FUNCTIONAL ANALYSIS OF TRANSCRIPTION FACTORS IN HIV-1

人类免疫缺陷病毒（HIV）的转录， 艾滋病，是由细胞因子NF-κ B，它结合到艾滋病毒 增强剂 NF-kB，最初被描述为结合到 免疫球蛋白k增强子，参与转录调节， 许多基因。 本项目的目标是确定 NF-kB在HIV转录中的作用，以及 与相同同源物结合的相关因子H2 TF 1、MBP-1和KBF 1 NF-kB序列。 为了实现后一个目标，平行研究 这些相关因素都是必要的。 这些因素可能存在 同时在核环境中，因此，它仍然是 确定哪些因素实际上涉及积极或消极的 体内转录调控。 NF-kB和H2 TF 1的cDNA克隆将 这是项目的第一步。 NF-κ B的部分蛋白质序列， 将使用通过DNA亲和层析从HeLa细胞纯化的H2 TF 1 分离cDNA克隆。 并行方法将包括筛选 利用寡核苷酸探针构建cDNA表达文库的方法 在夏普博士的实验室里 NF-kB和H2 TF 1的功能结构域将被 通过转染的细胞中的cDNA表达构建体的缺失分析作图， 细胞，使用报告质粒方法。 一套抗体试剂 对每个因子具有特异性的蛋白质将从从 cDNA克隆。 这些试剂将用于确定NF-kB是否 和H2 TF 1在体内结合到HIV增强子上， 生理条件。 该方法将涉及UV交联， 然后进行免疫沉淀和Southern印迹分析， 沉淀的DNA 这种方法已被用来衡量定量 热休克基因RNA聚合酶II（pol II）密度的差异 热休克前后hsp 70的变化。 上述试剂也将用于 比较研究NF-κ B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。 这个项目是一个长期的 长期的方法来了解艾滋病毒基因表达的调控， 最终的目标是发展新的方法， 艾滋病的治疗和预防。

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.