Mapping Chromosome Accessability to Transposition Using Microarrays

已经开发了两种相关的方法来研究活细菌内部的染色体结构。一种称为Mubrinting的技术使用PCR反应来产生噬菌体Mu在染色体中插入的高分辨率图谱。通过比较特定DNA区域的体外和体内Mubrints，可以监测阻遏物、染色体分隔蛋白和RNA聚合酶的结合。一种新的方法利用DNA微阵列和生物信息学来产生染色体结构的全基因组扫描。微阵列“快照”鉴定具有非常高和非常低频率的转座子插入的染色体位置。本项目的三个目标是：（i）制作细胞染色体的基因组快照，所述细胞生长于90分钟倍增时间（最低葡萄糖培养基）、125分钟倍增时间（最低丙氨酸）和300分钟倍增时间（最低琥珀酸盐培养基）。当与来自细胞以最大速度生长的快照相结合时，这些信息显示了染色体结构如何响应细胞代谢需求而变化。(ii)明确增强和掩盖DNA对Mu转座可及性的分子机制。将使用体外和体内高分辨率Mubrint反应研究顺式和反式调节因子参与遗传控制的DNA位点。(iii)E. coli和革兰氏阳性菌S.将比较鼠伤寒菌的转座模式，以了解进化如何改变63%的基因中的转座子，这些基因自从这两种细菌从共同祖先分化以来一直是保守的。这些研究为理解染色体结构提供了一种新的生物化学模型，并允许从高中到研究生院的学生研究两种遗传相关细菌的进化差异。

Two related methods have been developed to study chromosome structure inside living bacteria. One technique called Muprinting uses PCR reactions to generate high resolution maps of phage Mu insertions in chromosomes. By comparing in vitro and in vivo Muprints of specific DNA regions, the binding of repressors, chromosome partitioning proteins, and RNA polymerase can be monitored. A new method exploits DNA microarrays and bioinformatics to generate genome-wide scans of chromosome structure. Microarray "snapshots" identify chromosomal locations with very high and very low frequencies of transposon insertions. Three goals of the current project are: (i) Make genome snapshots of chromosomes for cells growing with 90 min. doubling time (minimal glucose medium), 125 min doubling time (minimal alanine) , and 300 min. doubling time (minimal succinate medium). When combined with snapshots from cells growing at maximal speed, this information shows how chromosome structure changes in response to cellular metabolic needs. (ii) Define molecular mechanisms that enhance and cloak DNA accessibility to Mu transposition. DNA sites where cis- and trans-regulatory factors are implicated in genetic control will be studied using high resolution in vitro and in vivo Muprint reactions. (iii) E. coli and S. typhimurium transposition patterns will be compared to learn how evolution has altered transposon in the 63% of genes that have been conserved since divergence of these two bacteria from common ancestors. These studies provide a new biochemical model to understand chromosome structure and allow students from high school to graduate school to study evolutionary differences in two genetically related bacteria.