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Characterization of Tn10d-Cam: A transposition-defective Tn10 specifying chloramphenicol resistance

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Summary

We have constructed a small, transposition-defective derivative of the transposon Tn10 that carries the chloramphenicol acetyltransferase gene of pACYC184. This new genetic element, Tn10d-Cam, transposes when Tn10 transposase is provided from a multi-copy plasmid. Transposon insertion mutagenesis of Salmonella typhimurium was performed by using a strain carrying a Tn10d-Cam insertion in an Escherichia coli F' episome as the donor in transductional crosses into recipients that carried a plasmid expressing Tn10 transposase. Tn10d-Cam insertion mutations were also generated by complementation in cis of Tn10d-Cam by a cotransducible Tn10 element that overproduces transposase. Here, transposase was provided only transiently, and the Tn10d-Cam insertion mutations were recovered in a transposase-free strain. Cis complementation was used for mutagenesis of a plasmid target. The site specificity of insertion and the effect of insertions on expression of a downstream gene were investigated, using Tn10d-Cam insertions in a plasmid carrying a segment of the histidine operon.

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References

  • Amman E, Brosius J, Ptashne M (1983) Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 25:167–178

    Google Scholar 

  • Bellofatto V, Shapiro L, Hodgson DA (1984) Generation of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus. Proc Natl Acad Sci USA 81:1035–1039

    Google Scholar 

  • Bender J, Kleckner N (1986) Genetic evidence that Tn10 transposes by a nonreplicative mechanism. Cell 45:801–815

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    Google Scholar 

  • Casadaban MJ, Cohen SN (1979) Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci USA 76:4530–4533

    Google Scholar 

  • Casadaban MJ, Chou J, Cohen SN (1980) In vitro gene fusions that join an enzymatically active β-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol 143:971–980

    Google Scholar 

  • Castilho BA, Olfson P, Casadaban MJ (1984) Plasmid insertion mutagenesis and lac gene fusion with mini-Mu bacteriophage transposons. J Bacteriol 158:488–495

    Google Scholar 

  • Chaconas G, deBruijn FJ, Casadaban MJ, Lupski JR, Kwoh TJ, Harshey RM, DuBow MS, Bukhari AI (1981) In vitro and in vivo manipulations of bacteriophage Mu DNA: cloning of Mu ends and construction of mini-Mu's carrying selectable markers. Gene 13:37–46

    Google Scholar 

  • Ciampi MS, Roth JR (1988) Polarity effects in the hisG gene of Salmonella require a site that is within the coding sequence. Genetics 118:193–202

    Google Scholar 

  • Ciampi MS, Schmid MB, Roth JR (1982) Transposon Tn10 provides a promoter for transcription of adjacent sequences. Proc Natl Acad Sci USA 79:5016–5020

    Google Scholar 

  • Davis RW, Botstein D, Roth JR (1980) Advanced Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • DeBoer HA, Comstock LJ, Vasser M (1983) The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci USA 80:21–25

    Google Scholar 

  • Elliott T, Geiduschek EP (1984) Defining a bacteriophage T4 late promoter: absence of a “-35” region. Cell 36:211–219

    Google Scholar 

  • Enomoto M, Stocker BAD (1974) Transduction by phage P1kc in Salmonella typhimurium. Virology 60:503–514

    Google Scholar 

  • Foster TJ (1977) Insertion of the tetracycline resistance translocation unit Tn10 in the lac operon of Escherichia coli K12. Mol Gen Genet 154:305–309

    Google Scholar 

  • Foster TJ, Davis MA, Roberts DE, Takeshita K, Kleckner N (1981a) Genetic organization of transposon Tn10. Cell 23:201–213

    Google Scholar 

  • Foster TJ, Lundblad V, Hanley-Way S, Halling SM, Kleckner N (1981b) Three Tn10-associated excision events: relationship to transposition and role of direct and inverted repeats. Cell 23:215–227

    Google Scholar 

  • Gill RE, Heffron F, Falkow S (1979) Identification of the protein encoded by the transposable element Tn3 which is required for its transposition. Nature 282:797–801

    Google Scholar 

  • Grindley ND, Joyce CM (1981) Analysis of the structure and function of the kanamycin-resistance transposon Tn903. Cold Spring Harbor Symp Quant Biol 45:125–133

    Google Scholar 

  • Halling SM, Kleckner N (1982) A symmetrical six-base-pair target site sequence determines Tn10 insertion specificity. Cell 28:155–163

    Google Scholar 

  • Hughes KT, Roth JR (1984) Conditionally transposition-defective derivative of Mu d1(Amp Lac). J Bacteriol 159:130–137

    Google Scholar 

  • Hughes KT, Roth JR (1988) Transitory cis complementation: a method for providing transposition functions to defective transposons. Genetics 119:9–12

