Positional cloning of the taste receptor for sucrose oct

Diabetes, Digestive, Kidney Diseases

Investigators

Abstract

Our laboratory is interested in understanding the molecular recognition of ingested nutrients that results in the secretion of gastrointestinal hormones regulating digestion and gut motility necessary for absorption of these nutrients. Amino acids, short chain fatty acids, carbohydrates and changes in intraluminal pH are known potent stimulators of gastrin, cholecystokinin and secretin, some of the most important gastrointestinal hormones regulating digestion, secretion and motility. The cells responsible for chemosensation of these nutrients are presumed to be scattered along the mucosal layer lining the gastrointestinal tract and possibly among the neurons within the enteric nervous system. Because of the inherent dispersed nature of these chemosensory cells and the lack of any know markers for their identification, we chose to approach the problem by studying other more well known and better characterized chemosensory cells that are likely to possess related mechanisms for molecular recognition. The gustatory system recognizes at least four basic taste modalities, bitter, sweet, salty, sour and umami, the taste of glutamate. These include the recognition of amino acids, sugars and hydrogen ions utilizing putative G protein-coupled receptors and ion channels in ways that might be shared by the gastrointestinal system. It is also know that certain toxic compounds, that are usually perceived as bitter, can be sensed within the gastrointestinal system resulting in emesis. Therefore, we undertook the task of identifying cell surface molecules that would recognize known tastants with the hope that their identity would reveal a family of related molecules underlying the known diversity for taste recognition that would either be shared by the gastrointestinal system or at least be closely related. The number of taste receptor cells are rather small and present only in a small number of tastebuds limiting the amount of tissue available for experimental use. Although the taste receptors for bitter and sweet tastants are presumbed to be G protein-coupled receptors, some controvery still remains. The discovery of any orphan taste receptor would ultimately require the identification of the corresponding specific tastant in order to prove its function. Because of these difficulties, we decided to take advantage of a discovery made 30 years ago of an inbred strain of mice capable of tasting a bitter tastant, sucrose octaacetate, SOA, at far lower concentrations than nearly all other inbred strains. This phenotypic trait has been shown to be result of a single autosomal dominant genetic locus, termed the SOA locus, which had been grossly localized to a region approximately 63 cM on the distal end of mouse chromosome 6. To overcome these obstacles, we undertook a positional cloning approach toward the discovery of the basis for bitter taste recognition in SOA tasting SWR/J mice. To more finely map the SOA locus, we first evaluated 150 mice from three lines of CH3HeB/SWR/J congenic SOA taster mice provided in collaboration with Glayde Whitney. Using at least 30 polymorphic markers within a13 cM interval centered around the Prp locus, we were able to link the taste phenotype to a more narrow genetic interval of 1.4 cM. To further reduce the genetic interval containing the SOA locus, we set up a backcross between the SWR/J, taster and Castaneous nontaster strains. Using 20 polymorphic markers within the 1.4 cM interval established with the congenic lines, we first evaluated 500 backcross mice. Unfortunately, this allowed us to reduce the interval only 0.3 cM. Despite genotyping and phenotyping an additional 1000 mice, we were unable to decrease the genetic locus for SOA. From our data and from the literature reporting the linkage data for positional cloning of the nearby CMV 1 locus in several different strains of mice, we believe that our genetic linkage studies failed to achieve the expected mapping resolution because of recombinational "hot spots" that we encountered. With the soon expected completion of the human and mouse genomes and the strong possibility that a GPCR was a likely candidate for SOA, we undertook the physical mapping of the 1.1 cM genetic interval. PCR screening of a mouse YAC library, WI/MIT820, identified four nonchimeric, overlapping YACs (without deletions) that defined a 2 Mb interval containing the SOA locus. A combination of established polymorphic markers, YAC end sequence and new sequence from pulse field gel electrophoresis separated YAC restriction digest fragments were used to screen a mouse BAC library, RPCI-23, by filter hybridization. Further screening with newly acquired BAC end sequence allowed us to define a minimal contig of 12 overlapping BACs. These BACS are being screened for candidate GPCR receptors that may code for the SOA gene.