REGULATION OF CYTOCHROME P-450 BIOSYNTHESIS

这项研究的总体目标是了解受管制的 兔肝微粒体细胞色素P-450的生物合成。在……里面 本研究探讨了苯巴比妥的诱导和作用机制 P450IIC亚家族的组织特异性表达将被研究。 P450IIC亚家族至少包含五个成员， 对苯巴比妥和苯巴比妥的反应有显著差异 不同的组织特异性表达模式。这位将军 方法将是尝试检测在 染色质是组织特异性的或与 用苯巴比妥治疗。这种方法允许初始的 对基因进行“远程”分析，以确定潜在的调控因素 基因的区域，并最终将导致分离和 结合的“反式作用”调节因子的特征 基因。第二种方法是评估体外试验的效果。 转基因后基因表达的诱变研究 哺乳动物细胞。这种方法应该识别“顺位代理”。 对转录很重要的因素。 P450IIC基因的分离和鉴定将是 完成。肝染色质中的DNase I超敏区 从苯巴比妥处理的兔和对照组的兔身上检测到 通过使用间接末端标记的探针进行Southern杂交。蛋白 超敏区内的结合位点将通过以下方式检测 经DNase I处理的DNA的改良S1核酸酶图谱 原子核。P450IIC基因中的核蛋白结合部位将 也可以通过体外足迹和凝胶迁移率变化来确定 化验。基因的表达将通过转染法进行检测 融合到肝脏来源的细胞中的基因5‘侧翼区 与细菌氯霉素乙酰基酶基因有关。删除和 定点突变将被用来定义调控区域 并将核蛋白位置与基因表达联系起来。 细胞色素P450催化大量的 药物、有毒化合物和致癌物以及内源性 类固醇和前列腺素等化合物。监管 因此，外源生物对细胞色素P-450水平的调节作用 对药物或有毒化合物的反应。这些研究都是关于 细胞色素P-450基因的表达应增加我们的 了解这一重要的临床问题。

The overall goal of this research is to understand the regulated biosynthesis of the rabbit liver microsomal cytochromes P-450. In this proposal the mechanisms of the phenobarbital induction and tissue-specific expression of the P450IIC subfamily will be studied. The P450IIC subfamily contains at least five members, which exhibit striking differences in responses to phenobarbital and different patterns of tissue-specific expression. The general approach will be to try to detect structural alterations in the chromatin that are tissue-specific or that correlate with treatment with phenobarbital. This approach permits an initial "long range" analysis of the genes to identify potential regulatory regions of the gene and will ultimately result in the isolation and characterization of "trans-acting" regulatory factors that bind to the gene. The second approach is to assess the effects of in vitro mutagenesis on the expression of the gene after transfection into mammalian cells. This approach should identify "cis-acting" factors important for transcription. The isolation and characterization of the P450IIC genes will be completed. DNase I hypersensitive regions in liver chromatin from phenobarbital-treated and control rabbits will be detected by Southern blotting using indirect end-labelled probes. Protein binding sites within the hypersensitive regions will be detected by a modified S1 nuclease mapping of DNA from DNase I treated nuclei. Nuclear protein binding sites in the P450IIC genes will also be deteted by in vitro footprinting and the gel mobility shift assay. Expression of the genes will be assayed by transfection into liver-derived cells of the 5' flanking region of the genes fused to the bacterial chloramphenicol acetylase gene. Deletion and site-specific mutagenesis will be used to define regulatory regions and to correlate nuclear protein sites with gene expression. Cytochromes P450 catalyze the oxidation of a large number of drugs, toxic compounds and carcinogens, as well as endogenous compounds such as steroids and prostaglandins. The regulation of cytochrome P-450 levels by xenobiotics, therefore, can modulate the response to a drug or toxic compound. These studies on the expression of the cytochrome P-450 gene should increase our understanding of this important clinical problem.