1887

Abstract

We analysed the DNA rearrangements that occurred during the integration and amplification of an Epstein-Barr virus (EBV)-simian virus 40 (SV40) hybrid shuttle vector in human cells. The human HeLa cell line was episomally transformed with the EBV-SV40 p205-GTI plasmid. After a 2 month culture in a selective medium, a HeLa cell-derived population (H-G1 cells) was obtained in which the p205-GTI vector was integrated as a single intact copy deleted in the EBV latent origin of replication (OriP). Sequencing data showed that the endpoints of the plasmid sequences, at the plasmid-cell DNA junctions, are located within the two essential elements of EBV OriP, which may form several secondary structures. This result suggests that a specific DNA sequence (OriP) or palindromic structures could play a role in this integration process. This represents the first fully characterized site of integration of an EBV vector in human cells. The transient expression of the SV40 large T antigen in H-G1 cells leads to the appearance of episomal molecules with an extremely heterogeneous size pattern. Individual analysis of these episomes after rescue in bacteria indicated that they retained sequences of both the p205-GTI plasmid and cellular DNA. Comparison of the structure of these circular DNAs with those of the integrated p205-GTI copy indicated that large T antigen expression in human cells leads to the amplification of the integrated shuttle vector according to the ‘onion skin’ model developed for transformed rodent cells. Indeed, amplified sequences were collinear with the integrated p205-GTI copy and its surrounding cellular sequences, distributed almost equally around the SV40 replication origin, and circularized by illegitimate recombination which did not involve specific nucleotide sequences. This system is of interest in that it enables easy recovery of individual recombined molecules in host bacteria. Each isolated clone contains a unique recombination junction which is easily and rapidly characterized and sequenced.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-73-7-1679
1992-07-01
2021-10-24
Loading full text...

Full text loading...

/deliver/fulltext/jgv/73/7/JV0730071679.html?itemId=/content/journal/jgv/10.1099/0022-1317-73-7-1679&mimeType=html&fmt=ahah

