Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1981 Dec;1(12):1077–1083. doi: 10.1128/mcb.1.12.1077

Relationship of amplified dihydrofolate reductase genes to double minute chromosomes in unstably resistant mouse fibroblast cell lines.

P C Brown, S M Beverley, R T Schimke
PMCID: PMC369734  PMID: 6287217

Abstract

Murine 3T6 selected in increasing concentrations of methotrexate were unstable with respect to dihydrofolate reductase overproduction and methotrexate resistance when they are cultured in the absence of methotrexate. An analysis of the karyotypes of these resistant cells revealed the presence of numerous double minute chromosomes. We observed essentially identical kinetics of loss of dihydrofolate reductase gene sequences in total deoxyribonucleic acid and in deoxyribonucleic acid from fractions enriched in double minute chromosomes and in the numbers of double minute chromosomes per cell during reversion to methotrexate sensitivity, and this suggested that unstably amplified gene sequences were localized on double minute chromosomes. This conclusion ws also supported by an analysis of cell populations sorted according to dihydrofolate reductase enzyme contents, in which relative gene amplification and double minute chromosome content were related proportionally.

Full text

PDF
1077

Images in this article

1079Image
on p.1079
1080Image
on p.1080

Selected References

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

  1. Balaban-Malenbaum G., Gilbert F. Double minute chromosomes and the homogeneously staining regions in chromosomes of a human neuroblastoma cell line. Science. 1977 Nov 18;198(4318):739–741. doi: 10.1126/science.71759. [DOI] [PubMed] [Google Scholar]
  2. Barker P. E., Hsu T. C. Double minutes in human carcinoma cell lines, with special reference to breast tumors. J Natl Cancer Inst. 1979 Feb;62(2):257–262. [PubMed] [Google Scholar]
  3. Barker P. E., Stubblefield E. Ultrastructure of double minutes from a human tumor cell line. J Cell Biol. 1979 Dec;83(3):663–666. doi: 10.1083/jcb.83.3.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blumenthal A. B., Dieden J. D., Kapp L. N., Sedat J. W. Rapid isolation of metaphase chromosomes containing high molecular weight DNA. J Cell Biol. 1979 Apr;81(1):255–259. doi: 10.1083/jcb.81.1.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brunk C. F., Jones K. C., James T. W. Assay for nanogram quantities of DNA in cellular homogenates. Anal Biochem. 1979 Jan 15;92(2):497–500. doi: 10.1016/0003-2697(79)90690-0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Chen T. R. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res. 1977 Feb;104(2):255–262. doi: 10.1016/0014-4827(77)90089-1. [DOI] [PubMed] [Google Scholar]
  8. Cowell J. K. A new chromosome region possibly derived from double minutes in an in vitro transformed epithelial cell line. Cytogenet Cell Genet. 1980;27(1):2–7. doi: 10.1159/000131458. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Kapuściński J., Skoczylas B. Simple and rapid fluorimetric method for DNA microassay. Anal Biochem. 1977 Nov;83(1):252–257. doi: 10.1016/0003-2697(77)90533-4. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Kaufman R. J., Schimke R. T. Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line. Mol Cell Biol. 1981 Dec;1(12):1069–1076. doi: 10.1128/mcb.1.12.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kellems R. E., Morhenn V. B., Pfendt E. A., Alt F. W., Schimke R. T. Polyoma virus and cyclic AMP-mediated control of dihydrofolate reductase mRNA abundance in methotrexate-resistant mouse fibroblasts. J Biol Chem. 1979 Jan 25;254(2):309–318. [PubMed] [Google Scholar]
  16. 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]
  17. Marinello M. J., Bloom M. L., Doeblin T. D., Sandberg A. A. Double minute chromosomes in human leukemia. N Engl J Med. 1980 Sep 18;303(12):704–704. doi: 10.1056/NEJM198009183031221. [DOI] [PubMed] [Google Scholar]
  18. McGrogan M., Spector D. J., Goldenberg C. J., Halbert D., Raskas H. J. Purification of specific adenovirus 2 RNAs by preparative hybridization and selective thermal elution. Nucleic Acids Res. 1979 Feb;6(2):593–607. doi: 10.1093/nar/6.2.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nilsen T., Baglioni C. Unusual base-pairing of newly synthesized DNA in HeLa cells. J Mol Biol. 1979 Sep 25;133(3):319–338. doi: 10.1016/0022-2836(79)90396-6. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Quinn L. A., Moore G. E., Morgan R. T., Woods L. K. Cell lines from human colon carcinoma with unusual cell products, double minutes, and homogeneously staining regions. Cancer Res. 1979 Dec;39(12):4914–4924. [PubMed] [Google Scholar]
  23. Reichmann A., Riddell R. H., Martin P., Levin B. Double minutes in human large bowel cancer. Gastroenterology. 1980 Aug;79(2):334–339. [PubMed] [Google Scholar]
  24. Schimke R. T., Brown P. C., Kaufman R. J., McGrogan M., Slate D. L. Chromosomal and extrachromosomal localization of amplified dihydrofolate reductase genes in cultured mammalian cells. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 2):785–797. doi: 10.1101/sqb.1981.045.01.097. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES