Mapping Chromosome Accessability to Transposition Using Microarrays

使用微阵列绘制染色体易接近性至转座的图谱

• 批准号: 0615953
• 负责人: Norman Higgins
• 金额: --
• 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: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0615953&HistoricalAwards=false
• 关键词: Mapping; Chromosome; Accessability; Transposition; Using

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.

目前已有两种相关的方法来研究活细菌内部的染色体结构。一种叫做多重印刷的技术使用聚合酶链反应来生成染色体中噬菌体Mu插入的高分辨率地图。通过比较体外和体内特定DNA区域的多印体，可以监测抑制因子、染色体分配蛋白和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”可识别转座子插入频率非常高和非常低的染色体位置。当前项目的三个目标是：(i)为90分钟倍增时间（最小葡萄糖培养基）、125分钟倍增时间（最小丙氨酸）和300分钟倍增时间（最小琥珀酸培养基）生长的细胞制作染色体基因组快照。当与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）定义增强和掩盖DNA对Mu转位的可及性的分子机制。在遗传控制中涉及顺式和反式调控因子的DNA位点将使用高分辨率的体外和体内Muprint反应进行研究。（iii）将比较大肠杆菌和鼠伤寒沙门氏菌的转座子模式，以了解进化如何改变这两种细菌从共同祖先分化以来保存的63%基因中的转座子。这些研究为理解染色体结构提供了一个新的生化模型，并允许从高中到研究生的学生研究两种遗传相关细菌的进化差异。