Probing Chromosome Structure and Function With Phage Mu Transposition

利用噬菌体 Mu 转座探测染色体结构和功能

• 批准号: 9604875
• 负责人: Norman Higgins
• 金额: $ 28.5万
• 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: 1997
• 资助国家: 美国
• 起止时间: 1997-03-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604875&HistoricalAwards=false
• 关键词: Probing; Chromosome; Structure; Function; Phage

Higgins 9604875 A new method has been developed to probe the structure of bacterial chromosomes inside living cells. This technique, which is called Muprinting, uses PCR reactions to generate a high-resolution picture of the transposition target sites in a bacterial population infected with phage MU. With Muprints, it is possible to detect the location of sequence-specific DNA binding proteins like repressors and RNA polymerases, to identify hot spots and cold spots for transposon insertion, and to follow changes in chromosome structure when cells are placed in different physiological environments. Two primary objectives will be pursued. First, detailed Muprint maps will be made for a 40 kb segment of the S. typhimurium chromosome, which includes the cob and pdu operons (1% of the whole genome). These operons are tightly repressed under aerobic growth conditions but become actively transcribed and translated under anaerobic growth conditions. The Muprint profile will provide detailed information about the binding of proteins involved in repression or activation of these operons, including ArcA, ArcB, PocR, and CRP. The second objective is to use the Muprint mapping to discern how a large segment of DNA can become shielded from transposition. Transposition immunity means that when a copy of phage Mu already exists at one chromosomal site, the frequency of a second Mu transposon moving near the first is very low. Thus, as a chromosome becomes infiltrated with replicating Mu elements, immunity guarantees that the replicating Mu elements become spaced relatively evenly over the bacterial chromosome. The immunity distance will be measured for a Mu insertion in the cobT gene. Genetic factors and DNA sites that influence how and how far immunity spreads through a chromosome will be defined. Developing methods to systematically investigate the mechanics of protein-DNA movement inside living cells has been one of the most challenging and perplexing problems in modern molecular biology. T he information gained from Muprinting technology will provide a perspective on chromosome structure that may stimulate new genetic and biochemical models for genome function. It will also provide a model for understanding processes that generate stochastic gradients along the DNA sequence like silencing in yeast and immunoglobulin recombination in higher vertebrates.

希金斯9604875已经开发了一种新方法来探测活细胞内细菌染色体的结构。 该技术称为muprinting，使用PCR反应来产生感染噬菌体MU的细菌种群中的换位目标位点的高分辨率图片。 使用默蒙图，可以检测序列特异性DNA结合蛋白（如阻遏物和RNA聚合酶）的位置，以鉴定用于转座子插入的热点和冷点，并遵循将细胞放置在不同生理环境中的细胞时染色体结构的变化。 将实现两个主要目标。 首先，将为鼠伤寒链球菌染色体的40 kb段制作详细的默认图，其中包括COB和PDU操纵子（占整个基因组的1％）。 这些操纵子在有氧生长条件下严重抑制，但在厌氧生长条件下会积极转录和翻译。 Muprint曲线将提供有关与ARCA，ARCB，POCR和CRP在内的蛋白质结合或激活涉及的蛋白质的详细信息。 第二个目标是使用默特图映射来辨别大部分DNA如何避免转置。 转座免疫意味着，当噬菌体MU的副本已经存在于一个染色体部位时，第二个MU转座子的频率在第一个染色体附近移动的频率非常低。 因此，随着染色体被复制的MU元素浸润，免疫力可以保证复制的MU元素在细菌染色体上的间隔相对均匀。 将测量在COBT基因中插入MU的免疫距离。 将定义影响免疫力如何以及如何通过染色体扩散的遗传因素和DNA位点。 开发系统研究活细胞内蛋白-DNA运动的力学的方法一直是现代分子生物学中最具挑战性和最困惑的问题之一。 从默许技术中获得的信息将提供有关染色体结构的观点，该结构可能刺激基因组功能的新遗传和生化模型。 它还将提供一个模型，以理解沿着DNA序列产生随机梯度的过程，例如酵母中的沉默和较高脊椎动物中的免疫球蛋白重组。