Patrick Higgins N
Department of Biochemistry and Molecular Genetics, The University of Alabama at Birmingham, Kaul Human Genetics Building, Room 524A, 720, 20th Street South, Birmingham, AL, 35294-0024, USA.
Methods Mol Biol. 2018;1681:303-314. doi: 10.1007/978-1-4939-7343-9_22.
Bacteriophage Mu infects a broad range of gram-negative bacteria. After infection, Mu amplifies its DNA through a coupled transposition/replication cycle that inserts copies of Mu throughout all domains of the folded chromosome. Mu has the most relaxed target specificity of the known transposons (Manna et al., J Bacteriol 187: 3586-3588, 2005) and the Mu DNA packaging process, called "headful packaging", incorporates 50-150 bp of host sequences covalently bound to its left end and 2 kb of host DNA linked to its right end into a viral capsid. The combination of broad insertion coverage and easy phage purification makes Mu ideal for analyzing chromosome dynamics and DNA structure inside living cells. "Mu printing" (Wang and Higgins, Mol Microbiol 12: 665-677, 1994; Manna et al., J Bacteriol 183: 3328-3335, 2001) uses the polymerase chain reaction (PCR) to generate a quantitative fine structure map of Mu insertion sites within specific regions of a bacterial chromosome or plasmid. A complementary technique uses microarray platforms to provide quantitative insertion patterns covering a whole bacterial genome (Manna et al., J Bacteriol 187: 3586-3588, 2005; Manna et al., Proc Natl Acad Sci U S A 101: 9780-9785, 2004). These two methods provide a powerful complementary system to investigate chromosome structure inside living cells.
噬菌体Mu可感染多种革兰氏阴性细菌。感染后，Mu通过一个耦合转座/复制循环来扩增其DNA，该循环会在折叠染色体的所有区域插入Mu的拷贝。Mu具有已知转座子中最宽松的靶标特异性（Manna等人，《细菌学杂志》187: 3586 - 3588，2005年），并且Mu的DNA包装过程，即“头部充满式包装”，会将50 - 150碱基对共价连接于其左端的宿主序列以及连接于其右端的2千碱基对宿主DNA纳入病毒衣壳。广泛的插入覆盖范围与简便的噬菌体纯化方法相结合，使得Mu成为分析活细胞内染色体动态和DNA结构的理想工具。“Mu印记法”（Wang和Higgins，《分子微生物学》12: 665 - 677，1994年；Manna等人，《细菌学杂志》183: 3328 - 3335，2001年）利用聚合酶链反应（PCR）生成细菌染色体或质粒特定区域内Mu插入位点的定量精细结构图。一项互补技术利用微阵列平台来提供覆盖整个细菌基因组的定量插入模式（Manna等人，《细菌学杂志》187: 3586 - 3588，2005年；Manna等人，《美国国家科学院院刊》101: 9780 - 9785，2004年）。这两种方法为研究活细胞内的染色体结构提供了一个强大的互补系统。
