Hayakawa Y, Matsubara K
Mol Gen Genet. 1979 Jan 16;169(1):107-12. doi: 10.1007/BF00267551.
A derivative of plasmid F which is packageable in lambda phage coat was constructed using techniques of in vitro recombination. This plasmid is composed of three DNA fragments generated by restriction enzyme EcoRI: a miniF fragment (fragment f5 of F'lac) which is able to replicate autonomously, a DNA fragment from Staphylococcus plasmid that carries the beta-lactamase gene, and a portion of guaA (B) transducing lambda phage DNA carrying lambda cohesive ends (cos site) along with almost all the late genes but devoid of all those genes and sites that are needed for replication, regulation, and recombination. The hybrid plasmid has a molecular weight of 2.7 x 10(7) daltons, about 84% size of lambda phage genome, and can be packaged in lambda coat when helper phage replicates in the plasmid-carrier cell. The packaged plasmid and the helper lambda phage particles are separated by CsCl density gradient centrifugation. The replication characteristics of the recombinant plasmid are all those of F including the copy number, incompatibility, and curing with acidine orange. The packaged plasmid is injected into an F- cell and establishes a plasmid state with normal efficiency. In F+ or Hfr cells, the resident F factor hinders this process.
利用体外重组技术构建了一种可包装在λ噬菌体外壳中的质粒F衍生物。该质粒由限制性内切酶EcoRI产生的三个DNA片段组成：一个能够自主复制的miniF片段（F'lac的f5片段）、一个来自葡萄球菌质粒且携带β-内酰胺酶基因的DNA片段，以及一部分guaA（B）转导λ噬菌体DNA，该片段携带λ粘性末端（cos位点）以及几乎所有晚期基因，但缺少复制、调控和重组所需的所有那些基因和位点。该杂种质粒的分子量为2.7×10⁷道尔顿，约为λ噬菌体基因组大小的84%，当辅助噬菌体在携带质粒的细胞中复制时，它可以被包装在λ外壳中。通过氯化铯密度梯度离心将包装好的质粒和辅助λ噬菌体颗粒分离。重组质粒的复制特性与F质粒的所有特性相同，包括拷贝数、不相容性以及用吖啶橙消除。包装好的质粒被注入F⁻细胞并以正常效率建立质粒状态。在F⁺或Hfr细胞中，常驻F因子会阻碍这一过程。
