Summary
In vitro joining of the two small multicopy plasmidsRsc11 andColE1 by a poly dAdT linker resulted in hybrid plasmids, which determine resistance to ampicillin and immunity to colicin E1. Isolation of the plasmid DNA from single colonies revealed that a variety of hybrid plasmids was formed. Cleavage of these plasmids with restriction endonucleasesHincII,HindIII,EcoR1,SmaI andBamI and hybridization withColE1 demonstrated that they contain different parts of the parent plasmids,Rsc11 andColE1. Their copy number in the cell is between 6 and 15 per chromosome depending on the plasmid. None of these plasmids can replicate inpolA mutants. Replication continues in the presence of chloramphenicol. This suggests that replication can only occur from theColE1 origin and that the replication function of theRsc11 part is lost. The hybrid plasmids are compatible withRsc11 but not withColE1. The comparison of the physical maps of theseRsc11-ColE1 hybrids with their functions allows a partial determination of the location of ampicillin resistance, replication and incompatibility on theRsc11 genome.
Similar content being viewed by others
References
Denhardt, D.T.: A membrane-filter technique for the detection of complementary DNA. Biochem. biophys. Res. Commun.23, 641–646 (1966)
Gautier, F., Mayer, H., Goebel, W.: Cloning of calf thymus satellite I DNA inEscherichia coli. Molec. gen. Genet.149, 23–31 (1976)
Goebel, W., Bonewald, R.: Class of small multicopy plasmids originating from the mutant antibiotic resistance factor R1drd19-B2. J. Bact.123, 658–665 (1975)
Hedgepeth, J., Goodman, H.M., Boyer, H.W.: DNA nucleotide sequence restricted by the RI endonuclease. Proc. nat. Acad. Sci. (Wash.)69, 3448–3452 (1972)
Hersfield, V., Boyer, H.W., Chow, L., Helinski, D.R.: Characterization of a mini-ColE1 plasmid. J. Bact.126, 447–453 (1976)
Humphreys, G.O., Willshaw, G.A., Anderson, E.S.: A simple method for the preparation of large quantities of pure plasmid DNA. Biochem. biophys. Acta (Amst.)383, 457–463 (1975)
Jackson, D., Symons, R., Berg, P.: Biochemical method for inserting new genetic information into DNA of simian virus 40: circular SV40 DNA molecules containing lamda phages genes and the galactose operon ofEscherichia coli. Proc. nat. Acad. Sci. (Wash.)69, 2904–2909 (1972)
Kato, K.L., Goncalves, J.M., Houts, G.E., Bollum, F.J.: Deoxynucleotide-polymerizing enzymes of calf thymus gland. J. biol. Chem.242, 2780–2789 (1967)
Landy, A., Ruedisueli, E., Robinson, L., Foeller, C., Ross, W.: Digestion of deoxyribonucleic acids from bacteriophage T7, λ and ϕ 80h with site-specific nucleases fromHemophilus influenzae strain Rc and strain Rd. Biochem.13, 2134–2142 (1974)
Little, J.W., Lehmann, I.R., Kaiser, A.D.: An exonuclease induced by bacteriophage λ. I. Preparation of the crystalline enzyme. J. biol. Chem.242, 672–678 (1967)
Luibrand, G., Blohm, D., Mayer, H., Goebel, W.: Characterization of small ampicillin resistance plasmids (Rsc) originating from the mutant antibiotic resistance factor R1drd19-B2. Molec. gen. Genet. in press
Mayer, H., Luibrand, G., Goebel, W.: Replication of the mini-R1 plasmid Rsc11 and Rsc11 hybrid plasmids. Molec. gen. Genet. in press
Nordström, K., Ingram, L.C., Lundbäck, A.: Mutations in R-factors ofEscherichia coli causing an increased number of R-factor copies per chromosome. J. Bact.110, 562–569 (1972)
Polisky, B., Greene, P., Garfin, D.E., Carthy, B.J., Goodman, H.M., Boyer, H.W.: Specificity of substrate recognition by theEcoRI restriction endonuclease. Proc. nat. Sci. (Wash.)72, 3310–3314 (1975)
Smith, H.O., Wilcox, K.W.: A restriction enzyme fromHemophilus influenzae. I. Purification and general properties. J. molec. Biol.51, 379–391 (1970)
So, M., Gill, R., Falkow, S.: The Generation of ColEl-Apr Cloning Vehicle which Allows Detection of Inserted DNA. Molec. gen. Genet.142, 239–249 (1975)
Timmis, K., Cabello, F., Cohen, S.N.: Utilization of two distinct modes of replication by a hybrid plasmid constructured in vitro from separate replicons. Proc. nat. Acad. Sci. (Wash.)71, 4556–4560 (1974)
Wensink, P.C., Finnegan, D.J., Donelson, J.E., Hogness, D.S.: A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster. Cell3, 315–325 (1974)
Wilson, G.A., Young, F.E.: Isolation of a sequence-specific endonuclease (BamI) fromBacillus amyloliquefaciens H. J. molec. Biol.97, 123–125 (1975)
Author information
Authors and Affiliations
Additional information
Communicated by F. Kaudewitz
Schering AG, Müllerstr. 170–178, D-1000 Berlin 65
Gesellschaft für Biotechnologische Forschung mbH, Abt. Genetik, Mascheroder Weg 1, D-3300 Braunschweig-Stöckheim
Rights and permissions
About this article
Cite this article
Boidol, W., Siewert, G., Lindenmaier, W. et al. Properties of hybrid plasmids, consisting of parts of the mini-R1 factorRsc11 andColE1. Molec. Gen. Genet. 152, 231–237 (1977). https://doi.org/10.1007/BF00693075
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00693075