GENOMIC SCALE CLONING OF NUCLEAR DNA BINDING PROTEINS

描述(改编自《调查者摘要》)：特定序列 DNA结合核蛋白在基因调控中起着基础性作用 表情。因此，开发一种允许 基因组规模的这种分子的差异克隆。目前的方法，如 差异筛选和消减文库构建失败 区分哪些差异表达的基因编码核 DNA结合蛋白。为了识别这种蛋白质，研究人员建议 使用功能基因组学方法：第一步称为 “核捕获”由分离的核蛋白组成，核蛋白由一种 随机引物消减cDNA文库侧翼带有C-末端标签 编码6个组氨酸(His)残基。将该文库转入 细胞系将允许转录和翻译具有 完成5‘端。其中一些cDNA编码的核蛋白将 定位于细胞核，在那里可以从核提取液中纯化它们 镍-琼脂糖珠通过其His标记的C-末端。第二 分步偶联多轮特异性亲和浓缩和聚合酶 用于识别识别的DNA序列的链式反应(PCR)选择 通过分离的His标记的核蛋白池。最后，入选的 靶序列池可用于克隆编码蛋白质的cDNA 绑定到这些站点的。调查人员将参考聚合酶链式反应亲和力 使用首字母缩写PASEC的选择表达克隆。 这项提案的目标是执行一个试点项目，该项目将评估 开发允许基因组规模差异的技术的可行性 结合双链(DS)的核蛋白编码基因的克隆 DNA以特定于序列的方式。尽管可行性研究将是 使用果蝇的一种黑腹果蝇卵巢蛋白作为标记进行实验 核定位和DNA结合的特异性得到了很好的表征， 该方法是通用的，可适用于任何真核系统。 具体目标包括：1.评估核捕获的可行性 通过替换编码卵巢转录因子的单个cdna CF2-II蛋白(而不是差异消减的cDNA文库) 该程序的转染期和组氨酸标签纯化阶段。2. 使用His标记的CF2-II评估PASEC手术的可行性 从转基因细胞系的细胞核中提纯的蛋白质。3.执行20 几轮特异性亲和力浓缩和聚合酶链式反应扩增以获得池 中包含的位点特异性DNA结合蛋白识别的DNA 果蝇卵巢核提取物。由于CF2-II在 对于卵泡细胞的细胞核，研究人员将确定 任何扩增的DNA池都含有已知的CF2结合位点。

DESCRIPTION (Adapted from the Investigator's Abstract): Sequence-specific DNA-binding nuclear proteins play a fundamental role in regulating gene expression. It would thus be valuable to develop a method permitting genomic scale differential cloning of such molecules. Current methods such as differential screening and construction of subtracted cDNA libraries fail to discriminate which of the differentially expressed genes encode nuclear DNA-binding proteins. To identify such proteins, the investigators propose using a functional genomics approach: The first step referred to as "nuclear trapping" consists of isolating nuclear proteins encoded by a random primed subtracted cDNA library flanked with a C-terminal tag that encodes six histidine (His) residues. Transfection of this library into cell-lines will permit transcription and translation of cDNAs that have complete 5' ends. Nuclear proteins encoded by some of these cDNAs will localize to the nucleus where they can be purified from a nuclear extract with nickel-agarose beads through their His-tagged C-terminus. The second step couples multiple rounds of specific affinity enrichment and polymerase chain reaction (PCR) selection to identify DNA sequences that are recognized by the isolated pool of His-tagged nuclear proteins. Finally, the selected pool of target sequences can be used to clone cDNAs that encode proteins that bind to these sites. The investigators will refer to PCR affinity selection expression cloning using the acronym PASEC. The goal of this proposal is to perform a pilot project that will assess the feasibility of developing technology permitting genomic scale differential cloning of cDNAs encoding nuclear proteins that bind double stranded (ds) DNA in a sequence-specific manner. Though feasibility studies will be performed using as a marker a Drosophila melanogaster ovarian protein whose nuclear localization and DNA-binding specificity are well characterized, the method is versatile and can be adapted for use in any eukaryotic system. The specific aims include: 1. Assess the feasibility of nuclear trapping by substituting a single cDNA that encodes the ovarian transcription factor CF2-II protein (instead of a differential subtracted cDNA library) during the transfection and His-tag purification phase of the procedure. 2. Assess the feasibility of the PASEC procedure using the His-tagged CF2-II protein purified from the nuclei of transfected cell lines. 3. Perform 20 rounds of specific affinity enrichment and PCR amplification to obtain pools of DNAs recognized by site-specific DNA binding proteins contained within Drosophila ovarian nuclear extracts. Since CF2-II is expressed in the nuclei of ovarian follicle cells, the investigators will determine whether any of the amplified pools of DNA contain known CF2-binding sites.