ACCESSORY CELL ACTIVATION IN THE IMMUNE RESPONSE

免疫反应中的辅助细胞激活

• 批准号: 2763400
• 负责人: PHILIP E AURON
• 金额: $ 30.38万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-25 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2763400
• 关键词: cell differentiation; cyclic AMP; cytokine; gene induction /repression; gene mutation; genetic promoter element; genetic regulatory element; interleukin 1; leukocyte activation /transformation; lipopolysaccharides; monocyte; phorbols; polymerase chain reaction; tissue /cell culture; transcription factor; transfection

The object of this proposal is to elucidate the mechanisms for the earliest stages of gene induction in activated or differentiating monocytes. The primary focus will be on gene regulation, paying particular attention to inducible transcription factors responsible for mediating differentiation and cell activation. The approach is to use prointerleukin 1-beta (IL1B) and a small number of other inducible monocyte-specific genes as examples for investigating the mechanisms of immediate early gene induction in monocytes. Evaluation of transiently transfected portions of these genes will be studied along with the binding of specific protein factors in order to gain insight into the regulatory mechanisms. The cDNA sequences for several transcription factors which appear to be important for monocyte activation and differentiation will be used in cotransfection studies aimed at overexpression in order to evaluate specific effects. Similarly, dominant-negative mutants of these factors will also be investigated. Specific Aims are: (I) to elucidate the functional mechanism of action for the IL1B Upstream Induction Sequence (UIS) which targets cell type-independent induction of the IL1B gene; (2) to examine the function of the tissue-restricted Spi-I/PU.I transcription factor, which binds to the promoter proximal sequence in the IL1B gene and other monocyte-specific genes to confer tissue-specific expression. Our previous studies demonstrated that human cytomegalovirus (HCMV) IE1 protein trans-activation of the IL1B gene requires an intact Spi-I/PU.I binding site. Therefore, the relationship between Spi-I/PU.I and HCMV IE1 will be investigated using these two proteins in functional and structural studies.

本提案的目的是阐明 基因诱导的最早阶段， 单核细胞 主要重点将是基因调控，支付 特别注意诱导型转录因子， 介导分化和细胞活化。 方法是使用 前白细胞介素1-β（IL 1B）和少量其他可诱导的 单核细胞特异性基因作为研究的机制的例子， 单核细胞中立即早期基因诱导。 瞬态评估 将沿着研究这些基因的转染部分， 特定蛋白质因子，以深入了解调节 机制等 几种转录因子的cDNA序列， 似乎对单核细胞活化和分化很重要， 用于旨在过表达的共转染研究， 评估具体效果。 类似地，这些基因的显性阴性突变体 还将调查各种因素。 具体目标是：（一）阐明 IL 1B上游诱导的功能性作用机制 靶向IL 1B的细胞类型非依赖性诱导的序列（UIS） （2）检测组织限制性Spi-I/PU.I 转录因子，其结合到启动子近端序列中， IL 1B基因和其他单核细胞特异性基因赋予组织特异性 表情 我们以前的研究表明，人巨细胞病毒 IL 1B基因的（HCMV）IE 1蛋白反式激活需要完整的 Spi-I/PU.I结合位点。 因此，Spi-I/PU.I 和HCMV IE 1将使用这两种蛋白进行功能研究。 结构研究。