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. 1981 Dec;1(12):1069–1076. doi: 10.1128/mcb.1.12.1069

Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line.

R J Kaufman, R T Schimke
PMCID: PMC369733  PMID: 7346712

Abstract

During stepwise increases in the methotrexate concentration in culture medium, we selected Chinese hamster ovary cells that contained elevated dihydrofolate reductase levels which were proportional to the number of dihydrofolate reductase gene copies (i.e., gene amplification). We studied the dihydrofolate reductase levels in individual cells that underwent the initial steps of methotrexate resistance by using the fluorescence-activated cell sorter technique. Such cells constituted a heterogeneous population with differing dihydrofolate reductase levels, and they characteristically lost the elevated enzyme levels when they were grown in the absence of methotrexate. The progeny of individual cells with high enzyme levels behaved differently and could lose all or variable numbers of the amplified genes.

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Selected References

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  1. Alt F. W., Kellems R. E., Bertino J. R., Schimke R. T. Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J Biol Chem. 1978 Mar 10;253(5):1357–1370. [PubMed] [Google Scholar]
  2. Alt F. W., Kellems R. E., Schimke R. T. Synthesis and degradation of folate reductase in sensitive and methotrexate-resistant lines of S-180 cells. J Biol Chem. 1976 May 25;251(10):3063–3074. [PubMed] [Google Scholar]
  3. Biedler J. L., Spengler B. A. Metaphase chromosome anomaly: association with drug resistance and cell-specific products. Science. 1976 Jan 16;191(4223):185–187. doi: 10.1126/science.942798. [DOI] [PubMed] [Google Scholar]
  4. Brown P. C., Beverley S. M., Schimke R. T. Relationship of amplified dihydrofolate reductase genes to double minute chromosomes in unstably resistant mouse fibroblast cell lines. Mol Cell Biol. 1981 Dec;1(12):1077–1083. doi: 10.1128/mcb.1.12.1077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang A. C., Nunberg J. H., Kaufman R. J., Erlich H. A., Schimke R. T., Cohen S. N. Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase. Nature. 1978 Oct 19;275(5681):617–624. doi: 10.1038/275617a0. [DOI] [PubMed] [Google Scholar]
  6. Dolnick B. J., Berenson R. J., Bertino J. R., Kaufman R. J., Nunberg J. H., Schimke R. T. Correlation of dihydrofolate reductase elevation with gene amplification in a homogeneously staining chromosomal region in L5178Y cells. J Cell Biol. 1979 Nov;83(2 Pt 1):394–402. doi: 10.1083/jcb.83.2.394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dugaiczyk A., Boyer H. W., Goodman H. M. Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures. J Mol Biol. 1975 Jul 25;96(1):171–184. doi: 10.1016/0022-2836(75)90189-8. [DOI] [PubMed] [Google Scholar]
  8. Gershey E. L., D'Alisa R. M., Zucker R. M. Characterization of a CV-1 cell cycle. IV. No critical size for S phase entry. Exp Cell Res. 1979 Aug;122(1):9–14. doi: 10.1016/0014-4827(79)90554-8. [DOI] [PubMed] [Google Scholar]
  9. Kaufman R. J., Bertino J. R., Schimke R. T. Quantitation of dihydrofolate reductase in individual parental and methotrexate-resistant murine cells. Use of a fluorescence activated cell sorter. J Biol Chem. 1978 Aug 25;253(16):5852–5860. [PubMed] [Google Scholar]
  10. 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]
  11. Kaufman R. J., Brown P. C., Schimke R. T. Loss and stabilization of amplified dihydrofolate reductase genes in mouse sarcoma S-180 cell lines. Mol Cell Biol. 1981 Dec;1(12):1084–1093. doi: 10.1128/mcb.1.12.1084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Levan A., Levan G. Have double minutes functioning centromeres? Hereditas. 1978;88(1):81–92. doi: 10.1111/j.1601-5223.1978.tb01606.x. [DOI] [PubMed] [Google Scholar]
  14. Levan G., Mandahl N., Bregula U., Klein G., Levan A. Double minute chromosomes are not centromeric regions of the host chromosomes. Hereditas. 1976;83(1):83–90. doi: 10.1111/j.1601-5223.1976.tb01573.x. [DOI] [PubMed] [Google Scholar]
  15. Loken M. R., Herzenber L. A. Analysis of cell populations with a fluorescence-activated cell sorter. Ann N Y Acad Sci. 1975 Jun 30;254:163–171. doi: 10.1111/j.1749-6632.1975.tb29166.x. [DOI] [PubMed] [Google Scholar]
  16. Melera P. W., Lewis J. A., Biedler J. L., Hession C. Antifolate-resistant Chinese hamster cells. Evidence for dihydrofolate reductase gene amplification among independently derived sublines overproducing different dihydrofolate reductases. J Biol Chem. 1980 Jul 25;255(14):7024–7028. [PubMed] [Google Scholar]
  17. Nunberg J. H., Kaufman R. J., Chang A. C., Cohen S. N., Schimke R. T. Structure and genomic organization of the mouse dihydrofolate reductase gene. Cell. 1980 Feb;19(2):355–364. doi: 10.1016/0092-8674(80)90510-3. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Parks D. R., Bryan V. M., Oi V. T., Herzenberg L. A. Antigen-specific identification and cloning of hybridomas with a fluorescence-activated cell sorter. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1962–1966. doi: 10.1073/pnas.76.4.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Schimke R. T., Kaufman R. J., Nunberg J. H., Dana S. L. Studies on the amplification of dihydrofolate reductase genes in methotrexate-resistant cultured mouse cells. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):1297–1303. doi: 10.1101/sqb.1979.043.01.148. [DOI] [PubMed] [Google Scholar]
  22. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  23. Wiedemann L. M., Johnson L. F. Regulation of dihydrofolate reductase synthesis in an overproducing 3T6 cell line during transition from resting to growing state. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2818–2822. doi: 10.1073/pnas.76.6.2818. [DOI] [PMC free article] [PubMed] [Google Scholar]

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