Scherzinger E, Lauppe H F, Voll N, Wanke M
Nucleic Acids Res. 1980 Mar 25;8(6):1287-305. doi: 10.1093/nar/8.6.1287.
Two full-length contiguous HpaI fragments of the 0 to 18.2% region of T7 H DNA (HpF-H and HpG) were inserted into plasmids pHV14 or pC194 using oligo(dG . dC) connectors or synthetic HindIII adaptors. Amplification of the two early T7 fragments was achieved by transforming lysostaphin-treated S. aureus W57 with the hybrid plasmids. Experimental evidence is presented suggesting that neither of these T7 segments can be cloned in an intact form in E. coli. One of the hybrids, pHV14-HpF-H, proved to be unstable even in B. subtilis 168. The supercoiled recombinant plasmids were tested for their capacity to support RNA synthesis by purified E. coli or T7 RNA polymerases and to serve as templates in a cell-free T7 DNA replication system. The results of these in vitro studies indicate the presence of active "early" promoters in the cloned fragment HpF-H and active "late" promoters, as well as a functional origin of replication in the cloned fragment HpG.
利用寡聚（dG·dC）连接子或合成的HindIII接头，将T7 H DNA 0至18.2%区域的两个全长相邻HpaI片段（HpF-H和HpG）插入质粒pHV14或pC194中。通过用杂交质粒转化经溶葡萄球菌素处理的金黄色葡萄球菌W57，实现了两个早期T7片段的扩增。实验证据表明，这两个T7片段均无法以完整形式克隆到大肠杆菌中。其中一个杂种质粒pHV14-HpF-H，甚至在枯草芽孢杆菌168中也被证明是不稳定的。对超螺旋重组质粒进行了测试，以检测其支持纯化的大肠杆菌或T7 RNA聚合酶进行RNA合成的能力，以及在无细胞T7 DNA复制系统中作为模板的能力。这些体外研究结果表明，克隆片段HpF-H中存在活性“早期”启动子，克隆片段HpG中存在活性“晚期”启动子以及功能性复制起点。
