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. 1982 Oct;2(10):1229–1237. doi: 10.1128/mcb.2.10.1229

Revertants of an S49 cell mutant that expresses altered cyclic AMP-dependent protein kinase.

T van Daalen Wetters, P Coffino
PMCID: PMC369922  PMID: 6294499

Abstract

Dibutyryl adenosine 3',5'-phosphate (Bt2cAMP)-sensitive (Bt2cAMPS) revertants were isolated from a resistant S49 cell mutant carrying a structural gene lesion in the regulatory subunit of cAMP-dependent protein kinase (cA-PK). This was accomplished with a counter-selection in which, first, Bt2cAMP was used to reversibly arrest revertants, and then a sequence of treatments with bromodeoxyuridine, 33258 Hoechst dye, and white light was used to kill cycling mutant cells. Reversion rates in nonmutagenized cultures could not be accurately measured, but spontaneous revertants do occur and with frequencies of less than 10(-7) to 10(-5). The mutagens ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitro-soguanidine (MNNG), and ICR191 increased the reversion frequency. In all cases, reversion to Bt2cAMP sensitivity was associated with restoration of wild-type levels and apparent activation constant for cAMP of cA-PK. MNNG induced revertants whose cell extracts contained cA-PK activity distinguishable from that of wild type by thermal liability. EMS did not. The counter-selection effectively isolates rare phenotypes and is therefore a useful tool in further somatic genetic experiments. The association of reversion with alterations in cA-PK function supports all previous data from this and other laboratories implicating cA-PK as the intracellular mediator of cAMP effects. Reversion is probably the result of a mutational event. Induction of reversion by ICR191 suggests the existence of a novel mechanism for generating revertants in somatic cells.

