Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1984 Jan;4(1):69–76. doi: 10.1128/mcb.4.1.69

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.

W F Flintoff, E Livingston, C Duff, R G Worton
PMCID: PMC368659  PMID: 6700586

Abstract

In previous studies, we have described several classes of methotrexate-resistant Chinese hamster ovary cell lines. Although the RI class is resistant because of an altered target enzyme, dihydrofolate reductase, the RIII class derived from RI cells is somewhat more resistant because of a moderate amplification of the altered dhfr structural gene (Flintoff et al., Mol. Cell. Biol. 2:275-285, 1982). In one RIII line, a translocation between the short arm (p) of chromosome 2 and the long arm (q) of chromosome 5 was observed, and the amplified RIII gene complex was mapped to the p arm of the 2p-marker chromosome derived from the translocation (Worton et al., Mol. Cell. Biol. 1:330-335, 1981). We tested the hypothesis that chromosomal translocation is a general feature of RIII cells and that such translocation involves a site at or near the dhfr structural gene. Thus, we examined four independently derived RIII-type mutants and found that each had a moderate amplification of the dhfr gene sequences, and karyotype analysis revealed that each carried a translocation involving the 2p arm at or near band 2p25. That this chromosomal rearrangement involves a site near the dhfr locus was demonstrated by mapping the altered but unamplified structural gene coding for the RI phenotype to the short arm of an unaltered chromosome 2. This suggests that a highly specific rearrangement involving an exchange at or near the site of the unamplified gene is a necessary prerequisite for the amplification process. A model for gene amplification involving chromosomal rearrangements and sister chromatid exchange is described.

