Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1988 Dec;8(12):5268–5279. doi: 10.1128/mcb.8.12.5268

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.

J E Looney 1, C Ma 1, T H Leu 1, W F Flintoff 1, W B Troutman 1, J L Hamlin 1
PMCID: PMC365629  PMID: 3244355

Abstract

We have previously cloned and characterized two different dihydrofolate reductase amplicon types from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). The largest of these (the type I amplicon) is 273 kilobases (kb) in length. In the present study, we utilized clones from the type I amplicon as probes to analyze the size and variability of the amplified DNA sequences in five other independently isolated methotrexate-resistant Chinese hamster cell lines. Our data indicated that the predominant amplicon types in all but one of these cell lines are larger than the 273-kb type I sequence. In-gel renaturation experiments as well as hybridization analysis of large SfiI fragments separated by pulse-field gradient gel electrophoresis showed that two highly resistant cell lines (A3 and MK42) have amplified very homogeneous core sequences that are estimated to be at least 583 and 653 kb in length, respectively. Thus, the sizes of the major amplicon types can be different in different drug-resistant Chinese hamster cell lines. However, there appears to be less heterogeneity in size and sequence arrangement within a given methotrexate-resistant Chinese hamster cell line than has been reported for several other examples of DNA sequence amplification in mammalian systems.

Full text

PDF
5269

Images in this article

Selected References

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

  1. Alitalo K., Schwab M., Lin C. C., Varmus H. E., Bishop J. M. Homogeneously staining chromosomal regions contain amplified copies of an abundantly expressed cellular oncogene (c-myc) in malignant neuroendocrine cells from a human colon carcinoma. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1707–1711. doi: 10.1073/pnas.80.6.1707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ardeshir F., Giulotto E., Zieg J., Brison O., Liao W. S., Stark G. R. Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate. Mol Cell Biol. 1983 Nov;3(11):2076–2088. doi: 10.1128/mcb.3.11.2076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Biedler J. L., Spengler B. A. A novel chromosome abnormality in human neuroblastoma and antifolate-resistant Chinese hamster cell lives in culture. J Natl Cancer Inst. 1976 Sep;57(3):683–695. doi: 10.1093/jnci/57.3.683. [DOI] [PubMed] [Google Scholar]
  4. Caizzi R., Bostock C. J. Gene amplification in methotrexate-resistant mouse cells. IV. Different DNA sequences are amplified in different resistant lines. Nucleic Acids Res. 1982 Nov 11;10(21):6597–6618. doi: 10.1093/nar/10.21.6597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Debatisse M., Hyrien O., Petit-Koskas E., de Saint-Vincent B. R., Buttin G. Segregation and rearrangement of coamplified genes in different lineages of mutant cells that overproduce adenylate deaminase. Mol Cell Biol. 1986 May;6(5):1776–1781. doi: 10.1128/mcb.6.5.1776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Federspiel N. A., Beverley S. M., Schilling J. W., Schimke R. T. Novel DNA rearrangements are associated with dihydrofolate reductase gene amplification. J Biol Chem. 1984 Jul 25;259(14):9127–9140. [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  8. Flintoff W. F., Davidson S. V., Siminovitch L. Isolation and partial characterization of three methotrexate-resistant phenotypes from Chinese hamster ovary cells. Somatic Cell Genet. 1976 May;2(3):245–261. doi: 10.1007/BF01538963. [DOI] [PubMed] [Google Scholar]
  9. Flintoff W. F., Essani K. Methotrexate-resistant Chinese hamster ovary cells contain a dihydrofolate reductase with an altered affinity for methotrexate. Biochemistry. 1980 Sep 2;19(18):4321–4327. doi: 10.1021/bi00559a027. [DOI] [PubMed] [Google Scholar]
  10. Flintoff W. F., Livingston E., Duff C., Worton R. G. Moderate-level gene amplification in methotrexate-resistant Chinese hamster ovary cells is accompanied by chromosomal translocations at or near the site of the amplified DHFR gene. Mol Cell Biol. 1984 Jan;4(1):69–76. doi: 10.1128/mcb.4.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Flintoff W. F., Weber M. K., Nagainis C. R., Essani A. K., Robertson D., Salser W. Overproduction of dihydrofolate reductase and gene amplification in methotrexate-resistant Chinese hamster ovary cells. Mol Cell Biol. 1982 Mar;2(3):275–285. doi: 10.1128/mcb.2.3.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ford M., Fried M. Large inverted duplications are associated with gene amplification. Cell. 1986 May 9;45(3):425–430. doi: 10.1016/0092-8674(86)90328-4. [DOI] [PubMed] [Google Scholar]
  13. Gardiner K., Laas W., Patterson D. Fractionation of large mammalian DNA restriction fragments using vertical pulsed-field gradient gel electrophoresis. Somat Cell Mol Genet. 1986 Mar;12(2):185–195. doi: 10.1007/BF01560665. [DOI] [PubMed] [Google Scholar]
  14. Giulotto E., Saito I., Stark G. R. Structure of DNA formed in the first step of CAD gene amplification. EMBO J. 1986 Sep;5(9):2115–2121. doi: 10.1002/j.1460-2075.1986.tb04474.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gross-Bellard M., Oudet P., Chambon P. Isolation of high-molecular-weight DNA from mammalian cells. Eur J Biochem. 1973 Jul 2;36(1):32–38. doi: 10.1111/j.1432-1033.1973.tb02881.x. [DOI] [PubMed] [Google Scholar]
  16. Gupta R. S., Flintoff W. F., Siminovitch L. Purification and properties of dihydrofolate reductase from methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells. Can J Biochem. 1977 Apr;55(4):445–452. doi: 10.1139/o77-062. [DOI] [PubMed] [Google Scholar]
  17. Hamlin J. L., Biedler J. L. Replication pattern of a large homogenously staining chromosome region in antifolate-resistant Chinese hamster cell lines. J Cell Physiol. 1981 Apr;107(1):101–114. doi: 10.1002/jcp.1041070112. [DOI] [PubMed] [Google Scholar]
  18. Hamlin J. L., Milbrandt J. D., Heintz N. H., Azizkhan J. C. DNA sequence amplification in mammalian cells. Int Rev Cytol. 1984;90:31–82. doi: 10.1016/s0074-7696(08)61487-4. [DOI] [PubMed] [Google Scholar]