References

  1. Ambinder R. F., Shah W. A., Rawlins D. R., Hayward G. S., Hayward S. D. 1990; Definition of the sequence requirement for binding of the EBNA-1 protein to its palindromic target sites in Epstein-Barr virus DNA. Journal of Virology 64:2369–2379
    [Google Scholar]
  2. Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Séguin C., Tuffnell P. S., Barrell B. G. 1984; DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature, London 310:207–211
    [Google Scholar]
  3. Botchan M., Topp W., Sambrook J. 1979; Studies on simian virus 40 excision from cellular chromosomes. Cold Spring Harbor Symposia on Quantitative Biology 43:709–719
    [Google Scholar]
  4. Botchan M., Stringer J., Mitchison T., Sambrook J. 1980; Integration and excision of SV40 DNA from the chromosome of a transformed cell. Cell 20:143–152
    [Google Scholar]
  5. Brouillette S., Chartrand P. 1987; Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions. Molecular and Cellular Biology 7:2248–2255
    [Google Scholar]
  6. Croce C. M. 1987; Role of chromosome translocations in human neoplasia. Cell 49:155–156
    [Google Scholar]
  7. Gerard R. D., Gluzman Y. 1985; New host cell system for regulated simian virus 40 DNA replication. Molecular and Cellular Biology 5:3231–3240
    [Google Scholar]
  8. Hanahan D. 1983; Studies on the transformation of Escherichia coli with plasmids. Journal of Molecular Biology 166:557–580
    [Google Scholar]
  9. Heinzel S. S., Krysan P. J., Calos M. P., DuBridge R. B. 1988; Use of simian virus 40 replication to amplify Epstein-Barr virus shuttle vector in human cells. Journal of Virology 62:3738–3746
    [Google Scholar]
  10. Hurley E. A., Agger S., McNeil J. A., Lawrence J. B., Calendar A., Lenoir G., Thorley-Lawson D. A. 1991; When Epstein-Barr virus persistently infects B-cell lines, it frequently integrates. Journal of Virology 65:1245–1254
    [Google Scholar]
  11. Hyrien O., Debatisse M., Buttin G., Robert de Saint Vincent B. 1987; A hotspot for novel amplification joints in a mosaic of Alu- like repeats and palindromic A + T-rich DNA. EMBO Journal 6:2401–2408
    [Google Scholar]
  12. Hyrien O., Debatisse M., Buttin G., Robert de Saint Vincent B. 1988; The multicopy appearance of a large inverted duplication and the sequence at the inversion joint suggest a new model for gene amplification. EMBO Journal 7:407–417
    [Google Scholar]
  13. Karlin S. 1986; Significant potential secondary structures in the Epstein-Barr virus genome. Proceedings of the National Academy of Sciences, U.S.A 83:6915–6919
    [Google Scholar]
  14. Krawinkel U., Zoebelein G., Bothwell A. L. M. 1986; Palindromic sequences are associated with sites of DNA breakage during gene conversion. Nucleic Acids Research 14:3871–3882
    [Google Scholar]
  15. Lupton S., Levine A. J. 1985; Mapping genetic elements of Epstein-Barr virus that facilitate extrachromosomal persistence of Epstein-Barr virus-derived plasmids in human cells. Molecular and Cellular Biology 5:2533–2542
    [Google Scholar]
  16. Menck C. F. M., Sarasin A., James M. R. 1987; SV40-based Escherichia coli shuttle vectors infectious for monkey cells. Gene 53:21–29
    [Google Scholar]
  17. Meuth M. 1989; Illegitimate recombination in mammalian cells. In Mobile DNA pp 833–860 Edited by Berg D. E., Howe M. M. Washington, D.C.: American Society for Microbiology;
    [Google Scholar]
  18. Mounts P., Kelly T. J. 1984; Rearrangements of host and viral DNA in mouse cells transformed by simian virus 40. Journal of Molecular Biology 177:431–460
    [Google Scholar]
  19. Murnane J. P., Yezzi M. J., Young B. R. 1990; Recombination events during integration of transfected DNA into normal human cells. Nucleic Acids Research 18:2733–2738
    [Google Scholar]
  20. Nalbantoglu J., Hartley D., Phear G., Tear G., Meuth M. 1986; Spontaneous deletion formation at the aprt locus of hamster cells: the presence of short sequence homologies and dyad symmetries at deletion termini. EMBO Journal 5:1199–1204
    [Google Scholar]
  21. Nicholls R. D., Fishel-Ghodsian N., Higgs D. R. 1987; Recombination at the human alpha-globin gene cluster: sequence features and topological constraints. Cell 49:369–378
    [Google Scholar]
  22. Orlowski R., Miller G. 1991; Single-stranded structures are present within plasmids containing the Epstein-Barr virus latent origin of replication. Journal of Virology 65:677–686
    [Google Scholar]
  23. Rawlins D. R., Milman G., Hayward S. D., Hayward G. S. 1985; Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region. Cell 42:859–868
    [Google Scholar]
  24. Reisman D., Yates J., Sugden B. 1985; A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components. Molecular and Cellular Biology 5:1822–1832
    [Google Scholar]
  25. Romani M., De Ambrosis A., Alhadeff B., Purrello M., Gluzman Y., Siniscalco M. 1990; Preferential integration of the ad5/SV40 hybrid virus at the highly recombinogenic human chromosomal site 1p36. Gene 95:231–241
    [Google Scholar]
  26. Roth D., Wilson J. 1988; Illegitimate recombination in mammalian cells. In Genetic Recombination pp 621–653 Edited by Kucherlapati R., Smith G. R. Washington, D.C.: American Society for Microbiology;
    [Google Scholar]
  27. Ruley H. E., Fried M. 1983; Clustered illegitimate recombination events in mammalian cells involving very short sequence homologies. Nature, London 304:181–184
    [Google Scholar]
  28. Skalka A. M. 1989; Integrative recombination of retroviral DNA. In Genetic Recombination pp 701–724 Edited by Kucherlapati R., Smith G. R. Washington, D.C.: American Society for Microbiology;
    [Google Scholar]
  29. Stary A., James M. R., Sarasin A. 1989; High recombination rate of an Epstein-Barr virus–simian virus 40 hybrid shuttle vector in human cells. Journal of Virology 63:3837–3843
    [Google Scholar]
  30. Stark G. R., Debatisse M., Giulotto E., Wahl G. M. 1989; Recent progress in understanding mechanisms of mammalian DNA amplification. Cell 57:901–908
    [Google Scholar]
  31. Wigler M., Pellicer A., Silverstein S., Axel R. 1978; Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell 14:725–731
    [Google Scholar]
  32. Yates J. L., Warren N., Sugden B. 1985; Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature, London 313:812–815
    [Google Scholar]
  33. Zheng H., Wilson J. H. 1990; Gene targeting in normal and amplified cell lines. Nature, London 344170–173
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-73-7-1679
Loading
/content/journal/jgv/10.1099/0022-1317-73-7-1679
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error