TRANSCRIPTIONAL ACTIVATION OF APOLIPOPROTEIN B GENE

载脂蛋白B是低密度脂蛋白的唯一蛋白质组分，它介导 通过低密度脂蛋白受体分解代谢低密度脂蛋白。他们的观点有力地证明了 该蛋白合成调控结构的改变 在动脉粥样硬化的发病机制中起主要作用。因此，HIGH或 低水平的载脂蛋白B或低密度脂蛋白与风险的增加或降低有关， 分别为冠状动脉疾病。该应用程序的目标是 目的：研究载脂蛋白B基因的转录调控机制。这个 具体目标是：1)识别核的识别序列 参与人类载脂蛋白B基因转录激活的因素。 这些识别序列将通过以下方式识别：a)定点定向 诱变；b)竞争足迹分析；c)分级 肝素琼脂糖柱上的核抽提物及其各种成分的分析 通过DNase I足迹和凝胶滞留实验分析其组分。2)至 确定不同的积极或消极转录因子 调节人载脂蛋白B基因在肝脏的表达。这些因素将是 鉴定方法：a)凝胶延迟竞争实验；b)生化 DNA亲和层析纯化。纯化后的因子将是 用于获取有限的蛋白质序列信息；b)分离和 Cdna和基因组克隆的特征。克隆将涉及初始 用人工合成的寡核苷酸筛选肝脏cDNA文库 与所述氨基酸序列对应或筛选表达文库 与寡核苷酸识别序列结合。基因组序列将 通过用该cDNA探针筛选基因组文库来确定；c) 从产生的CRNAs中鉴定该因子的性质 体外培养。要全面了解载脂蛋白B基因的调控，最终 要求对所有涉及的转录因子进行纯化， 确定他们相互作用的手段或他们的活动 受细胞分化事件的调节。理解规则 ApoB的表达可能在不久的将来提供新的途径 控制我们的血浆低密度脂蛋白和载脂蛋白B水平。

Apolipoprotein B is the sole protein component of LDL and mediates the catabolism of LDL via the LDL receptor. Their is strong evidence that alterations in the structure of regulation of synthesis of this protein play a major role in the pathogenesis of atherosclerosis. Thus, high or low levels of apoB or LDL are associated with increases or decreased risk, respectively, of coronary artery disease. The goals of the application are to study the mechanism of transcriptional regulation of the apoB gene. The specific aims are: 1) to identify the recognition sequences of nuclear factors involved in the transcriptional activation of the human apoB gene. These recognition sequences will be identified by: a) site-directed mutagenesis; b) competition footprinting analysis; c) fractionation of nuclear extracts on a heparin sepharose column and analysis of the various fractions by DNAse I footprinting and gel retardation assays. 2) to identify the different positive or negative transcriptional factors which regulate the hepatic expression of human apoB gene. The factors will be identified by: a) gel retardation competition experiments; b) biochemical purification by DNA affinity chromatography. The purified factor will be used to obtain limited protein sequence information; b) isolation and characterization of cDNA and genomic clones. Cloning will involve initial screening of a liver cDNA library with synthetic oligonucleotides corresponding to the amino acid sequence or screening an expression library with the oligonucleotide recognition sequence. The genomic sequence will be determined by screening a genomic library with the cDNA probe; c) identification of the properties of this factor from the cRNAs produced in vitro. To fully understand the apoB gene regulation will eventually require the purification of all transcriptional factors involved and the identification of the means which they interact or their activity is modulated by events of cell differentiation. Understanding the regulation of expression of apoB may provide in the near future new approaches toward controlling our plasma LDL and apoB levels.