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Mutagenesis

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Advanced Molecular Genetics

Abstract

Bacterial plasmids are autonomously replicating mini chromosomes (replicons) which contain genetic information for many different properties, including the production of toxins and adhesion antigens, resistance to antibiotics, and restriction and modification of DNA. The analysis of these and other properties is greatly faciliated by the isolation of plasmid derivatives carrying mutations in relevant genes. Conditional plasmid mutants affected in plasmid maintenance have, for example, greatly facilitated investigations on the mechanism and control of plasmid replication. Treatment of plasmid-harboring bacteria with nitrosoguanidine was first used to obtain mutant plasmids defective in replication (Kingsbury and Helinski 1973, Collins et al. 1978), but this method did not prove to be useful for multicopy plasmids. Subsequently, the exposure in vitro of purified plasmid DNA to mutagens, such as hydroxylamine (HA), and introduction of the mutagenized DNA into bacteria by transformation, was found to be very effective for the isolation of mutants of low and high copy number plasmids (Hashimoto-Gotoh and Sekiguchi 1976, Humphreys et al. 1976, Eichenlaub 1979, Eichenlaub and Wehlmann 1980). The crucial element in this procedure is transformation, which enables the biological separation of individual plasmid DNA molecules and their subsequent propagation as pure clones; mutagenesis may be carried out equally well in vivo, provided that the mutagenized plasmid DNA is subsequently isolated and used to transform a non-mutagenized host.

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References

  • Collins J, Yanofsky S, Helinski DR (1978) Involvement of the DNA-protein complex in the replication of plasmid ColEl. Mol Gen Genet 167: 21–28

    PubMed  CAS  Google Scholar 

  • Delcuve G, Cabezón T, Ghysen A, Herzog A, Bollen A (1977) Amber mutations in Escherichia coli essential genes: Isolation of mutants affected in the ribosome. Mol Gen Genet 157: 149–153

    Article  PubMed  CAS  Google Scholar 

  • Eichenlaub R (1979) Mutants of the mini-F plasmid pML31 thermosensitive in replication. J Bacteriol 138: 559–566

    PubMed  CAS  Google Scholar 

  • Eichenlaub R, Wehlmann H (1980) Amber mutants of plasmid mini-F defective in replication. Mol Gen Genet 180: 201–204

    Article  CAS  Google Scholar 

  • Freese E, Bautz E, Bautz-Freese E (1961) The chemical and mutagenic specificity of hydroxylamine. Proc Natl Acad Sei USA 47: 845–855

    Article  CAS  Google Scholar 

  • Hashimoto-Gotoh T, Sekiguchi M (1976) Isolation of temperature-sensitive mutants of R plasmid by in vitro mutagenesis with hydroxylamine. J Bacteriol 127: 1561–1563

    Google Scholar 

  • Humphreys GD, Willshaw GA, Smith HR, Anderson ES (1976) Mutagenesis of plasmid DNA with hydroxylamine: Isolation of mutants of multi-copy plasmids. Mol Gen Genet 145: 101–108

    Article  PubMed  CAS  Google Scholar 

  • Kingsbury DT, Helinski DR (1973) Temperature-sensitive mutants for the replication of plasmids in Escherichia coli I. Isolation and specificity of host and plasmid mutations. Genetics 74: 17–31

    Google Scholar 

  • Lovett MA, Helinski DR (1976) Method for the isolation of the replication region of a bacterial replicón: Construction of a mini-F Km plasmid. J Bacteriol 127: 982–987

    CAS  Google Scholar 

  • Bächi B, Arber W (1977) Physical mapping of Bglll, 5amHI, £coRI, Hindll and Pstl restriction fragments of bacteriophage PI DNA. Mol Gen Genet 153: 311–324

    Google Scholar 

  • Bächi B, Reiser J, Pirotta V (1979) Methylation and cleavage sequences of the EcoYl restriction-modification enzyme. J Mol Biol 128: 143–163

