Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1984 Jul;4(7):1411–1415. doi: 10.1128/mcb.4.7.1411

Detection of deletion mutations in pSV2gpt-transformed cells.

K R Tindall, L F Stankowski Jr, R Machanoff, A W Hsie
PMCID: PMC368924  PMID: 6095070

Abstract

We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient CHO cells that have been transformed by the plasmid vector pSV2gpt. One isolated transformant, designated AS52, carries a single copy of the Escherichia coli gpt gene stably integrated into the high-molecular-weight DNA and expresses the bacterial gene for the enzyme xanthine-guanine phosphoribosyltransferase. Mutants deficient in this enzyme can be induced in the AS52 cell line by a variety of mutagens, and spontaneous or induced mutants can be selected for resistance to 6-thioguanine (Tgr). Two Tgr clones derived from the AS52 line were analyzed by Southern blot hybridization and were found to contain deletions involving at least a portion of the gpt gene. Because of the small size and stability of the integrated pSV2gpt plasmid, and the well-defined selection protocol for mutant isolation, the AS52 line offers promise as a system suitable for the study of mutation at the molecular level in CHO cells.

Full text

PDF
1411

Images in this article

1414Image
on p.1414

Selected References

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

  1. Albertini A. M., Hofer M., Calos M. P., Miller J. H. On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions. Cell. 1982 Jun;29(2):319–328. doi: 10.1016/0092-8674(82)90148-9. [DOI] [PubMed] [Google Scholar]
  2. Calos M. P., Lebkowski J. S., Botchan M. R. High mutation frequency in DNA transfected into mammalian cells. Proc Natl Acad Sci U S A. 1983 May;80(10):3015–3019. doi: 10.1073/pnas.80.10.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Davis R. W., Thomas M., Cameron J., St John T. P., Scherer S., Padgett R. A. Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 1980;65(1):404–411. doi: 10.1016/s0076-6879(80)65051-4. [DOI] [PubMed] [Google Scholar]
  4. Efstratiadis A., Posakony J. W., Maniatis T., Lawn R. M., O'Connell C., Spritz R. A., DeRiel J. K., Forget B. G., Weissman S. M., Slightom J. L. The structure and evolution of the human beta-globin gene family. Cell. 1980 Oct;21(3):653–668. doi: 10.1016/0092-8674(80)90429-8. [DOI] [PubMed] [Google Scholar]
  5. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  6. Hsie A. W., Brimer P. A., Mitchell T. J., Gosslee D. G. The dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells. Somatic Cell Genet. 1975 Jul;1(3):247–261. doi: 10.1007/BF01538449. [DOI] [PubMed] [Google Scholar]
  7. Hsie A. W., Brimer P. A., Mitchell T. J., Gosslee D. G. The dose-response relationship for ultraviolet-light-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster ovary cells. Somatic Cell Genet. 1975 Oct;1(4):383–389. doi: 10.1007/BF01538669. [DOI] [PubMed] [Google Scholar]
  8. Hsie A. W., O'Neill J. P., Couch D. B., SanSebastian J. R., Brimer P. A., Machanoff R., Fuscoe J. C., Riddle J. C., Li A. P., Forbes N. L. Quantitative analyses of radiation- and chemical-induced lethality and mutagenesis in Chinese hamster ovary cells. Radiat Res. 1978 Dec;76(3):471–492. [PubMed] [Google Scholar]
  9. Miller R. L., Ramsey G. A., Krenitsky T. A., Elion G. B. Guanine phosphoribosyltransferase from Escherichia coli, specificity and properties. Biochemistry. 1972 Dec 5;11(25):4723–4731. doi: 10.1021/bi00775a014. [DOI] [PubMed] [Google Scholar]
  10. Mulligan R. C., Berg P. Expression of a bacterial gene in mammalian cells. Science. 1980 Sep 19;209(4463):1422–1427. doi: 10.1126/science.6251549. [DOI] [PubMed] [Google Scholar]
  11. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. O'Neill J. P., Brimer P. A., Machanoff R., Hirsch G. P., Hsie A. W. A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): development and definition of the system. Mutat Res. 1977 Oct;45(1):91–101. doi: 10.1016/0027-5107(77)90047-1. [DOI] [PubMed] [Google Scholar]
  13. O'Neill J. P., Couch D. B., Machanoff R., San Sebastian J. R., Brimer P. A., Hsie A. W. A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): utilization with a variety of mutagenic agents. Mutat Res. 1977 Oct;45(1):103–109. doi: 10.1016/0027-5107(77)90048-3. [DOI] [PubMed] [Google Scholar]
  14. Perucho M., Hanahan D., Wigler M. Genetic and physical linkage of exogenous sequences in transformed cells. Cell. 1980 Nov;22(1 Pt 1):309–317. doi: 10.1016/0092-8674(80)90178-6. [DOI] [PubMed] [Google Scholar]
  15. Razzaque A., Mizusawa H., Seidman M. M. Rearrangement and mutagenesis of a shuttle vector plasmid after passage in mammalian cells. Proc Natl Acad Sci U S A. 1983 May;80(10):3010–3014. doi: 10.1073/pnas.80.10.3010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  17. Robins D. M., Axel R., Henderson A. S. Chromosome structure and DNA sequence alterations associated with mutation of transformed genes. J Mol Appl Genet. 1981;1(3):191–203. [PubMed] [Google Scholar]
  18. 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]
  19. Thacker J., Debenham P. G., Stretch A., Webb M. B. The use of a cloned bacterial gene to study mutation in mammalian cells. Mutat Res. 1983 Sep;111(1):9–23. doi: 10.1016/0027-5107(83)90003-9. [DOI] [PubMed] [Google Scholar]
  20. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]

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

RESOURCES