Probing Chromosome Structure with Phage Mu

已经开发出两种相关的方法来探测活细胞内的细菌染色体结构。一种技术被称为互打印，它使用PCR反应来生成感染噬菌体Mu的细菌种群中转座靶点的高分辨率图像。通过比较体外裸DNA和体内蛋白质结合DNA的PCR图谱，有可能检测到序列特异性DNA结合的位置。用Muprint检测到的蛋白质包括阻遏物和RNA聚合酶。最近开发的一种名为Mu-Screen的新方法可以识别在体内复制Mu转座过程中有效使用的转座靶标。现在可以识别转座子插入的热点和冷点，并在细胞放置在不同的生理环境中时跟踪这些结构的变化。我们将追求两个主要目标。首先，Mu筛选将与大肠杆菌中含有4115/4238个ORF的基因微阵列结合使用，以确定大肠杆菌和鼠伤寒沙门氏菌基因组中最有效的转座靶标。其次，将克隆Mu转座热点，并进行体内和体外Muprint分析，以准确识别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。需要新的方法系统地研究活细胞内的染色体结构，以解决现代分子进化中具有挑战性的问题。随着有效获取几乎任何生物体的全部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-Screen和Muprint技术获得的信息有望为深入了解染色体进化领域和细菌染色质的结构问题提供帮助。微阵列驱动的结构分析是对基因表达研究的补充，应该会激发新的染色体功能的遗传和生化模型。

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.