The research goals of the Section of Molecular Neuroscience are to define the molecular mechanisms underlying the development and function of mammalian chemosensory systems. Research efforts this past year have been directed towards 1) establishing functional assays for bitter taste receptors, 2) determining the DNA regulatory sequences required to direct taste cell gene expression, and 3) identifying novel genes selectively expressed in taste cells. 1) To better understand the specificities and signal transduction pathways of the G-protein coupled bitter receptors, we have cloned and constructed expression vectors with the 23 identified human bitter receptors. Preliminary expression studies indicate that expression of at least one of the bitter receptors conferred the ability of a heterologous cell to respond to bitter compounds. Panels of bitter-tasting compounds will be used in both cell-based and in vitro reconstitution assays to determine additional ligand/receptor pairs and to characterize the specificities and coupling properties of bitter receptors. 2) Previously, we identified T1R3, a mammalian sweet receptor, and demonstrated that it was specifically expressed in a subset of taste cells. To define the DNA regulatory sequences responsible for directing the taste cell expression of T1R3, we have determined the genomic structure of the T1R3 gene and engineered a series of constructs with varying extents of the T1R3 upstream region driving the expression of green fluorescent protein. Transgenic mice carrying these constructs have been derived and are currently being analyzed. 3) In an attempt to identify novel genes involved in taste perception, we generated a normalized, subtracted cDNA library from taste tissue. Sequence analyses of 10000 clones from this library indicate that it is highly enriched in taste receptor cell specific genes. In situ hybridization expression studies with selected clones have led to the identification of several genes specifically expressed in taste cells. Two of these genes encode putative components of the taste cell signal transduction pathway including a novel ion channel and a novel G-protein coupled receptor.
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