REGULATION OF CYTOCHROME P450 BIOSYNTHESIS

描述(调查人员摘要)：本提案的总体目标是 为了了解经典诱导剂的分子机制， 苯巴比妥(PB)，增加细胞色素P450的基因表达。这个 本提案的具体目标是确定和描述 介导苯巴比妥诱导的顺式和反式作用因子 细胞色素P450 2 B和细胞色素P2 2 C亚家族。这一领域的进展受到了 缺乏用于苯巴比妥诱导的连续细胞培养模型系统。 然而，实验室已经证明，将DNA导入大鼠体内 原位肝移植已被证明是一种简单和可重复性的方法 CYP基因的表达，并用其证实了一段CYP2B2 DNA片段 可以调节苯巴比妥的反应。删除和站点特定 突变将被用来鉴定苯巴比妥反应元件 (PBRE)。经典重组DNA技术 将被用来确定对监管重要的地区。的影响 还将分析苯巴比妥对PBRE染色质结构的影响， 天然染色质中的调节因子的结合将是 下定决心。小鼠的同源重组将被用来评估In 已鉴定元素的活体功能。兔和小鼠的CYP2C基因将 通过序列相似性和原位杂交来筛选PBRE 转基因试验，并最终通过在小鼠中进行靶向缺失。 苯巴比妥对小鼠脑组织细胞色素P450-2C基因的诱导特性 与CYP2B基因进行比较，以确定不同的诱导机制 都与这两个基因有关。这些研究应该提供详细的 在分子水平上对功能性PBRE的理解。

DESCRIPTION (Investigator's Abstract): The overall goal of this proposal is to understand the molecular mechanisms by which the classical inducer, phenobarbital (PB), increases gene expression of cytochromes P450. The specific objectives of this proposal are to identify and characterize the cis-and trans-acting factors that mediate phenobarbital induction in the CYP2B and CYP2C subfamilies. Progress in this area has been hindered by the lack of continuous cell culture model systems for phenobarbital induction. However, the laboratory has demonstrated that transfection of DNA into rat liver in situ has been shown to be a simple and reproducible method for expression of CYP genes and was used to confirm that a CYP2B2 DNA fragment could mediate phenobarbital responsiveness. Deletion and site-specific mutation will be used to identify the phenobarbital-responsive elements (PBRE) in the CYP2B2 DNA fragment. Classical recombinant DNA techniques will be used to identify regions important for regulation. The effect of phenobarbital on the chromatin structure of the PBRE will also be analyzed, and binding of the regulatory factors in native chromatin will be determined. Homologous recombination in mice will be used to assess the in vivo function of the identified elements. Rabbit and mouse CYP2C genes will be screened for PBRE's by sequence similarity and by the in situ transfection assay, and ultimately by targeted deletions in mice. Characteristics of phenobarbital induction of CYP2C genes in the mouse will be compared with CYP2B genes to determine if different induction mechanisms are involved for the two genes. These studies should provide a detailed understanding of a functional PBRE at the molecular level.