Summary
A study has been made of the insertional properties of transposon Tn7, a 14 kilobase transposable element encoding resistances to trimethoprim, streptomycin and specitinomycin. It has previously been shown that Tn7 transposes at a low frequency and with low specificity into multiple sites in large transmissible plasmids. However, Tn7 transposes with extrame specificity and at high efficiency into the E. coli chromosome. In all cases we have studied, insertion of Tn7 into the chromosome has occurred at a unique site and with a unique orientation. A combination of genetic and biochemical techniques have been used to precisely locate this site on the E. coli chromosome to minute 82 on the linkage map between markers glmS and uncA.
To investigate the nature of this highly specific transpositional event, a small region of the E. coli chromosome that includes the unique site, was cloned into the plasmid vector pBR322. Subsequently a lkb restriction fragment, including the Tn7 insertion site, was sub-cloned from this plasmid into the plasmid pACYC184. We show that Tn7 transposes into both these plasmid recombinants with the frequency and specificity characteristie of the E. coli chromosome.
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References
Allet B, Katagiri KJ, Gesteland RF (1973) Characterization of polypeptides made in vitro from bacteriophage lambda DNA. J Mol Biol 78:589–600
Barth PT, Datta N, hedges RW, Grinter NS (1976) Transposition of a deoxyribonucleic acid sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons. J Bacteriol 125:800–810
Barth PT, Datta N (1977) Two naturally occurring transposons indistinguishable from Tn7. J Gen Microbiol 102:129–134
Barth PT, Grinter NJ (1977) Map of RP4 derived by insertion of transposon C. J Mol Biol 113:455–474
Barth PT, Grinter NJ, Bradley DE (1978) Conjugal transfer system of plasmid RP4: Analysis of transposon 7 insertion. J Bacteriol 133:43–52
Bachmann BJ, Low KB, Taylor AL (1976) Recalibrated linkage map of Escherichia coli K-12. Bacteriol Rev 40:116–167
Bedbrook JR, Lehrach H, Ausubel FM (1979) Directive segregation is the basis of ColE1 plasmid incompatibility. Nature 281:447–452
Bolivar F, Rodriquez RL, Greene PJ, Betlach MC, Hegresjer HL, Boyer HW (1977) Construction and characterization of new cloning vehicles. I Ampicillin resistant derivatives of plasmid pMB9. Gene 2:75–93
Brenner S, Cesareni G, Karn J (1981) Phasmids: Hybrids between ColEl Plasmids and Bacteriophage Lambda. J Mol Biol (in press)
Calos MP, Miller JH (1980) Transposable elements. Cell 20:579–595
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Clewell DB, Helinski DR (1970) Properties of a supercoiled deoxyribonucleic acid — protein relaxation complex and strand specificity of the relaxation event. Biochemistry 9, 4428–4440
Datta N, Hedges RW (1972) Trimethoprim resistance conferred by W plasmids in enterobacteriaceae. J Gen Microbiol 72:349–355
Fennewald MA, Shapiro JA (1979) Transposition of Tn7 in Pseudomonas aeruginosa and isolation of alk:: Tn7 mutations. J Bacteriol 139:264–269
Hedges RW, Datta N (1973) Plasmid determining I pili constitute a compatibility complex. J Gen Microbiol 77:19–25
Lichtenstein CP, Brenner S (1981) The unique insertion site of Tn7 in the E. coli chromosome. Nature (manuscript submitted)
Mandel M, Higa A (1970) Calcium-dependent bacteriophage DNA infection. J Mol Biol 53:159–162
Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208–218
Miki T, Hiraga S, Nagata T, Yura T (1978) Bacteriophage λ carrying the Escherichia coli Chromosomal region of the replication origin. Proc Natl Acad Sci USA 75:5099–5103
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, New York
Reznikoff WS, Winter RB, Hurley CK (1974) The location of the repressor binding sites in the lac operon. Proc Natl Acad Sci USA 71:2314–2318
Rigby PWJ, Dieckman M, Rhodes C, Berg P (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251
Sharpe PA, Sugden B, Sambrook J (1973) Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose — Ethidium bromide electrophoresis. Biochemistry 12:3054–3063
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Wahl GM, Stern M, Stark GR (1979) Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid identification by using dextran sulphate. Proc Natl Acad Sci USA 76:3683–3687
Warren G, Sherratt D (1978) Incompatibility and transforming efficiency of ColE1 and related plasmids. Mol Gen Genet 161:39–47
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Lichtenstein, C., Brenner, S. Site-specific properties of Tn7 transposition into the E. coli Chromosome. Molec. Gen. Genet. 183, 380–387 (1981). https://doi.org/10.1007/BF00270644
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DOI: https://doi.org/10.1007/BF00270644