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PMID: 2320001 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line.

Molecular and cellular biology ·Vol. 10 ·No. 4 ·1990-04-00 ·Pages 1338-46

Ma C, Leu TH, Hamlin JL

Abstract

We recently showed that replication initiates in the early S period at two closely spaced zones in the 240-kilobase (kb) dihydrofolate reductase (DHFR) amplicon of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Both of these initiation loci (ori-beta and ori-gamma) have previously been cloned in a recombinant cosmid. In this study, we identified a third early-firing initiation locus (ori-alpha) in the much larger DHFR amplicon of the independently isolated methotrexate-resistant Chinese hamster cell line DC3F-A3/4K (A3/4K). We describe the molecular cloning of this newly identified locus and demonstrate by chromosomal walking that ori-alpha lies approximately 240 kb upstream from ori-beta. Using overlapping cosmid clones for more than 450 kb of DNA sequence from this region of the DHFR domain, we have monitored the replication pattern of the amplicons in synchronized A3/4K cells. These studies suggest that ori-alpha, ori-beta, and ori-gamma are the only early-firing initiation sites in this 450-kb sequence. In addition, we have been able to roughly localize the termini between ori-alpha and ori-beta and between ori-alpha and the next origin in the 5' direction. Thus, we have now isolated the equivalent of three early-firing replicons (including their origins) from a well-characterized chromosomal domain. With these tools, it should be possible to determine those properties that are shared by the origins and termini of different replicons and which are therefore likely to be functionally significant.

