Pátek Miroslav, Muth Günther, Wohlleben Wolfgang
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, CZ-14220 Prague 4, Czech Republic.
J Biotechnol. 2003 Sep 4;104(1-3):325-34. doi: 10.1016/s0168-1656(03)00159-7.
The function of seven promoters from Corynebacterium glutamicum, P-hom, P-leuA, P-per, P-aes1, P-aes2, P-45, and P-104, was analyzed in a heterologous background. DNA fragments carrying the promoters were cloned into shuttle promoter-probe vectors replicating in Escherichia coli and C. glutamicum (pET2), Streptomyces lividans (pGL7011) and Bacillus subtilis (pRB394). With the exception of P-hom, P-leuA and P-104 in B. subtilis, all promoters were found to be active in all species. Non-radioactive methods of primer-extension analysis and of S1-nuclease protection assay using automatic sequencer were developed to determine the respective transcriptional start points (TSPs). All TSPs were determined by primer extension and in two promoters (P-45 and P-hom) the main TSPs were confirmed by S1-mapping. While the main TSPs were identical in all four species, utilization of multiple TSPs varied among the species and additional TSPs were detected in S. lividans. Knowledge of the efficiency of promoters and of exact respective TSPs may be of practical value for the construction of expression systems in a heterologous background.
在异源背景下分析了谷氨酸棒杆菌的7个启动子（P-hom、P-leuA、P-per、P-aes1、P-aes2、P-45和P-104）的功能。将携带这些启动子的DNA片段克隆到穿梭启动子探针载体中，这些载体可在大肠杆菌和谷氨酸棒杆菌（pET2）、天蓝色链霉菌（pGL7011）和枯草芽孢杆菌（pRB394）中复制。除了枯草芽孢杆菌中的P-hom、P-leuA和P-104外，所有启动子在所有物种中均有活性。开发了使用自动测序仪的引物延伸分析和S1核酸酶保护测定的非放射性方法来确定各自的转录起始点（TSP）。所有TSP均通过引物延伸确定，在两个启动子（P-45和P-hom）中，主要TSP通过S1作图得到证实。虽然所有四个物种中的主要TSP相同，但多个TSP的利用在不同物种间有所不同，并且在天蓝色链霉菌中检测到了额外的TSP。启动子效率和各自确切TSP的知识对于在异源背景下构建表达系统可能具有实际价值。
