Probing Chromosome Structure and Function With Phage Mu Transposition

希金斯9604875开发了一种新的方法来探测活细胞内的细菌染色体结构。这项技术被称为互打印，使用PCR反应来生成感染噬菌体MU的细菌种群中转座靶点的高分辨率图像。利用Muprint，可以检测到抑制物和RNA聚合酶等序列特异性DNA结合蛋白的位置，识别转座子插入的热点和冷点，并跟踪细胞放置在不同生理环境中时染色体结构的变化。我们将追求两个主要目标。首先，将为鼠伤寒沙门氏菌染色体的40kb片段绘制详细的Muprint图谱，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被严格抑制，但在无氧生长条件下变得活跃转录和翻译。Muprint图谱将提供有关参与抑制或激活这些操纵子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图谱来识别如何保护一大段DNA不受转座影响。转座免疫是指当噬菌体Mu的一个拷贝已经存在于一个染色体位置时，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元素渗透时，免疫保证了复制的Mu元素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。影响免疫如何以及在多大程度上通过染色体传播的遗传因素和DNA位点将被定义。发展系统地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。从多点打印技术获得的信息将提供关于染色体结构的观点，这可能会刺激新的基因组功能的遗传和生化模型。它还将为理解沿着DNA序列产生随机梯度的过程提供一个模型，如酵母中的沉默和高等脊椎动物中的免疫球蛋白重组。

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.