Summary
The 30 megadalton (Mdal)-conjgaative, fi- plasmid pRSD1 determines inducible tetracycline resistance (Tc) in Escherichia coli. As shown by restriction analysis, a 3.5 Mdal-EcoRI fragment of pRSD1 spliced into the small plasmid pRSD2124 comprises the entire Tc determinant (tet) region. A restriction map of pRSD1 is presented which includes the location of the tet region and of an “underwound” loop not related to Tc (Burkardt et al., 1978). Selective amplification of tet genes is demonstrated by three lines of evidence. (i) The resistance level of cell harbouring pRSD1 increases approximately tenfold by induction with 10μg/ml of tetracycline. Further groth in the presence of 100 μg/ml of the drug (“tet-racycline stress”) selects for cells with even higher resistance levels (about 300 μg/ml) in rec + cells. In a recA strain, a smaller proportion of cells attains these high resistance levels suggesting the involvement of host recombination. (ii) Electron micrographs of pRSD1-DNA isolated from tetracycline-stressed cells reveal a heterogeneous population of circular DNA molecules ranging between 1.7 and 21.6 μm. The distribution of contour lengths shows a discrete pattern ascribed to the presence of autonomous single-and multiple-copy Tc determinants and to intact plasmids containing zero to six tet regions in tandem repeats. (iii) This interpretation is supported by heteroduplex and restriction analyses which demonstrate the presence of multiple copies of the 3.5 Mdal-element encompassing the tet region in pRSD1 molecules selected by tetracycline stress. It has been concluded that gene amplification leading to tandem repetition of the tet region ensues in pRSD1. Such plasmids confer increased tetracycline resistance and can, therefore, be selected by high doses of the drug.
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Bächi, B., Arber, W.: Physical mapping of BglII, BamHI, EcoRI, HindIII and PstI restriction fragments of bacteriophage P1 DNA. Mol. Gen. Genet. 153, 311–324 (1977)
Burkardt, H.J., Mattes, R., Schmid, K., Schmitt, R.: Properties of two conjugative plasmids mediating tetracycline resistance, raffinose catabolism and hydrogen sulfide production in Escherichia coli. Mol. Gen. Genet. 166, 75–84 (1978)
Clewell, D.B., Yagi, Y., Bauer, B.: Plasmid-determined tetracycline resistance in Streptococcus faecalis: evidence for gene amplification during growth in the presence of tetracycline. Proc. Natl. Acad. Sci. U.S.A. 72, 1720–1724 (1975)
Cohen, S.N.: Transposable genetic elements and plasmid evolution. Nature 263, 731–738 (1976)
Davies, J.E., Rownd, R.: Transmissible multiple drug resistance in enterobacteriaceae. Science 176, 758–768 (1972)
Greene, P.J., Betlach, M.C., Boyer, H.W., Goodman, H.M.: The EcoRI restriction endonuclease, pp. 87–111. In: Meth. in molec. biol., Vol. 7 (R.B. Wickner, ed.). New York: Marcel Dekker 1974
Grinsted, J., Bennett, P.M., Richmond, M.H.: A restriction enzyme map of the R-factor RP. Plasmid 1, 34–37 (1977)
Hashimoto, H., Rownd, R.H.: Transition of the R-factor NR1 in Proteus mirabilis: level of drug resistance of nontransitioned and transitioned cells. J. Bacteriol. 123, 56–68 (1975)
Hu, S., Ohtsubo, E., Davidson, N., Saedler, H.: Electron microscope heteroduplex studies of sequence relations among bacterial plasmids: identification and mapping of insertion sequences IS1 and IS2 in F and R plasmids. J. Bacteriol. 122, 764–775 (1975)
Humphreys, G.O., Willshaw, G.A., Anderson, E.S.: A simple procedure for the preparation of large quantities of pure plasmid DNA. Biochim. Biophys. Acta 383, 457–463 (1975)
Lang, D.: Molecular weights of coliphages and coliphage DNA. III. Contour length and molecular weight of DNA from bacteriophages T4, T5, and T7, and from bovine papilloma virus. J. Mol. Biol. 54, 557–565 (1970)
Normark, S., Edlund, T., Grundström, T., Bergström, S., Wolf-Watz, H.: Escherichia coli K-12 mutants hyperproducing chromosomal beta-lactamase by gene repetitions. J. Bacteriol. 132, 912–922 (1977)
Rownd, R., Perlman, D., Hashimoto, H., Mickel, E., Applebaum, E., Taylor, D.: Dissociation and reassociation of the transfer factor and resistance determinants of R factors as a mechanism of gene amplification in bacteria, pp. 115–128. In: Cellular modification and genetic transformation by exogenous nucleic acids. Proc. 6th Miles Int. Symp. Molec. Biol. Baltimore: John Hopkins Press 1973
Schmitt, R.: Analysis of melibiose mutants deficient in α-galactosidase and thiomethylgalactoside permeaseII in Escherichia coli K12. J. Bacteriol. 96, 462–471 (1968)
Smith, D.J., Blattner, F.R., Davies, J.: The isolation and partial characterization of a new restriction endonuclease from Providencia stuartii. Nucleic Acids Res. 3, 343–353 (1976)
So, M., Gill, R., Falkow, S.: The generation of a ColE1-Ap cloning vehicle which allows detection of inserted DNA. Mol. Gen. Genet. 142, 239–249 (1975)
Tanaka, T., Weisblum, B.: Construction of a colicin El-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. J. Bacteriol. 121, 354–362 (1975)
Taylor, D.O., Greenberg, J., Rownd, R.H.: Generation of miniplasmids from copy number mutants of the R plasmid NR1. J. Bacteriol. 132, 986–995 (1977)
Yagi, Y., Clewell, D.B.: Plasmid-determined tetracycline resistance in Streptococcus faecalis: tandemly repeated resistance determinants in amplified forms of pAMα1 DNA. J. Mol. Biol. 102, 583–600 (1976)
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Mattes, R., Burkardt, H.J. & Schmitt, R. Repetition of tetracycline resistance determinant genes on R plasmid pRSD1 in Escherichia coli . Molec. Gen. Genet. 168, 173–184 (1979). https://doi.org/10.1007/BF00431443
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DOI: https://doi.org/10.1007/BF00431443