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。一种技术被称为Mupringing，它使用PCR反应来生成感染噬菌体Mu的细菌群体中转座靶位点的高分辨率图像。通过比较体外裸DNA和体内蛋白结合DNA的PCR谱，可以检测序列特异性DNA结合的位置。用Mupringing检测的蛋白质包括阻遏物和RNA聚合酶。最近开发的一种称为Mu-筛选的新方法允许鉴定在复制Mu转座期间在体内有效使用的转座靶点。现在可以识别转座子插入的热点和冷点，并在细胞置于不同的生理环境中时跟踪这些结构的变化。将追求两个主要目标。首先，Mu筛选将与含有E.大肠杆菌中，以确定最有效的转座目标的基因组中的E.大肠杆菌和沙门氏菌血清型鼠伤寒。其次，Mu转座热点将被克隆并进行体内和体外Muprint分析，以精确鉴定活细胞中蛋白质结合的区域。最终，与Mu热点结合的蛋白质将通过生物化学和遗传学方法进行表征。为了解决现代分子进化中的挑战性问题，需要系统地研究活细胞内染色体结构的新方法。随着有效获取几乎任何生物体的整个序列的能力的惊人进步，现在迫切需要新的方法来了解染色体的功能和结构。从Mu筛选和Mupringing技术获得的信息有望为染色体进化领域和细菌染色质结构问题提供深入了解。微阵列驱动的结构分析补充了基因表达研究，并应刺激新的遗传和生化模型的染色体功能。