Role of conserved sequences in odorant receptor genes

气味受体基因中保守序列的作用

• 批准号: 6795156
• 负责人: ANDREA ROTHMAN
• 金额: $ 8.43万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6795156
• 关键词: DNA binding protein; binding sites; biochemical evolution; chemical structure function; chemoreceptors; gene expression; gene targeting; genetic promoter element; genetic regulation; genetically modified animals; laboratory mouse; nucleic acid sequence; olfactions; olfactory nerve; point mutation; site directed mutagenesis; transfection

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.

描述（由申请人提供）：嗅觉感觉神经元表达约1000个气味受体（OR）基因的单个等位基因的机制尚不清楚。为了设计直接解决OR基因选择机制的实验，必须首先确定OR基因表达所涉及的元件。在最近的一项研究中显示，对于两个小鼠OR基因，异位整合在小鼠基因组中的小标记基因组片段（转基因）再现了OR基因表达的特性。这些片段含有一个同源结构域和一个O/E样位点，这些位点在小鼠、大鼠、人和斑马鱼的许多OR基因的推定启动子区中是保守的。本申请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基因表达中起作用。M71 OR基因的推定启动子区中存在的保守同源结构域和O/E样序列的必需残基已经突变。这些突变的影响已经在与内源性M71基因座（在转基因小鼠中）分离的情况下以及在内源性M71基因座（在基因靶向敲入小鼠中）的情况下进行了研究。转基因和敲入相结合的方法揭示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，这些点突变不会消除这些位点的结合特性，但会降低或改变它们的结合特异性。在进行这些研究时，转基因和敲入结果之间的差异表明，额外的序列有助于M71的表达。同时，额外的同源结构域和O/E-样网站已被确定约800 bp的保守同源结构域和O/E-样网站的上游。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。为此目的，将使用所述的组合的转基因和敲入方法。在这里提出的体内研究应继续提供新的见解或基因表达的调控。