Development And Function Of Mammalian Chemosensory Syste

分子神经科学部分的研究目标是确定哺乳动物化学感觉系统发展和功能的分子机制。在过去的一年里，研究工作主要集中在1)建立苦味受体的功能分析，2)确定指导味觉细胞基因表达所需的DNA调控序列，以及3)寻找在味觉细胞中选择性表达的新基因。 1)为了更好地了解G蛋白偶联苦味受体的特异性和信号转导途径，我们克隆并构建了23个已鉴定的人苦味受体的表达载体。初步的表达研究表明，至少一个苦味受体的表达赋予了异源细胞对苦味化合物做出反应的能力。苦味化合物的面板将用于基于细胞的和体外重建分析，以确定额外的配体/受体对，并表征苦味受体的特异性和偶联特性。 2)之前，我们鉴定了哺乳动物甜味受体T1R3，并证明它在味觉细胞亚群中特异表达。为了确定负责指导T1R3味觉细胞表达的DNA调控序列，我们确定了T1R3基因的基因组结构，并设计了一系列具有不同程度的T1R3上游区域的结构来驱动绿色荧光蛋白的表达。携带这些结构的转基因小鼠已经被衍生出来，目前正在进行分析。 3)为了寻找与味觉相关的新基因，我们从味觉组织中构建了一个归一化的消减文库。对该文库中的10000个克隆的序列分析表明，该文库高度富含味觉受体细胞特异性基因。对选定克隆的原位杂交表达研究导致了几个在味觉细胞中特异表达的基因的鉴定。其中两个基因编码味觉细胞信号转导通路的可能成分，包括一个新的离子通道和一个新的G蛋白偶联受体。

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.