Biochemical Genetics

, Volume 18, Issue 7–8, pp 655–667 | Cite as

Repetitive DNA sequences in methotrexate- and methasquin-sensitive and -resistant Chinese hamster cell lines

  • Debra J. Wolgemuth
  • June L. Biedler
  • Peter W. Melera


DNA purified from a Chinese hamster cell line of lung fibroblast origin (DC83F) was analyzed by density gradient centrifugation and by gel electrophoresis after restriction endonuclease digestion in order to fractionate discrete repetitive fractions within the total DNA. No obvious satellite DNAs were resolved using the CsCl or Ag-Cs2SO4 density gradient conditions described herein. However, analysis of the digestion products of a battery of restriction endonucleases indicated that three of these enzymes, EcoR1, HaeIII, and XhoI, yielded discrete fragments which could be visualized with EtBr staining or identified by scintillation counting of [ 3 H] DNA. DNAs from several highly (≥ hundredfold increased resistance) antifolate-resistant sublines of DC-3F, characterized by a large homogeneously staining region (HSR) in the chromosome complement, were examined with both techniques and compared to the parental, antifolate-sensitive cell line DNA. The density gradient profiles and electrophoretic patterns of restriction endonuclease digests were identical among all the cell lines examined and were indistinguishable from those of the parental DC-3F DNA.