    Article  PubMed  Google Scholar 

  • Datta N, Hedges RW, Shaw EJ, Sykes RB, Richmond MH (1971) Properties of a R factor from Pseudomonas aeruginosa. J Bacteriol 108 (3): 1244–1249

    PubMed  CAS  Google Scholar 

  • Habermann A (1974) The bacteriophage PI restriction endonuelease. J Mol Biol 89: 545–563

    Article  Google Scholar 

  • Hedges RW, Jacob AE (1974) Transposition of ampicillin resistance from RP4 to other replicons. Mol Gen Genet 132: 31–40

    Article  PubMed  CAS  Google Scholar 

  • Heffron F, McCarthy BJ, Ohtsubo H, Ohtsubo E (1979) DNA sequence analysis of the transpson Tn3: Three genes and three sites involved in transposition of Tn3. Cell 18: 1153–1163

    Article  PubMed  CAS  Google Scholar 

  • Heilmann H (1979) Molekulare Analyse des Restriktionsgens des Phagen PI, Dissertation, Univ Erlangen

    Google Scholar 

  • Heilmann H, Pühler A, Brukardt HJ, Reeve JN (1980) Transposon mutagenesis of the gene encoding the bacteriophage PI restriction endonuelease: Colinearity of the gene and gene product. J Mol Biol 144: 387–396

    Article  PubMed  CAS  Google Scholar 

  • Ikeda H, Tomizawa J (1968) Prophage PI, an extrachromosomal replication unit. Symp Quant Biol Cold Spring Harbor 33: 791–798

    CAS  Google Scholar 

  • Jacob F, Monod J (1961) Genetic regulatory mechanism in the synthesis of proteins. J Mol Biol 3: 318

    Article  PubMed  CAS  Google Scholar 

  • Johnsrud L (1979) DNA sequence of the transposable element IS1. Mol Gen Genet 169: 213–218

    Article  PubMed  CAS  Google Scholar 

  • Lennox ES (1955) Transduction of linked genetic characters of the host by bacteriophage PI. Virology 1: 190–206

    Article  PubMed  CAS  Google Scholar 

  • Meselson M, Yuan R (1968) DNA restriction enzyme from E. coli. Nature 217: 1110–1114

    CAS  Google Scholar 

  • Ohtsubo H, Ohtsubo E (1978) Nucleotide sequence of an insertion element, IS1. Proc Natl Acad Sei USA 75: 615–619

    Article  CAS  Google Scholar 

  • Priefer UB, Burkardt HJ, Klipp W, Pühler A (1981) ISR1: An insertion element isolated from the soil bacterium Rhizobium lupini Cold Spring Harbor Symp Quant Biol 45: 87–91

    CAS  Google Scholar 

  • Pühler A, Krauss G (1977) Transposition of the ampicillin resistance gene from the RP4 factor to the bacteriophage PI and to the Escherichia coli fertility factor. In: Mutsuhashi S, Rosival L, Krcmery V (eds) Plasmids 3rd Int Symp Antibiotic Resistance-Avicenum Praque. Springer, Berlin Heidelberg New York, pp 151–160

    Google Scholar 

  • Rosner JL (1972) Formation, induction and curing of bacteriophage PI lysogens. Virology 48: 679–689

    Article  PubMed  CAS  Google Scholar 

  • Simon R (1980) Inkompatibilität und Replikation des Resistenzplasmids RP4. Dissertation, Univ Erlangen

    Google Scholar 

  • Alton NK, Vapnek D (1979) Nucleotide sequence analysis of chloramphenicol resistance transposon Tn9. Nature 268: 864–869

    Article  Google Scholar 

  • Barth PT (1979) RP4 and R300B as wide host-range plasmid cloning vehicles. In Timmis KN, Pühler A (eds) Plasmids of medical, environmental and commercial importance. Elsevier/North-Holland Biomedical Press, Amsterdam New York Oxford, pp 399–401

    Google Scholar 

  • Barth PT, GrinterNJ (1977) Map of plasmid RP4 derived by insertion of transposon C. J Mol Biol 113: 455