Full text

PDF
69

Images in this article

71Image
on p.71
72Image
on p.72
74Image
on p.74

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. Andrulis I. L., Duff C., Evans-Blackler S., Worton R., Siminovitch L. Chromosomal alterations associated with overproduction of asparagine synthetase in albizziin-resistant Chinese hamster ovary cells. Mol Cell Biol. 1983 Mar;3(3):391–398. doi: 10.1128/mcb.3.3.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beach L. R., Palmiter R. D. Amplification of the metallothionein-I gene in cadmium-resistant mouse cells. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2110–2114. doi: 10.1073/pnas.78.4.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  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. Brennand J., Chinault A. C., Konecki D. S., Melton D. W., Caskey C. T. Cloned cDNA sequences of the hypoxanthine/guanine phosphoribosyltransferase gene from a mouse neuroblastoma cell line found to have amplified genomic sequences. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1950–1954. doi: 10.1073/pnas.79.6.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brown D. D., Blackler A. W. Gene amplification proceeds by a chromosome copy mechanism. J Mol Biol. 1972 Jan 14;63(1):75–83. doi: 10.1016/0022-2836(72)90522-0. [DOI] [PubMed] [Google Scholar]
  9. Brown D. D., Dawid I. B. Specific gene amplification in oocytes. Oocyte nuclei contain extrachromosomal replicas of the genes for ribosomal RNA. Science. 1968 Apr 19;160(3825):272–280. doi: 10.1126/science.160.3825.272. [DOI] [PubMed] [Google Scholar]
  10. Cowan K. H., Goldsmith M. E., Levine R. M., Aitken S. C., Douglass E., Clendeninn N., Nienhuis A. W., Lippman M. E. Dihydrofolate reductase gene amplification and possible rearrangement in estrogen-responsive methotrexate-resistant human breast cancer cells. J Biol Chem. 1982 Dec 25;257(24):15079–15086. [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Flintoff W. F., Spindler S. M., Siminovitch L. Genetic characterization of methotrexate-resistant chinese hamster ovary cells. In Vitro. 1976 Nov;12(11):749–757. doi: 10.1007/BF02835450. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Kafatos F. C., Jones C. W., Efstratiadis A. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res. 1979 Nov 24;7(6):1541–1552. doi: 10.1093/nar/7.6.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kaufman R. J., Sharp P. A., Latt S. A. Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences. Mol Cell Biol. 1983 Apr;3(4):699–711. doi: 10.1128/mcb.3.4.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Latt S. A. Sister chromatid exchange formation. Annu Rev Genet. 1981;15:11–55. doi: 10.1146/annurev.ge.15.120181.000303. [DOI] [PubMed] [Google Scholar]
  20. Lewis J. A., Biedler J. L., Melera P. W. Gene amplification accompanies low level increases in the activity of dihydrofolate reductase in antifolate-resistant Chinese hamster lung cells containing abnormally banding chromosomes. J Cell Biol. 1982 Aug;94(2):418–424. doi: 10.1083/jcb.94.2.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McBurney M. W., Whitmore G. F. Isolation and biochemical characterization of folate deficient mutants of Chinese hamster cells. Cell. 1974 Jul;2(3):173–182. doi: 10.1016/0092-8674(74)90091-9. [DOI] [PubMed] [Google Scholar]
  22. Melera P. W., Wolgemuth D., Biedler J. L., Hession C. Antifolate-resistant chinese hamster cells. Evidence from independently derived sublines for the overproduction of two dihydrofolate reductases encoded by different mRNAs. J Biol Chem. 1980 Jan 25;255(2):319–322. [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Ray M., Mohandas T. Proposed banding nomenclature for the Chinese hamster chromosomes (Cricetulus griseus). Cytogenet Cell Genet. 1976;16(1-5):83–91. doi: 10.1159/000130559. [DOI] [PubMed] [Google Scholar]
  26. Roberts J. M., Axel R. Gene amplification and gene correction in somatic cells. Cell. 1982 May;29(1):109–119. doi: 10.1016/0092-8674(82)90095-2. [DOI] [PubMed] [Google Scholar]
  27. Roberts J. M., Buck L. B., Axel R. A structure for amplified DNA. Cell. 1983 May;33(1):53–63. doi: 10.1016/0092-8674(83)90334-3. [DOI] [PubMed] [Google Scholar]
  28. Schimke R. T., Kaufman R. J., Alt F. W., Kellems R. F. Gene amplification and drug resistance in cultured murine cells. Science. 1978 Dec 8;202(4372):1051–1055. doi: 10.1126/science.715457. [DOI] [PubMed] [Google Scholar]
  29. Smith G. P. Unequal crossover and the evolution of multigene families. Cold Spring Harb Symp Quant Biol. 1974;38:507–513. doi: 10.1101/sqb.1974.038.01.055. [DOI] [PubMed] [Google Scholar]
  30. Spradling A. C., Mahowald A. P. Amplification of genes for chorion proteins during oogenesis in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1096–1100. doi: 10.1073/pnas.77.2.1096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stanners C. P., Eliceiri G. L., Green H. Two types of ribosome in mouse-hamster hybrid cells. Nat New Biol. 1971 Mar 10;230(10):52–54. doi: 10.1038/newbio230052a0. [DOI] [PubMed] [Google Scholar]
  32. Tartof K. D. Redundant genes. Annu Rev Genet. 1975;9:355–385. doi: 10.1146/annurev.ge.09.120175.002035. [DOI] [PubMed] [Google Scholar]
  33. Wahl G. M., Padgett R. A., Stark G. R. Gene amplification causes overproduction of the first three enzymes of UMP synthesis in N-(phosphonacetyl)-L-aspartate-resistant hamster cells. J Biol Chem. 1979 Sep 10;254(17):8679–8689. [PubMed] [Google Scholar]
  34. Worton R. G., Duff C. Karyotyping. Methods Enzymol. 1979;58:322–344. doi: 10.1016/s0076-6879(79)58148-8. [DOI] [PubMed] [Google Scholar]
  35. Worton R. G., Duff C. The use of selection and counterselection in microcell-mediated transfer of dominant genetic markers. Cytogenet Cell Genet. 1981;29(3):184–188. doi: 10.1159/000131567. [DOI] [PubMed] [Google Scholar]
  36. Worton R., Duff C., Flintoff W. Microcell-mediated cotransfer of genes specifying methotrexate resistance, emetine sensitivity, and chromate sensitivity with Chinese hamster chromosome 2. Mol Cell Biol. 1981 Apr;1(4):330–335. doi: 10.1128/mcb.1.4.330. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES