HEPATIC REGULATION OF THE HUMAN APO-B GENE

人类 APO-B 基因的肝脏调节

• 批准号: 2225557
• 负责人: HRIDAY K DAS
• 金额: $ 15.26万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2225557
• 关键词: DNA binding protein; DNA footprinting; Escherichia coli; HeLa cells; affinity chromatography; apolipoproteins; cholesterol; coronary disorder; gene expression; genetic transcription; hyperlipidemia; ion exchange chromatography; laboratory rabbit; laboratory rat; liver metabolism; molecular cloning; molecular weight; nucleic acid sequence; polymerase chain reaction; recombinant proteins; restriction mapping; structural genes; transcription factor; transfection

Heart disease is a lethal disease in the United States. Atherosclerosis develops as a result of high serum cholesterol. Cholesterol is transported through the circulation as lipoprotein particles and the protein components of these particles are called apolipoproteins. ApoB, the only protein of LDL, is synthesized primarily in the liver, and plasma levels of LDL cholesterol and apoB correlate directly with atherosclerosis susceptibility. Therefore, factors regulating apoB gene expression should participate in the regulation of plasma cholesterol level, and may be involved in the development of hyperlipidemia and heart diseases. Since traps-acting protein factors interact with cis-acting DNA elements and with themselves to mediate liver specific expression of the human apoB gene, it is necessary to purify and characterize these factors and clone their structural genes to understand the mechanism of hyperlipidemia. To achieve this goal, two such apoB gene regulatory factors BRF-1 and BRF-2, which bind to the apoB gene regulatory elements (-84 to -60) and (-128 to -85) respectively have been purified to apparent homogeneity by DNA- specific affinity chromatography. The molecular weights of these proteins have been determined to be 68 kDa and 120 kDa respectively by SDS/PAGE. The NH2- terminal sequence of BRF-l has been determined to be NH2-Gly-Arg- Ser-Ala-Gly-Ala-Phe-Gly-Arg-Val-Arg-Ile-Glu. A polyclonal anti serum against this peptide has also been raised in rabbit to clone cDNA encoding BRF-1. Factor(s) BRF-3/BRF-4 which produced footprints~(+30 to +41) and (+44 to +53) on the apoB promoter, have also been partially purified. Factors BRF-3/BRF-4 will be purified by DNA-specific affinity chromatography and characterized by SDS/PAGE, DNaseI footprinting and in vitro transcription. Genes encoding these factors will be cloned using antibody probes. Full or partial length cDNAs will be used to synthesize full size or truncated forms of proteins in E. Coli or in an in vitro transcription-translation system. Recombinant proteins could be used to study the role of these factors in apoB gene transcription. Truncated proteins synthesized in vitro could be used to map their domains for DNA- protein, and protein-protein interactions as well as to delineate domains needed for the formation of oligomeric structure. The possible role of these factors in the transcription of other apolipoprotein genes will also be studied.

心脏病是美国的致命疾病。动脉粥样硬化 由于高血清胆固醇而发展。胆固醇被运输 通过循环作为脂蛋白颗粒和蛋白质 这些颗粒的成分称为载脂蛋白。 apob，唯一 LDL的蛋白质主要在肝脏中合成，血浆水平合成 LDL胆固醇和APOB与动脉粥样硬化直接相关 敏感性。因此，调节APOB基因表达的因素应 参与血浆胆固醇水平的调节，可能是 参与高脂血症和心脏病的发展。自从 陷阱作用蛋白因子与顺式作用DNA元件相互作用， 与自己介导人apob的肝脏特异性表达 基因，有必要净化和表征这些因素并克隆 它们的结构基因了解高脂血症的机制。到 实现这一目标，两个这样的APOB基因调节因子BRF-1和BRF-2， 结合APOB基因调节元件（-84至-60）和（-128） -85）分别已通过DNA纯化为明显的同质性 特定亲和力色谱。这些蛋白质的分子量 已确定通过SDS/PAGE分别确定为68 kDa和120 kDa。 BRF-L的NH2-末端序列已被确定为NH2-Gly-arg- ser-ala-gly-ala-phe-gly-arg-arg-arg-arg-ile-glu。多克隆抗血清 反对这种肽也已在兔子中升高到克隆cDNA编码 BRF-1。因子（S）BRF-3/BRF-4产生足迹〜（+30至+41）和 （+44至+53）在APOB启动子上也已部分纯化。 BRF-3/BRF-4的因素将通过DNA特异性亲和力纯化 色谱和以SDS/PAGE，DNASEI足迹为特征 体外转录。编码这些因素的基因将使用 抗体探针。完整或部分长度的cDNA将用于合成 大肠杆菌或体外中的全尺寸或截短的蛋白质形式 转录翻译系统。重组蛋白可用于 研究这些因素在APOB基因转录中的作用。截断 在体外合成的蛋白质可用于绘制其域的DNA- 蛋白质和蛋白质 - 蛋白质相互作用以及描述结构域 形成寡聚结构所需的。可能的作用 其他载脂蛋白基因转录中的这些因素也将 被研究。