    Article  PubMed  CAS  Google Scholar 

  • Barth PT, Datta N, Hedges RW, Grinter NJ (1976) Transposition of a deoxyribonucleic acid sequence encoding trimethoprim and streptomycin resistance from R483 to other replicons. J Bacteriol 125: 800

    PubMed  CAS  Google Scholar 

  • Barth PT, Grinter NJ, Bradley DE (1978) Conjugal transfer system of plasmid RP§: analysis bym transposon 7 insertion. J Bacteriol 113: 43

    Google Scholar 

  • Berg DE, Davies J, Allet B, Rodraix J (1975) Transposition of R factor genes to bacteriophage X. Proc Natl Acad Sei USA 72: 3628

    Article  CAS  Google Scholar 

  • Beringer JE, Beynon JL, Buchanan-Wollaston AV, Johnston AWB (1978) Transfer of the drug-resistance transposon Tn5 to Rhizobium. Nature 276: 633

    Article  Google Scholar 

  • Beringer JG (1974) R factor transfer in Rhizobium leguninosarum. J Gen Microbiol 84: 188

    PubMed  CAS  Google Scholar 

  • Boucher C, Bergeron B, Bertalmio MB, Denarie J (1977) Introduction of bacteriophage Mu into Pseudomonas solanacearum and Rhizobium meliloti using the R factor RP4. J Gen Microbiol 98: 253–263

    PubMed  CAS  Google Scholar 

  • Bukhari AI (1976) Bacteriophage Mu as a transposition element. Annu Rev Genet 10: 389

    Article  PubMed  CAS  Google Scholar 

  • Burkardt HJ, Rieß G, Pühler A (1979) Relationship of group PI plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical. J Gen Microbiol 114: 341

    PubMed  CAS  Google Scholar 

  • Campbell A, Berg D, Botstein D, Lederberg E, Novick R, Starlinger P, Szybalski W (1977) Nomenclature of transposable elements in prokaryotes. In: Bukhari AI, Shapiro JA, Adhya SL (eds) DNA insertion elements, plasmids and episomes. Cold Spring Harbor Lab, Cold Spring Harbor, pp 15–22

    Google Scholar 

  • Datta N, Hedges RW (1972) Host ranges of R factors. J Gen Microbiol 70: 453

    PubMed  CAS  Google Scholar 

  • Datta N, Hedges RW, Shaw EJ, Sykes RB, Richmond MH (1971) Properties of an R factor from Pseudomonas aeruginosa. J Bacteriol 108: 1244–1245

    PubMed  CAS  Google Scholar 

  • Denarie J, Rosenberg C, Bergeron B, Boucher C, Michel M, de Bertalmio MB (1977) Potential of RP4::Mu plasmids for in vivo genetic engineering of Gram-negative Bacteria. In Bukhari AI, Shapiro JA, Adhya SL (eds) DNA insertion elements, plasmids and episomes. Cold Spring Harbor Lab, Cold Spring Harbor New York, pp 507–520

    Google Scholar 

  • Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Braod host range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti Proc Natl Acad S Sei USA 77: 7347

    CAS  Google Scholar 

  • Figurski DH, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sei USA 76: 1648

    Article  CAS  Google Scholar 

  • Figurski D, Meyer R, Miller DS, Helinski DR (1976) Generation in-vitro of deletions in the broad host range plasmid RK2 using phage Mu insertions and restriction endonucleases. Gene 1: 107

    Article  PubMed  CAS  Google Scholar 

  • Foster TJ, Howe GB, Richmond KMV (1975) Translocation of the tetracycline resistance determinant from R100–1 to the Escherichia coli Kl2 chromosome. J Bacteriol 124: 1153

    PubMed  CAS  Google Scholar 

  • Gottesmann M, Rosner JL (1975) Acquisition of a determinant for chloramphenicol resistance by coliphage X. Proc Natl Acad Sei USA 72: 5041

    Article  Google Scholar 

  • Hedges RW, Jacob AE (1974) Transposition of ampicillin resistance from RP4 to other replicons. Mol Gen Genet 132: 31

