Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1982 Nov 11;10(21):6597–6618. doi: 10.1093/nar/10.21.6597

Gene amplification in methotrexate-resistant mouse cells. IV. Different DNA sequences are amplified in different resistant lines.

R Caizzi, C J Bostock
PMCID: PMC326952  PMID: 6294610

Abstract

DNA was purified from double minutes isolated from MTX-resistant EL4/8 mouse lymphoma cells, digested to completion with Bam H1 restriction endonuclease and cloned in lambda-1059. The properties of the library suggest that the DNA from which it was made was not detectably contaminated with non-dm chromosome material, and that the library is essentially complete for sequences contained in Bam H1 restriction fragments between 9 and 19 kb. The inserts of some selected lambda-recombinants were subcloned in pBR328 or pAT153 to separate sequences of differing repetition frequency. Clones representative of different classes of sequences were used as probes to Southern transfers of Bam H1 digested total nuclear DNAs of various MTX-resistant cell lines. The results clearly show that the amplified unit of each cell line has a unique structure, and that different amplified units differ widely in their sequence composition.

Full text

PDF

Images in this article

Selected References

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

  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. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [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. Bostock C. J., Clark E. M., Harding N. G., Mounts P. M., Tyler-Smith C., van Heyningen V., Walker P. M. The development of resistance to methotrexate in a mouse melanoma cell line. I. Characterisation of the dihydrofolate reductases and chromosomes in sensitive and resistant cells. Chromosoma. 1979;74(2):153–177. doi: 10.1007/BF00292270. [DOI] [PubMed] [Google Scholar]
  5. Bostock C. J., Clark E. M. Satellite DNA in large marker chromosomes of methotrexate-resistant mouse cells. Cell. 1980 Mar;19(3):709–715. doi: 10.1016/s0092-8674(80)80047-x. [DOI] [PubMed] [Google Scholar]
  6. Bostock C. J., Tyler-Smith C. Gene amplification in methotrexate-resistant mouse cells. II. Rearrangement and amplification of non-dihydrofolate reductase gene sequences accompany chromosomal changes. J Mol Biol. 1981 Dec 5;153(2):219–236. doi: 10.1016/0022-2836(81)90275-8. [DOI] [PubMed] [Google Scholar]
  7. Courtenay V. D., Robins A. B. Loss of resistance to methotrexate in L5178Y mouse leukemia grown in vitro. J Natl Cancer Inst. 1972 Jul;49(1):45–53. [PubMed] [Google Scholar]
  8. 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]
  9. George D. L., Powers V. E. Amplified DNA sequences in Y1 mouse adrenal tumor cells: association with double minutes and localization to a homogeneously staining chromosomal region. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1597–1601. doi: 10.1073/pnas.79.5.1597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. George D. L., Powers V. E. Cloning of DNA from double minutes of Y1 mouse adrenocortical tumor cells: evidence for gene amplification. Cell. 1981 Apr;24(1):117–123. doi: 10.1016/0092-8674(81)90507-9. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Karn J., Brenner S., Barnett L., Cesareni G. Novel bacteriophage lambda cloning vector. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5172–5176. doi: 10.1073/pnas.77.9.5172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  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 A., Levan G., Mitelman F. Chromosomes and cancer. Hereditas. 1977;86(1):15–30. doi: 10.1111/j.1601-5223.1977.tb01208.x. [DOI] [PubMed] [Google Scholar]
  17. Maniatis T., Jeffrey A., Kleid D. G. Nucleotide sequence of the rightward operator of phage lambda. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1184–1188. doi: 10.1073/pnas.72.3.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Masters J., Keeley B., Gay H., Attardi G. Variable content of double minute chromosomes is not correlated with degree of phenotype instability in methotrexate-resistant human cell lines. Mol Cell Biol. 1982 May;2(5):498–507. doi: 10.1128/mcb.2.5.498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Morrison D. A. Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol. 1979;68:326–331. doi: 10.1016/0076-6879(79)68023-0. [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. Scalenghe F., Turco E., Edström J. E., Pirrotta V., Melli M. Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes. Chromosoma. 1981;82(2):205–216. doi: 10.1007/BF00286105. [DOI] [PubMed] [Google Scholar]
  23. Sillar R., Young B. D. A new method for the preparation of metaphase chromosomes for flow analysis. J Histochem Cytochem. 1981 Jan;29(1):74–78. doi: 10.1177/29.1.6162882. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Tyler-Smith C., Alderson T. Gene amplification in methotrexate-resistant mouse cells. I. DNA rearrangement accompanies dihydrofolate reductase gene amplification in a T-cell lymphoma. J Mol Biol. 1981 Dec 5;153(2):203–218. doi: 10.1016/0022-2836(81)90274-6. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Young B. D., Ferguson-Smith M. A., Sillar R., Boyd E. High-resolution analysis of human peripheral lymphocyte chromosomes by flow cytometry. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7727–7731. doi: 10.1073/pnas.78.12.7727. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES