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. 1988 Feb;7(2):303–308. doi: 10.1002/j.1460-2075.1988.tb02813.x

Replication termini in the rDNA of synchronized pea root cells (Pisum sativum)

P Hernández 1, S S Lamm 1, C A Bjerknes 1, J Van't Hof 1
PMCID: PMC454311  PMID: 16453826

Abstract

In synchronized root cells of Pisum sativum (cv. Alaska) the joining of nascent replicons is delayed until cells reach the S–G2 boundary or early G2 phase. To determine if the delayed ligation of nascent chains occurs at specific termination sites, we mapped the location of arrested forks in the ribosomal DNA (rDNA) repeats from cells in late S and G2 phases. Two-dimensional (neutral-alkaline) agarose electrophoresis and Southern blot hybridization with specific rDNA sequences show that only cells located at the S–G2 boundary and early G2 phase produce alkali-released rDNA fragments of discrete size. The released fragments are from a particular restriction fragment, demonstrating that the replication forks stop non-randomly within the rDNA repeats. Indirect end-labeling with probes homologous to one or the other end of the fork-containing restriction fragment shows that there are two termination regions, T1 and T2, where forks stop. T1 is located in the non-transcribed spacer and T2 is at the junction between the non-transcribed spacer and the 18S gene. The two termini are separated by ˜1.3 kb. Replication forks stop at identical sites in both the 8.6- and 9.0-kb rDNA repeat size classes indicating that these sites are sequence determined.

