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Genetics, Vol 130, 27-36, Copyright © 1992
INVESTIGATIONS |
Promoters of the Broad Host Range Plasmid RK2: Analysis of Transcription (Initiation) in Five Species of Gram-Negative Bacteria
A. Greener, S. M. Lehman and D. R. Helinski
Present address: Stratagene, Inc., 11099 North Torrey Pines Road, La Jolla, California 92037.
A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.
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