MeSH Terms
Animals Bromodeoxyuridine/metabolism Cell Line Cosmids Cricetinae Cricetulus DNA/genetics,isolation & purification DNA Replication Drug Resistance/genetics Female Gene Amplification Methotrexate/pharmacology Ovary Tetrahydrofolate Dehydrogenase/genetics
Chemicals
DNA Tetrahydrofolate Dehydrogenase Bromodeoxyuridine Methotrexate
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Ma C
Department of Biochemistry, University of Virginia School of Medicine, Charlottesville 22908.
Leu T H
Hamlin J L
References (57)
57 references, click to expand
  1. The localization of replication origins on ARS plasmids in S. cerevisiae.
    Cell. 1987 Nov 6;51(3):463-71 PMID: 2822257
  2. A simple, rapid, and sensitive DNA assay procedure.
    Anal Biochem. 1980 Mar 1;102(2):344-52 PMID: 6158890
  3. Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line.
    Mol Cell Biol. 1988 Jun;8(6):2316-27 PMID: 2841578
  4. A replication fork barrier at the 3' end of yeast ribosomal RNA genes.
    Cell. 1988 Nov 18;55(4):637-43 PMID: 3052854
  5. Organization of replication of ribosomal DNA in Saccharomyces cerevisiae.
    Mol Cell Biol. 1988 Nov;8(11):4927-35 PMID: 3062373
  6. The dihydrofolate reductase amplicons in different methotrexate-resistant Chinese hamster cell lines share at least a 273-kilobase core sequence, but the amplicons in some cell lines are much larger and are remarkably uniform in structure.
    Mol Cell Biol. 1988 Dec;8(12):5268-79 PMID: 3244355
  7. 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):5398-409 PMID: 3244360
  8. 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):523-31 PMID: 2710115
  9. 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):532-40 PMID: 2710116
  10. Isolation of human sequences that replicate autonomously in human cells.
    Mol Cell Biol. 1989 Mar;9(3):1026-33 PMID: 2542763
  11. Identification and characterization of a gene that is coamplified with dihydrofolate reductase in a methotrexate-resistant CHO cell line.
    Mol Cell Biol. 1989 Mar;9(3):1137-47 PMID: 2725490
  12. Mapping replication units in animal cells.
    Cell. 1989 Jun 16;57(6):909-20 PMID: 2544294
  13. The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication.
    Cell. 1989 Aug 11;58(3):527-35 PMID: 2547525
  14. Temporal order of gene replication in Chinese hamster ovary cells.
    Mol Cell Biol. 1989 Jul;9(7):2881-9 PMID: 2476659
  15. Limited DNA synthesis in the absence of protein synthesis in Physarum polycephalum.
    J Cell Biol. 1966 Dec;31(3):577-83 PMID: 4226393
  16. On the mechanism of DNA replication in mammalian chromosomes.
    J Mol Biol. 1968 Mar 14;32(2):327-41 PMID: 5689363
  17. Isolation of high-molecular-weight DNA from mammalian cells.
    Eur J Biochem. 1973 Jul 2;36(1):32-8 PMID: 4200179
  18. Evaluation of S phase synchronization by analysis of DNA replication in 5-bromodeoxyuridine.
    Exp Cell Res. 1973 Nov;82(1):137-42 PMID: 4270895
  19. Supercoiled loops and eucaryotic DNA replicaton.
    Cell. 1980 Nov;22(1 Pt 1):79-85 PMID: 7428042
  20. Replication pattern of a large homogenously staining chromosome region in antifolate-resistant Chinese hamster cell lines.
    J Cell Physiol. 1981 Apr;107(1):101-14 PMID: 6163787
  21. 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):6043-7 PMID: 6273843
  22. The remarkable instability of replication loops provides a general method for the isolation of origins of DNA replication.
    Cell. 1981 Nov;27(1 Pt 2):155-63 PMID: 6276021
  23. Smooth muscle cell hypertrophy versus hyperplasia in hypertension.
    Proc Natl Acad Sci U S A. 1981 Dec;78(12):7759-63 PMID: 6950415
  24. 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):4083-7 PMID: 6955792
  25. The temporal order of replication of murine immunoglobulin heavy chain constant region sequences corresponds to their linear order in the genome.
    Nucleic Acids Res. 1982 Nov 11;10(21):6887-902 PMID: 6294619
  26. Organization of a Chinese hamster ovary dihydrofolate reductase gene identified by phenotypic rescue.
    Mol Cell Biol. 1983 Jul;3(7):1266-73 PMID: 6310370
  27. Detection and mapping of homologous, repeated and amplified DNA sequences by DNA renaturation in agarose gels.
    Nucleic Acids Res. 1983 Aug 25;11(16):5413-31 PMID: 6310499
  28. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
    Anal Biochem. 1983 Jul 1;132(1):6-13 PMID: 6312838
  29. Amplification and molecular cloning of murine adenosine deaminase gene sequences.
    J Biol Chem. 1983 Dec 25;258(24):15179-85 PMID: 6197412
  30. Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.
    Mol Cell Biol. 1983 Nov;3(11):2076-88 PMID: 6656763
  31. Replication timing of genes and middle repetitive sequences.
    Science. 1984 May 18;224(4650):686-92 PMID: 6719109
  32. Gene amplification in cultured animal cells.
    Cell. 1984 Jul;37(3):705-13 PMID: 6378386
  33. Novel DNA rearrangements are associated with dihydrofolate reductase gene amplification.
    J Biol Chem. 1984 Jul 25;259(14):9127-40 PMID: 6086622
  34. Gene amplification.
    Annu Rev Biochem. 1984;53:447-91 PMID: 6383198
  35. DNA sequence amplification in mammalian cells.
    Int Rev Cytol. 1984;90:31-82 PMID: 6389416
  36. Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells.
    Mol Cell Biol. 1985 Apr;5(4):619-27 PMID: 2985962
  37. Rapid transfer of DNA from agarose gels to nylon membranes.
    Nucleic Acids Res. 1985 Oct 25;13(20):7207-21 PMID: 4059056
  38. Large inverted duplications are associated with gene amplification.
    Cell. 1986 May 9;45(3):425-30 PMID: 2870814
  39. Permanent attachment of replication origins to the nuclear matrix in BHK-cells.
    Nucleic Acids Res. 1986 Apr 25;14(8):3241-9 PMID: 3703674
  40. Autonomous replicating sequences from mouse cells which can replicate in mouse cells in vivo and in vitro.
    Mol Cell Biol. 1987 Jan;7(1):1-6 PMID: 3031448
  41. Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer.
    Proc Natl Acad Sci U S A. 1987 Apr;84(8):2160-4 PMID: 3031668
  42. Autonomous replication of plasmids bearing monkey DNA origin-enriched sequences.
    Proc Natl Acad Sci U S A. 1987 Oct;84(19):6668-72 PMID: 3477798
  43. The relationship between chromosomal origins of replication and the nuclear matrix during the cell cycle.
    Exp Cell Res. 1986 Jun;164(2):426-36 PMID: 2940105
  44. The role of the nuclear matrix in the organization and function of DNA.
    Annu Rev Biophys Biophys Chem. 1986;15:457-75 PMID: 3013231
  45. 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):7790-4 PMID: 3094015
  46. Replication origins are attached to the nuclear skeleton.
    Nucleic Acids Res. 1986 Oct 24;14(20):8189-207 PMID: 3774556
  47. Structure of DNA formed in the first step of CAD gene amplification.
    EMBO J. 1986 Sep;5(9):2115-21 PMID: 3023056
  48. Polarity of DNA replication through the avian alpha-globin locus.
    Mol Cell Biol. 1986 Apr;6(4):976-84 PMID: 3785167
  49. Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells.
    Mol Cell Biol. 1987 Feb;7(2):569-77 PMID: 3821723
  50. Analysis of the replication pattern of Chinese hamster chromosomes using 5-bromodeoxyuridine suppression of 33258 Hoechst fluorescence.
    Chromosoma. 1975 Dec 10;53(3):209-21 PMID: 53132
  51. A novel chromosome abnormality in human neuroblastoma and antifolate-resistant Chinese hamster cell lives in culture.
    J Natl Cancer Inst. 1976 Sep;57(3):683-95 PMID: 62055
  52. Conformational constraints in nuclear DNA.
    J Cell Sci. 1976 Nov;22(2):287-302 PMID: 1033937
  53. The units of DNA replication in the mammalian chromosomes: evidence for a large size of replication units.
    Chromosoma. 1977 Apr 19;60(3):253-67 PMID: 870295
  54. Eucaryotic DNA: organization of the genome for replication.
    Cell. 1978 Oct;15(2):317-25 PMID: 719745
  55. Amplified dihydrofolate reductase genes are localized to a homogeneously staining region of a single chromosome in a methotrexate-resistant Chinese hamster ovary cell line.
    Proc Natl Acad Sci U S A. 1978 Nov;75(11):5553-6 PMID: 281704
  56. The asymmetric segregation of parental nucleosomes during chrosome replication.
    Cell. 1979 Oct;18(2):439-49 PMID: 227608
  57. Replication program of active and inactive multigene families in mammalian cells.
    Mol Cell Biol. 1988 May;8(5):2149-58 PMID: 3386634
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.10.4.1338-1346.1990
Published
1990-04-00
Pages
1338-46
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC362235
Subset
IM
Grants
NIGMS NIH HHS · GM26108 · United States
External Links
PubMed source DOI: 10.1128/mcb.10.4.1338-1346.1990 PMC Full Text View journal details
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