Probing Chromosome Structure with Phage Mu

用噬菌体 Mu 探测染色体结构

• 批准号: 0110675
• 负责人: Norman Higgins
• 金额: $ 32.88万
• 依托单位: University of Alabama at Birmingham
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-11-15 至 2004-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0110675&HistoricalAwards=false
• 关键词: Probing; Chromosome; Structure; Phage; Mu

Two related methods have been developed to probe the structure of bacterial chromosomes inside living cells. One technique is called Muprinting, and it uses PCR reactions to generate a high-resolution picture of the transposition target sites in a bacterial population infected with phage Mu. By comparing the PCR profiles of naked DNA in vitro and protein bound DNA in vivo, it is possible to detect the location of sequence-specific DNA binding. Proteins detected with Muprinting include repressors and RNA polymerases. A new method recently developed called Mu-screening allows the identification of the transposition targets that are used efficiently in vivo during replicative Mu transposition. It is now possible to identify hot spots and cold spots for transposon insertion, and to follow changes in these structure when cells are placed in different physiological environments. Two primary objectives will be pursued. First, Mu-screening will be used in conjunction with gene micro-arrays containing 4115/4238 ORFs in E. coli to identify the most efficient transposition targets in the genomes of E. coli and Salmonella enterica serovar typhimurium. Second, Mu transposition hotspots will be cloned and subjected to in vivo and in vitro Muprint analysis to precisely identify the regions where proteins are bound in living cells. Ultimately, the proteins bound to Mu hotspots will be characterized by biochemical and genetic methods. New methods to systematically investigate chromosome structure inside living cells are required to address the challenging problems in modern molecular evolution. With the stunning advances in the ability to efficiently acquire entire sequences of virtually any organism, new methods to understand chromosome function and structure are now urgent. Information gained from Mu-screening and Muprinting technology promises to provide insight into the area of chromosome evolution and into the question of structure in bacterial chromatin. Micro-array-driven structure analysis complements gene expression studies and should stimulate new genetic and biochemical models of chromosome function.

已经开发了两种相关的方法来探测活细胞内细菌染色体的结构。一种技术称为muprinting，它使用PCR反应来产生感染噬菌体MU的细菌种群中换位目标位点的高分辨率图片。通过在体内比较体外和蛋白质结合的DNA的PCR谱，可以检测序列特异性DNA结合的位置。用默认打印检测到的蛋白质包括阻遏物和RNA聚合酶。最近开发的一种称为MU屏幕的新方法允许鉴定在复制MU转座期间有效地在体内使用的换位靶标。现在可以识别用于转座子插入的热点和冷点，并在将细胞放置在不同的生理环境中时遵循这些结构的变化。将实现两个主要目标。首先，将使用含有4115/4238 ORF的基因微阵列使用MU筛选，以鉴定大肠杆菌和肠肠球菌typhimurium的基因组中最有效的转位靶标。其次，MU转位热点将被克隆并进行体内和体外默认分析，以精确地识别蛋白质与活细胞结合的区域。最终，与MU热点结合的蛋白质将以生化和遗传学方法为特征。需要系统研究活细胞内部染色体结构的新方法，以解决现代分子进化中具有挑战性的问题。由于能够有效地获取几乎任何生物的整个序列的能力的惊人进步，现在紧急了解染色体功能和结构的新方法。从MU筛选和穆布打印技术中获得的信息有望洞悉染色体进化区域，并将其介绍为细菌染色质中的结构问题。微阵列驱动的结构分析补充了基因表达研究，并应刺激染色体功能的新遗传和生化模型。