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Selected References

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

  1. Ames B. N., Whitfield H. J., Jr Frameshift mutagenesis in Salmonella. Cold Spring Harb Symp Quant Biol. 1966;31:221–225. doi: 10.1101/sqb.1966.031.01.030. [DOI] [PubMed] [Google Scholar]
  2. Ayusawa D., Iwata K., Seno T., Koyama H. Conditional thymidine auxotrophic mutants of mouse FM3A cells due to thermosensitive thymidylate synthase and their prototrophic revertants. J Biol Chem. 1981 Dec 10;256(23):12005–12012. [PubMed] [Google Scholar]
  3. Chasin L. A., Feldman A., Konstam M., Urlaub G. Reversion of a Chinese hamster cell auxotrophic mutant. Proc Natl Acad Sci U S A. 1974 Mar;71(3):718–722. doi: 10.1073/pnas.71.3.718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Coffino P., Baumal R., Laskov R., Scharff M. D. Cloning of mouse myeloma cells and detection of rare variants. J Cell Physiol. 1972 Jun;79(3):429–440. doi: 10.1002/jcp.1040790313. [DOI] [PubMed] [Google Scholar]
  5. Coffino P., Bourne H. R., Tomkins G. M. Somatic genetic analysis of cyclic AMP action: selection of unresponsive mutants. J Cell Physiol. 1975 Jun;85(3):603–610. doi: 10.1002/jcp.1040850312. [DOI] [PubMed] [Google Scholar]
  6. Coffino P., Gray J. W., Tomkins G. M. Cyclic AMP, a nonessential regulator of the cell cycle. Proc Natl Acad Sci U S A. 1975 Mar;72(3):878–882. doi: 10.1073/pnas.72.3.878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Creech H. J., Preston R. K., Peck R. M., O'Connell A. P. Antitumor and mutagenic properties of a variety of heterocyclic nitrogen and sulfur mustards. J Med Chem. 1972 Jul;15(7):739–746. doi: 10.1021/jm00277a011. [DOI] [PubMed] [Google Scholar]
  8. Daniel V., Bourne H. R., Tomkins G. M. Altered metabolism and endogenous cyclic AMP in cultured cells deficient in cyclic AMP-binding proteins. Nat New Biol. 1973 Aug 8;244(136):167–169. doi: 10.1038/newbio244167a0. [DOI] [PubMed] [Google Scholar]
  9. Daniel V., Litwack G., Tomkins G. M. Induction of cytolysis of cultured lymphoma cells by adenosine 3':5'-cyclic monophosphate and the isolation of resistant variants. Proc Natl Acad Sci U S A. 1973 Jan;70(1):76–79. doi: 10.1073/pnas.70.1.76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Drake J. W., Baltz R. H. The biochemistry of mutagenesis. Annu Rev Biochem. 1976;45:11–37. doi: 10.1146/annurev.bi.45.070176.000303. [DOI] [PubMed] [Google Scholar]
  11. Fenwick R. G., Jr, Caskey C. T. Mutant chinese hamster cells with a thermosensitive hypoxanthine-guanine phosphoribosyltransferase. Cell. 1975 Jun;5(2):115–122. doi: 10.1016/0092-8674(75)90019-7. [DOI] [PubMed] [Google Scholar]
  12. Friedrich U., Coffino P. Mutagenesis in S49 mouse lymphoma cells: induction of resistance to ouabain, 6-thioguanine, and dibutyryl cyclic AMP. Proc Natl Acad Sci U S A. 1977 Feb;74(2):679–683. doi: 10.1073/pnas.74.2.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gupta R. S., Siminovitch L. Genetic markers for quantitative mutagenesis studies in Chinese hamster ovary cells: characteristics of some recently developed selective systems. Mutat Res. 1980 Jan;69(1):113–126. doi: 10.1016/0027-5107(80)90181-5. [DOI] [PubMed] [Google Scholar]
  14. Gutmann N. S., Rae P. A., Schimmer B. P. Altered cyclic AMP-dependent protein kinase activity in a mutant adrenocortical tumor cell line. J Cell Physiol. 1978 Dec;97(3 Pt 2 Suppl 1):451–460. doi: 10.1002/jcp.1040970320. [DOI] [PubMed] [Google Scholar]
  15. Hochman J., Insel P. A., Bourne H. R., Coffino P., Tomkins G. M. A structural gene mutation affecting the regulatory subunit of cyclic AMP-dependent protein kinase in mouse lymphoma cells. Proc Natl Acad Sci U S A. 1975 Dec;72(12):5051–5055. doi: 10.1073/pnas.72.12.5051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Horibata K., Harris A. W. Mouse myelomas and lymphomas in culture. Exp Cell Res. 1970 Apr;60(1):61–77. doi: 10.1016/0014-4827(70)90489-1. [DOI] [PubMed] [Google Scholar]
  17. Insel P. A., Bourne H. R., Coffino P., Tomkins G. M. Cyclic AMP-dependent protein kinase: pivotal role in regulation of enzyme induction and growth. Science. 1975 Nov 28;190(4217):896–898. doi: 10.1126/science.171770. [DOI] [PubMed] [Google Scholar]
