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Plasmid cloning vectors that can be nicked at a unique site

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We describe ColE1-type plasmids, with relaxed DNA replication, based on pMB9, and carrying the CmR determinant of R1, in addition to the TcR determinant of pMB9. One of the plasmids, pPH207, has unique sites for EcoRI, HindIII, BamI, SalI and Hpal. Insertion of foreign DNA into all but the last of these inactivates cither the CmR or the TcR determinant.

The original CmR TcR plasmid (pCM2) contains a copy of IS1 which produces deletions to left and to right. Most of these inactivate either the CmR or the TcR determinant. An internal 280 bp deletion of IS1 DNA in pPH207 greatly reduces the frequency at which deletions are observed.

The main feature of these plasmids is a site that is cleaved by some preparations of EcoRI in only one strand of the DNA duplex (the EcoRIn site). This site facilitates strand separation of sequences inserted at the HindIII, BamI and SalI sites of the TcR gene, and also of any inserted at the true EcoRI site by a method that destroys that site. Since the oricntation of the EcoRIn site is known, the orientation of sequences inserted at the neighbouring sites can be easily determined.

Plasmid pPH207 is not mobilised by a Hfr, but its mobilisation is promoted by ColE1. It is therefore Mob - bom +. Experiments with minicells show that it directs the copious synthesis of chloramphenicol transacetylase.

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  1. Alton NK, Vapnek D (1970) Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature 282:864

  2. Bazaral M, Helinski DR (1970) Replication of a bacterial plasmid and an episome in E. coli. Biochemistry 9:399

  3. Benton WD, Davis RW (1977) Screening λgt recombinant clones by hybridisation to single plaques in situ. Science 196:180

  4. Bishop JO (1979) A DNA sequence cleaved by restriction endonuclease R. EcoRI in only one strand. J Mol Biol 128:545

  5. Bolivar F, Betlach MC, Heyneker HL, Shine J, Rodriguez RL, Boyer HW (1977) Origin of replication of pBR345 plasmid DNA. Proc Natl Acad Sci USA 74:5265

  6. Chang ACY, Cohen SN (1978) Construction and characterization of amplifyable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141

  7. Clewell DB, Evenchik B, Cranston JW (1972) Direct inhibition of ColE1 plasmid DNA replication in E. coli by rifampicin. Nature (London) New Biol 237:29

  8. Davis RW, Simon M, Davidson N (1971) Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. In: Methods in enzymology, vol 21. Academic Press, New York, p413

  9. Gosden JR, Irving MI, Bishop JO (1971) Galactose-specific messenger RNA contents in E. coli. Biochem J 121:109

  10. Green RJ, Poonian MS, Nussbaum AL, Tobias L, Garfin DE, Boyer HW, Goodman HM (1975) Restriction and modification of a self-complementary octanucleotide containing the EcoRI substrate. J Mol Biol 99:237

  11. Hirsch HJ, Saedler H, Starlinger P (1972) Insertion mutations in the control region of the gal operon in E. coli. II. Physical characterisation of the mutations. Mol Gen Genet 115:266

  12. Hu S, Ohtsubo E, Davidson N (1975) Electron microscope heteroduplex studies of sequence relations among bacterial plasmids. XII. Identification and mapping of the insertion sequences of IS1 and IS2 in F and R plasmids. J Bacteriol 122:764

  13. Inselburg J, Ware P (1979) A complementation analysis of mobilization deficient mutants of the plasmid ColE1. Mol Gen Genet 172:211

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

  15. Jovin TH, Englund PT, Bertsch LL (1969) Physical and chemical studies of a homogeneous DNA polymerase. J Biol Chem 244:2996

  16. Mandel M, Higa A (1970) Calcium-dependent bacteriophage DNA infection. J Mol Biol 53:159

  17. Maniatis T, Jeffrey A, van de Sande H (1975) Chain length determination of small double and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry 14:3787

  18. Meagher RB, Tait RD, Betlach M, Boyer HW (1977) Protein expression in E. coli minicells by recombinant plasmids. Cell 10:521

  19. Messing J, Standenbauer WL, Hofschneider PH (1972) Inhibition of minicircular DNA replication in Escherichia coli 15 by rifampicin. Nature (London) New Biol 238:202

