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. 1984 Aug;3(8):1731–1735. doi: 10.1002/j.1460-2075.1984.tb02039.x

Cloning the origin of transfer region of the resistance plasmid R1.

E Ostermann, F Kricek, G Högenauer
PMCID: PMC557589  PMID: 6383819

Abstract

The insertion of a 7.7-kb EcoRI fragment of the resistance plasmid R1 into pBR325 yielded a plasmid which is mobilizable by pDB12, a multicopy derivative of R1drd-19 lacking most of the resistance determinants. The vector alone was not mobilizable in this system. From this observation we conclude that we have cloned the origin of transfer (oriT) of R1. After inserting a 5.3-kb PvuII-EcoRI fragment of the 7.7-kb region into pUC9 the DNA was cleaved randomly with DNaseI and BamHI linkers were attached to the ends. A subsequent BamHI digestion and electrophoretic separation of the resulting DNA molecules by their size allowed us to generate an ordered series of stepwise shortened plasmids. Plasmids with a deletion of approximately 3400 bp could no longer be mobilized. Since the next larger plasmid with 284 additional base pairs could be mobilized, we are able to confine the oriT location within this extra nucleotide stretch. The DNA sequence of this region was determined. Dominant features within the DNA region are a high AT content and five inverted repeats, which might function as recognition or substrate sites for proteins of the conjugational transfer system.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bastia D. Determination of restriction sites and the nucleotide sequence surrounding the relaxation site of ColE1. J Mol Biol. 1978 Oct 5;124(4):601–639. doi: 10.1016/0022-2836(78)90174-2. [DOI] [PubMed] [Google Scholar]
  2. Blohm D., Goebel W. Restriction map of the antibiotic resistance plasmid R1drd-19 and its derivatives pKN102 (R1drd-19B2) and R1drd-16 for the enzymes BamHI, HindIII, EcoRI and SalI. Mol Gen Genet. 1978 Nov 29;167(2):119–127. doi: 10.1007/BF00266905. [DOI] [PubMed] [Google Scholar]
  3. Clerget M., Chandler M., Caro L. The structure of R1drd19: a revised physical map of the plasmid. Mol Gen Genet. 1981;181(2):183–191. doi: 10.1007/BF00268425. [DOI] [PubMed] [Google Scholar]
  4. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Everett R., Willetts N. Characterisation of an in vivo system for nicking at the origin of conjugal DNA transfer of the sex factor F. J Mol Biol. 1980 Jan 15;136(2):129–150. doi: 10.1016/0022-2836(80)90309-5. [DOI] [PubMed] [Google Scholar]
  6. Everett R., Willetts N. Cloning, mutation, and location of the F origin of conjugal transfer. EMBO J. 1982;1(6):747–753. doi: 10.1002/j.1460-2075.1982.tb01241.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Finnegan D., Willetts N. The site of action of the F transfer inhibitor. Mol Gen Genet. 1973 Dec 31;127(4):307–316. doi: 10.1007/BF00267101. [DOI] [PubMed] [Google Scholar]
  8. Frischauf A. M., Garoff H., Lehrach H. A subcloning strategy for DNA sequence analysis. Nucleic Acids Res. 1980 Dec 11;8(23):5541–5549. doi: 10.1093/nar/8.23.5541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guiney D. G., Yakobson E. Location and nucleotide sequence of the transfer origin of the broad host range plasmid RK2. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3595–3598. doi: 10.1073/pnas.80.12.3595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  11. Johnson D., Everett R., Willetts N. Cloning of F DNA fragments carrying the origin of transfer oriT and the fertility inhibition gene finP. J Mol Biol. 1981 Dec 5;153(2):187–202. doi: 10.1016/0022-2836(81)90273-4. [DOI] [PubMed] [Google Scholar]
  12. Lovett M. A., Guiney D. G., Helinski D. R. Relaxation complexes of plasmids ColE1 and ColE2: unique site of the nick in the open circular DNA of the relaxed complexes. Proc Natl Acad Sci U S A. 1974 Oct;71(10):3854–3857. doi: 10.1073/pnas.71.10.3854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  15. Meynell E., Datta N. Mutant drug resistant factors of high transmissibility. Nature. 1967 May 27;214(5091):885–887. doi: 10.1038/214885a0. [DOI] [PubMed] [Google Scholar]
  16. Nordström K., Ingram L. C., Lundbäck A. Mutations in R factors of Escherichia coli causing an increased number of R-factor copies per chromosome. J Bacteriol. 1972 May;110(2):562–569. doi: 10.1128/jb.110.2.562-569.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sharp P. A., Cohen S. N., Davidson N. Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. II. Structure of drug resistance (R) factors and F factors. J Mol Biol. 1973 Apr 5;75(2):235–255. doi: 10.1016/0022-2836(73)90018-1. [DOI] [PubMed] [Google Scholar]
  18. Warren G., Sherratt D. Complementation of transfer deficient ColE1 mutants. Mol Gen Genet. 1977 Mar 7;151(2):197–201. doi: 10.1007/BF00338695. [DOI] [PubMed] [Google Scholar]
  19. Willetts N. S. Location of the origin of transfer of the sex factor F. J Bacteriol. 1972 Nov;112(2):773–778. doi: 10.1128/jb.112.2.773-778.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Willetts N., Skurray R. The conjugation system of F-like plasmids. Annu Rev Genet. 1980;14:41–76. doi: 10.1146/annurev.ge.14.120180.000353. [DOI] [PubMed] [Google Scholar]
  21. Willetts N. The transcriptional control of fertility in F-like plasmids. J Mol Biol. 1977 May 5;112(1):141–148. doi: 10.1016/s0022-2836(77)80161-7. [DOI] [PubMed] [Google Scholar]

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