Alignment of Deduced Amino Acid Sequences of T1R3 and T1R2 from Five Species

<p>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.</p

Protein Expression of Cat T1R2 and T1R3

<p>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).</p

Gene Structures of Cat <i>Tas1r3,</i> Human <i>TAS1R3</i>, and Cat <i>Tas1r2</i>, Human <i>TAS1R2</i>

<p>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 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010003#pgen-0010003-g003" target="_blank">Figure 3</a>) are shown by the horizontal lines labeled “P1” and “P2” under the sketch of the cat <i>Tas1r3</i> and cat <i>Tas1r2</i>. Boundaries of sequence used to generate peptide antigens for immunohistochemical studies (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010003#pgen-0010003-g004" target="_blank">Figure 4</a>) are shown by the horizontal lines labeled “A” under the sketch of the cat <i>Tas1r3</i> and cat <i>Tas1r2.</i> 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 <i>Tas1r2</i> introns. Under each human exon is the percent similarity between each human exon and its cat counterpart at the nucleotide level (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010003#pgen-0010003-g002" target="_blank">Figure 2</a>B). The exons for cat <i>Tas1r2</i> refer to parts corresponding to human exons. The spade symbol (♠) indicates the position of microdeletion in exon 3 of cat <i>Tas1r2</i>. Asterisks (*) indicate the stop codon positions in exon 4 and 6 of cat <i>Tas1r2</i>. Note that nucleotide numbers of the exon 3 in human <i>TAS1R2</i> and cat <i>Tas1r2</i> are not identical.</p

RNA Expression of Cat <i>Tas1r2</i> and <i>Tas1r3</i> from Circumvallate Papillae

<p>Digoxigenin-labeled sense and antisense cRNA probes corresponding to exons 3 and 6 of cat <i>Tas1r2</i> and <i>Tas1r3</i> were synthesized using DIG RNA labeling kit (Roche Applied Science, Indianapolis, Indiana, United States) (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010003#pgen-0010003-g002" target="_blank">Figure 2</a> for the locations of in situ probes, and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010003#pgen-0010003-t003" target="_blank">Table 3</a> for identity of primers.) Hybridizations were carried as described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010003#pgen-0010003-b39" target="_blank">39</a>]. Panel (A) shows result of antisense probes for <i>Tas1r3</i>, whereas panel (B) shows the result of the sense probes for <i>Tas1r3</i>. Panel (C) shows results of the antisense probes for <i>Tas1r2</i> whereas panel (D) shows results of the sense probes. Scale bar, shown only in panel (A), = 60 μm for (A), (B), (C), and (D).</p