Nieuwlandt D T, Daniels C J
Department of Microbiology, Ohio State University, Columbus 43210.
J Bacteriol. 1990 Dec;172(12):7104-10. doi: 10.1128/jb.172.12.7104-7110.1990.
The recent development of an efficient transformation method and shuttle vectors for Haloferax volcanii has set the stage for rapid progress in archaebacterial molecular biology. We describe a shuttle-expression vector that can be selected for and maintained in either H. volcanii or Escherichia coli and permits the expression of cloned genes in H. volcanii. The vector, pWL204, was constructed by incorporating an H. volcanii tRNA(Lys) gene promoter into a derivative of the H. volcanii-E. coli shuttle vector pWL102. The vector has been used to express a modified, intron-containing, H. mediterranei tRNA(Trp) gene (tRNA(Trp)-O167). Transcription from the tRNA(Lys) gene promoter in vivo was detected by Northern (RNA) analysis with an oligonucleotide probe complementary to the unique intron sequence of tRNA(Trp)-O167. Dependence of transcription on the tRNA(Lys) promoter was demonstrated by the absence of transcription when the promoter sequence was deleted from the vector and by mapping the transcription initiation site by primer extension.
用于嗜盐栖热袍菌的高效转化方法和穿梭载体的最新进展为古细菌分子生物学的快速发展奠定了基础。我们描述了一种穿梭表达载体，它可以在嗜盐栖热袍菌或大肠杆菌中进行选择和维持，并允许在嗜盐栖热袍菌中表达克隆基因。载体pWL204是通过将嗜盐栖热袍菌tRNA（Lys）基因启动子整合到嗜盐栖热袍菌-大肠杆菌穿梭载体pWL102的衍生物中构建而成的。该载体已用于表达修饰的、含内含子的嗜盐栖地中海嗜盐菌tRNA（Trp）基因（tRNA（Trp）-O167）。通过用与tRNA（Trp）-O167独特内含子序列互补的寡核苷酸探针进行Northern（RNA）分析，检测了体内tRNA（Lys）基因启动子的转录。当从载体中删除启动子序列时转录缺失，以及通过引物延伸对转录起始位点进行定位，证明了转录对tRNA（Lys）启动子的依赖性。
