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. 1977 Apr;130(1):148–153. doi: 10.1128/jb.130.1.148-153.1977

Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation.

D D Womble, D P Taylor, R H Rownd
PMCID: PMC235186  PMID: 323223

Abstract

A method that gives high recovery of deoxyribonucleic acid (DNA) from crude bacterial lysates using ethidium bromide-cesium chloride density gradient centrifugation is presented. After Pronase digestion and shearing of the lysate, essentially 100% recovery of chromosomal DNA and a reproducible recovery of covalently closed circular (CCC) plasmid DNA is obtained for a specific plasmid in a given strain. This method should be useful for comparing the CCC plasmid/chromosome ratio of various plasmid-host combinations.

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Selected References

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

  1. Bauer W., Vinograd J. The interaction of closed circular DNA with intercalative dyes. I. The superhelix density of SV40 DNA in the presence and absence of dye. J Mol Biol. 1968 Apr 14;33(1):141–171. doi: 10.1016/0022-2836(68)90286-6. [DOI] [PubMed] [Google Scholar]
  2. Bazaral M., Helinski D. R. Circular DNA forms of colicinogenic factors E1, E2 and E3 from Escherichia coli. J Mol Biol. 1968 Sep 14;36(2):185–194. doi: 10.1016/0022-2836(68)90374-4. [DOI] [PubMed] [Google Scholar]
  3. Bazaral M., Helinski D. R. Replication of a bacterial plasmid and an episome in Escherichia coli. Biochemistry. 1970 Jan 20;9(2):399–406. doi: 10.1021/bi00804a029. [DOI] [PubMed] [Google Scholar]
  4. Clewell D. B., Yagi Y., Dunny G. M., Schultz S. K. Characterization of three plasmid deoxyribonucleic acid molecules in a strain of Streptococcus faecalis: identification of a plasmid determining erythromycin resistance. J Bacteriol. 1974 Jan;117(1):283–289. doi: 10.1128/jb.117.1.283-289.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clowes R. C. Molecular structure of bacterial plasmids. Bacteriol Rev. 1972 Sep;36(3):361–405. doi: 10.1128/br.36.3.361-405.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cress D. E., Kline B. C. Isolation and characterization of Escherichia coli chromosomal mutants affecting plasmid copy number. J Bacteriol. 1976 Feb;125(2):635–642. doi: 10.1128/jb.125.2.635-642.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Freifelder D., Folkmanis A., Kirschner I. Studies on Escherichia coli sex factors: evidence that covalent circles exist within cells and the general problem of isolation of covalent circles. J Bacteriol. 1971 Mar;105(3):722–727. doi: 10.1128/jb.105.3.722-727.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Helinski D. R., Clewell D. B. Circular DNA. Annu Rev Biochem. 1971;40:899–942. doi: 10.1146/annurev.bi.40.070171.004343. [DOI] [PubMed] [Google Scholar]
  9. Helinski D. R. Plasmid determined resistance to antibiotics: molecular properties of R factors. Annu Rev Microbiol. 1973;27:437–470. doi: 10.1146/annurev.mi.27.100173.002253. [DOI] [PubMed] [Google Scholar]
  10. Hershberger C., Mickel S., Rownd R. Asymmetric distribution of guanine plus thymine between complementary strands of deoxyribonucleic acid of members of the Enterobacteriaceae. J Bacteriol. 1971 Apr;106(1):238–242. doi: 10.1128/jb.106.1.238-242.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kasamatsu H., Rownd R. Replication of R-factors in Proteus mirabilis: replication under relaxed control. J Mol Biol. 1970 Aug;51(3):473–489. doi: 10.1016/0022-2836(70)90002-1. [DOI] [PubMed] [Google Scholar]
  12. Kupersztoch-Portnoy Y. M., Miklos G. L., Helinski D. R. Properties of the relaxation complexes of supercoiled deoxyribonucleic acid and protein of the R plasmids R64, R28K, and R6K. J Bacteriol. 1974 Oct;120(1):545–548. doi: 10.1128/jb.120.1.545-548.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Morris C. F., Hashimoto H., Mickel S., Rownd R. Round of replication mutant of a drug resistance factor. J Bacteriol. 1974 Jun;118(3):855–866. doi: 10.1128/jb.118.3.855-866.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Morris C. F., Hershberger C. L., Rownd R. Strand-specific nick in open circular R-factor deoxyribonucleic acid: attachment of the linear strand to a proteinaceous cellular component. J Bacteriol. 1973 Apr;114(1):300–308. doi: 10.1128/jb.114.1.300-308.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nakaya R., Rownd R. Transduction of R factors by a Proteus mirabilis bacteriophage. J Bacteriol. 1971 Jun;106(3):773–783. doi: 10.1128/jb.106.3.773-783.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nisioka T., Mitani M., Clowes R. C. Molecular recombination between R-factor deoxyribonucleic acid molecules in Escherichia coli host cells. J Bacteriol. 1970 Jul;103(1):166–177. doi: 10.1128/jb.103.1.166-177.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Perlman D., Rownd R. H. Transition of R factor NR1 in Proteus mirabilis: molecular structure and replication of NR1 deoxyribonucleic acid. J Bacteriol. 1975 Sep;123(3):1013–1034. doi: 10.1128/jb.123.3.1013-1034.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Radloff R., Bauer W., Vinograd J. A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc Natl Acad Sci U S A. 1967 May;57(5):1514–1521. doi: 10.1073/pnas.57.5.1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schubach W. H., Whitmer J. D., Davern C. I. Genetic control of DNA initiation in Escherichia coli. J Mol Biol. 1973 Feb 25;74(2):205–221. doi: 10.1016/0022-2836(73)90107-1. [DOI] [PubMed] [Google Scholar]
  20. Tanak N., Cramer J. H., Rownd R. H. EcoRI restriction endonuclease map of the composite R plasmid NR1. J Bacteriol. 1976 Jul;127(1):619–636. doi: 10.1128/jb.127.1.619-636.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Womble D. D., Warren R. L., Rownd R. H. Partially supercoiled replication intermediates of R plasmid DNA are resistant to relaxation. Nature. 1976 Dec 16;264(5587):676–678. doi: 10.1038/264676a0. [DOI] [PubMed] [Google Scholar]

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