    Article  PubMed  CAS  Google Scholar 

  • Heffron F, McCarthy BJ (1979) DNA sequence analysis of the transposon TnJ: three genes and three sites involved in transposition of TnJ. Cell 18: 1153–1163

    Article  PubMed  CAS  Google Scholar 

  • Heffron F, Sublett R, Hedges RW, Jacob A, Falkow S (1975) Origin of the TEM beta lactamase gene found on plasmids. J Bacteriol 122: 250

    PubMed  CAS  Google Scholar 

  • Holloway BW (1979) Plasmids that mobilize bacterial chromosome. Plasmid 2: 1–19

    Article  PubMed  CAS  Google Scholar 

  • Kleckner N (1977) Translocatable elements in prokaryotes. Cell 11: 11–13

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Olsen RH, Shipley P (1973) Host range and properties of the Pseudomonas R factor R1822. J Bacteriol 113: 772

    PubMed  CAS  Google Scholar 

  • Ruvkun GB, Ausubel FM (1980) Interspecies homology of nitrogenase genes. Proc Natl Acad Sei USA 77: 191

    Article  CAS  Google Scholar 

  • Ruvkun GB, Ausubel FM (1981) A general method for site–directed mutagenesis in prokaryotes. Nature 289: 85

    Article  PubMed  CAS  Google Scholar 

  • Thomas CM, Stalker D, Guiney D, Helinski DR (1979) Essential regions for the replication and conjugal transfer of the broad host range plasmid RK2. In: Timmis KN, Pühler A (eds) Plasmids of medical, environmental and commercial importance. Elsevier/North-Holland Biomedical Press, Amsterdam New York Oxford, pp 375–386

    Google Scholar 

  • Van Vliet F, Silva B, van Montagu M, Schell J (1978) Transfer of RP4::Mu plasmids to Agrobacterium tumefaciens. Plasmid 1: 446–455

    Article  PubMed  Google Scholar 

  • Bagdasarian M, Timmis KN (1982) Host-vector systems for gene cloning in Pseudomonas. Gurr Top Microbiol Immunol 96: 47–67

    CAS  Google Scholar 

  • Bagdasarian M, Bagdasarian MM, Lurz R, Nordheim A, Frey J, Timmis KN (1982) Molecular and functional analysis of the broad host range plasmid RSF1010 and construction of vectors for gene cloning in Gram-negative bacteria. In: Mitsuhashi S (ed) Bacterial drug resistance. Jpn Sci Soc Press Tokyo, pp 183–197

    Google Scholar 

  • Bagdasarian M, Lurz R, Riickert B, Franklin RCH, Bagdasarian MM, Frey J, Timmis KN (1981) specific–purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host vector system for gene cloning in Pseudomonas. Gene 16: 237–247

    Google Scholar 

  • Barth PT, Grinter NG (1974) Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids carrying linked resistance to streptomycin and sulfonamides. J Bacterid 120: 618–630

    CAS  Google Scholar 

  • Carbon J, Shenk TE, Berg P (1975) Biochemical procedure for production of small deletions in simian virus DNA. Proc Natl Acad Sci USA 72: 1392–1396

    Article  PubMed  CAS  Google Scholar 

  • Covey C, Richardson D, Carbon J (1976) A method for deletion of restriction sites in bacterial plasmid deoxyribonucleic acid. Mol Gen Genet 145: 155–158

    Article  PubMed  CAS  Google Scholar 

  • Eckhardt T (1978) A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria. Plasmid 1: 584–588

    Article  PubMed  CAS  Google Scholar 

  • Gray Jr, Ostander DA, Halnett JL, Legerski RJ, Robberson P (1975) Extracellular nucleases of Pseudomonas BAL31. I. Characterization of single-strand specific deoxyribonuclease and double strand deoxyribonuclease activities. Nuclei Acids Res 2: 1459–1492

