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. 1986 Feb;6(2):549–558. doi: 10.1128/mcb.6.2.549

Inducible gene expression by DNA rearrangements in human cells.

J P Murnane
PMCID: PMC367545  PMID: 2431271

Abstract

A permanent human cell line, cell line LM205, was established by transforming primary human fibroblasts with a plasmid containing both simian virus 40 sequences with a defective origin of replication and a G418 resistance gene (neo) that lacked a eucaryotic transcriptional promoter. G418-resistant cells appeared spontaneously in clonal populations of LM205 cells at a frequency of approximately 10(-5) cell per cell plated in the presence of 400 micrograms of G418 per ml. G418 resistance was stable and correlated with the appearance of neo-specific RNA. Characterization of the neo gene in the G418-sensitive parental cell line by both a Southern blot analysis and a restriction map analysis of cloned sequences demonstrated that there was a stable integration site containing a single neo coding sequence. A Southern blot analysis of five G418-resistant subclones indicated that there were heterogeneous DNA rearrangements in the region of the neo gene that were unique in each subclone. Restriction mapping of a fragment containing the neo gene isolated from one of the resistant subclones demonstrated that the rearrangement was a tandem duplication that resulted in the relocation of the simian virus 40 bidirectional transcriptional promoter 5' to the neo gene. Tandem duplication was also consistent with the Southern blot polymorphisms observed in the other resistant subclones, suggesting that there were heterogeneous sites of recombination with respect to both the neo gene and the simian virus 40 promoter. Although these rearrangements resulted in an increase in neo gene copy number per cell, amplification showed no correlation quantitatively with the large increase in neo-specific RNA in these cells. Therefore, G418-resistant colony formation in cell line LM205 provides a method for studying both the mechanisms involved in this type of recombination and the factors influencing its frequency.

