Kiss A, Sain B, Kiss I, Boros I, Udvardy A, Venetianer P
Gene. 1978 Oct;4(2):137-52. doi: 10.1016/0378-1119(78)90026-4.
The DNA of the specialized transducing phage lambdarifd18, which carries a bacterial rRNA transcription unit, was digested with restriction enzymes EcoRI and/or BamHI. Attempts were made to clone fragments containing the presumed rRNA promoter region or the entire rRNA gene in RSF2124 or pBR313 plasmid vectors with the following results: (1) We failed to clone an EcoRI fragment with the rRNA promoter region in plasmid RSF2124. (2) A smaller EcoRI-BamHI fragment with the rRNA promoter was also unclonable by itself, but one recombinant was found containing this fragment together with another large (7 Mdaltons) fragment, derived from phage lambda. The presence of this large fragment proved to be essential. The identity of these DNA fragments in the recombinant clone was confirmed by redigestion with several restriction enzymes, hybridization with rRNA, and in vitro transcription experiments, which showed preferential rRNA transcription. (3) A BamHI fragment encompassing the entire rRNA gene was easily cloned. Such stable clones carried a doubled number of rRNA genes. In vitro transcription using the recombinant plasmid resulted in 70% rRNA transcription. These recombinant clones allow the easy purification of the relevant DNA fragments for further investigation including sequencing.
携带细菌rRNA转录单位的特异性转导噬菌体λrifd18的DNA用限制性内切酶EcoRI和/或BamHI进行消化。尝试将含有假定的rRNA启动子区域或整个rRNA基因的片段克隆到RSF2124或pBR313质粒载体中，结果如下：（1）我们未能在质粒RSF2124中克隆到带有rRNA启动子区域的EcoRI片段。（2）一个带有rRNA启动子的较小的EcoRI - BamHI片段本身也无法克隆，但发现一个重组体含有该片段以及另一个来自噬菌体λ的大（7兆道尔顿）片段。事实证明这个大片段的存在至关重要。通过用几种限制性内切酶重新消化、与rRNA杂交以及体外转录实验（显示出优先的rRNA转录），证实了重组克隆中这些DNA片段的身份。（3）一个包含整个rRNA基因的BamHI片段很容易被克隆。这样的稳定克隆携带了双倍数量的rRNA基因。使用重组质粒进行体外转录产生了70%的rRNA转录。这些重组克隆便于纯化相关的DNA片段以进行包括测序在内的进一步研究。
