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MOLECULAR BIOLOGY OF THE SYNAPSE

突触的分子生物学

基本信息

批准号：
3415791
负责人：
EILEEN M. LAFER
金额：
$ 11.92万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-04-01 至 1993-07-31
项目状态：
已结题

项目摘要

Experiments-aimed at elucidating the biological role of Z-DNA have taken two forms. Natural sequences which can form Z-DNA have been identified and proteins have been purified which bind better to Z- DNA than B-DNA in vitro. However, the sequences have not been shown to form Z-DNA in vivo, nor have the proteins been shown to bind Z-DNA in vivo. We recently purified three proteins from coli which bind specifically to Z-DNA in vitro, prepared monoclonal antibodies against them, and cloned their genes. For the first time reagents are available that make possible molecular analyses of ,genes encoding Z-DNA binding proteins and extensive biochemical studies of these proteins. The goal of these studies is to elucidate the biological role of Z-DNA in E. coli by determining the function of these Z-DNA binding proteins. We will sequence their genes and locate them on the E. coli map, and analyze the migration of the proteins on 2-D PACE. Comparison of this information with available data banks will reveal if data on these proteins or genes has been collected. Using allelic replacement and constructs overexpressing or producing antisense mRNA for these genes we will generate null mutants or conditional lethals and strains which overexpress these genes ;or produce reduced levels of these proteins. We will examine the mutants for defects in processes executed at the DNA level: replication, recombination, repair, supercoiling, and transcription. If we obtain conditional lethals we will clone mutants in other genes which suppress the original mutant phenotype. Using highly purified protein from strains overexpressing these genes we will carry out an extensive study of the interaction of these proteins with Z-DNA. We will precisely quantitate the specificity of these proteins for Z-DNA. Chemical modification experiments will reveal details, at the base pair level, of the interaction. We will identify the in vivo binding sites of these proteins by immunoprecipitating UV cross- linked protein-DNA complexes from cell extracts and analyzing the precipitated DNA by cloning and sequencing. Purified proteins will also be assayed for NTPase, nuclease, topoisomerase, and recombination activities. Since so little is known about what Z- DNA does in a cell, we believe that E. coli, which is amenable to genetic analysis and is one of the simplest and best characterized laboratory organisms, is an ideal system in which to begin studying the function of Z-DNA. These studies will have far reaching implications since they will provide clues to the function of Z- DNA in organisms other than E. coli.

旨在阐明Z-DNA生物学作用的实验已经采取了两种形式。可以形成Z-DNA的天然序列已经被已经鉴定并纯化了与Z-结合更好的蛋白质， DNA比B-DNA在体外。然而，这些序列并没有被显示在体内形成Z-DNA，也没有显示蛋白质在体内结合Z-DNA。我们最近从大肠杆菌中纯化了三种蛋白质其在体外特异性结合Z-DNA，并克隆了它们的基因。第一时间试剂可以使分子分析成为可能基因编码Z-DNA结合蛋白和广泛的生化研究这些蛋白质。这些研究的目的是阐明了Z-DNA在大肠杆菌中的生物学作用。通过测定大肠杆菌这些Z-DNA结合蛋白的功能我们将测序他们的基因，并将其定位在E。大肠杆菌图谱，并分析蛋白质在二维PACE上的迁移。与此相比，现有数据库的信息将揭示，蛋白质或基因已经被收集。使用等位基因替换以及过表达或产生这些反义mRNA的构建体基因，我们将产生无效突变体或条件致死，过度表达这些基因的菌株;或产生水平降低的菌株这些蛋白质。我们将检查突变体的缺陷，在DNA水平上执行的过程：复制，重组，修复、超螺旋和转录。如果我们得到条件我们将在其他基因中克隆突变体，原始突变表型。使用高度纯化的蛋白质我们将对过度表达这些基因的菌株进行广泛的研究这些蛋白质与Z-DNA的相互作用。我们将精确定量这些蛋白质对Z-DNA的特异性。化学改性实验将揭示细节，在基地对水平，相互作用。我们将在体内鉴定这些蛋白质的结合位点的免疫沉淀紫外交叉，连接的蛋白质-DNA复合物的细胞提取物和分析，通过克隆和测序沉淀DNA。纯化的蛋白质将还可以测定NTR、核酸酶、拓扑异构酶，重组活动因为我们对Z- DNA在细胞中的作用，我们相信E。大肠杆菌，这是顺应遗传分析，是最简单和最好的特点之一，实验室生物，是一个理想的系统，在其中开始研究 Z-DNA的功能这些研究将产生深远的影响。的影响，因为他们将提供线索的功能Z- 除了E.杆菌