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. 1986 Dec;6(12):4594–4601. doi: 10.1128/mcb.6.12.4594

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.

J J Dermody, B E Wojcik, H Du, H L Ozer
PMCID: PMC367244  PMID: 3796611

Abstract

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

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Selected References

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  1. Basilico C. Temperature-sensitive mutations in animal cells. Adv Cancer Res. 1977;24:223–266. doi: 10.1016/s0065-230x(08)61016-7. [DOI] [PubMed] [Google Scholar]
  2. Burstin S. J., Meiss H. K., Basilico C. A temperature-sensitive cell cycle mutant of the BHK cell line. J Cell Physiol. 1974 Dec;84(3):397–408. doi: 10.1002/jcp.1040840308. [DOI] [PubMed] [Google Scholar]
  3. Challberg M. D., Kelly T. J., Jr Adenovirus DNA replication in vitro. Proc Natl Acad Sci U S A. 1979 Feb;76(2):655–659. doi: 10.1073/pnas.76.2.655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chu E. H., Brimer P., Jacobson K. B., Merriam E. V. Mammalian cell genetics. I. Selection and characterization of mutations auxotrophic for L-glutamine or resistant to 8-azaguanine in Chinese hamster cells in vitro. Genetics. 1969 Jun;62(2):359–377. doi: 10.1093/genetics/62.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Colwill R. W., Sheinin R. ts A1S9 locus in mouse L cells may encode a novobiocin binding protein that is required for DNA topoisomerase II activity. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4644–4648. doi: 10.1073/pnas.80.15.4644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Esko J. D., Raetz C. R. Replica plating and in situ enzymatic assay of animal cell colonies established on filter paper. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1190–1193. doi: 10.1073/pnas.75.3.1190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Giles R. E., Ruddle F. H. X-linkage of a human genetic locus that corrects the DNA synthesis lesion in tsC1AGOH mouse cells. Genetics. 1979 Dec;93(4):975–996. doi: 10.1093/genetics/93.4.975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Gupta R. S. Random segretation of multiple genetic markers from CHO-CHO hybrids: evidence for random distribution of functional hemizygosity in the genome. Somatic Cell Genet. 1980 Jan;6(1):115–125. doi: 10.1007/BF01538700. [DOI] [PubMed] [Google Scholar]
  10. Guttman S. A., Sheinin R. Properties of ts Cl mouse L cells which exhibit temperature-sensitive DNA synthesis. Exp Cell Res. 1979 Oct 1;123(1):191–205. doi: 10.1016/0014-4827(79)90435-x. [DOI] [PubMed] [Google Scholar]
  11. Hirschberg J., Marcus M. Isolation by a replica-plating technique of Chinese hamster temperature-sensitive cell cycle mutants. J Cell Physiol. 1982 Oct;113(1):159–166. doi: 10.1002/jcp.1041130125. [DOI] [PubMed] [Google Scholar]
  12. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  13. Hodge L. D., Scharff M. D. Effect of adenovirus on host cell DNA synthesis in synchronized cells. Virology. 1969 Apr;37(4):554–564. doi: 10.1016/0042-6822(69)90273-6. [DOI] [PubMed] [Google Scholar]
  14. Jha K. K., Siniscalco M., Ozer H. L. Temperature-sensitive mutants of BALB/3T3 cells. III. Hybrids between ts2 and other mouse mutant cells affected in DNA synthesis and correction of ts2 defect by human X chromosome. Somatic Cell Genet. 1980 Sep;6(5):603–614. doi: 10.1007/BF01538640. [DOI] [PubMed] [Google Scholar]