    Google Scholar 

  • Heffron F, Bechinger P, Champoux JJ, Falkow S (1977) Deletions affecting the transposition of an antibiotic resistance gene. Proc Natl Acad Sci USA 74: 702–706

    Article  PubMed  CAS  Google Scholar 

  • Kushner SR (1978) An improved method for transformation of Escherichia coli with ColEl derived plasmids: In: BoyerHW, NocosiaS (eds) Genetic Engineering. Elsevier/North Holland, Amsterdam, pp 17–23

    Google Scholar 

  • Lai CJ, Nathans D (1974) Deletion mutants of simian virus 40 generated by the enzymatic excision of DNA segments from the viral genome. J Mol Biol 89: 179–193

    Article  PubMed  CAS  Google Scholar 

  • Legerski RJ, Halnett JL, Gray Jr (1978) Extracellular nucleases of Pseudomonas BAL31. III. Use of the double strand deoxyribonuclease activity as the basis of a conventional method for the mapping of fragments of DNA produced by cleavage with restriction enzymes. Nucleic Acid Res 5: 1445–1464

    Article  PubMed  CAS  Google Scholar 

  • Murray NE, Murray K (1974) Manipulation of restriction targets in phage X to form receptor chromosomes for DNA fragments. Nature 251: 476–481

    Article  PubMed  CAS  Google Scholar 

  • Nordheim A, Hashimoto-Gotoh T, Timmis KN (1980) Location of two relaxation nick sites in RK6 and single sites in pSClOl and RSF1010 close to origins of vegetative replication: Implications for conjugal transfer of plasmid DNA. J Bacteriol 144: 923–932

    Google Scholar 

  • Shenk TE, Carbon J, Berg P (1976) Construction and analysis of viable deletion mutants of simian virus 40. J Virology 18: 669–671

    Google Scholar 

  • Timmis KN (1981) Gene manipulation in vitro. Symp Soc Gen Microbiol 31: 49–109

    Google Scholar 

  • Timmis KN, Cabello F, Cohen SN (1975) Cloning, isolation and characterization of replication regions of complex plasmid genomes. Proc Natl Acad Sci USA 72: 2242–2246

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Institut für Allgemeine Botanik, Arbeitsbereich Genetik, Universität Hamburg, Ohnhorststr. 18, D-2000, Hamburg 52, Fed. Rep. of Germany

    R. Eichenlaub

  2. Lehrstuhl für Genetik, Universität Bielefeld, D-4800, Bielefeld, Fed. Rep. of Germany

    H. Heilmann

  3. Lehrstuhl für Genetik,Fakultät für Biologie, Universität Bielefeld, D-4800, Bielefeld l, Fed. Rep. of Germany

    R. Simon

  4. Department of Biochemistry, University of Geneva, CH-1211, Geneva, Switzerland

    J. Frey

  5. Max-Planck-Institute for Molecular Genetics, D-1000, Berlin-Dahlem, Fed. Rep. of Germany

    M. Bagdasarian

  6. Department of Medical Biochemistry, University of Geneva, CH-1211, Geneva, Switzerland

    K. N. Timmis

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  1. R. Eichenlaub
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  2. A. Pühler
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  3. V. Krishnapillai
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  4. H. Heilmann
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  5. R. Simon
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  6. J. Frey
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  7. M. Bagdasarian
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  8. K. N. Timmis
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Editors and Affiliations

  1. Fakultät für Biologie, Universität Bielefeld, Postfach 8640, D-4800, Bielefeld, Fed. Rep. of Germany

    Alfred Pühler (Professor) (Professor)

  2. Department of Medical Biochemistry, University Medical Centre, 9, Avenue de Champel, CH-1211, Geneva 4, Switzerland

    Kenneth N. Timmis (Professor) (Professor)

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© 1984 Springer-Verlag Berlin Heidelberg

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Eichenlaub, R. et al. (1984). Mutagenesis. In: Pühler, A., Timmis, K.N. (eds) Advanced Molecular Genetics. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-69305-2_2

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  • DOI: https://doi.org/10.1007/978-3-642-69305-2_2

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