Wild J, Hradecná Z, Pósfai G, Szybalski W
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 53706, USA.
Gene. 1996 Nov 7;179(1):181-8. doi: 10.1016/s0378-1119(96)00487-8.
A prerequisite for sequencing large genomes is to obtain 30- to 150-kb genomic DNA fragments in adequate quantity. Previously, we developed a system which enables one to excise and amplify in vivo such segments directly from the Escherichia coli genome. This system, which employed the yeast Flp/FRT elements for excision and the plasmid R6K-based replication machinery for DNA amplification, permits one to bypass conventional cloning [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392-2398]. To extend the applicability of such a system to many species, we describe here a broad-host-range (bhr) system in which the amplification of the excised DNA fragment depends on the oriV element and the Rep (TrfA) protein from the promiscuous RK2/RP4 plasmid. We have constructed insertion plasmids which carry the FRT and oriV sites. To introduce such plasmids into the appropriate position in the host genome, a short genomic sequence homologous to this position was cloned into the multiple cloning site (MCS) of the FRT/oriV insertion plasmid and then recombined into this position in the genome by RecA-mediated recombination. In such a manner, many strains with single FRT/oriV insertions at various positions could be generated. Subsequent genetic crosses or phage transduction allow two neighboring FRT/oriV sites (less than 150 kb apart) to be brought into a single genome. In the present report, the lacZ and phoB sites, which are 51 kb apart in the E. coli genome, were used for the introduction of the FRT/oriV sites. To deliver the Flp (excision) and Rep (amplification) functions in trans, the yeast FLP and RK2 plasmid trfA genes were placed under the control of the Ptet promoter/operator which is tightly regulated by the TetR repressor. The addition of heated chlortetracycline (cTc) inactivates TetR, turning on the synthesis of Flp and TrfA, which respectively, execute (i) excision of the 51-kb genomic segment between the two FRT sites (in lacZ and in phoB), and (ii) its amplification.
对大型基因组进行测序的一个先决条件是获得足够数量的30至150千碱基的基因组DNA片段。此前，我们开发了一种系统，该系统能够直接从大肠杆菌基因组中体内切除并扩增此类片段。该系统利用酵母Flp/FRT元件进行切除，并利用基于质粒R6K的复制机制进行DNA扩增，从而使人们能够绕过传统克隆方法[波斯法伊等人（1994年）《核酸研究》22卷，2392 - 2398页]。为了将这样一个系统的适用性扩展到许多物种，我们在此描述一种广宿主范围（bhr）系统，其中切除的DNA片段的扩增依赖于来自通用性RK2/RP4质粒的oriV元件和Rep（TrfA）蛋白。我们构建了携带FRT和oriV位点的插入质粒。为了将此类质粒引入宿主基因组的适当位置，将与该位置同源的短基因组序列克隆到FRT/oriV插入质粒的多克隆位点（MCS）中，然后通过RecA介导的重组将其重组到基因组中的该位置。通过这种方式，可以产生许多在不同位置有单个FRT/oriV插入的菌株。随后的遗传杂交或噬菌体转导可使两个相邻的FRT/oriV位点（相距小于150千碱基）进入单个基因组。在本报告中，大肠杆菌基因组中相距51千碱基的lacZ和phoB位点被用于引入FRT/oriV位点。为了反式提供Flp（切除）和Rep（扩增）功能，将酵母FLP和RK2质粒trfA基因置于由TetR阻遏物严格调控的Ptet启动子/操纵子的控制之下。加入加热的金霉素（cTc）会使TetR失活，开启Flp和TrfA的合成，它们分别执行（i）切除两个FRT位点（在lacZ和phoB中）之间的51千碱基基因组片段，以及（ii）其扩增。
