Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Lisbon, Portugal.
Appl Microbiol Biotechnol. 2010 Mar;86(2):671-9. doi: 10.1007/s00253-009-2339-3.
ColE1-like plasmids are widely used as expression vectors and as gene delivery vehicles. We have recently described a naturally occurring plasmid deletion phenomenon in the ColE1-type plasmid, pCI-neo, which leads to the detectable expression of an apparently promotorless kanamycin resistance gene. In the current work, we found that the expression of that aminoglycoside phosphotransferase (aph) gene is regulated by an RNAII preprimer promoter located within the plasmid ColE1 replication origin, as a consequence of the extension of the RNA II species for at least 1.5 kb, up to the aph gene. This mechanism is dependent on the nonformation and/or dissociation of the hybrid between plasmid DNA and RNA II preprimer transcript. This is the first in vivo description of RNA II transcription beyond ori in wild-type Escherichia coli strains and nonmutated RNAII plasmid sequences resulting in productive transcription of distant downstream genes. Our results raise questions about unwanted expression of genes from expression or cloning vectors of ColE1 type and highlight the importance of a more careful design of ColE1-derived plasmid vectors.
ColE1 样质粒被广泛用作表达载体和基因传递载体。我们最近描述了 ColE1 型质粒 pCI-neo 中一种自然发生的质粒缺失现象，导致可检测到明显无启动子的卡那霉素抗性基因的表达。在当前的工作中，我们发现该氨基糖苷磷酸转移酶 (aph) 基因的表达受到位于质粒 ColE1 复制起点内的 RNAII 前引物启动子的调控，这是由于 RNAII 种至少延伸了 1.5kb，直到 aph 基因。这种机制依赖于质粒 DNA 和 RNA II 前引物转录物之间杂种的非形成和/或解离。这是第一个在野生型大肠杆菌菌株中 RNA II 转录超越 ori 的体内描述，并且非突变 RNAII 质粒序列导致下游基因的有效转录。我们的结果引发了对 ColE1 型表达或克隆载体中基因意外表达的质疑，并强调了更仔细设计 ColE1 衍生质粒载体的重要性。
