An expression vector for the archaebacterium Haloferax volcanii.
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.
Nieuwlandt
D T
DT
Department of Microbiology, Ohio State University, Columbus 43210.
Daniels
C J
CJ
D001105:Archaea / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics; D012357:RNA, Transfer, Lys / Q000235:genetics; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012357:RNA, Transfer, Lys