Donoghue D J, Sharp P A
Gene. 1977 May;1(3-4):209-27. doi: 10.1016/0378-1119(77)90046-4.
An attenuated bacteriophage lambda has been prepared for proposed use as an EK2 vector. This phage, designated lambdagt vir Jam27 Zam718-lambdaB' can accomodate up to 11.10(6) daltons of foreign DNA inserted through Eco RI ends. The virulence mutations and nin 5 reduce the frequency of lysogen and/or plasmid formation. The mutations Jam27 and Zam718 require a suppressor in the bacterial host. The phage recombination functions contained in the EcoRIlambdaC fragment have been deleted, and only the EcoRIlambdaB fragment remains (in reverse orientation) in the center portion of the vector. In addition, this phage adsorbs to sensitive bacteria at a significantly reduced rate, conferring another block to the escape of free phage. Model recombinants have been constructed by in vitro recombination with an EcoRI fragment coding for kanamycin resistance (originally derived from R-factor R6-5). This fragment of DNA is 4.6.10(6) daltons in size, contains an inverted repeat, and also appears to contain a promoter for the kanamycin resistance gene. Using this model recombinant, the rate of transfer of kanamycin resistance to permissive and nonpermissive strains of E. coli has been measured.
已制备出一种减毒噬菌体λ，拟用作EK2载体。这种噬菌体命名为λgt vir Jam27 Zam718 - λB'，通过Eco RI末端插入的外源DNA容纳量可达11×10⁶道尔顿。毒力突变和nin 5降低了溶原菌形成和/或质粒形成的频率。Jam27和Zam718突变在细菌宿主中需要一个抑制基因。Eco RIλC片段中所含的噬菌体重组功能已被删除，载体中心部分仅保留Eco RIλB片段（方向相反）。此外，这种噬菌体以显著降低的速率吸附到敏感细菌上，为游离噬菌体的逸出提供了另一重阻碍。通过与编码卡那霉素抗性的Eco RI片段（最初来源于R因子R6 - 5）进行体外重组构建了模型重组体。该DNA片段大小为4.6×10⁶道尔顿，包含一个反向重复序列，似乎还含有卡那霉素抗性基因的启动子。利用这个模型重组体，已测定了卡那霉素抗性向大肠杆菌允许菌株和非允许菌株的转移速率。
