Cloning of Streptococcus pneumoniae DNA fragments in Escherichia coli requires vectors protected by strong transcriptional terminators.
Unstable recombinant plasmids are frequently encountered when cloning pneumococcal DNA into ordinary Escherichia coli plasmid vectors (e.g., pBR325, pMB9, pHC79). Stassi and Lacks [Gene 18 (1982) 319-328] have shown that the pneumococcal mal region, which exhibits strong promoter activity, interferes with maintenance of a recombinant plasmid in E. coli. In this paper, we report that random pneumococcal DNA fragments cloned in E. coli exhibited a higher frequency of strong promoter activity than did similarly cloned E. coli fragments. Furthermore, shotgun cloning yields for pneumococcal DNA were found to be higher with cloning vectors containing efficient transcriptional terminators surrounding the insertion site than with vectors lacking such protection. Therefore, vectors which carry an efficient transcriptional terminator are likely to be valuable for cloning pneumococcal DNA and stabilizing the recombinants. A new vector, pJDC9, was constructed, containing a lacZ alpha' multiple cloning site surrounded by transcriptional terminators, and an erythromycin-resistant marker expressed both in E. coli and Streptococcus. This plasmid was shown to be unusually effective for cloning of streptococcal genes in E. coli, and is designed to permit excisional cloning of streptococcal DNA in E. coli.
Chen
J D
JD
Laboratory for Cell, Molecular, and Developmental Biology, University of Illinois at Chicago 60680.
Morrison
D A
DA
D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005809:Genes, Regulator; D005822:Genetic Vectors; D011401:Promoter Regions, Genetic; D013296:Streptococcus pneumoniae / Q000235:genetics*; D013728:Terminator Regions, Genetic
D004269:DNA, Bacterial; D004274:DNA, Recombinant