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. 1974 Mar;117(3):1201–1209. doi: 10.1128/jb.117.3.1201-1209.1974

Size Distribution of the Closed Circular Deoxyribonucleic Acid Molecules of Bacillus megaterium: Sedimentation Velocity and Electron Microscope Measurements

Bruce C Carlton 1, Malcolm P W Smith 2
PMCID: PMC246602  PMID: 4205193

Abstract

The circular deoxyribonucleic acid (DNA) of Bacillus megaterium was fractionated by sedimentation velocity on preparative zonal gradients. The fractions obtained were characterized by analytical sedimentation velocity analysis on neutral and alkaline sucrose gradients, and by contour length measurement by electron microscopy. Each fraction was found to contain circular molecules of one to three discrete sizes, in various combinations of covalently closed and open circular forms. Estimations of the molecular masses of these sizes gave values of 3.9, 6.2, 16.0, 31, and 60 million daltons for the major molecular species. Minor amounts of molecules of 7.6, 11.7, 47, 89, and 112 million daltons were observed in the electron microscope analyses. Length measurements of almost 600 molecules from the various fractions showed that all except six could be placed in distinct size classes. The distribution of molecular sizes in unfractionated circular DNA was shown to consist primarily of the two smallest size classes, although the relative proportions by weight of five of the classes were roughly equivalent.

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Selected References

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

  1. Carlton B. C., Helinski D. R. Heterogeneous circular DNA elements in vegetative cultures of Bacillus megaterium. Proc Natl Acad Sci U S A. 1969 Oct;64(2):592–599. doi: 10.1073/pnas.64.2.592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Cuzin F., Vogt M., Dieckmann M., Berg P. Induction of virus multiplication in 3T3 cells transformed by a thermosensitive mutant of polyoma virus. II. Formation of oligometric polyoma DNA molecules. J Mol Biol. 1970 Feb 14;47(3):317–333. doi: 10.1016/0022-2836(70)90305-0. [DOI] [PubMed] [Google Scholar]
  5. Freifelder D. Molecular weights of coliphages and coliphage DNA. IV. Molecular weights of DNA from bacteriophages T4, T5 and T7 and the general problem of determination of M. J Mol Biol. 1970 Dec 28;54(3):567–577. doi: 10.1016/0022-2836(70)90127-0. [DOI] [PubMed] [Google Scholar]
  6. Godson G. N., Vapnek D. A simple method of preparing large amounts of phiX174 RF 1 supercoiled DNA. Biochim Biophys Acta. 1973 Apr 11;299(4):516–520. doi: 10.1016/0005-2787(73)90223-2. [DOI] [PubMed] [Google Scholar]
  7. Hedgpeth J., Goodman H. M., Boyer H. W. DNA nucleotide sequence restricted by the RI endonuclease. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3448–3452. doi: 10.1073/pnas.69.11.3448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Henneberry R. C., Carlton B. C. Characterization of the polydisperse closed circular deoxyribonucleic acid molecules of Bacillus megaterium. J Bacteriol. 1973 May;114(2):625–631. doi: 10.1128/jb.114.2.625-631.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hudson B., Clayton D. A., Vinograd J. Complex mitochondrial DNA. Cold Spring Harb Symp Quant Biol. 1968;33:435–442. doi: 10.1101/sqb.1968.033.01.050. [DOI] [PubMed] [Google Scholar]
  10. Lang D. Molecular weights of coliphages and coliphage DNA. 3. Contour length and molecular weight of DNA from bacteriophages T4, T5 and T7, and from bovine papilloma virus. J Mol Biol. 1970 Dec 28;54(3):557–565. doi: 10.1016/0022-2836(70)90126-9. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Roth T. F., Hayashi M. Allomorphic forms of bacteriophage phiX-174 replicative DNA. Science. 1966 Nov 4;154(3749):658–660. doi: 10.1126/science.154.3749.658. [DOI] [PubMed] [Google Scholar]
  13. STUDIER F. W. SEDIMENTATION STUDIES OF THE SIZE AND SHAPE OF DNA. J Mol Biol. 1965 Feb;11:373–390. doi: 10.1016/s0022-2836(65)80064-x. [DOI] [PubMed] [Google Scholar]
  14. Yamamoto K. R., Alberts B. M., Benzinger R., Lawhorne L., Treiber G. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology. 1970 Mar;40(3):734–744. doi: 10.1016/0042-6822(70)90218-7. [DOI] [PubMed] [Google Scholar]

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