  19. Hand R. Eucaryotic DNA: organization of the genome for replication. Cell. 1978 Oct;15(2):317–325. doi: 10.1016/0092-8674(78)90001-6. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Heintz N. H., Milbrandt J. D., Greisen K. S., Hamlin J. L. Cloning of the initiation region of a mammalian chromosomal replicon. 1983 Mar 31-Apr 6Nature. 302(5907):439–441. doi: 10.1038/302439a0. [DOI] [PubMed] [Google Scholar]
  22. Hyrien O., Debatisse M., Buttin G., de Saint Vincent B. R. A hotspot for novel amplification joints in a mosaic of Alu-like repeats and palindromic A + T-rich DNA. EMBO J. 1987 Aug;6(8):2401–2408. doi: 10.1002/j.1460-2075.1987.tb02518.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kaufman R. J., Brown P. C., Schimke R. T. Amplified dihydrofolate reductase genes in unstably methotrexate-resistant cells are associated with double minute chromosomes. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5669–5673. doi: 10.1073/pnas.76.11.5669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kinzler K. W., Zehnbauer B. A., Brodeur G. M., Seeger R. C., Trent J. M., Meltzer P. S., Vogelstein B. Amplification units containing human N-myc and c-myc genes. Proc Natl Acad Sci U S A. 1986 Feb;83(4):1031–1035. doi: 10.1073/pnas.83.4.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Looney J. E., Hamlin J. L. Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells. Mol Cell Biol. 1987 Feb;7(2):569–577. doi: 10.1128/mcb.7.2.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ma C., Looney J. E., Leu T. H., Hamlin J. L. 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):2316–2327. doi: 10.1128/mcb.8.6.2316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Milbrandt J. D., Azizkhan J. C., Hamlin J. L. Amplification of a cloned Chinese hamster dihydrofolate reductase gene after transfer into a dihydrofolate reductase-deficient cell line. Mol Cell Biol. 1983 Jul;3(7):1274–1282. doi: 10.1128/mcb.3.7.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Montoya-Zavala M., Hamlin J. L. Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells. Mol Cell Biol. 1985 Apr;5(4):619–627. doi: 10.1128/mcb.5.4.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nunberg J. H., Kaufman R. J., Schimke R. T., Urlaub G., Chasin L. A. 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):5553–5556. doi: 10.1073/pnas.75.11.5553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Reed K. C., Mann D. A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. doi: 10.1093/nar/13.20.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  33. Roninson I. B., Abelson H. T., Housman D. E., Howell N., Varshavsky A. Amplification of specific DNA sequences correlates with multi-drug resistance in Chinese hamster cells. Nature. 1984 Jun 14;309(5969):626–628. doi: 10.1038/309626a0. [DOI] [PubMed] [Google Scholar]
  34. Roninson I. B. Detection and mapping of homologous, repeated and amplified DNA sequences by DNA renaturation in agarose gels. Nucleic Acids Res. 1983 Aug 25;11(16):5413–5431. doi: 10.1093/nar/11.16.5413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schimke R. T. Gene amplification in cultured animal cells. Cell. 1984 Jul;37(3):705–713. doi: 10.1016/0092-8674(84)90406-9. [DOI] [PubMed] [Google Scholar]
  36. Schwab M., Alitalo K., Varmus H. E., Bishop J. M., George D. A cellular oncogene (c-Ki-ras) is amplified, overexpressed, and located within karyotypic abnormalities in mouse adrenocortical tumour cells. Nature. 1983 Jun 9;303(5917):497–501. doi: 10.1038/303497a0. [DOI] [PubMed] [Google Scholar]
  37. Stark G. R., Wahl G. M. Gene amplification. Annu Rev Biochem. 1984;53:447–491. doi: 10.1146/annurev.bi.53.070184.002311. [DOI] [PubMed] [Google Scholar]
  38. Tyler-Smith C., Bostock C. J. Gene amplification in methotrexate-resistant mouse cells. III. Interrelationships between chromosome changes and DNA sequence amplification or loss. J Mol Biol. 1981 Dec 5;153(2):237–256. doi: 10.1016/0022-2836(81)90276-x. [DOI] [PubMed] [Google Scholar]
  39. Van der Bliek A. M., Van der Velde-Koerts T., Ling V., Borst P. Overexpression and amplification of five genes in a multidrug-resistant Chinese hamster ovary cell line. Mol Cell Biol. 1986 May;6(5):1671–1678. doi: 10.1128/mcb.6.5.1671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wahl G. M., Vitto L., Padgett R. A., Stark G. R. Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization. Mol Cell Biol. 1982 Mar;2(3):308–319. doi: 10.1128/mcb.2.3.308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zieg J., Clayton C. E., Ardeshir F., Giulotto E., Swyryd E. A., Stark G. R. Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate. Mol Cell Biol. 1983 Nov;3(11):2089–2098. doi: 10.1128/mcb.3.11.2089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. de Saint Vincent B. R., Delbrück S., Eckhart W., Meinkoth J., Vitto L., Wahl G. The cloning and reintroduction into animal cells of a functional CAD gene, a dominant amplifiable genetic marker. Cell. 1981 Dec;27(2 Pt 1):267–277. doi: 10.1016/0092-8674(81)90410-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES