Shimamoto T, Noguchi K, Kuroda M, Tsuda M, Tsuchiya T
Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Nucleic Acids Symp Ser. 1988(19):171-3.
We constructed a vector to evaluate terminator or attenuator of transcription quantitatively. This vector is a plasmid possessing lac promoter-polylinker-CAT(chloramphenicol acetyltransferase) gene. DNA fragment of interest can be inserted into the polylinker site, and the effect of the DNA fragment on the expression of the downstream gene is evaluated from the CAT activity. We analyzed gene expression of the melibiose operon of Escherichia coli using this plasmid, and found that a DNA fragment containing a large stem-loop structure with boxA sequence in it, which was present between melA and melB, caused strong reduction of gene expression. This DNA portion seems to be responsible for reduced expression of melB, the second gene of the melibiose operon.
我们构建了一个用于定量评估转录终止子或衰减子的载体。该载体是一种含有乳糖启动子-多克隆位点-氯霉素乙酰转移酶（CAT）基因的质粒。感兴趣的DNA片段可插入多克隆位点，通过CAT活性评估该DNA片段对下游基因表达的影响。我们使用该质粒分析了大肠杆菌蜜二糖操纵子的基因表达，发现位于melA和melB之间的一个含有大茎环结构且其中带有boxA序列的DNA片段，导致基因表达大幅降低。这个DNA部分似乎是蜜二糖操纵子第二个基因melB表达降低的原因。
