Groisman E A, Casadaban M J
Gene. 1987;51(1):77-84. doi: 10.1016/0378-1119(87)90476-8.
A mini-Mu bacteriophage, containing the cohesive-end packaging site (cos) from a lambda-phi 80 hybrid phage, a high-copy-number plasmid replicon, and a kanamycin-resistance gene for independent selection, was constructed to clone genes in vivo. This mini-Mu element can be derepressed to transpose at a high frequency. DNA segments that become flanked by copies of this mini-Mu element in the same orientation can be packaged by a helper lambda phage. The resulting lambda lysate can be used to infect recipient cells where the injected DNA can circularize by annealing at the cos termini. Drug-resistant transductants obtained carry the mini-Mu-replicon cosmid element with inserts of different nucleotide sequences. These are analogous to recombinant DNA clones generated in vitro with restriction endonuclease cutting and ligase joining reactions replaced by the Mu transposition process. Clones of particular genes were isolated by their ability to complement specific mutations. Both recA+ and recA- recipient cells can be used with equal efficiency. Clones obtained with a helper lambda phage require the presence of the cos site in the mini-Mu replicon. They carry larger inserts than those isolated with the same mini-Mu element and Mu as a helper phage. The mini-Mu replicon-cosmid bacteriophage contains a lac-gene fusing segment for isolating fusions of lac operon DNA to gene control regions in the cloned sequences. Independent clones of a particular gene can be used to prepare a restriction map of the gene and its flanking regions.
构建了一种微型 Mu 噬菌体，它含有来自 λ-φ80 杂交噬菌体的粘性末端包装位点（cos）、高拷贝数质粒复制子以及用于独立筛选的卡那霉素抗性基因，用于体内基因克隆。这种微型 Mu 元件可以去阻遏并以高频转座。在同一方向上被这种微型 Mu 元件的拷贝侧翼包围的 DNA 片段可以被辅助 λ 噬菌体包装。产生的 λ 裂解物可用于感染受体细胞，注入的 DNA 可在 cos 末端退火而环化。获得的抗药转导子携带带有不同核苷酸序列插入片段的微型 Mu 复制子粘粒元件。这些类似于通过 Mu 转座过程取代体外限制性内切酶切割和连接酶连接反应而产生的重组 DNA 克隆。通过其互补特定突变的能力分离特定基因的克隆。recA+和 recA-受体细胞都可以以相同的效率使用。用辅助 λ 噬菌体获得的克隆需要微型 Mu 复制子中存在 cos 位点。它们携带的插入片段比用相同的微型 Mu 元件和 Mu 作为辅助噬菌体分离的插入片段更大。微型 Mu 复制子粘粒噬菌体含有一个 lac 基因融合片段，用于分离 lac 操纵子 DNA 与克隆序列中基因控制区域的融合。特定基因的独立克隆可用于制备该基因及其侧翼区域的限制性图谱。
