T2R genes (genes implicated in the bitter response) were obtained through RFLP analysis comparison of individuals who varied in their response to bitter tastants. The genes were identified by monitoring conserved sequences in individuals who did not exhibit a strong bitter response. The predicted amino acid sequences are shown for the genes that have been predicted to be involved bitter perception. The sequence regions where at least half of the amino acids are identical are shaded darkly. Gray areas indicate conservative amino acid substitutions. The sequences are from different T2R taste receptors in human, mouse and rat. An abundance of predicted protein sequences are listed for human and rat. Only ten T2R protein sequences have been predicted for mouse. Predicted transmembrane domains are indicated by solid bars above the protein sequences. Seven transmembrane domains have been predicted for this class of T2R proteins.
The sequence relationships of T2R proteins were depicted with respect to similarity between human, rat and mouse. The similarities between human and mouse protein sequences ranged from 46% to 67% for three potential bitter proteins. Sequence similarities between rat and mouse ranged from 74% to 92% for two taste receptors. The percentage of protein sequence similarities were also depicted between these taste receptors and opsin and Vomeronasal proteins 1-3. These proteins most likely got into the figure because they are sensory proteins.
This figure demonstrates homologous protein sequences of T2R for mouse and human. Chromosomes 5 and 15 of human and mouse, respectively, shared a homologous region encoding a T2R protein. Chromosome 6 of human and 7 of mouse also have homologous T2R protein expression. This region, moreover, has been implicated in bitter perception, strengthening the claim that the T2R group could be involved in bitter perception. The T2R expression on mouse chromosome 7 has been elucidated further in a figure on the bottom of the page. Chromosome 12 of human and 6 of mouse bear a homologous chromosomal region expressing T2R. Both of these regions have been further explained in the figures below, where direction of transcription of various T2Rís is indicated. Boxed regions of T2R mean that the order of these genes in the chromosome is unknown. Dots that are offset in the figure represent a palindromic sequence found in many T2R DNAís. Grey regions of the chromosomes in the figure represent genes that are not translated (pseudogenes.) The number of pseudogenes detected, coupled with information retrieved from databases on potential T2R genes, led the group to conclude that approximately 40-80 T2R genes are present in the genome. CYX, QUI, RUA and SOA all represent bitter tastants detected by T2R bitter genes clustering on mouse chromosome 6. This figure shows that T2Rís were clustered next to each other in different pats of the genome. Such affirmation was attained through recombination studies not described here. Chromosomal location of the genes was most likely determined through fluorescent in situ hybridization.
If T2Rís are involved in bitter taste, then these genes should be transcribed in taste buds. Hence in situ hybridization was performed with an anti-sense RNA probe from rat on circumvallate papillae, foliate, geschmachstreifen, and epiglottis in rat. Rat circumvallate taste buds were shown in figure 5 to hybridize to rT2R-7,8,3,2,4. The foliate papillae of rat hybridized to rT2R-7. The geschmachstreifen and epiglottis hybridized to rT2R-3 and rT2R-7, respectively. Fungiform papillae hybridized to T2R cRNA in a clustered fashion like the other oral regions. Only 10% of the papillae hybridized, however, which is less than the percentage of papillae that hybridized to T2Rís in other regions. This demonstrates that T2R is predominantly transcribed in the posterior region of the oral cavity. 17 different mT2R probes hybridized in comparable sections in mouse but this data was not shown. The authors suggested that the clustering of T2Rís in the fungiform and papillae could lead to discoveries of the neural processes of taste buds.
To answer the question of whether a taste cell makes more than one receptor, they labeled taste cells with multiple probes through in situ hybridization and showed that 20% of the cells hybridized to multiple probes whereas 15% hybridized to individual probes. The similar expression locations of T2R-3 and 7 was demonstrated through double fluorescent in situ hybridization. These two facts lead one to conclude that taste cells in the oral cavity are producing multiple T2Rís.
To support their claim that T2R proteins coupled with gustducin, they tried to show that these two proteins were made in the same cells. Thus, in panel A, they performed in situ hybridization for T2R with a green fluorescent probe. In panel B, in situ hybridization was performed to detect gustducin with a red fluorescent probe. Panel C demonstrated that gustducin and T2R are expressed in the same set of cells since yellow fluorescence appeared as a result of a mixture of red and green fluorescence. A negative control appears in panel D. No yellow fluorescence is observed when TR1 is labeled with a green probe by in situ hybridization. This is consistent with previous research that showed that gustducin and TR1 are expressed in a different subset of cells. Panels C and D, like A and B, are images of in situ hybridization. The difference in the images are due to an interference background contrast.
The authors mentioned that gustducin was not coexpressed in similar locations as T2R in the fungiform. This was not due to the absence of T2R, however. Their explanation for this was that there might be another class of receptors expressed in the fungiform that couple with gustducin. The multiple functionality of gustducin also explains why T2Rís in the fungiform may not couple with gustducin.
Although the authors have shown that T2R and gustducin have overlapping expression, they have not shown that gustducin functionally binds to T2R. They also have not shown that gustducin functionally mediates the phenotype for tasting bitter food. In order to show that gustducin functionally binds to T2R, one should construct a two hybrid expression system. The first plasmid should contain a Gal4 binding domain fused to gustducin and a His+ locus so as to select for transformants in His- medium. The second plasmid should contain a Gal4 activating sequence fused to T2R cDNA and a leu+ locus so as to select for transformants on leu- medium. Negative controls should have the host cell (yeast) containing the first plasmid and not the second plasmid and vice versa. For the experimental group, the activating vector should contain different T2Rís for separate experiments since gustducin may not bind to every T2R. If gustducin functionally binds to a T2R cDNA, then the experimental group should turn blue on medium containing lac-Z after taking in both plasmids. This should not occur in controls. If the experimental group does not turn blue here, one might question whether a bitter substance such as cycloheximide mediates binding between gustducin and a T2R protein. As such, one could manipulate the previous experiment by adding cycloheximide to the medium. If blue colonies formed for the experimental group in this experiment, then one could conclude that a bitter substance like cycloheximide mediates binding between gustducin and T2R.
An experiment that would test whether gustducin does, indeed, mediate taste reception for bitter substances would be to knock out the gustducin gene and substitute a gene that has a unique restriction site and that makes mice hair turn black. (The subject mice must be all white and not carry the allele for black hair.) After injecting the knockout vector into embryonic stem cells, one could determine the success of the recombination by amplifying the region of recombination and running a restriction digest on a gel with the restriction enzyme that is present on the knockout vector. This should produce a predictable banding pattern. The appearance of black hair in the offspring would also confirm that recombination took place. If the recombination event was successful, then one could measure the tolerance of the chimeric mouse for bitter substances such as cycloheximide. If the tolerance for bitter substances is considerably higher for the chimeric mouse than the wild type control, then one could logically conclude that gustducin does, indeed, modify the bitter response.
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