicate that cat Tas1r3 is an expressed and likely functional receptor, whereas cat Tas1r2 is an unexpressed pseudogene.