    Google Scholar 

  • Isberg R, Syvanen M (1981) Replicon fusions promoted by the inverted repeats of Tn5. J Mol Biol 150:15–32

    Google Scholar 

  • Johnson RC, Reznikoff WS (1983) DNA sequences at the ends of transposon Tn5 required for transposition. Nature 304:280–282

    Google Scholar 

  • Joyce CM, Grindley ND (1984) Method for determining whether a gene of Escherichia coli is essential: application to the polA gene. J Bacteriol 158:636–643

    Google Scholar 

  • Kleckner N, Chan RK, Tye B-K, Botstein D (1975) Mutagenesis by insertion of a drug-resistance element carrying an inverted repetition. J Mol Biol 97:561–575

    Google Scholar 

  • Kleckner N, Roth J, Botstein D (1977) Genetic engineering in vivo using translocatable drug-resistance elements. New methods in bacterial genetics. J Mol Biol 116:125–159

    Google Scholar 

  • Kleckner N, Steele DA, Reichardt K, Botstein D (1979) Specificity of insertion by the translocatable tetracycline resistance element Tn10. Genetics 92:1023–1040

    Google Scholar 

  • Kleckner N, Foster TJ, Davis MA, Hanley-Way S, Halling SM, Lundblad V, Takeshita N (1981) Genetic organization of Tn10 and analysis of Tn10-associated excision events. Cold Spring Harbor Symp Quant Biol 45:225–238

    Google Scholar 

  • Kroos L, Kaiser D (1984) Construction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into Myxococcus xanthus. Proc Natl Acad Sci USA 81:5816–5820

    Google Scholar 

  • Machida Y, Machida C, Ohtsubo H, Ohtsubo E (1982) Factors determining frequency of plasmid cointegration mediated by the insertion sequence IS1. Proc Natl Acad Sci USA 79:277–281

    Google Scholar 

  • Manoil C, Beckwith J (1985) TnphoA: A transposon probe for protein export signals. Proc Natl Acad Sci USA 82:8129–8133

    Google Scholar 

  • Menzel R, Roth J (1981) Regulation of the genes for proline utilization in Salmonella typhimurium: autogenous repression by the putA gene product. J Mol Biol 148:21–44

    Google Scholar 

  • Miller JH, Calos MP, Galas DJ (1981) Genetic and sequencing studies of the specificity of transposition in to the lac region of E. coli. Cold Spring Harbor Symp Quant Biol 45:243–257

    Google Scholar 

  • Morisato D, Way JC, Kim H-J, Kleckner N (1983) Tn10 transposase acts preferentially on nearby transposon ends in vivo. Cell 32:799–807

    Google Scholar 

  • Morrison DA (1979) Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol 68:326–331

    Google Scholar 

  • Orbach MJ, Jackson EN (1982) Transfer of chimeric plasmids among Salmonella typhimurium strain by P22 transduction. J Bacteriol 149:985–994

    Google Scholar 

  • Schmieger H (1971) A method for detection of phage mutants with altered transducing ability. Mol Gen Genet 110:378–381

    Google Scholar 

  • Simons RW, Hoopes BC, McClure WR, Kleckner N (1983) Three promoters near the termini of IS10: pIN, pOUT and pIII. Cell 34:673–682

    Google Scholar 

  • Smith HO, Levine M (1967) A phage P22 gene controlling integration of prophage. Virology 31:207–216

    Google Scholar 

  • Vogel HJ, Bonner DM (1956) Acetylornithase of Escherichia coli: partial purification and some properties. J Biol Chem 218:97–106

    Google Scholar 

  • Way JC, Kleckner N (1984) Essential sites at transposon Tn10 termini. Proc Natl Acad Sci USA 81:3452–3456

    Google Scholar 

  • Way JC, Davis MA, Morisato D, Roberts DE, Kleckner N (1984) New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene 32:369–379

    Google Scholar 

  • Youngman P, Zuber P, Perkins JB, Sandman K, Igo M, Losick R (1985) New ways to study developmental genes in sporeforming bacteria. Science 228:285–291

    Google Scholar 

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  1. Thomas Elliott

    Present address: Department of Microbiology, University of Alabama at Birmingham, 35294, Birmingham, AL, USA

Authors and Affiliations

  1. Department of Biology, University of Utah, 84112, Salt Lake City, UT, USA

    Thomas Elliott & John R. Roth

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  1. Thomas Elliott
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  2. John R. Roth
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Communicated by W. Arber

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Elliott, T., Roth, J.R. Characterization of Tn10d-Cam: A transposition-defective Tn10 specifying chloramphenicol resistance. Mol Gen Genet 213, 332–338 (1988). https://doi.org/10.1007/BF00339599

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  • DOI: https://doi.org/10.1007/BF00339599

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