FUNCTIONAL ANALYSIS OF TRANSCRIPTION FACTORS IN HIV-1

人类免疫缺陷病毒(HIV)的转录，其病原体是 艾滋病是由与HIV结合的细胞因子NF-kB调节的 增强剂。核因子-kB，最初被描述为一种与 免疫球蛋白K增强子参与转录调控 无数的基因。这个项目的目标是确定结构 核因子-kB的表达及其在HIV转录中的作用 与同源基因结合的相关因子H_2TF1、MBP-1和KBF1 序列为核因子-kB。为了实现后一项目标，平行研究 这些相关因素将是必要的。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因素 体内转录调控。核因子-kB和H_2TF1基因的克隆 是项目的第一步。核因子-kB的部分蛋白序列和 将使用DNA亲和层析从HeLa细胞中纯化的H2TF1 分离cDNA3个克隆。抵押品方法将涉及筛选 一种改进的寡核苷酸探针表达文库方法 在夏普博士的实验室里。核因子-kB和H_2TF1的功能域将是 基因表达载体缺失分析定位的研究 细胞，使用报告质粒法。一套抗体试剂 每种因子的特异性将产生于过度表达的蛋白质 CDNA3克隆。这些试剂将用于确定核因子-kB 和H2TF1结合到体内的HIV增强子，在各种不同的 生理条件。该方法将涉及紫外光交联， 然后进行免疫沉淀和Southern印迹分析 沉淀的DNA。这种方法已经被用来测量定量 热休克基因RNA聚合酶II(PolII)密度的差异 热休克前后HSP70的变化。上述试剂也将用于 比较研究核因子-kB在免疫球蛋白基因调控中的作用 正常B细胞发育过程中的转录。这个项目是一个漫长的项目的一部分 理解HIV基因表达调控和IS的术语方法 最终指向开发新的方法，以 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.