Cloning DNA from Rhizobium Meliloti Using a New Broad Host Range, Binary Vehicle System
Molecular cloning techniques now permit the dissection of DNA to almost any desired degree in order to study both structure and function of individual genes. An important strategy for analyzing cloned DNAs involves reintroducing them into their host of origin under various conditions designed to affect gene function. This procedure is greatly facilitated if the primary cloning vector is capable of autonomous replication in multiple hosts. Double vectors, capable of replication in more than one host, have been developed for use in Escherichia coli/yeast (Struhl et al., 1979) and Bacillus subtilis/Staphylococcus aureus (Lofdahl et al., 1978). There are many other organisms of medical, agricultural, and economic importance, however, for which no such dual purpose vectors exits. We have therefore developed a broad host range cloning system for gram-negative bacteria from the naturally-occurring drug resistance plasmid RK2. This system, whose possible development was first suggested some time ago (Meyer et al., 1977), has proved exceedingly valuable to our laboratory in working with the nitrogen-fixing alfalfa symbiont, Rhizobium meliloti.
KeywordsGene Bank Broad Host Range Helper Plasmid Transfer Frequency Acinetobacter Calcoaceticus
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- Ditta, G., Stanfield, S., Corbin, D., and Helinski, D. R., 1980, A new broad host range cloning system for Gram-negative bacteria and its use in constructing a gene bank of Rhizobium meliloti Google Scholar
- Figurski, D., and Helinski, D. R., 1979, Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans, Proc. Natl. Acad. Sci. USA, 74: 1658.Google Scholar
- Guiney, D. G., and Helinski, D. R., 1979, The DNA-protein relaxation complex of the plasmid RK2: location of the site-specific nick in the region of the proposed origin of transfer, Mol. Gen. Genet., 1976:183.Google Scholar
- Kahn, M., Kolter, R., Thomas, C., Figurski, D., Meyer, R., Remaut, E., and Helinski, D. R., 1980, Plasmid cloning vehicles derived from plasmids Col El, F, R6K and RK2, in: “Methods in Enzymology, Vol. 68, Recombinant DNA,” R. Wu, ed., Academic Press, New York.Google Scholar
- Meyer, R. J., Figurski, D., and Helinski, D. R., 1977, Properties of the plasmid RK2 as a cloning vehicle, in: “DNA Insertion Elements, Plasmids, and Episomes,” A. I. Bukhari, J. A. Shapiro, and S. L. Adhya, eds., Cold Springs Harbor Laboratory.Google Scholar
- Thomas, C. M., and Helinski, D. R., 1979, Regions of the broad host range plasmid RK2 that are required for replication and maintenance, J. Bact., 141:213.Google Scholar