  20. Ohtsubo H, Ohtsubo E (1978) Nucleotide sequence of an insertion element, IS1. Proc Natl Acad Sci, USA 75:615

  21. Perlman S, Phillips C, Bishop JO (1976) A study of foldback DNA. Cell 8:33

  22. Polisky B, Green P, Garfin DE, McCarthy BJ, Goodman HM, Boyer HW (1975) Specificity of substrate recognition by the Eco.RI restriction endonuclease. Proc Natl Acad Sci USA 72:3310

  23. Reif HJ, Saedler H (1975) IS1 is involved in deletion formation in the gal region of E. coli K12. Mol Gen Genet 137:17

  24. Roberts RJ, Myers PA, Morrison A, Murray K (1975) A second specific endonuclease from Haemophilus aegyptius. J Mol Biol 91:121

  25. Rodriguez RL, Bolivar F, Goodman HM, Boyer HW, Betlach M (1976) Construction and characterisation of cloning vehicles. In: Molecular mechanisms in the control of gene expression. Academic Press, New York, p471

  26. Saedler H, Heiss B (1973) Multiple copies of the insertion DNA sequences IS1 and IS2 in the chromosome of E. coli K12. Mol Gen Genet 122:267

  27. Shaw WV (1967) The enzymatic acetylation of chloramphenicol by extracts of R factor-resistant Escherichia coli. J Biol Chem 242:687

  28. Shaw WV, Packman LC, Burleigh BD, Dell A, Morris HR, Hartley BS (1979) Primary structure of a chloramphenicol acetyltransferase specified by R plasmids. Nature 282:870

  29. Shenk J, Rhodes C, Rigby P, Berg P (1975) Biochemical method for mapping mutational alterations in DNA with S1 nuclease: the location of deletions and temperature-sensitive mutations in SV40. Proc Natl Acad Sci USA 72:989

  30. Smith HO, Birnstiel ML (1976) A simple method for DNA restriction site mapping. Nucleic Acids Res 3:2387

  31. Smith DI, Blattner FR, Davies J (1976) The isolation and partial characterisation of a new restriction endonuclease from Providencia stuartii. Nucleic Acids Res 3:343

  32. Suzuki Y, Okamoto S (1967) The enzymatic acetylation of chloramphenicol by the multiple drug resistant Escherichia coli carrying R factor. J Biol Chem 242:4722

  33. Tikchonenko TL, Karamov EV, Zavizion BA, Naroditsky BS (1978) EcoRI* activity: enzyme modification or activation of accompanying endonuclease? Gene 4:195

  34. Timmis K, Cabello F, Cohen SN (1975) Clining, isolation and characterization of replication regions of complex plasmid genomes. Proc Natl Acad Sci USA 72:2242

  35. Vogt VM (1973) Purification and further properties of singlestranded-specific nuclease from Aspergillus oryzae. Eur J Biochem 33:192

  36. Warren GJ, Twigg AJ, Sherratt DJ (1978) ColE1 plasmid mobility and relaxation complex. Nature 274:259

  37. Weiss B, Jacquemin-Sablon A, Live TR, Fareed GC, Richardson CC (1968) Further purification and properties of polynucleotide kinase from Escherichia coli infected with bacteriophage T4. J Biol Chem 243:4543

  38. Wilson GA, Young FE (1975) Isolation of a sequence-specific endonuclease from Bacillus amyloliquefaciens H. J Mol Biol 97:123

  39. Yoshimori RN (1971) A genetic and biochemical analysis of the restriction and modification of DNA by resistance transfer factors. Ph D thesis, University of California, San Francisco

  40. Zimmerman SB, Sandeen G (1966) The ribonuclease activity of crystallised, pancreatic deoxyribonuclease. Anal Biochem 14:269

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Correspondence to J. O. Bishop.

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Communicated by E. Bautz

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Bishop, J.O., Davies, J.A. Plasmid cloning vectors that can be nicked at a unique site. Molec. Gen. Genet. 179, 573–580 (1980). https://doi.org/10.1007/BF00271747

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  • Chloramphenicol
  • Cloning Vector
  • EcoRI Site
  • Plasmid Cloning
  • Unique Site