CONTROL OF COLONY STIMULATING FACTOR 1 GENE EXPRESSION

集落刺激因子1基因表达的控制

• 批准号: 2182298
• 负责人: MAUREEN A. HARRINGTON
• 金额: $ 21.8万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182298
• 关键词: DNA binding protein; DNA footprinting; Sertoli cells; antibody; biological signal transduction; chloramphenicol acetyltransferase; colony stimulating factor; cytokine receptors; fibroblasts; gel mobility shift assay; gene expression; genetic regulation; genetic regulatory element; genetic transcription; immunoprecipitation; interleukin 1; methylation; monocyte; nucleic acid sequence; polymerase chain reaction; receptor binding; site directed mutagenesis; transcription factor; transfection; vascular endothelium

This project is aimed at understanding cellular and molecular mechanisms that govern colony stimulating factor-1 (CSF-1) gene expression. Our hypothesis is that a basal set of trans-acting factors is bound to the CSF-1 gene transcription, by growth-arrest (decrease) or stimulation of growth-arrested fibroblasts (re-initiate) is mediated by changes in the basal set of factors bound or by the addition of stimulus specific factors. CSF-1-CAT reporter constructs in transient transfection assays will be used to identify genomic sequences that affect transcriptional activity; DNase I protection, electrophoretic mobility shift and methylating interference assays will be used to identify putative cis- acting elements. CSF-1-CAT constructs containing mutated cis-acting elements will be used in transient transection assays to determine if the mutation affects transcriptional activity. As cis-acting elements that affect CSF-1-CAT reporter construct activity are identified, cognate trans-acting factors will be identified, by antibody or by molecular cloning. We will extend our hypothesis to include other cells types to determine if mechanisms used to control CSF-1 gene expression in fibroblasts are unique or represent common non-tissue specific regulatory mechanisms. We will determine if cis-acting element(s) that affect CSF- 1-CAT reporter construct activity in fibroblasts affect CSF-1-CAT activity in monocytes, endothelial cells and sertoli cells. We want to include in our hypothesis how a signal at the plasma membrane in growth- arrested fibroblasts results in activation of CSF-1 gene transcription. We propose to use immunoprecipitates of the IL-1 receptors in fibroblasts. Identification of bound protein(s) will be done by sequence analysis of cloned cDNA inserts or bound protein or alternatively by immunoreactivity. Identification of trans-acting factors that affect CSF-1 gene transcription and signaling pathways that modulate trans- acting factor binding activity will identify targets for therapeutic intervention in situations, such as cancer, atherosclerosis or infertility, where control over CSF-1 gene expression may be desirable.

该项目旨在了解细胞和分子机制 控制刺激因子1（CSF-1）基因表达的菌落。 我们的 假设是一组基础跨作用因子与 CSF-1基因转录，通过生长暂停（减少）或刺激 生长降落的成纤维细胞（重新分泌）是由变化的变化 基础因子集合或通过添加刺激特异性 因素。 瞬态转染测定中的CSF-1-CAT报告基因构建体 将用于识别影响转录的基因组序列 活动; DNase I保护，电泳移动性转移和 甲基化干扰测定将用于识别推定的顺式 表演元素。 CSF-1-CAT构建体包含突变的顺式作用 元素将用于瞬态横向测定法，以确定是否是否 突变会影响转录活性。 作为顺式作用元素 影响CSF-1-CAT报告基因构建活性，同源 将通过抗体或分子鉴定跨作用因子 克隆。 我们将将我们的假设扩展到包括其他单元类型的 确定是否用于控制CSF-1基因表达的机制是否用于 成纤维细胞是唯一的或代表常见的非组织特异性调节 机制。 我们将确定影响CSF-的顺式作用元素是否存在 成纤维细胞中的1-CAT报告基因构建活性影响CSF-1-CAT 单核细胞，内皮细胞和Sertoli细胞的活性。 我们想 在我们的假设中包括在生长中质膜处的信号如何 滞留的成纤维细胞导致CSF-1基因转录的激活。 我们建议在IL-1受体中使用IL-1受体的免疫沉淀物 成纤维细胞。 结合蛋白的识别将通过序列完成 分析克隆的cDNA插入物或结合蛋白，或者通过 免疫反应性。 识别影响影响的跨性别因素 CSF-1基因转录和信号传导途径调节反式 作用因子结合活性将识别治疗目标 干预情况，例如癌症，动脉粥样硬化或 不孕症，对CSF-1基因表达的控制可能是可取的。