Grambow N J, Birkeland N K, Anders D L, Christie G E
Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond 23298-0678.
Gene. 1990 Oct 30;95(1):9-15. doi: 10.1016/0378-1119(90)90407-i.
We have fused the promoter (PF) for the P2 late FETUD operon to the gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a plasmid vector. Synthesis of CAT in Escherichia coli strains carrying this plasmid requires the product of the P2 ogr gene or the satellite phage P4 transactivation gene, delta. Our results demonstrate that these phage-encoded transcriptional regulatory proteins are necessary and sufficient for activation of P2 late transcription in this reporter plasmid. Positive regulation of cloned PF is severely impaired in a host strain carrying the rpoA109 mutation. Expression from the cloned promoter thus approximates those features of P2 late transcription that have been shown to occur during normal P2 infection. To define sequences required for promoter function, sequential upstream deletions of PF were generated using BAL 31 nuclease, and the mutant promoters were assayed for cat expression. A sequence between nucleotides -69 and -64 from the transcription start point was found to be essential for promoter activity. This coincides with a region of homology conserved among all four P2 late gene promoters and the two P4 late promoters, and includes an element of dyad symmetry.
我们已将P2晚期FETUD操纵子的启动子（PF）与编码氯霉素乙酰转移酶（CAT）的基因（cat）在质粒载体中融合。携带该质粒的大肠杆菌菌株中CAT的合成需要P2 ogr基因的产物或卫星噬菌体P4反式激活基因δ。我们的结果表明，这些噬菌体编码的转录调节蛋白对于该报告质粒中P2晚期转录的激活是必要且充分的。在携带rpoA109突变的宿主菌株中，克隆的PF的正调控受到严重损害。因此，克隆启动子的表达类似于正常P2感染期间已显示出的P2晚期转录的那些特征。为了确定启动子功能所需的序列，使用BAL 31核酸酶对PF进行了连续的上游缺失，并对突变启动子进行了cat表达检测。发现转录起始点核苷酸-69至-64之间的序列对于启动子活性至关重要。这与所有四个P2晚期基因启动子和两个P4晚期启动子中保守的同源区域一致，并且包括一个二元对称元件。
