Role of conserved sequences in odorant receptor genes

描述(申请人提供)：嗅觉感觉神经元表达大约1000个气味受体(OR)基因的单个等位基因的机制尚不清楚。要设计直接解决OR基因选择机制的实验，首先必须确定OR基因表达所涉及的元件。最近的一项研究表明，对于两个小鼠OR基因，异位整合到小鼠基因组(转基因)中的小标记基因组片段复制了OR基因表达的特性。这些片段包含一个同源结构域和一个类似O/E的位点，它们在小鼠、大鼠、人类和斑马鱼的许多OR基因的可能启动子区中保守。这项应用的第一个目的是确定保守的同源结构域和O/E样位点是否在OR基因表达中发挥作用。M71OR基因启动子区域的同源同源结构域和类似O/E的保守序列的基本残基发生了突变。这些突变的影响已经从内源性M71基因座中分离出来(在转基因小鼠中)，并在内源性M71基因座的背景下(在基因靶向敲入小鼠中)进行了研究。转基因和敲入相结合的方法已经发现了保守的同源结构域和O/E样位点在OR基因表达中的重要作用。在这里，这些位点的作用将通过产生新的点突变来进一步定义，这些点突变不会取消这些位点的结合属性，但会降低或改变它们的结合特异性。在进行这些研究时，转基因和转基因结果之间的差异表明，额外的序列有助于M71的表达。同时，在保守的同源结构域和O/E样位点的上游约800bp处发现了额外的同源结构域和O/E样位点。本申请的第二个目的是测试这些位点在M71表达中的功能作用。为此，将使用所述的转基因和转基因相结合的方法。这里提出的体内研究应该继续为OR基因表达的调控提供新的见解。

DESCRIPTION (provided by applicant): The mechanisms by which an olfactory sensory neuron expresses a single allele of the approximately 1000 odorant receptor (OR) genes is not known. To design experiments that address directly the mechanism of OR gene choice it is essential to first identify the elements involved in OR gene expression. In a recent study it was shown, for two mouse OR genes, that small tagged genomic fragments ectopically integrated in the mouse genome (transgenes) reproduce the properties of OR gene expression. These fragments contain a homeodomain and an O/E-like site that are conserved in putative promoter regions of many OR genes in mouse, rat, human, and zebrafish. The first aim of this application is to determine if the conserved homeodomain and O/E-like sites have a role in OR gene expression. Essential residues of conserved homeodomain and O/E-like sequences present in the putative promoter region of the M71 OR gene have been mutated. The effect of these mutations has been studied in isolation from the endogenous M71 locus (in transgenic mice), and in the context of the endogenous M71 locus (in gene-targeted knock-in mice). The transgenic and knock-in approaches combined have uncovered important roles for the conserved homeodomain and O/E-like sites in OR gene expression. Here, the role of these sites will be further defined by generating new point mutations that do not abolish the binding properties of these sites but reduce or alter their binding specificity. While carrying out these studies the discrepancies between the transgenic and knock-in results have suggested that additional sequences contribute to the expression of M71. Concurrently, additional homeodomain and O/E-like sites have been identified about 800 bp upstream of the conserved homeodomain and O/E-like sites. The second aim of this application is to test the functional role of these sites in expression of M71. For this purpose the combined transgenic and knock-in approaches described will be used. The in vivo studies proposed here should continue to provide novel insights into the regulation of OR gene expression.