Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Sep;16(9):4915–4922. doi: 10.1128/mcb.16.9.4915

Composite patterns in neutral/neutral two-dimensional gels demonstrate inefficient replication origin usage.

R F Kalejta 1, J L Hamlin 1
PMCID: PMC231493  PMID: 8756650

Abstract

The neutral/neutral two-dimensional (2-D) gel replicon mapping technique has been used to great advantage to localize and characterize origins of replication. Interestingly, many yeast origins display a composite pattern consisting of both a bubble arc and a single-fork arc. Moreover, in every instance in which neutral/neutral 2-D gels have been used to analyze origins in higher eukaryotic cells, two or more adjacent fragments display these composite patterns. We believe that composite patterns signal inefficient origin usage in yeast cells because the replicators in question are not active in every cell cycle and in higher eukaryotic replicons because initiation sites are chosen from among many potential sites lying within a zone. However, others have suggested that the single-fork arcs in these composite gel patterns arise from nicking activity that converts replication bubbles to branched structures that comigrate with bona fide single forks. Here, we have used three different replicon mapping strategies to show that broken simian virus 40 replication bubbles trace unique arcs that are clearly distinguishable from classic, intact single forks. Thus, it is likely that composite 2-D gel patterns represent origins that are inefficiently utilized.

Full Text

The Full Text of this article is available as a PDF (662.4 KB).

