Summary
By the use of a restriction enzyme digestion of gently lysed E. coli or B. subtilis cells, it is possible to isolate a minute fraction of the total DNA that has an unusually high sedimentation coefficient. Upon inspection of this DNA in the electron microscope, branched DNA fragments are observed. Single branched DNA fragments were analyzed by restriction enzyme and partial denaturation mapping techniques. The fragments appear to have the properties of growing forks excised from in vivo replicating intermediates. In B. subtilis, the minute fraction of DNA was also investigated by transformation assays and found to be greatly enriched for a marker near the origin and slightly enriched for a terminus marker.
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References
Aguinaga M d P (1979) Master's Thesis. Seccion Bioquimica, Universitdad Peruana Cayetano Heredia, Lima, Peru
Bott KF, Wilson GA (1967) Development of competence in the B. subtilis transformation system. J Bacteriol 94:562–570
Boylan RJ, Mendelson NH, Brooks D, Young FE (1972) Regulation of the bacterial cell wall: Analysis of a mutant of B. subtilis defective in biosynthesis of teichoic acid. J Bacteriol 110:281–290
Chattoraj DK, Inman RB (1973) Origin and direction of replication of bacteriophage 186 DNA. Proc Natl Acad Sci USA 70:1768–1771
Clark PO, Leach FR (1980) Stimulation of B. subtilis transformation by sepermidine. Molec Gen Genet 178:21–26
Ganesan AT, Lederberg J (1965) A cell membrane bound fraction of DNA. Biochem Biophys Res Commun 18:824–835
Henner DJ, Hoch JA (1980) The Bacillus subtilis chromosome. Microbiol Rev 44:57–82
Inman RB, Schnös M (1971) Structure of branch points in replicating DNA: Presence of single stranded connections in λ DNA branch points. J Mol Biol 56:319–325
Ogasawara N, Seiki M, Yoshikawa H (1979) Effect of novobiocin on initiation of DNA replication in Bacillus subtilis. Nature 281:702–704
Parker DL, Glaser DA (1975) Effect of growth conditions on DNA membrane attachment in Escherichia coli. Proc Natl Acad Sci USA 72:2446–2450
Schnös M, Inman RB (1971) Staring point and direction of replication in P2 DNA. J Mol Biol 55:31–38
Smith DW, Hanawalt PC (1967) Properties of the growing point region in the bacterial chromosome. Biochim Biophys Acta 149:519–531
Sueoka N, Quinn WG (1968) Membrane attachment of the chromosome replication origin in Bacillus subtilis. Cold Spring Harbor Symp Quant Biol 33:695–705
Sueoka N, Hammers JM (1974) Isolation of DNA-membrane complex in Bacillus subtilis. Proc Natl Acad Sci USA 71:4787–4791
Valenzuela MS, Inman RB (1975) Visualization of a novel junction in bacteriophage λ DNA. Proc Natl Acad Sci USA 72:3024–3028
Valenzuela MS, Inman RB (1978) Restriction enzyme cleavage of DNA resulting from gently lysed Escherichia coli. Mol Gen Genet 166:245–249
Yamaguchi K, Yoshikawa H (1977) Chromosome-membrane association in B. subtilis. III. Isolation and characterization of a DNA-protein complex carrying replication origin markers. J Mol Biol 110:219–253
Yasuda S, Hirota Y (1977) Cloning and mapping of the replication origin of Escherichia coli. Proc Natl Acad Sci USA 74:5458–5462
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Communicated by F.W. Stahl
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Valenzuela, M.S., Aguinaga, M.D.P. & Imman, R.B. Isolation of DNA fragments containing replicating growing forks from both E. coli and B. subtilis . Molec. Gen. Genet. 181, 241–247 (1981). https://doi.org/10.1007/BF00268432
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DOI: https://doi.org/10.1007/BF00268432