Keywords: replication termini, rDNA, Pisum sativum

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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., Germino J., Crosa J. H., Hale P. Molecular cloning of the replication terminus of the plasmid R6K. Gene. 1981 Jun-Jul;14(1-2):81–89. doi: 10.1016/0378-1119(81)90150-5. [DOI] [PubMed] [Google Scholar]
  2. Burr B., Burr F. A. Controlling-element events at the shrunken locus in maize. Genetics. 1981 May;98(1):143–156. doi: 10.1093/genetics/98.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cullis C. A., Davies D. R. Ribosomal DNA Amounts in PISUM SATIVUM. Genetics. 1975 Nov;81(3):485–492. doi: 10.1093/genetics/81.3.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Funderud S., Andreassen R., Haugli F. Size distribution and maturation of newly replicated DNA through the S and G2 phases of Physarum polycephalum. Cell. 1978 Dec;15(4):1519–1526. doi: 10.1016/0092-8674(78)90074-0. [DOI] [PubMed] [Google Scholar]
  5. Germino J., Bastia D. Termination of DNA replication in vitro at a sequence-specific replication terminus. Cell. 1981 Mar;23(3):681–687. doi: 10.1016/0092-8674(81)90431-1. [DOI] [PubMed] [Google Scholar]
  6. Hanley P. J., Carrigan C. M., Rowe D. B., Wake R. G. Breakdown and quantitation of the forked termination of replication intermediate of Bacillus subtilis. J Mol Biol. 1987 Aug 5;196(3):721–727. doi: 10.1016/0022-2836(87)90043-x. [DOI] [PubMed] [Google Scholar]
  7. Hill T. M., Henson J. M., Kuempel P. L. The terminus region of the Escherichia coli chromosome contains two separate loci that exhibit polar inhibition of replication. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1754–1758. doi: 10.1073/pnas.84.7.1754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ingle J., Sinclair J. Ribosomal RNA genes and plant development. Nature. 1972 Jan 7;235(5332):30–32. doi: 10.1038/235030a0. [DOI] [PubMed] [Google Scholar]
  9. Kolter R., Helinski D. R. Activity of the replication terminus of plasmid R6K in hybrid replicons in Escherichia coli. J Mol Biol. 1978 Sep 25;124(3):425–441. doi: 10.1016/0022-2836(78)90180-8. [DOI] [PubMed] [Google Scholar]
  10. Kowalski J., Cheevers W. P. Synthesis of high molecular weight DNA strands during S phase. J Mol Biol. 1976 Jul 5;104(3):603–615. doi: 10.1016/0022-2836(76)90123-6. [DOI] [PubMed] [Google Scholar]
  11. Kuempel P. L., Duerr S. A. Chromosome replication in Escherichia coli is inhibited in the terminus region near the rac locus. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):563–567. doi: 10.1101/sqb.1979.043.01.062. [DOI] [PubMed] [Google Scholar]
  12. Kuempel P. L., Duerr S. A., Seeley N. R. Terminus region of the chromosome in Escherichia coli inhibits replication forks. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3927–3931. doi: 10.1073/pnas.74.9.3927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Louarn J., Patte J., Louarn J. M. Evidence for a fixed termination site of chromosome replication in Escherichia coli K12. J Mol Biol. 1977 Sep 25;115(3):295–314. doi: 10.1016/0022-2836(77)90156-5. [DOI] [PubMed] [Google Scholar]
  14. Schvartzman J. B., Chenet B., Bjerknes C., Van't Hof J. Nascent replicons are synchronously joined at the end of S phase or during G2 phase in peas. Biochim Biophys Acta. 1981 Apr 27;653(2):185–192. doi: 10.1016/0005-2787(81)90154-4. [DOI] [PubMed] [Google Scholar]
  15. Schvartzman J. B., Krimer D. B., Van't Hof J. The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment. Exp Cell Res. 1984 Feb;150(2):379–389. doi: 10.1016/0014-4827(84)90581-0. [DOI] [PubMed] [Google Scholar]
  16. Tapper D. P., DePamphilis M. L. Preferred DNA sites are involved in the arrest and initiation of DNA synthesis during replication of SV40 DNA. Cell. 1980 Nov;22(1 Pt 1):97–108. doi: 10.1016/0092-8674(80)90158-0. [DOI] [PubMed] [Google Scholar]
  17. Thompson J. E. Peer review in an American College of Nurse-Midwives' local chapter. J Nurse Midwifery. 1986 Nov-Dec;31(6):289–295. doi: 10.1016/0091-2182(86)90040-6. [DOI] [PubMed] [Google Scholar]
  18. Walters R. A., Tobey R. A., Hildebrand C. E. Chain elongation and joining of DNA synthesized during hydroxyurea treatment of Chinese hamster cells. Biochim Biophys Acta. 1976 Sep 20;447(1):36–44. doi: 10.1016/0005-2787(76)90093-9. [DOI] [PubMed] [Google Scholar]
  19. Watson J. C., Kaufman L. S., Thompson W. F. Developmental regulation of cytosine methylation in the nuclear ribosomal RNA genes of Pisum sativum. J Mol Biol. 1987 Jan 5;193(1):15–26. doi: 10.1016/0022-2836(87)90622-x. [DOI] [PubMed] [Google Scholar]
  20. Weaver D. T., Fields-Berry S. C., DePamphilis M. L. The termination region for SV40 DNA replication directs the mode of separation for the two sibling molecules. Cell. 1985 Jun;41(2):565–575. doi: 10.1016/s0092-8674(85)80029-5. [DOI] [PubMed] [Google Scholar]
  21. Weiss A. S., Wake R. G. A unique DNA intermediate associated with termination of chromosome replication in Bacillus subtilis. Cell. 1984 Dec;39(3 Pt 2):683–689. doi: 10.1016/0092-8674(84)90475-6. [DOI] [PubMed] [Google Scholar]
  22. Weiss A. S., Wake R. G., Inman R. B. An immobilized fork as a termination of replication intermediate in Bacillus subtilis. J Mol Biol. 1986 Mar 20;188(2):199–205. doi: 10.1016/0022-2836(86)90304-9. [DOI] [PubMed] [Google Scholar]
  23. de Massy B., Béjar S., Louarn J., Louarn J. M., Bouché J. P. Inhibition of replication forks exiting the terminus region of the Escherichia coli chromosome occurs at two loci separated by 5 min. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1759–1763. doi: 10.1073/pnas.84.7.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]

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