Honigman A, Mahajna J, Altuvia S, Koby S, Teff D, Locker-Giladi H, Hyman H, Kronman C, Oppenheim A B
Gene. 1985;36(1-2):131-41. doi: 10.1016/0378-1119(85)90077-0.
We have constructed synthetic operons in which two genes (cat and lacZ or cat and galK) were placed in tandem under the control of the bacteriophage lambda oLpL operator and promoter. Restriction sites were introduced between the promoter and the proximal cat gene or between the cat and lacZ or galK genes. In the latter case, introduction of a transcriptional terminator between the two structural genes should affect only the distal gene. Thus, following induction, the expression of the cat gene serves as an internal control, compensating for changes due to plasmid copy number or possible decrease in transcription initiation. We used these plasmids to select a lambda DNA fragment which includes the N-unresponsive tJ transcriptional terminator. This DNA fragment was inserted between the cat and galK genes. Enzymatic assays of these two gene activities following induction indicate that transcripts initiated at the pL promoter under N+ conditions terminate at tJ between the two genes. S1-nuclease analysis showed that these transcripts terminate at several sites in the tJ region. Similar results were obtained whether the host cells were RNaseIII+ or RNaseIII-. As a control, we showed a complete antitermination of the lambda t'I terminator under similar conditions, indicating that a sufficient amount of the N gene product is made from one N gene copy to suppress terminators carried on multicopy plasmids.
我们构建了合成操纵子，其中两个基因（cat和lacZ或cat和galK）串联置于噬菌体λoLpL操纵子和启动子的控制之下。在启动子与近端cat基因之间或cat与lacZ或galK基因之间引入了限制性酶切位点。在后一种情况下，在两个结构基因之间引入转录终止子应仅影响远端基因。因此，诱导后，cat基因的表达可作为内部对照，补偿由于质粒拷贝数变化或转录起始可能降低而导致的变化。我们使用这些质粒筛选出一个包含对N不敏感的tJ转录终止子的λDNA片段。该DNA片段插入到cat和galK基因之间。诱导后对这两种基因活性的酶促分析表明，在N+条件下从pL启动子起始的转录本在两个基因之间的tJ处终止。S1核酸酶分析表明，这些转录本在tJ区域的几个位点终止。无论宿主细胞是RNaseIII+还是RNaseIII-，都得到了类似的结果。作为对照，我们发现在类似条件下λt'I终止子完全抗终止，这表明从一个N基因拷贝产生的足够量的N基因产物能够抑制多拷贝质粒上携带的终止子。