  18. Jarvik J., Botstein D. Conditional-lethal mutations that suppress genetic defects in morphogenesis by altering structural proteins. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2738–2742. doi: 10.1073/pnas.72.7.2738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Krebs E. G., Beavo J. A. Phosphorylation-dephosphorylation of enzymes. Annu Rev Biochem. 1979;48:923–959. doi: 10.1146/annurev.bi.48.070179.004423. [DOI] [PubMed] [Google Scholar]
  20. Krebs E. G. Protein kinases. Curr Top Cell Regul. 1972;5:99–133. [PubMed] [Google Scholar]
  21. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  22. 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]
  23. MacInnes M. A., Friedrich U., van Daalen Wetters T., Coffino P. Quantitative forward-mutation specificity of mono-functional alkylating agents, ICR-191, and aflatoxin B1 in mouse lymphoma cells. Mutat Res. 1982 Aug;95(2-3):297–311. doi: 10.1016/0027-5107(82)90266-4. [DOI] [PubMed] [Google Scholar]
  24. Puck T. T., Kao F. T. Genetics of somatic mammalian cells. V. Treatment with 5-bromodeoxyuridine and visible light for isolation of nutritionally deficient mutants. Proc Natl Acad Sci U S A. 1967 Sep;58(3):1227–1234. doi: 10.1073/pnas.58.3.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rosen N., Piscitello J., Schneck J., Muschel R. J., Bloom B. R., Rosen O. M. Properties of protein kinase and adenylate cyclase-deficient variants of a macrophage-like cell line. J Cell Physiol. 1979 Jan;98(1):125–136. doi: 10.1002/jcp.1040980114. [DOI] [PubMed] [Google Scholar]
  26. Rosenstraus M. J., Chasin L. A. Mutants of Chinese hamster ovary cells with altered glucose-6-phosphate dehydrogenase activity. Somatic Cell Genet. 1977 May;3(3):323–333. doi: 10.1007/BF01538750. [DOI] [PubMed] [Google Scholar]
  27. Simantov R., Sachs L. Temperature sensitivity of cyclic adenosine 3':5'-monophosphate-binding proteins and the regulation of growth and differentiation in neuroblastoma cells. J Biol Chem. 1975 May 10;250(9):3236–3242. [PubMed] [Google Scholar]
  28. Singh T. J., Roth C., Gottesman M. M., Pastan I. H. Characterization of cyclic AMP-resistant Chinese hamster ovary cell mutants lacking type I protein kinase. J Biol Chem. 1981 Jan 25;256(2):926–932. [PubMed] [Google Scholar]
  29. Steinberg R. A., O'Farrell P. H., Friedrich U., Coffino P. Mutations causing charge alterations in regulatory subunits of the cAMP-dependent protein kinase of cultured S49 lymphoma cells. Cell. 1977 Mar;10(3):381–391. doi: 10.1016/0092-8674(77)90025-3. [DOI] [PubMed] [Google Scholar]
  30. Steinberg R. A., van Daalen Wetters T., Coffino P. Kinase-negative mutants of S49 mouse lymphoma cells carry a trans-dominant mutation affecting expression of cAMP-dependent protein kinase. Cell. 1978 Dec;15(4):1351–1361. doi: 10.1016/0092-8674(78)90060-0. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Thompson L. H., Baker R. M. Isolation of mutants of cultured mammalian cells. Methods Cell Biol. 1973;6:209–281. doi: 10.1016/s0091-679x(08)60052-7. [DOI] [PubMed] [Google Scholar]
  33. Thompson L. H., Lofgren D. J., Adair G. M. Evidence for structural gene alterations affecting aminoacyl-tRNA synthetases in CHO cell mutants and revertants. Somatic Cell Genet. 1978 Jul;4(4):423–435. doi: 10.1007/BF01538864. [DOI] [PubMed] [Google Scholar]
  34. Urlaub G., Chasin L. A. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4216–4220. doi: 10.1073/pnas.77.7.4216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wims L. A., Morrison S. L. ICR-191 and ethyl methanesulfonate induced mutagenesis at the immunoglobulin locus in the Y5606 cultured myeloma cell line. Mutat Res. 1981 Apr;81(2):215–228. doi: 10.1016/0027-5107(81)90036-1. [DOI] [PubMed] [Google Scholar]
  36. Witt J. J., Roskoski R., Jr Rapid protein kinase assay using phosphocellulose-paper absorption. Anal Biochem. 1975 May 26;66(1):253–258. doi: 10.1016/0003-2697(75)90743-5. [DOI] [PubMed] [Google Scholar]
  37. Yanofsky C., Ito J., Horn V. Amino acid replacements and the genetic code. Cold Spring Harb Symp Quant Biol. 1966;31:151–162. doi: 10.1101/sqb.1966.031.01.023. [DOI] [PubMed] [Google Scholar]

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