Jin H, Wang Y
Institute of Medicinal Biotechnology, CAMS, TUMC, Beijing.
Wei Sheng Wu Xue Bao. 1994 Dec;34(6):415-21.
A DNA fragment containing promoter activity has been cloned from midecamycin producing strain (S. mycarofaciens 1748)., using promoter-probe plasmid vector pIJ486. The molecular size of this fragment was 2.3kb as shown by restriction analysis. A HindIII-HindIII 2.08KB DNA fragment obtained from the original fragment has been analysed by subcloning it into polylinker of vector pIJ486/7 which have opposite direction. The result showed that HindIII-HindIII 2.08kb DNA fragment has promoter activity in both direction. Transformants of plasmid containing this fragment in vector pIJ487 in S. lividans TK24 were resistant to Km in the level of 20mg/ml, but in vector pIJ 486 were resistant to the level of 3mg/ml. It indicated that a rather strong promoter activity region was in the HindIII/XbaI-HindIII direction. BamHI fragments (A-0.79kg, B-0.67kb, C-0.62kb) in 2.08kb DNA fragment have been studied in regards of their promoter activity. The result suggested that A-0.79kb region has the same promoter activity as in HindIII-HindIII 2.08kb DNA fragment.
利用启动子探针质粒载体pIJ486，从麦迪霉素产生菌（弗氏链霉菌1748）中克隆出一段具有启动子活性的DNA片段。经限制性酶切分析表明，该片段的分子大小为2.3kb。从原始片段中获得的一个HindIII - HindIII 2.08KB DNA片段，通过亚克隆到具有相反方向的载体pIJ486/7的多克隆位点中进行了分析。结果表明，HindIII - HindIII 2.08kb DNA片段在两个方向上均具有启动子活性。含有该片段的质粒在变铅青链霉菌TK24的载体pIJ487中的转化子对卡那霉素的抗性水平为20mg/ml，而在载体pIJ486中则为3mg/ml。这表明在HindIII/XbaI - HindIII方向上存在一个相当强的启动子活性区域。对2.08kb DNA片段中的BamHI片段（A - 0.79kb、B - 0.67kb、C - 0.62kb）的启动子活性进行了研究。结果表明，A - 0.79kb区域具有与HindIII - HindIII 2.08kb DNA片段相同的启动子活性。