Selected References

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

  1. Anachkova B., Hamlin J. L. Replication in the amplified dihydrofolate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element. Mol Cell Biol. 1989 Feb;9(2):532–540. doi: 10.1128/mcb.9.2.532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BLACK P. H., CRAWFORD E. M., CRAWFORD L. V. THE PURIFICATION OF SIMIAN VIRUS 40. Virology. 1964 Nov;24:381–387. doi: 10.1016/0042-6822(64)90175-8. [DOI] [PubMed] [Google Scholar]
  3. Bell L., Byers B. Separation of branched from linear DNA by two-dimensional gel electrophoresis. Anal Biochem. 1983 Apr 15;130(2):527–535. doi: 10.1016/0003-2697(83)90628-0. [DOI] [PubMed] [Google Scholar]
  4. Brewer B. J., Fangman W. L. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell. 1987 Nov 6;51(3):463–471. doi: 10.1016/0092-8674(87)90642-8. [DOI] [PubMed] [Google Scholar]
  5. Brun C., Dijkwel P. A., Little R. D., Hamlin J. L., Schildkraut C. L., Huberman J. A. Yeast and mammalian replication intermediates migrate similarly in two-dimensional gels. Chromosoma. 1995 Nov;104(2):92–102. doi: 10.1007/BF00347691. [DOI] [PubMed] [Google Scholar]
  6. Burhans W. C., Selegue J. E., Heintz N. H. Isolation of the origin of replication associated with the amplified Chinese hamster dihydrofolate reductase domain. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7790–7794. doi: 10.1073/pnas.83.20.7790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burhans W. C., Selegue J. E., Heintz N. H. Replication intermediates formed during initiation of DNA synthesis in methotrexate-resistant CHOC 400 cells are enriched for sequences derived from a specific, amplified restriction fragment. Biochemistry. 1986 Jan 28;25(2):441–449. doi: 10.1021/bi00350a025. [DOI] [PubMed] [Google Scholar]
  8. Burhans W. C., Vassilev L. T., Caddle M. S., Heintz N. H., DePamphilis M. L. Identification of an origin of bidirectional DNA replication in mammalian chromosomes. Cell. 1990 Sep 7;62(5):955–965. doi: 10.1016/0092-8674(90)90270-o. [DOI] [PubMed] [Google Scholar]
  9. Delidakis C., Kafatos F. C. Amplification enhancers and replication origins in the autosomal chorion gene cluster of Drosophila. EMBO J. 1989 Mar;8(3):891–901. doi: 10.1002/j.1460-2075.1989.tb03450.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dershowitz A., Newlon C. S. The effect on chromosome stability of deleting replication origins. Mol Cell Biol. 1993 Jan;13(1):391–398. doi: 10.1128/mcb.13.1.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dijkwel P. A., Hamlin J. L. Initiation of DNA replication in the dihydrofolate reductase locus is confined to the early S period in CHO cells synchronized with the plant amino acid mimosine. Mol Cell Biol. 1992 Sep;12(9):3715–3722. doi: 10.1128/mcb.12.9.3715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dijkwel P. A., Hamlin J. L. Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells. Mol Cell Biol. 1988 Dec;8(12):5398–5409. doi: 10.1128/mcb.8.12.5398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dijkwel P. A., Hamlin J. L. The Chinese hamster dihydrofolate reductase origin consists of multiple potential nascent-strand start sites. Mol Cell Biol. 1995 Jun;15(6):3023–3031. doi: 10.1128/mcb.15.6.3023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dijkwel P. A., Vaughn J. P., Hamlin J. L. Mapping of replication initiation sites in mammalian genomes by two-dimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix. Mol Cell Biol. 1991 Aug;11(8):3850–3859. doi: 10.1128/mcb.11.8.3850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dijkwel P. A., Vaughn J. P., Hamlin J. L. Replication initiation sites are distributed widely in the amplified CHO dihydrofolate reductase domain. Nucleic Acids Res. 1994 Nov 25;22(23):4989–4996. doi: 10.1093/nar/22.23.4989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ferguson B. M., Brewer B. J., Reynolds A. E., Fangman W. L. A yeast origin of replication is activated late in S phase. Cell. 1991 May 3;65(3):507–515. doi: 10.1016/0092-8674(91)90468-e. [DOI] [PubMed] [Google Scholar]
  17. Gaudette M. F., Benbow R. M. Replication forks are underrepresented in chromosomal DNA of Xenopus laevis embryos. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5953–5957. doi: 10.1073/pnas.83.16.5953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Greenfeder S. A., Newlon C. S. A replication map of a 61-kb circular derivative of Saccharomyces cerevisiae chromosome III. Mol Biol Cell. 1992 Sep;3(9):999–1013. doi: 10.1091/mbc.3.9.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Handeli S., Klar A., Meuth M., Cedar H. Mapping replication units in animal cells. Cell. 1989 Jun 16;57(6):909–920. doi: 10.1016/0092-8674(89)90329-2. [DOI] [PubMed] [Google Scholar]
  20. Heck M. M., Spradling A. C. Multiple replication origins are used during Drosophila chorion gene amplification. J Cell Biol. 1990 Apr;110(4):903–914. doi: 10.1083/jcb.110.4.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heintz N. H., Hamlin J. L. An amplified chromosomal sequence that includes the gene for dihydrofolate reductase initiates replication within specific restriction fragments. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4083–4087. doi: 10.1073/pnas.79.13.4083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Heinzel S. S., Krysan P. J., Tran C. T., Calos M. P. Autonomous DNA replication in human cells is affected by the size and the source of the DNA. Mol Cell Biol. 1991 Apr;11(4):2263–2272. doi: 10.1128/mcb.11.4.2263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  24. Huberman J. A., Riggs A. D. On the mechanism of DNA replication in mammalian chromosomes. J Mol Biol. 1968 Mar 14;32(2):327–341. doi: 10.1016/0022-2836(68)90013-2. [DOI] [PubMed] [Google Scholar]
  25. Hyrien O., Méchali M. Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos. EMBO J. 1993 Dec;12(12):4511–4520. doi: 10.1002/j.1460-2075.1993.tb06140.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. JACOB F., BRENNER S. [On the regulation of DNA synthesis in bacteria: the hypothesis of the replicon]. C R Hebd Seances Acad Sci. 1963 Jan 2;256:298–300. [PubMed] [Google Scholar]
