Probing Chromosome Structure with Phage Mu

University Of Alabama At Birmingham, Birmingham AL

Investigators

Abstract

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.