TRANSCRIPTIONAL ACTIVATION OF THE APOLIPOPROTEIN B GENE

载脂蛋白B是LDL的唯一蛋白质组分，并介导LDL的代谢。 通过LDL受体催化LDL。 这是有力的证据， 这种蛋白质合成调节结构的改变 在动脉粥样硬化的发病机制中起主要作用。 因此，高或 低水平的载脂蛋白B或低密度脂蛋白与风险的增加或减少有关， 分别是冠状动脉疾病。 应用程序的目标是 研究apoB基因的转录调控机制。 的 具体目标是：1）确定核的识别序列， 参与人apoB基因转录激活的因子。 这些识别序列将通过以下方式鉴定： 诱变; B）竞争足迹分析; c） 在肝素琼脂糖凝胶柱上的核提取物和各种 通过DNA酶I足迹法和凝胶阻滞测定法测定组分。2)到 鉴定不同的正或负转录因子， 调节人apoB基因在肝脏的表达。 这些因素将是 通过：a）凝胶阻滞竞争实验; B）生物化学 通过DNA亲和层析纯化。 纯化因子将是 用于获得有限的蛋白质序列信息; B）分离和 cDNA和基因组克隆的表征。 克隆将涉及初始 用合成寡核苷酸筛选肝脏cDNA文库 或筛选表达文库 与寡核苷酸识别序列连接。 基因组序列将 通过用cDNA探针筛选基因组文库来确定; c） 从产生的cRNA中鉴定该因子的性质， 体外 为了充分了解apoB基因的调控， 需要纯化所有涉及的转录因子， 确定它们相互作用或活动的方式， 由细胞分化事件调节。 了解法规 在不久的将来，apoB的表达可能会提供新的方法， 控制我们的血浆LDL和apoB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.