Huan L, Sun H, Chen X, Zhuang Z, Xue Y
Institute of Microbiology, Chinese Academy of Sciences, Beijing.
Yi Chuan Xue Bao. 1997 Oct;24(5):471-9.
Promoter and signal peptide function fragments from Lactococcus lactis were cloned in E. coli using a promoter-signal sequence probe vector pGPB14. Forty-two clones were obtained, whose level of resistance to ampicillin ranged from 100 to 8000 micrograms/ml. Eight clones were selected for beta-lactamase distribution assay. The beta-lactamase activity was found mainly in the periplasm, which indicated successful secretion of the enzyme. Southern hybridization test demonstrated that the inserted fragments were indeed from L. lactis. Restriction enzyme analysis revealed that the size of inserted fragments ranged from 80bp to 400 bp, four of which were sequenced on the vector pGEM-3Zf. It was found that the inserted fragments of pSEQ8 and pSEQ12 turned out to be part of the inserted fragments of pSEQ4 and pSEQ17 respectively. Promoter and translation initiation codon were found among all four fragments signal sequenced. One typical S. D. sequence and one atypical signal sequence were found in pHSB4 and pHSB8, while the other two contained no typical S. D. sequence and signal peptide sequence. In addition, it was found that the upstream regions of the promoter contributed to the efficiency of transcription initiation.
利用启动子 - 信号序列探针载体pGPB14，将乳酸乳球菌的启动子和信号肽功能片段克隆到大肠杆菌中。获得了42个克隆，其对氨苄青霉素的抗性水平在100至8000微克/毫升之间。选择了8个克隆进行β-内酰胺酶分布测定。发现β-内酰胺酶活性主要存在于周质中，这表明该酶成功分泌。Southern杂交试验表明插入片段确实来自乳酸乳球菌。限制性内切酶分析显示插入片段的大小在80bp至400bp之间，其中4个在载体pGEM - 3Zf上进行了测序。发现pSEQ8和pSEQ12的插入片段分别原来是pSEQ4和pSEQ17插入片段的一部分。在所有四个信号测序的片段中都发现了启动子和翻译起始密码子。在pHSB4和pHSB8中发现了一个典型的SD序列和一个非典型信号序列，而另外两个则没有典型的SD序列和信号肽序列。此外，发现启动子的上游区域有助于转录起始的效率。
