Everett R, Willetts N
EMBO J. 1982;1(6):747-53. doi: 10.1002/j.1460-2075.1982.tb01241.x.
pED806 , a pBR322 derivative carrying the origin of transfer ( oriT ) of F, was rapidly lost from cells carrying an F tra+ plasmid. Instability was increased in a RecA- host, and depended in particular upon the Ftra YZ genes that produce the nick at oriT at which transfer is initiated. Instability was also correlated with the orientation of the oriT fragment in the vector plasmid. Mutants of pED806 selected as being stable in the presence of Flac proved to carry cis-dominant oriT mutations. The oriT site was subcloned from pED806 on a HaeII fragment including a HaeII-Bg/II segment of F DNA approximately 385 base pair (bp) long into the 2.25 kilobase (kb) vector plasmid pED825 , giving pED822 . pED822 was fully proficient for oriT function, and recircularised in recipient cells by a recA- and tra-independent oriT -specific ligation/recombination event. ' Phasmids ' constructed by cloning pED806 or an oriT - mutant into a lambda vector were used to confirm that the nick site in lambda oriT phages grown in the presence of Flac tra+ is indeed at oriT . The nick site in a further lambda oriT phage (ED lambda 102) was then located 140 +/- 20 bp from the Bg/II site forming one terminus of the F fragment cloned in pED806 and pED822 .
pED806是一种携带F因子转移起始点（oriT）的pBR322衍生质粒，在携带F tra⁺质粒的细胞中会迅速丢失。在RecA⁻宿主中，其不稳定性增加，并且特别依赖于F tra YZ基因，该基因在oriT处产生转移起始时的切口。不稳定性还与oriT片段在载体质粒中的方向有关。在Flac存在下被选择为稳定的pED806突变体被证明携带顺式显性oriT突变。oriT位点从pED806亚克隆到一个HaeII片段上，该片段包括一段约385个碱基对（bp）长的F DNA的HaeII - Bg/II片段，进入2.25千碱基（kb）的载体质粒pED825，得到pED822。pED822在oriT功能方面完全正常，并且通过一个不依赖RecA和tra的oriT特异性连接/重组事件在受体细胞中重新环化。通过将pED806或一个oriT突变体克隆到λ载体中构建的“噬菌粒”被用于证实，在Flac tra⁺存在下生长的λ oriT噬菌体中的切口位点确实在oriT处。然后，另一个λ oriT噬菌体（EDλ102）中的切口位点位于距Bg/II位点140±20 bp处，Bg/II位点形成了克隆在pED806和pED822中的F片段的一个末端。