Key words

methotrexate resistance restriction endonuclease analysis repetitive DNA HSRs 


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  1. Alt, F. W., Kellems, R. E., Bertino, J. R., and Schimke, R. T. (1978). Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J. Biol. Chem. 2531357.Google Scholar
  2. Arrighi, F. E., Mandel, M., Bergendahl, J., and Hsu, T. C. (1970). Buoyant densities of DNA of mammals. Biochem. Genet. 4367.Google Scholar
  3. Benz, R. D., and Burki, H. J. (1978). The distribution of moderately repeated DNA sequences among Chinese hamster chromosomes. Exp. Cel Res. 112155.Google Scholar
  4. Biedler, J. L., and Spengler, B. A. (1976a). Metaphase chromosome anomaly: Association with drug resistance and cell-specific products. Science 191185.Google Scholar
  5. Biedler, J. L., and Spengler, B. A. (1976b). Quantitative relationship between a chromosome abnormality (HSR) and antifolate resistance associated with enzyme overproduction. J. Cell Biol. 70:117a.Google Scholar
  6. Biedler, J. L., Albrecht, A. M., Hutchison, D. J., and Spengler, B. A. (1972). Drug response, dihydrofolate reductase, and cytogenetics of amethopterin-resistant Chinese hamster cells in vitro. Cancer Res. 32153.Google Scholar
  7. Biedler, J. L., Albrecht, A. M., and Spengler, B. A. (1978). Biochemical and karyological properties of cells resistant to the quinazoline antifolate, methasquin, Eur. J. Cancer 1441.Google Scholar
  8. Biedler, J. L., Melera, P. W., and Spengler, B. A. (1980). Specifically altered metaphase chromosomes in antifolate-resistant Chinese hamster cells that overproduce dihydrofolate reductase. Cancer Genet. and Cytogenet. 247.Google Scholar
  9. Bostock, C. J., Gosden, J. R., and Mitchell, A. R. (1978). Localization of a male-specific DNA fragment to a sub-region of the human Y chromosome. Nature 272324.Google Scholar
  10. Brown, D. D., and Dawid, I. B. (1968). Specific gene amplification in oocytes. Science 160272.Google Scholar
  11. Cooke, H. (1976). Repeated sequences specific to human males. Nature 262182.Google Scholar
  12. Corneo, G., Ginelli, E., and Polli, E. (1967). A satellite DNA isolated from human tissues. J. Mol. Biol. 23619.Google Scholar
  13. Corneo, G., Ginelli, E., and Polli, E. (1968). Isolation of the complementary strands of a human satellite DNA. J. Mol. Biol. 33331.Google Scholar
  14. Cortadas, J., Macaya, G., and Bernardi, G. (1977). An analysis of the bovine genome by density gradient centrifugation: Fractionation in Cs2SO4/3,6-bis(acetatomercurimethyl)dioxane density gradient. Eur. J. Biochem. 7613.Google Scholar
  15. Deaven, L. L., and Peterson, D. F. (1973). The chromosomes of CHO, an aneuploid Chinese hamster cell line: G-band, C-band, and autoradiographic analyses. Chromosoma (Berlin) 41129.Google Scholar
  16. Filipski, J., Thiery, J.-P., and Bernardi, G. (1973). An analysis of the bovine genome by Cs2SO4-Ag+ density gradient centrifugation. J. Mol. Biol. 80177.Google Scholar
  17. Gall, J. G. (1968). Differential synthesis of the genes for ribosomal RNA during amphibian oogenesis. Proc. Natl. Acad. Sci. 60553.Google Scholar
  18. Gosden, J. R., Mitchell, A. R., Buckland, R. A., Clayton, R. P., and Evans, H. J. (1975). The location of four human satellite DNAs on human chromosomes. Exp. Cell Res. 92148.Google Scholar
  19. Hsu, T. C., and Arrighi, F. E. (1971). Distribution of constitutive heterochromatin in mammalian chromosomes. Chromosoma (Berlin) 34243.Google Scholar
  20. Jensen, R. H., and Davidson, N. (1966). Spectrophotometric, potentiometric, and density gradient ultracentrifugation studies of the binding of silver ion by DNA. Biopolymers 417.Google Scholar
  21. Jones, K. W., and Corneo, G. (1971). Location of satellite and homogeneous DNA sequences on human chromosomes. Nature New Biol. 233268.Google Scholar
  22. Jones, K. W., Prosser, J., Corneo, G., and Ginelli, E. (1973). The chromosomal location of human satellite DNA III. Chromosoma (Berlin) 42445.Google Scholar
  23. Kit, S. (1961). Equilibrium sedimentation in density gradients of DNA preparations from animal tissues. J. Mol. Biol. 3711.Google Scholar
  24. Loveday, K. S., and Latt, S. A. (1978). Search for DNA interchange corresponding to sister chromatid exchanges in Chinese hamster ovary cells. Nucleic Acids Res. 54087.Google Scholar
  25. Manuelidis, L. (1976). Repeating restriction fragments of human DNA. Nucleic Acids Res. 33063.Google Scholar
  26. Manuelidis, L. (1977). A simplified method for the preparation of mouse satellite DNA. Anal. Biochem. 78561.Google Scholar
  27. Manuelidis, L. (1978a). Complex and simple sequences in human repeated DNAs. Chromosoma (Berlin) 661.Google Scholar
  28. Manuelidis, L. (1978b). Chromosomal localization of complex and simple repeated human DNAs. Chromosoma (Berlin) 6623.Google Scholar
  29. Melera, P. W., Wolgemuth, D. J., Hession, C., and Biedler, J. L. (1980). Antifolate resistant Chinese hamster cells: Evidence for the overproduction of two dihydrofolate reductases encoded by separate mRNAs. J. Biol. Chem. 255319.Google Scholar
  30. Miller, O. J., Tantravahi, R., Miller, D. A., Yu, L.-C., Szabo, P., and Prensky, W. (1979). Marked increase in ribosomal RNA gene multiplicity in a rat hepatoma cell line. Chromosoma (Berlin) 71183.Google Scholar
  31. Moar, M. H., and Natarajan, A. T. (1977). Chromosomal locations of satellite DNA in MSWBS ascites tumor cell lines. J. Natl. Cancer Inst. 581151.Google Scholar
  32. Nunberg, J. H., Kaufman, R. J., Schimke, R. T., Urlaub, G., and Chasin, L. A. (1978). 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. 755553.Google Scholar
  33. Nunberg, J. H., Kaufman, R. J., Chang, A. C. Y., Erlich, H. A., Cohen, S. N., and Schimke, R. T. (1979). Structure of dihydrofolate reductase genes in methotrexate-resistant mouse lines: Expression of mouse dihydrofolate reductase in E. coli. J. Supramol. Struct. Suppl. 369.Google Scholar
  34. Parker, D. L., Rothblum, L. I., and Busch, H. (1978). A 3.6 megadalton EcoR 1 restriction fragment found in abundance in the genome of the Novikoff hepatoma and in few copies in the normal rat liver. J. Cell Biol. 79:137a.Google Scholar
  35. Rinehart, F. P., and Schmid, C. W. (1976). The effect of silver ion binding and pH on the buoyant density of DNA and its use in fractionating heterogeneous DNA. Biochim. Biophys. Acta 425451.Google Scholar
  36. Saunders, G. F., Hsu, T. C., Getz, M. J., Simes, E. L., and Arrighi, F. E. (1972). Locations of a human satellite DNA in human chromosomes. Nature New Biol. 236244.Google Scholar
  37. Schildkraut, C. L., and Maio, J. J. (1969). Fractions of the HeLa DNA differing in their content of guanine + cytosine. J. Mol. Biol. 46305.Google Scholar
  38. Schimke, R. T., Alt, F. W., Kellems, R. E., Kaufman, R. J., and Bertino, J. R. (1977). Amplification of bihydrofolate reductase genes in methotrexateresistant cultured mouse cells. C.S.H.S.Q.B. 42649.Google Scholar
  39. Schimke, R. T., Brown, P., Kaufman, R., Nunberg, J., and Setzer, D. (1979). Gene amplification and methotrexate resistance in cultured mammalian cells. J. Supramol. Struct. Suppl. 341.Google Scholar
  40. Skinner, D. (1977). Satellite DNA's. Bioscience 27790.Google Scholar
  41. Tapiero, H., Canera, R., and Schildkraut, C. L. (1972). Fractions of Chinese hamster DNA differing in their content of guanine + cytosine and evidence for the presence of single-stranded DNA. Biochim. Biophys. Acts 272350.Google Scholar
  42. Thiery, J.-P., Macaya, G., and Bernardi, G. (1976). An analysis of eukaryotic genomes by density gradient centrifugation. J. Mol. Biol. 108219.Google Scholar
  43. Votavova, H., and Sponar, J. (1975). Identification and separation of components of calf thymus DNA using a CsCl-netropsin density gradient. Nucleic Acids Res. 2431.Google Scholar
  44. Weinstock, R., Sweet, R., Weiss, M., Cedar, H., and Axel, R. (1978). Intragenic DNA spacers interrupt the ovalbumin gene. Proc. Natl. Acad. Sci. 751299.Google Scholar
  45. Yunis, J. J., Kuo, M. T., and Saunders, G. F. (1977). Localization of sequences specifying messenger RNA to light-staining G-bands of human chromosomes. Chromosoma (Berlin) 61335.Google Scholar

Copyright information

© Plenum Publishing Corporation 1980

Authors and Affiliations

  • Debra J. Wolgemuth
    • 1
  • June L. Biedler
    • 1
  • Peter W. Melera
    • 1
  1. 1.Laboratories of RNA Synthesis and Regulation and Cellular and Biochemical GeneticsSloan-Kettering Institute for Cancer ResearchRye

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