A bacterial one-hybrid system for analyzing DNA-binding specificities

一种用于分析 DNA 结合特异性的细菌单杂交系统

• 批准号: 7230270
• 负责人: SCOT A WOLFE
• 金额: $ 15.78万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-18 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7230270
• 关键词: bacterial; hybrid; system; analyzing; DNA

DESCRIPTION (provided by applicant): An important goal of the human genome project is to completely describe the cis-regulatory sequences and trans-acting factors that control gene transcription in complex genomes. A significant remaining impediment to this goal is the paucity of well-characterized DNA-binding specificities for transcription factors. A high-throughput, inexpensive method is needed to facilitate the analysis of the DNA-binding specificity of transcription factors on a genome-wide scale. We have developed a bacterial 1-hybrid system that can characterize the DNA-binding specificity of transcription factors. This technology has advantages over existing in vitro methods: only basic molecular biology reagents and techniques are required, and no protein purification is necessary. In this application, we propose to evaluate our bacterial 1-hybrid system as a high-throughput method for determining the DNA-binding specificities of transcription factors. In Aim 1, we will optimize the bacterial 1-hybrid system for the high-throughput analysis of factors and establish a standard set of selection conditions that will yield high quality binding site motifs for transcription factors with a broad range of affinities. In Aim 2, we will perform a pilot screen of 30 Cys2His2 zinc finger proteins and 20 other transcription factors from the model organism Drosophila. In Aim 3, we will assess the quality of the DNA-binding specificities determined using the bacterial 1-hybrid system. If this project is successful, we anticipate that the bacterial 1-hybrid system will be used by ourselves and adopted by others to perform genome-wide surveys of transcription factors in Drosophila and a variety of other model organisms. The resulting database of information on transcription factors will be valuable for deciphering the mechanisms of gene regulation in higher eukaryotes.

描述（由申请人提供）：人类基因组项目的一个重要目标是完全描述控制基因组中基因转录的顺式调节序列和反式作用因子。对该目标的剩余障碍是转录因子缺乏特征良好的DNA结合特异性。需要一种高通量，廉价的方法来促进在全基因组量表上分析转录因子的DNA结合特异性。我们已经开发了一种细菌1杂交系统，可以表征转录因子的DNA结合特异性。该技术比现有的体外方法具有优势：仅需要基本的分子生物学试剂和技术，并且不需要蛋白质纯化。在此应用中，我们建议评估我们的细菌1杂交系统作为确定转录因子DNA结合特异性的高通量方法。在AIM 1中，我们将优化细菌1 Hybrid系统，以对因子进行高通量分析，并建立一组标准的选择条件，这些条件将产生高质量的结合位点基序，用于具有广泛亲和力的转录因子。在AIM 2中，我们将执行30个Cys2HIS2锌指蛋白和20个其他转录因子的试验屏幕。在AIM 3中，我们将评估使用细菌1杂交系统确定的DNA结合特异性的质量。如果这个项目成功，我们预计我们自己将使用细菌1杂交系统，并被其他人采用，以对果蝇和各种其他模型生物的转录因子进行全基因组进行调查。所得的有关转录因子的信息数据库对于破译较高真核生物中基因调节的机制是有价值的。