  15. Kafatos F. C., Jones C. W., Efstratiadis A. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res. 1979 Nov 24;7(6):1541–1552. doi: 10.1093/nar/7.6.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kao F. T., Puck T. T. Genetics of somatic mammalian cells. IV. Properties of Chinese hamster cell mutants with respect to the requirement for proline. Genetics. 1967 Mar;55(3):513–524. doi: 10.1093/genetics/55.3.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Killary A. M., Lugo T. G., Fournier R. E. Isolation of thymidine kinase-deficient rat hepatoma cells by selection with bromodeoxyuridine, Hoechst 33258, and visible light. Biochem Genet. 1984 Apr;22(3-4):201–213. doi: 10.1007/BF00484224. [DOI] [PubMed] [Google Scholar]
  18. Kimelman D., Miller J. S., Porter D., Roberts B. E. E1a regions of the human adenoviruses and of the highly oncogenic simian adenovirus 7 are closely related. J Virol. 1985 Feb;53(2):399–409. doi: 10.1128/jvi.53.2.399-409.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. La Bella F., Ozer H. L. Differential replication of SV40 and polyoma DNAs in Chinese hamster ovary cells. Virus Res. 1985 Jun;2(4):329–344. doi: 10.1016/0168-1702(85)90029-2. [DOI] [PubMed] [Google Scholar]
  20. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6973–6977. doi: 10.1073/pnas.81.22.6973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Longiaru M., Horwitz M. S. Chinese hamster ovary cells replicate adenovirus deoxyribonucleic acid. Mol Cell Biol. 1981 Mar;1(3):208–215. doi: 10.1128/mcb.1.3.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Marcus M., Fainsod A., Diamond G. The genetic analysis of mammalian cell-cycle mutants. Annu Rev Genet. 1985;19:389–421. doi: 10.1146/annurev.ge.19.120185.002133. [DOI] [PubMed] [Google Scholar]
  23. McCracken A. A. A temperature-sensitive DNA synthesis mutant isolated from the Chinese hamster ovary cell line. Somatic Cell Genet. 1982 Mar;8(2):179–195. doi: 10.1007/BF01538676. [DOI] [PubMed] [Google Scholar]
  24. Meiss H. K., Basilico C. Temperature sensitive mutants of BHK 21 cells. Nat New Biol. 1972 Sep 20;239(90):66–68. doi: 10.1038/newbio239066a0. [DOI] [PubMed] [Google Scholar]
  25. Meuth M., Gonçalves O., Thom P. A selection system specific for the Thy mutator phenotype. Somatic Cell Genet. 1982 Jul;8(4):423–432. doi: 10.1007/BF01538705. [DOI] [PubMed] [Google Scholar]
  26. Murakami Y., Yasuda H., Miyazawa H., Hanaoka F., Yamada M. Characterization of a temperature-sensitive mutant of mouse FM3A cells defective in DNA replication. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1761–1765. doi: 10.1073/pnas.82.6.1761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nishimoto T., Basilico C. Analysis of a method for selecting temperature-sensitive mutants of BHK cells. Somatic Cell Genet. 1978 May;4(3):323–340. doi: 10.1007/BF01542846. [DOI] [PubMed] [Google Scholar]
  28. Nishimoto T., Eilen E., Basilico C. Premature of chromosome condensation in a ts DNA- mutant of BHK cells. Cell. 1978 Oct;15(2):475–483. doi: 10.1016/0092-8674(78)90017-x. [DOI] [PubMed] [Google Scholar]
  29. Nishimoto T., Sekiguchi T., Kai R., Yamashita K., Takahashi T., Sekiguchi M. Large-scale selection and analysis of temperature-sensitive mutants for cell reproduction from BHK cells. Somatic Cell Genet. 1982 Nov;8(6):811–824. doi: 10.1007/BF01543021. [DOI] [PubMed] [Google Scholar]
  30. Ohno K., Okuda A., Ohtsu M., Kimura G. Genetic analysis of control of proliferation in fibroblastic cells in culture. I. Isolation and characterization of mutants temperature-sensitive for proliferation or survival of untransformed diploid rat cell line 3Y1. Somat Cell Mol Genet. 1984 Jan;10(1):17–28. doi: 10.1007/BF01534469. [DOI] [PubMed] [Google Scholar]