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Selected References

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

  1. Bender M. A., Brockman W. W. Rearrangement of integrated viral DNA sequences in mouse cells transformed by simian virus 40. J Virol. 1981 Jun;38(3):872–879. doi: 10.1128/jvi.38.3.872-879.1981. [DOI] [PMC free article] [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. Colbère-Garapin F., Horodniceanu F., Kourilsky P., Garapin A. C. A new dominant hybrid selective marker for higher eukaryotic cells. J Mol Biol. 1981 Jul 25;150(1):1–14. doi: 10.1016/0022-2836(81)90321-1. [DOI] [PubMed] [Google Scholar]
  4. Crawford B. D., Barrett J. C., Ts'o P. O. Neoplastic conversion of preneoplastic Syrian hamster cells: rate estimation by fluctuation analysis. Mol Cell Biol. 1983 May;3(5):931–945. doi: 10.1128/mcb.3.5.931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davies J., Jimenez A. A new selective agent for eukaryotic cloning vectors. Am J Trop Med Hyg. 1980 Sep;29(5 Suppl):1089–1092. doi: 10.4269/ajtmh.1980.29.1089. [DOI] [PubMed] [Google Scholar]
  6. Davies J., Smith D. I. Plasmid-determined resistance to antimicrobial agents. Annu Rev Microbiol. 1978;32:469–518. doi: 10.1146/annurev.mi.32.100178.002345. [DOI] [PubMed] [Google Scholar]
  7. DeVries P. J., Davidson R. L., Clough D. W. Ultraviolet-induced reactivation, amplification, and hypomethylation of a herpes simplex virus thymidine kinase gene. Somat Cell Mol Genet. 1984 Nov;10(6):625–632. doi: 10.1007/BF01535228. [DOI] [PubMed] [Google Scholar]
  8. Economou-Pachnis A., Lohse M. A., Furano A. V., Tsichlis P. N. Insertion of long interspersed repeated elements at the Igh (immunoglobulin heavy chain) and Mlvi-2 (Moloney leukemia virus integration 2) loci of rats. Proc Natl Acad Sci U S A. 1985 May;82(9):2857–2861. doi: 10.1073/pnas.82.9.2857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Emerman M., Temin H. M. Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell. 1984 Dec;39(3 Pt 2):449–467. [PubMed] [Google Scholar]
  10. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  11. Feinstein S. C., Ross S. R., Yamamoto K. R. Chromosomal position effects determine transcriptional potential of integrated mammary tumor virus DNA. J Mol Biol. 1982 Apr 15;156(3):549–565. doi: 10.1016/0022-2836(82)90266-2. [DOI] [PubMed] [Google Scholar]
  12. Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
  13. GIRARDI A. J., JENSEN F. C., KOPROWSKI H. SV40-INDUCED TRANFORMATION OF HUMAN DIPLOID CELLS: CRISIS AND RECOVERY. J Cell Physiol. 1965 Feb;65:69–83. doi: 10.1002/jcp.1030650110. [DOI] [PubMed] [Google Scholar]
  14. Godson G. N., Vapnek D. A simple method of preparing large amounts of phiX174 RF 1 supercoiled DNA. Biochim Biophys Acta. 1973 Apr 11;299(4):516–520. doi: 10.1016/0005-2787(73)90223-2. [DOI] [PubMed] [Google Scholar]
  15. Hiscott J. B., Murphy D., Defendi V. Instability of integrated viral DNA in mouse cells transformed by simian virus 40. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1736–1740. doi: 10.1073/pnas.78.3.1736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hiscott J., Murphy D., Defendi V. Amplification and rearrangement of integrated SV40 DNA sequences accompany the selection of anchorage-independent transformed mouse cells. Cell. 1980 Nov;22(2 Pt 2):535–543. doi: 10.1016/0092-8674(80)90363-3. [DOI] [PubMed] [Google Scholar]
  17. Hwang S. P., Kucherlapati R. S. Events preceding stable integration of SV40 genomes in a human cell line. Somatic Cell Genet. 1983 Jul;9(4):457–468. doi: 10.1007/BF01543046. [DOI] [PubMed] [Google Scholar]
  18. Klein G., Klein E. Oncogene activation and tumor progression. Carcinogenesis. 1984 Apr;5(4):429–435. doi: 10.1093/carcin/5.4.429. [DOI] [PubMed] [Google Scholar]
  19. Lin F. L., Sternberg N. Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome. Mol Cell Biol. 1984 May;4(5):852–861. doi: 10.1128/mcb.4.5.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liskay R. M., Stachelek J. L. Evidence for intrachromosomal gene conversion in cultured mouse cells. Cell. 1983 Nov;35(1):157–165. doi: 10.1016/0092-8674(83)90218-0. [DOI] [PubMed] [Google Scholar]
  21. Luria S. E., Delbrück M. Mutations of Bacteria from Virus Sensitivity to Virus Resistance. Genetics. 1943 Nov;28(6):491–511. doi: 10.1093/genetics/28.6.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  24. Mounts P., Kelly T. J., Jr Rearrangements of host and viral DNA in mouse cells transformed by simian virus 40. J Mol Biol. 1984 Aug 15;177(3):431–460. doi: 10.1016/0022-2836(84)90294-8. [DOI] [PubMed] [Google Scholar]
  25. Murnane J. P., Fuller L. F., Painter R. B. Establishment and characterization of a permanent pSV ori--transformed ataxia-telangiectasia cell line. Exp Cell Res. 1985 May;158(1):119–126. doi: 10.1016/0014-4827(85)90437-9. [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. Sager R., Anisowicz A., Howell N. Genomic rearrangements in a mouse cell line containing integrated SV40 DNA. Cell. 1981 Jan;23(1):41–50. doi: 10.1016/0092-8674(81)90268-3. [DOI] [PubMed] [Google Scholar]
  28. Singer M. F. Highly repeated sequences in mammalian genomes. Int Rev Cytol. 1982;76:67–112. doi: 10.1016/s0074-7696(08)61789-1. [DOI] [PubMed] [Google Scholar]
  29. Small M. B., Gluzman Y., Ozer H. L. Enhanced transformation of human fibroblasts by origin-defective simian virus 40. Nature. 1982 Apr 15;296(5858):671–672. doi: 10.1038/296671a0. [DOI] [PubMed] [Google Scholar]
  30. Smith A. J., Berg P. Homologous recombination between defective neo genes in mouse 3T6 cells. Cold Spring Harb Symp Quant Biol. 1984;49:171–181. doi: 10.1101/sqb.1984.049.01.020. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  33. Stark G. R., Wahl G. M. Gene amplification. Annu Rev Biochem. 1984;53:447–491. doi: 10.1146/annurev.bi.53.070184.002311. [DOI] [PubMed] [Google Scholar]
  34. Subramani S., Rubnitz J. Recombination events after transient infection and stable integration of DNA into mouse cells. Mol Cell Biol. 1985 Apr;5(4):659–666. doi: 10.1128/mcb.5.4.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vande Woude G. F., Oskarsson M., Enquist L. W., Nomura S., Sullivan M., Fischinger P. J. Cloning of integrated Moloney sarcoma proviral DNA sequences in bacteriophage lambda. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4464–4468. doi: 10.1073/pnas.76.9.4464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Varmus H. E. The molecular genetics of cellular oncogenes. Annu Rev Genet. 1984;18:553–612. doi: 10.1146/annurev.ge.18.120184.003005. [DOI] [PubMed] [Google Scholar]
  37. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]

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