CHOLESTEROL 7ALPHA-HYDROXYLASE IN TRANSGENIC MICE

胆固醇分解代谢和排泄的最重要途径 在哺乳动物中是胆汁酸的形成。对此不适当的监管 途径具有广泛的含义，因为 胆固醇在动脉粥样硬化等疾病中是一个非常重要的因素， 胆石症，以及一些脂肪堆积症。初始和速率 在这一途径中的限制步骤是胆固醇在 第7位，由胆固醇7α-羟基酶催化，a 微粒体细胞色素P450。这种酶受到反馈控制， 从而使高水平的胆汁酸通过 肠-肝循环抑制其活性，而胆固醇、 酶的底物，施加阳性对照。这两项规定都会发生 在RNA积累的水平上，通过与其他 参与胆固醇动态平衡的基因，在 它的基因转录。这项提案的总体目标是 了解胆固醇和胆汁的分子机制 酸对胆固醇7α-羟基酶表达的调节。我们 克隆了胆固醇7α-羟基酶基因，鉴定了其 转录起始点，并测定了其5‘侧翼1.6kb的序列 区域。通过转基因实验，我们已经证明了这一点 片段包含促进ITS所需的DNA元件 以肝脏特有的方式转录。然而，我们一直无法 证明胆固醇或胆汁酸依赖的基因调控 7α-羟基酶5‘侧翼区的转录调控 吉恩。因为这种监管似乎需要复杂的相互作用 整个器官系统，我们将开发出融合的转基因小鼠 含有7α-羟基酶启动子的基因。一旦我们获得了一个 在转基因小鼠中显示受调控表达的启动子的构建 将使用体内和体外相结合的方法来鉴定和 描述与胆固醇和胆汁酸有关的DNA元素- 介导7α-羟基酶表达的调节。我们将在以下时间完成这项工作 修剪7α-羟基酶基因的5‘侧翼区域， 创造将在体外进行分析的缺失突变体 转录系统，在将它们引入小鼠之前。 蛋白质/DNA结合研究将使我们能够定位启动子 地区(S)最有可能包含监管元素。这些地区(S)将 通过寡核苷酸突变技术进行突变，并由IN进行研究 上述体内和体外方法。这些研究应该提供 更好地了解与生物多样性有关的分子机制 胆汁酸合成的调控。

The most important pathway for the catabolism and excretion of cholesterol in mammals is the formation of bile acids. Improper regulation of this pathway has widespread implications because the rate of elimination of cholesterol is a very important factor in diseases such as atherosclerosis, gallstone disease, and some lipid storage diseases. The initial and rate limiting step in this pathway is the hydroxylation of cholesterol at position 7, and is catalized by cholesterol 7 alpha-hydroxylase, a microsomal cytochrome P450. This enzyme is subject to a feedback control, whereby high levels of bile acids returning to the liver via the enterohepatic circulation suppress its activity, and cholesterol, the substract of the enzyme, exerts a positive control. Both regulations occur at the level of RNA accumulation, presumably and by analogy with other genes involved in cholesterol homeostasis, at the level of the transcription of its gene. The overall goal of this proposal is to understand the molecular mechanisms involved in the cholesterol and bile acid-mediated regulation of cholesterol 7alpha-hydroxylase expression. We have cloned the cholesterol 7alpha-hydroxylase gene, characterized its transcriptional initiation site, and sequenced 1.6 kb of its 5'-flanking region. By transfection experiments we have demonstrated that this fragment contains the DNA elements necessary for the promotion of its transcription in a liver-specific manner. However, we have been unable to demonstrate either cholesterol or bile acid-dependent regulation of gene transcription mediated by the 5'-flanking region of the 7alpha-hydroxylase gene. Because that regulation seems to require the complex interaction of whole organ systems, we will develop transgenic mice that harbor fusion genes containing the 7alpha-hydroxylase promoter. Once we obtain a promoter construct that shows regulated expression in transgenic mice, we will use combined in vivo and in vitro approaches to identify and characterize the DNA elements involved in cholesterol and bile acid- mediated regulation of 7alpha-hydroxylase expression. We will do this by trimming down the 5'-flanking region of the 7alpha-hydroxylase gene, creating deletion mutants that will be analyzed in an in vitro transcription system, prior to introducing them into mice. Protein/DNA-binding studies will allow us to localize the promoter region(s) most likely to contain regulatory elements. These region(s) will be mutated by oligonucleotide-mutagenesis techniques and studied by the in vivo and in vitro approaches mentioned above. These studies should provide a better understanding of the molecular mechanisms involved in the regulation of bile acid synthesis.