  31. Pages J., Manteuil S., Stehelin D., Fiszman M., Marx M., Girard M. Relationship between replication of simian virus 40 DNA and specific events of the host cell cycle. J Virol. 1973 Jul;12(1):99–107. doi: 10.1128/jvi.12.1.99-107.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Sheinin Polyoma and cell DNA synthesis in mouse L cells temperature sensitive for the replication of cell DNA. J Virol. 1976 Mar;17(3):692–704. doi: 10.1128/jvi.17.3.692-704.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sheinin R., Fabbro J., Dubsky M. Mouse polyoma virus and adenovirus replication in mouse cells temperature-sensitive in DNA synthesis. Intervirology. 1985;24(3):174–180. doi: 10.1159/000149638. [DOI] [PubMed] [Google Scholar]
  35. Siminovitch L. On the nature of hereditable variation in cultured somatic cells. Cell. 1976 Jan;7(1):1–11. doi: 10.1016/0092-8674(76)90249-x. [DOI] [PubMed] [Google Scholar]
  36. Slater M. L., Ozer H. L. Temperature-sensitive mutants of Balb/3T3 cells: description of a mutant affected in cellular and polyoma virus DNA synthesis. Cell. 1976 Feb;7(2):289–295. doi: 10.1016/0092-8674(76)90028-3. [DOI] [PubMed] [Google Scholar]
  37. Srinivasan P. R., Gupta R. S., Siminovitch L. Studies on temperature-sensitive mutants of Chinese hamster ovary cells affected in DNA synthesis. Somatic Cell Genet. 1980 Sep;6(5):567–582. doi: 10.1007/BF01538638. [DOI] [PubMed] [Google Scholar]
  38. Stanley P., Siminovitch L. Selection and characterization of Chinese hamster ovary cells resistant to the cytotoxicity of lectins. In Vitro. 1976 Mar;12(3):208–215. doi: 10.1007/BF02796443. [DOI] [PubMed] [Google Scholar]
  39. Stetten G., Latt S. A., Davidson R. L. 33258 Hoechst enhancement of the photosensitivity of bromodeoxyuridine-substituted cells. Somatic Cell Genet. 1976 May;2(3):285–290. doi: 10.1007/BF01538967. [DOI] [PubMed] [Google Scholar]
  40. Tamanoi F., Stillman B. W. Function of adenovirus terminal protein in the initiation of DNA replication. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2221–2225. doi: 10.1073/pnas.79.7.2221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Thompson L. H., Harkins J. L., Stanners C. P. A mammalian cell mutant with a temperature-sensitive leucyl-transfer RNA synthetase. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3094–3098. doi: 10.1073/pnas.70.11.3094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Thompson L. H., Mankovitz R., Baker R. M., Till J. E., Siminovitch L., Whitmore G. F. Isolation of temperature-sensitive mutants of L-cells. Proc Natl Acad Sci U S A. 1970 Jun;66(2):377–384. doi: 10.1073/pnas.66.2.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Treisman R., Novak U., Favaloro J., Kamen R. Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein. Nature. 1981 Aug 13;292(5824):595–600. doi: 10.1038/292595a0. [DOI] [PubMed] [Google Scholar]
  44. Wissinger W. L., Wang R. J. Cell cycle mutants. Int Rev Cytol Suppl. 1983;15:91–113. doi: 10.1016/b978-0-12-364376-6.50010-7. [DOI] [PubMed] [Google Scholar]
  45. Wittes R. E., Ozer H. L. Characterization of a temperature-sensitive mutant of BALB-c 3T3 cells. Exp Cell Res. 1973 Jul;80(1):127–136. doi: 10.1016/0014-4827(73)90283-8. [DOI] [PubMed] [Google Scholar]
  46. Zeng G. C., Donegan J., Ozer H. L., Hand R. Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells. Mol Cell Biol. 1984 Sep;4(9):1815–1822. doi: 10.1128/mcb.4.9.1815. [DOI] [PMC free article] [PubMed] [Google Scholar]

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