STRUCTURE AND REGULATION OF LIVER ACUTE PHASE GENES

肝脏急性期基因的结构和调控

• 批准号: 3132948
• 负责人: GEORG H FEY
• 金额: $ 26.14万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-01 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3132948
• 关键词: DNA binding protein; acute phase protein; cell bank /registry; cell free system; chromosome deletion; gene expression; genetic library; genetic manipulation; genetic mapping; genetic transcription; genetically modified animals; glucocorticoids; hepatocellular carcinoma; hormone regulation /control mechanism; in situ hybridization; inflammation; interleukin 6; laboratory rabbit; laboratory rat; liver cells; macroglobulins; molecular cloning; nucleic acid sequence; protein sequence; transcription factor

Acute phase proteins are produced by hepatocytes and are important in the defense against tissue damage and infections. Alpha2- macroglobulin (alpha2M), a broad range proteinase inhibitor, is increased several 100 fold-in acute and chronic inflammations; alpha-inhibitor III (alpha1I3), a close structural relative is strongly decreased. Transcription of both genes is controlled by glucocorticoids and interleukin 6 (IL6) in opposite directions. We have isolated and characterized DNA clones for both genes and have located a glucocorticoid responsive element in the alpha1I3 gene 5' flanking region. We have identified suitable rat hepatoma cell lines to study the regulation of both genes in culture and have also isolated rat IL6 DNA clones. These will be used to produce recombinant IL6 in order to map the IL6 responsive control elements of both genes. ln this continuation we will characterize the cis- and trans-acting control elements of both genes responsible for their regulation by these two hormones. The cis-elements will be mapped by transfection studies in hepatoma cells or transgenic mice. The elements responsible for hepatic transcription of both genes will be studied by cell free transcription in liver nuclear extracts. The unique availability of rat liver will be exploited to purify microgram amounts of relevant trans-factors by specific ORA affinity chromatography. Their function as transcription factors will be established by mutagenesis and complementation. Partial amino acid sequences of the factors will be determined and corresponding DNA clones will be isolated. Studies of the tissue specific and developmental regulation oF the factor genes will be initiated. Recombinant factors will be expressed in suitable systems and an initial characterization of their biochemical functions will be attempted. The purpose of this research is to characterize the elements mediating the transient control of acute phase gene transcription and to compare them with the elements responsible for the stable. liver specific expression of these and other genes. We wish to learn whether acute phase control is achieved by different combinations of the same elements that cause hepatic transcription or whether it involves qualitatively different elements. This is attempted by focusing on a system that is biochemically feasible, biologically relevant and already well characterized. Improved knowledge of gene regulation by inflammatory mediators may facilitate the future design of control substances of inflammation.

急性时相蛋白由肝细胞产生， 在防御组织损伤和感染方面。 α 2- 巨球蛋白（α 2 M），一种广泛的蛋白酶抑制剂， 急性和慢性炎症增加数百倍; α-抑制剂III（alpha 1 I3），一种结构上的近亲， 大幅下降。 这两种基因的转录都是由 糖皮质激素和白细胞介素6（IL 6）在相反的方向。 我们已经分离和表征了这两种基因的DNA克隆， 已经在α 1 I3中定位了糖皮质激素反应元件， 基因5'侧翼区。 我们已经确定了合适的大鼠肝癌 细胞系，以研究这两种基因在培养物中的调节， 也分离了大鼠IL 6 DNA克隆。 这些将用于 产生重组IL 6，以定位IL 6应答对照 这两种基因的成分。 接下来我们将描述顺式和反式作用 这两个基因的控制元件负责他们的调节， 这两种荷尔蒙。 顺式元件将被映射为 在肝癌细胞或转基因小鼠中的转染研究。 的 负责这两种基因肝脏转录的元件将 通过在肝核提取物中的无细胞转录来研究。 将利用大鼠肝脏的独特可用性来纯化 特定ORA的相关反式因子的微克量 亲和层析 它们作为转录因子的功能 将通过诱变和互补来建立。 部分 将确定因子的氨基酸序列， 将分离相应的DNA克隆。 组织研究 这些因子基因的特异性和发育调节将被 启动。 重组因子将在合适的培养基中表达。 系统及其生化特性的初步表征 功能将被尝试。 这项研究的目的是表征元素 介导急性期基因转录的瞬时控制 并将其与稳定的元素进行比较。 肝脏特异性表达这些和其他基因。 我们希望 了解急性期控制是否通过不同的 引起肝转录的相同元件的组合 或者它是否涉及性质不同的元素。 这是 试图通过关注一个在生物化学上可行的系统， 生物学上相关的，已经很好地表征了。 改进 炎症介质对基因调控的认识， 便于将来设计控制炎症的物质。