FUNCTIONAL ANALYSIS OF TRANSCRIPTION FACTORS IN HIV-1

HIV-1转录因子的功能分析

• 批准号: 3080035
• 负责人: DAVID ALEXANDER POTTER
• 金额: $ 7.61万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-30 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3080035
• 关键词: AIDS; B lymphocyte; DNA directed RNA polymerase; DNA virus; HeLa cells; affinity chromatography; antiserum; chloramphenicol acetyltransferase; complementary DNA; crosslink; gene deletion mutation; gene expression; gene rearrangement; genetic enhancer element; genetic library; genetic mapping; genetic transcription; human immunodeficiency virus 1; immunogenetics; immunoglobulin genes; laboratory rabbit; molecular cloning; nucleic acid probes; oligonucleotides; plasmids; protein purification; protein sequence; reagent /indicator; southern blotting; stress proteins; tissue /cell culture; transcription factor; transfection; virus genetics; western blottings

Transcription of human immunodeficiency virus (HIV), the etiologic agent of AIDS, is regulated by the cellular factor NF-kB, which binds to the HIV enhancer. NF-kB, originally described as a factor binding to the immunoglobulin k enhancer, is implicated in transcriptional regulation of numerous genes. The goals of this project are to determine the structure of NF-kB, its role in HIV transcription, and the transcriptional roles of related factors H2TF1, MBP-1 and KBF1 which bind to the same cognate sequence as NF-kB. To accomplish the latter goals, parallel studies of these related factors will be necessary. These factors may be present simultaneously in the nuclear milieu and it therefore remains to be established which factors are actually involved in positive or negative regulation of transcription in vivo. cDNA cloning of NF-kB and H2TF1 will be the initial step of the project. Partial protein sequence of NF-kB and H2TF1 purified from HeLa cells by DNA affinity chromatography will be used to isolate cDNA clones. A collateral approach will involve screening of cDNA expression libraries with oligonucleotide probes, a method developed in Dr. Sharp's laboratory. Functional domains of NF-kB and H2TF1 will be mapped by deletion analysis of cDNA expression constructs in transfected cells, using a reporter plasmid method. A set of antibody reagents specific for each factor will be generated from proteins overexpressed from the cDNA clones. These reagents will be used to determine whether NF-kB and H2TF1 are bound to the HIV enhancer in vivo, under a variety of physiologic conditions. The methodology will involve UV-crosslinking, followed by immunoprecipitation and Southern blot analysis of the precipitated DNA. This approach has been used to measure quantitative differences in the density of RNA polymerase II (pol II) on heat shock gene hsp70, before and after heat shock. The above reagents will also be used to comparatively study the NF-kB role in regulation of immunoglobulin gene transcription in normal B cell development. This project is part of a long term approach to understand the regulation of HIV gene expression and is ultimately directed toward the development of new approaches to the treatment and prevention of AIDS.

人类免疫缺陷病毒（HIV）的转录，其病原体 艾滋病是由细胞因子 NF-kB 调节的，该因子与 HIV 结合 增强剂。 NF-kB，最初被描述为与 免疫球蛋白 k 增强子，参与转录调节 无数的基因。 该项目的目标是确定结构 NF-kB 的作用、其在 HIV 转录中的作用以及 NF-kB 的转录作用 与相同同源物结合的相关因子 H2TF1、MBP-1 和 KBF1 序列为 NF-kB。 为了实现后一个目标，并行研究 这些相关因素是必要的。 这些因素可能存在 同时在核环境中，因此仍有待 确定哪些因素实际上涉及积极或消极的影响 体内转录的调控。 NF-kB 和 H2TF1 的 cDNA 克隆将 是该项目的第一步。 NF-kB的部分蛋白序列和 将使用通过 DNA 亲和层析从 HeLa 细胞中纯化的 H2TF1 分离 cDNA 克隆。 附带方法将涉及筛选 开发了一种带有寡核苷酸探针的 cDNA 表达文库方法 在夏普博士的实验室里。 NF-kB 和 H2TF1 的功能域将是 通过转染中 cDNA 表达构建体的缺失分析绘制图谱 细胞，使用报告质粒方法。 抗体试剂一套 每种因子的特异性将从过表达的蛋白质中产生 cDNA 克隆。 这些试剂将用于确定 NF-kB 是否 和H2TF1在体内与HIV增强子结合，在多种条件下 生理条件。 该方法将涉及紫外线交联， 然后进行免疫沉淀和 Southern blot 分析 沉淀 DNA。 该方法已用于定量测量 热休克基因上 RNA 聚合酶 II (pol II) 密度的差异 hsp70，热休克前后。 还将使用上述试剂 比较研究NF-kB在免疫球蛋白基因调控中的作用 正常 B 细胞发育中的转录。 该项目是长期项目的一部分 理解 HIV 基因表达调控的术语方法是 最终旨在开发新的方法 艾滋病的治疗和预防。