  27. Kalejta R. F., Lin H. B., Dijkwel P. A., Hamlin J. L. Characterizing replication intermediates in the amplified CHO dihydrofolate reductase domain by two novel gel electrophoretic techniques. Mol Cell Biol. 1996 Sep;16(9):4923–4931. doi: 10.1128/mcb.16.9.4923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Krysan P. J., Calos M. P. Replication initiates at multiple locations on an autonomously replicating plasmid in human cells. Mol Cell Biol. 1991 Mar;11(3):1464–1472. doi: 10.1128/mcb.11.3.1464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Leu T. H., Hamlin J. L. High-resolution mapping of replication fork movement through the amplified dihydrofolate reductase domain in CHO cells by in-gel renaturation analysis. Mol Cell Biol. 1989 Feb;9(2):523–531. doi: 10.1128/mcb.9.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Liang C., Gerbi S. A. Analysis of an origin of DNA amplification in Sciara coprophila by a novel three-dimensional gel method. Mol Cell Biol. 1994 Feb;14(2):1520–1529. doi: 10.1128/mcb.14.2.1520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liang C., Spitzer J. D., Smith H. S., Gerbi S. A. Replication initiates at a confined region during DNA amplification in Sciara DNA puff II/9A. Genes Dev. 1993 Jun;7(6):1072–1084. doi: 10.1101/gad.7.6.1072. [DOI] [PubMed] [Google Scholar]
  32. Liang C., Weinreich M., Stillman B. ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome. Cell. 1995 Jun 2;81(5):667–676. doi: 10.1016/0092-8674(95)90528-6. [DOI] [PubMed] [Google Scholar]
  33. Linskens M. H., Huberman J. A. Ambiguities in results obtained with 2D gel replicon mapping techniques. Nucleic Acids Res. 1990 Feb 11;18(3):647–652. doi: 10.1093/nar/18.3.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Linskens M. H., Huberman J. A. Organization of replication of ribosomal DNA in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Nov;8(11):4927–4935. doi: 10.1128/mcb.8.11.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Little R. D., Platt T. H., Schildkraut C. L. Initiation and termination of DNA replication in human rRNA genes. Mol Cell Biol. 1993 Oct;13(10):6600–6613. doi: 10.1128/mcb.13.10.6600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ma C., Leu T. H., Hamlin J. L. Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line. Mol Cell Biol. 1990 Apr;10(4):1338–1346. doi: 10.1128/mcb.10.4.1338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Martin M. A., Axelrod D. SV40 gene activity during lytic infection and in a series of SV40 transformed mouse cells. Proc Natl Acad Sci U S A. 1969 Dec;64(4):1203–1210. doi: 10.1073/pnas.64.4.1203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Martín-Parras L., Hernández P., Martínez-Robles M. L., Schvartzman J. B. Initiation of DNA replication in ColE1 plasmids containing multiple potential origins of replication. J Biol Chem. 1992 Nov 5;267(31):22496–22505. [PubMed] [Google Scholar]
  39. Martín-Parras L., Hernández P., Martínez-Robles M. L., Schvartzman J. B. Unidirectional replication as visualized by two-dimensional agarose gel electrophoresis. J Mol Biol. 1991 Aug 20;220(4):843–853. doi: 10.1016/0022-2836(91)90357-c. [DOI] [PubMed] [Google Scholar]
  40. McWhinney C., Leffak M. Autonomous replication of a DNA fragment containing the chromosomal replication origin of the human c-myc gene. Nucleic Acids Res. 1990 Mar 11;18(5):1233–1242. doi: 10.1093/nar/18.5.1233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Milbrandt J. D., Heintz N. H., White W. C., Rothman S. M., Hamlin J. L. Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the dihydrofolate reductase gene. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6043–6047. doi: 10.1073/pnas.78.10.6043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Nawotka K. A., Huberman J. A. Two-dimensional gel electrophoretic method for mapping DNA replicons. Mol Cell Biol. 1988 Apr;8(4):1408–1413. doi: 10.1128/mcb.8.4.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rivier D. H., Rine J. An origin of DNA replication and a transcription silencer require a common element. Science. 1992 May 1;256(5057):659–663. doi: 10.1126/science.1585179. [DOI] [PubMed] [Google Scholar]
  44. Serwer P. Two-dimensional agarose gel electrophoresis without gel manipulation. Anal Biochem. 1985 Jan;144(1):172–178. doi: 10.1016/0003-2697(85)90100-9. [DOI] [PubMed] [Google Scholar]
  45. Serwer P., Watson R. H., Hayes S. J. Multidimensional analysis of intracellular bacteriophage T7 DNA: effects of amber mutations in genes 3 and 19. J Virol. 1987 Nov;61(11):3499–3509. doi: 10.1128/jvi.61.11.3499-3509.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Shinomiya T., Ina S. Analysis of chromosomal replicons in early embryos of Drosophila melanogaster by two-dimensional gel electrophoresis. Nucleic Acids Res. 1991 Jul 25;19(14):3935–3941. doi: 10.1093/nar/19.14.3935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shinomiya T., Ina S. DNA replication of histone gene repeats in Drosophila melanogaster tissue culture cells: multiple initiation sites and replication pause sites. Mol Cell Biol. 1993 Jul;13(7):4098–4106. doi: 10.1128/mcb.13.7.4098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Snapka R. M., Shin C. G., Permana P. A., Strayer J. Aphidicolin-induced topological and recombinational events in simian virus 40. Nucleic Acids Res. 1991 Sep 25;19(18):5065–5072. doi: 10.1093/nar/19.18.5065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sudo K., Ogata M., Sato Y., Iguchi-Ariga S. M., Ariga H. Cloned origin of DNA replication in c-myc gene can function and be transmitted in transgenic mice in an episomal state. Nucleic Acids Res. 1990 Sep 25;18(18):5425–5432. doi: 10.1093/nar/18.18.5425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Vassilev L. T., Burhans W. C., DePamphilis M. L. Mapping an origin of DNA replication at a single-copy locus in exponentially proliferating mammalian cells. Mol Cell Biol. 1990 Sep;10(9):4685–4689. doi: 10.1128/mcb.10.9.4685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Vaughn J. P., Dijkwel P. A., Hamlin J. L. Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain. Cell. 1990 Jun 15;61(6):1075–1087. doi: 10.1016/0092-8674(90)90071-l. [DOI] [PubMed] [Google Scholar]
  52. Wasserman S. A., Dungan J. M., Cozzarelli N. R. Discovery of a predicted DNA knot substantiates a model for site-specific recombination. Science. 1985 Jul 12;229(4709):171–174. doi: 10.1126/science.2990045. [DOI] [PubMed] [Google Scholar]
  53. Wohlgemuth J. G., Bulboaca G. H., Moghadam M., Caddle M. S., Calos M. P. Physical mapping of origins of replication in the fission yeast Schizosaccharomyces pombe. Mol Biol Cell. 1994 Aug;5(8):839–849. doi: 10.1091/mbc.5.8.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wu C., Friedlander P., Lamoureux C., Zannis-Hadjopoulos M., Price G. B. cDNA clones contain autonomous replication activity. Biochim Biophys Acta. 1993 Sep 23;1174(3):241–257. doi: 10.1016/0167-4781(93)90193-h. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES