Probing Chromosome Structure and Function With Phage Mu Transposition

Higgins 9604875一种新的方法已经开发出来，用于探测活细胞内细菌染色体的结构。 这种称为Mupringing的技术使用PCR反应来生成感染噬菌体MU的细菌群体中转座靶位点的高分辨率图像。 使用Muprints，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，识别转座子插入的热点和冷点，并在细胞置于不同生理环境时跟踪染色体结构的变化。 将追求两个主要目标。 首先，详细的Muprint地图将为一个40 kb的片段的S。鼠伤寒杆菌染色体，其中包括cob和pdu操纵子（占整个基因组的1%）。 这些操纵子在需氧生长条件下被紧密抑制，但在厌氧生长条件下被积极转录和翻译。 Muprint图谱将提供有关参与这些操纵子的抑制或激活的蛋白质结合的详细信息，包括ArcA，ArcB，PocR和CRP。 第二个目标是使用Muprint映射来辨别大的DNA片段如何被屏蔽而不被转座。 转座免疫意味着当噬菌体Mu的拷贝已经存在于一个染色体位点时，第二个Mu转座子移动到第一个转座子附近的频率非常低。 因此，当染色体被复制的Mu元件浸润时，免疫力保证了复制的Mu元件在细菌染色体上相对均匀地分布。 将测量cobT基因中Mu插入的免疫距离。 遗传因素和DNA位点影响免疫力如何以及如何通过染色体传播将被定义。 发展系统研究活细胞内蛋白质-DNA运动机制的方法一直是现代分子生物学中最具挑战性和最令人困惑的问题之一。 从Mupringing技术获得的信息将提供一个染色体结构的视角，可能会刺激新的基因组功能的遗传和生物化学模型。 它还将提供一个模型，用于理解过程中产生随机梯度沿着DNA序列一样沉默酵母和免疫球蛋白重组在高等脊椎动物。

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.