APOLIPOPROTEIN VARIATION AND HUMAN DISEASE

载脂蛋白变异与人类疾病

• 批准号: 2217419
• 负责人: VASSILIS I ZANNIS
• 金额: $ 28.13万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-09-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2217419
• 关键词: DNA binding protein; DNA footprinting; DNA replication; affinity chromatography; apolipoproteins; atherosclerosis; complementary DNA; disease /disorder proneness /risk; familial hyperlipoproteinemia type I; familial hyperlipoproteinemia type III; gel electrophoresis; gene expression; gene mutation; genetic disorder diagnosis; genetic library; genetic mapping; genetic promoter element; genetic transcription; inborn metabolism disorder diagnosis; liver cells; methylation; molecular cloning; nucleic acid sequence; oligonucleotides; protein purification; protein sequence; protein structure function; site directed mutagenesis; transcription factor

Our long-term objective is to understand the mechanism of transcriptional regulation of human apolipoprotein genes. It is our hypothesis that this regulation is controlled by protein-protein interactions of tissue-specific and ubiquitous nuclear proteins which recognize the apolipoprotein promoter. To understand this regulation we propose to identify the factors which bind to three apolipoprotein promoters (apoA-I, apoCIII, and apoA-II), to purify and clone unique factors, and to study in detail the mechanism of transcriptional regulation of human apoA-I. The specific aims are: 1) To determine the significance of the promoter elements for the transcription of the human apoA-I, apoCIII, and apoA-II genes by identifying the ubiquitous and tissue-specific transcription factors which bind to these elements and which regulate hepatic transcription. This will be accomplished by a) DNA binding gel electrophoresis, DNase I footprinting and methylation interference with normal and mutated promoter regions, and b) promoter (CAT) assays and in vitro transcription; 2) To purify and characterize new factors, the binding of which significantly affects the transcription of the human apoA-I, apoCIII, and apoA-II genes. Purification from nuclear extracts of rat liver will be accomplished by conventional procedures, followed by DNA sequence specific affinity chromatography. The DNA binding and specificity of the factors will be further tested by DNA binding, DNA protection, and in vitro transcription assays; 3) To clone and sequence cDNAs encoding new factors important for hepatic transcription of the human apoA-I, apoCIII, and apoA-II genes. This will be accomplished by a) screening of human liver cDNA libraries with synthetic oligonucleo- tides corresponding to the protein sequence of the factor, and b) screening expression libraries with the synthetic oligonucleotides corresponding to the recognition sequence of the factor and, 4) To express cDNAs encoding new transcription factors and to study in detail the mechanism of transcriptional regulation of the human apoA-I gene. Normal and mutated full length cDNAs encoding factors will be placed under the control of strong heterologous promoters and will be overexpressed in eukaryotic or bacterial cells in order to study the structure/function relationship of the corresponding factor. Co-trans- fection experiments will determine the importance of a factor for the activation or repression of transcription of target genes. The information which will emerge may then provide rational means of controlling plasma lipoprotein levels in ways that are protective against atherosclerosis.

我们的长期目标是了解转录的机制 调节人载脂蛋白基因。 我们的假设是 调节受蛋白质 - 蛋白质相互作用的控制 组织特异性和无处不在的核蛋白，识别 载脂蛋白启动子。 要了解这项法规，我们建议 确定与三个载脂蛋白启动子结合的因素 （apoa-i，apociii和apoa-ii），净化和克隆独特的因素，以及 详细研究人类的转录调节机制 apoa-i。 具体目的是：1）确定 人apoa-i，apocii和 通过鉴定无处不在的组织和组织特异性的apoA-II基因 与这些元素结合并调节的转录因子 肝转录。 这将通过a）DNA结合凝胶来完成 电泳，DNase I足迹和甲基化干扰 正常和突变的启动子区域，b）启动子（CAT）测定法和 体外转录； 2）净化和表征新因素， 其结合显着影响人类的转录 apoa-i，apocii和apoA-II基因。 从核提取物中纯化 大鼠肝脏将通过常规程序来完成 DNA序列特异性亲和色谱。 DNA结合和 DNA结合，DNA将进一步测试因素的特异性 保护和体外转录测定； 3）克隆和序列 编码新因素的cDNA对于肝转录很重要 人apoa-i，apociii和apoa-ii基因。 这将通过 a）用合成寡核 - 筛选人肝cDNA文库 对应于该因子的蛋白质序列的潮汐，b） 用合成寡核苷酸筛选表达库 对应于该因子的识别顺序，4）to 表达编码新的转录因子并详细研究的cDNA 人apoa-i基因转录调控的机制。 将放置正常和突变的全长cDNA编码因子 在强大异源启动子的控制下，将是 在真核或细菌细胞中过表达，以研究 相应因子的结构/功能关系。 二线 访问实验将确定一个因素的重要性 靶基因转录的激活或抑制。 这 然后将出现的信息可能提供合理的手段 以保护性的方式控制血浆脂蛋白水平 动脉粥样硬化。