GENOMIC SCALE CLONING OF NUCLEAR DNA BINDING PROTEINS

描述（改编自研究者摘要）：序列特异性 DNA结合核蛋白在基因调控中起着重要作用 表情 因此，开发一种方法， 这些分子的基因组规模差异克隆。 目前的方法，如 由于差异筛选和消减cDNA文库的构建失败 区分哪些差异表达基因编码核 DNA结合蛋白。 为了鉴定这些蛋白质，研究人员建议 使用功能基因组学方法：第一步称为 “核捕获”包括分离由一种蛋白质编码的核蛋白质， 随机引物消减cDNA文库，其侧翼为C-末端标签， 编码6个组氨酸残基。 将该文库转染入 细胞系将允许转录和翻译的cDNA， 完整的5'末端。 由这些cDNA编码的核蛋白将 定位于细胞核，在那里它们可以从细胞核提取物中纯化 在其His标记的C末端插入镍琼脂糖珠。 第二 步骤将多轮特异性亲和富集和聚合酶 链反应（PCR）选择以鉴定被识别的DNA序列 被分离出来的带His标签的核蛋白所吸引 最后，被选中的 靶序列库可用于克隆编码蛋白质的cDNA 与这些网站绑定的信息 研究者将参考PCR亲和力 使用首字母缩写PASEC选择表达克隆。 本提案的目标是开展一个试点项目， 开发允许基因组规模差异的技术的可行性 克隆编码结合双链（ds）的核蛋白的cDNA DNA以特定的方式。 虽然可行性研究将 使用黑腹果蝇卵巢蛋白作为标记进行， 核定位和DNA结合特异性得到了很好的表征， 该方法是通用的，可以适用于任何真核系统。 具体目标包括：1. 评估核俘获的可行性 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cDNA文库） 程序的转染和His标签纯化阶段。 2. 使用His标记的CF2-II评估PASEC程序的可行性 从转染细胞系的细胞核纯化的蛋白质。 3. 执行20 多轮特异性亲和富集和PCR扩增以获得池 的DNA识别的位点特异性DNA结合蛋白包含在 果蝇卵巢核提取物。 由于CF2-II在细胞中表达， 卵巢滤泡细胞核，研究人员将确定是否 任何扩增的DNA库都含有已知的CF 2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.