1887

Abstract

SUMMARY

A minor fraction of single-stranded DNA (ssDNA) was isolated by an improved method of hydroxylapatite chromatography (HAC) from the native nuclear DNA (nDNA) of SV-3T3 cells, non-productively transformed by SV40. Molecular hybridization, monitored by the use of S nuclease, HAC, isopycnic centrifugation and thermal melting showed that ssDNA from SV-3T3 cells (which amounts to 1.5 to 2% of the total nDNA) has the same characteristics as ssDNA previously isolated from other cell species. Only 27 to 28% of ssDNA can be self-hybridized but the greatest part can be reassociated to the non-repetitive portion of nDNA and up to 38% hybridized to homologous RNAs, as compared with 7 to 8% for bulk nDNA. Highly radioactive virus probes (SV40-H-cRNA synthesized in a cell-free system and the separated ‘early’ and ‘late’ strands of SV40 DNA labelled with I) were annealed to different excess amounts of cellular DNA. Both the quantities of each probe hybridized at saturation levels and the various reaction kinetics indicated that ssDNA is greatly enriched for virus sequences, mainly originating from the ‘early’ DNA strand which is predominantly expressed in SV-3T3 cells. The mode of formation of ssDNA is discussed in the light of other findings on the effects of DNA untwisting proteins and susceptibility of active animal genes to selective enzymic attacks.

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1979-06-01
2024-05-19
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References

  1. Affara N. A., Jacquft M., Jacob H., Jacob F., Gros F. 1977; Comparison of polyadenylated RNA from embryonal carcinoma and committed myogenic and erythropoetic cell lines. Cell 13:509–520
    [Google Scholar]
  2. Bick M. D., Lee C. S., Thomas C. A. Jun 1972; Local destabilization of DNA during transcription. Journal of Molecular Biology 71:1–9
    [Google Scholar]
  3. Champoux J. J. 1977; Strand breakage by the DNA untwisting enzyme results in covalent attachment of the enzyme to DNA. Proceedings of the National Academy of Sciences of the United States of America 74:3800–3804
    [Google Scholar]
  4. Cuzin F., Vogt M., Dieckmann, Berg P. 1970; Induction of virus multiplication of 3T3 cells transformed by a thermo-sensitive mutant of polyoma virus. II. Formation of oligomeric polyoma DNA molecules. Journal of Molecular Biology 47:317–333
    [Google Scholar]
  5. Franze Fernandez M. T., Pogo A. O. 1971; Regulation of the nucleolar-dependant RNA-polymerase by amino-acids in Ehrlich ascites tumor cells. Proceedings of the National Academy of Sciences of the United States of America 68:3030–3044
    [Google Scholar]
  6. Frenster J. H. 1976; Selective control of DNA helix opening during gene regulation. Cancer Research 36:3394–3398
    [Google Scholar]
  7. Garel A., Axel R. 1976; Selective digestion of transcriptionally active ovalbumin genes from oviduct nuclei. Proceedings of the National Academy of Sciences of the United States of America 73:3966–3970
    [Google Scholar]
  8. Gelb L. D., Kohne D. E., Martin M. A. 1971; Quantitation of SV40 sequences in African green monkey, mouse and virus transformed cell genomes. Journal of Molecular Biology 57:129–143
    [Google Scholar]
  9. Groner Y., Monroy M., Jacquet M., Hurwitz J. 1975; Chromatin as a template for RNA synthesis in vitro. Proceedings of the National Academy of Sciences of the United States of America 72:194–199
    [Google Scholar]
  10. Hanania N., Shaool D., Poncy C., Tapiero H., Harel J. 1977; Isolation of single-stranded transcription sites from human nuclear DNA. Cell Biology International Reports 1:309–315
    [Google Scholar]
  11. Hirt B. 1967; Selective extraction of polyoma DNA from infected mouse cells. Journal of Molecular Biology 26:365–369
    [Google Scholar]
  12. Khoury G., Byrne J., Martin M. 1972; Patterns of Simian virus 40 DNA transcription after acute infection of permissive and non permissive cells. Proceedings of the National Academy of Sciences of the United States of America 69:1925–1928
    [Google Scholar]
  13. Leibovitch S. A., Harel J. 1978; Active DNA transcription sites released from the genome of normal embryonic chicken cells. Nucleic Acids Research 5:777–787
    [Google Scholar]
  14. Leibovitch S. A., Tapiero H., Harel J. 1977; Single-stranded DNA from oncornavirus infected cells, enriched in virus specific DNA sequences. Proceedings of the National Academy of Sciences of the United States of America 74:3720–3724
    [Google Scholar]
  15. Levine A. J., Kang S. A., Billheimer F. E. 1970; DNA replication in SV40 infected cells. I. Analysis of SV40 DNA. Journal of Molecular Biology 50:549–568
    [Google Scholar]
  16. Ozanne B., Sharp P. A., Sambrook J. 1973; Transcription of Simian virus 40. II. Hybridization of RNA extracted from different lines of transformed cells to the separated strands of Simian virus 40 DNA. Journal of Virology 12:90–98
    [Google Scholar]
  17. Sambrook J., Sharp P. A., Keller W. 1972; Transcription of SV40 DNA. I. Separation of the strands of SV40 DNA and hybridization of the separated strands to RNA extracted from lytically infected and transformed cells. Journal of Molecular Biology 70:57–71
    [Google Scholar]
  18. Scherrer K. 1969 In Fundamental Techniques in Virology pp. 413–432 Edited by Habel K., Salzman I. N. P. New York: Academic Press;
    [Google Scholar]
  19. Sober H. A. 1968 In Handbook of Biochemistry. Selected data for molecular biology p 58 Cleveland, Ohio: The Technical Rubber Co;
    [Google Scholar]
  20. Tapiero H., Monier M. N., Shaool D., Harel J. 1974; Distribution of repetitious sequences in chick nuclear DNA. Nucleic Acids Research 1:309–322
    [Google Scholar]
  21. Tapiero H., Leibovitch S. A., Shaool D., Monier M. N., Harel J. 1976; Isolation of single-stranded DNA related to the transcriptional activity of animal cells. Nucleic Acids Research 3:953–963
    [Google Scholar]
  22. Tooze J. 1973 In The Molecular Biology of Tumour Viruses New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  23. Tournier P., Cassingena R., Wicker R., Coppey J., Suarez H. 1967; Etude du mécanisme de l’induction chez des cellules de hamster syrien transformées par le virus SV40. International Journal of Cancer 2:117–132
    [Google Scholar]
  24. Turler H. 1977; Interaction of polyoma and mouse DNAs. IV. Time course and extent of integration of polyoma DNA into mouse DNA during lytic infection. Journal of Virology 23:272–285
    [Google Scholar]
  25. Vogt V. 1969; Breaks in DNA stimulate transcription by core RNA polymerase. Nature, London 223:854–855
    [Google Scholar]
  26. Weintraub H., Groudine M. 1976; Chromosomal subunits in active genes have an altered conformation. Globin genes are digested by deoxyribonuclease I in red blood cell nuclei but not in fibroblast nuclei. Science 193:848–856
    [Google Scholar]
  27. Woo S. L. S., Rosen J. M., Liarakos C. D., Choi Y. C., Busch H., Means A. R., O’Malley B. W., Robberson D. L. 1975; Physical and chemical characterization of purified ovalbumin messenger RNA. The Journal of Biological Chemistry 250:7027–7039
    [Google Scholar]
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