Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1982 Dec;2(12):1558–1573. doi: 10.1128/mcb.2.12.1558

Temperature-Sensitive Chinese Hamster Fibroblast Mutant with a Defect in RNA Metabolism

Eric A Wong 1,, Immo E Scheffler 1
PMCID: PMC369964  PMID: 14582197

Abstract

We describe a new temperature-sensitive mutant of Chinese hamster cell fibroblasts. After a shift to the nonpermissive temperature of 40.5°C, the rates of DNA, RNA, and protein synthesis declined rapidly (to ≤50% within 12 h) and the progression of unsynchronized cells through the cell cycle was affected. We believe that DNA synthesis came to a halt after a short time, because cells no longer entered the S phase. The decrease in protein synthesis at 40.5°C was shown to be a consequence of a decrease in the number of polysomes, whereas free 80S ribosomes accumulated. We concluded that the components of the protein biosynthetic machinery were intact (ribosomes and soluble factors), but synthesis was limited by a shortage of mRNA. The decline in mRNA production had a significant effect on the synthesis of proteins (e.g., heat shock proteins) translated from short-lived messages. We observed that both polyadenylated and nonpolyadenylated RNA syntheses declined at 40.5°C, whereas the synthesis of small RNAs (4 to 5S) was less reduced. The argument is made that the temperature-sensitive phenotype is the result of a defect affecting mRNA synthesis.

Full text

PDF
1558

Images in this article

Selected References

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

  1. Ashburner M., Bonner J. J. The induction of gene activity in drosophilia by heat shock. Cell. 1979 Jun;17(2):241–254. doi: 10.1016/0092-8674(79)90150-8. [DOI] [PubMed] [Google Scholar]
  2. Bakay B., Nissinen E., Sweetman L. Analysis of radioactive and nonradioactive purine bases, nucleosides, and nucleotides by high-speed chromatography on a single column. Anal Biochem. 1978 May;86(1):65–77. doi: 10.1016/0003-2697(78)90319-6. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Breindl M., Holland J. J. Coupled in vitro transcription and translation of vesicular stomatitis virus messenger RNA. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2545–2549. doi: 10.1073/pnas.72.7.2545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Colombo B., Felicetti L., Baglioni C. Inhibition of protein synthesis in reticulocytes by antibiotics. I. Effects on polysomes. Biochim Biophys Acta. 1966 Apr 18;119(1):109–119. doi: 10.1016/0005-2787(66)90043-8. [DOI] [PubMed] [Google Scholar]
  7. DeFrancesco L., Werntz D., Scheffler I. E. Conditionally lethal mutations in chinese hamster cells. Characterization of a cell line with a possible defect in the Krebs cycle. J Cell Physiol. 1975 Apr;85(2 Pt 1):293–305. doi: 10.1002/jcp.1040850216. [DOI] [PubMed] [Google Scholar]
  8. Fan H., Penman S. Regulation of protein synthesis in mammalian cells. II. Inhibition of protein synthesis at the level of initiation during mitosis. J Mol Biol. 1970 Jun 28;50(3):655–670. doi: 10.1016/0022-2836(70)90091-4. [DOI] [PubMed] [Google Scholar]
  9. GIRARD M., LATHAM H., PENMAN S., DARNELL J. E. ENTRANCE OF NEWLY FORMED MESSENGER RNA AND RIBOSOMES INTO HELA CELL CYTOPLASM. J Mol Biol. 1965 Feb;11:187–201. doi: 10.1016/s0022-2836(65)80050-x. [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. Haralson M. A., Roufa D. J. A temperature-sensitive mutation affecting the mammalian 60 S ribosome. J Biol Chem. 1975 Nov 25;250(22):8618–8623. [PubMed] [Google Scholar]
  12. Harpold M. M., Evans R. M., Salditt-Georgieff M., Darnell J. E. Production of mRNA in Chinese hamster cells: relationship of the rate of synthesis to the cytoplasmic concentration of nine specific mRNA sequences. Cell. 1979 Aug;17(4):1025–1035. doi: 10.1016/0092-8674(79)90341-6. [DOI] [PubMed] [Google Scholar]
  13. Harpold M. M., Wilson M. C., Darnell J. E., Jr Chinese hamster polyadenylated messenger ribonucleic acid: relationship to non-polyadenylated sequences and relative conservation during messenger ribonucleic acid processing. Mol Cell Biol. 1981 Feb;1(2):188–198. doi: 10.1128/mcb.1.2.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hartwell L. H. Cell division from a genetic perspective. J Cell Biol. 1978 Jun;77(3):627–637. doi: 10.1083/jcb.77.3.627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hatzfeld J., Buttin G. Temperature-sensitive cell cycle mutants: a chinese hamster cell line with a reversible block in cytokinesis. Cell. 1975 Jun;5(2):123–129. doi: 10.1016/0092-8674(75)90020-3. [DOI] [PubMed] [Google Scholar]
  16. Herzberg M., Breitbart H. Block in the elongation of protein synthesis in rabbit reticulocyte by action of the ionophore valinomycin. Mol Biol Rep. 1980 Oct 31;6(3):163–167. doi: 10.1007/BF00775411. [DOI] [PubMed] [Google Scholar]
  17. Hutchison J. S., Moldave K. The effect of elevated temperature on protein synthesis in cell-free extracts of cultured Chinese hamster ovary cells. Biochem Biophys Res Commun. 1981 Mar 31;99(2):722–728. doi: 10.1016/0006-291x(81)91803-9. [DOI] [PubMed] [Google Scholar]
  18. Ingles C. J., Shales M. DNA-mediated transfer of an RNA polymerase II gene: reversion of the temperature-sensitive hamster cell cycle mutant TsAF8 by mammalian DNA. Mol Cell Biol. 1982 Jun;2(6):666–673. doi: 10.1128/mcb.2.6.666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ingles C. J. Temperature-sensitive RNA polymerase II mutations in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1978 Jan;75(1):405–409. doi: 10.1073/pnas.75.1.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kelley P. M., Schlesinger M. J. The effect of amino acid analogues and heat shock on gene expression in chicken embryo fibroblasts. Cell. 1978 Dec;15(4):1277–1286. doi: 10.1016/0092-8674(78)90053-3. [DOI] [PubMed] [Google Scholar]
  21. Kindle K. L., Firtel R. A. Identification and analysis of Dictyostelium actin genes, a family of moderately repeated genes. Cell. 1978 Nov;15(3):763–778. doi: 10.1016/0092-8674(78)90262-3. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  24. Landy-Otsuka F., Scheffler I. E. Enzyme induction in a temperature-sensitive cell cycle mutant of Chinese hamster fibroblasts. J Cell Physiol. 1980 Nov;105(2):209–220. doi: 10.1002/jcp.1041050204. [DOI] [PubMed] [Google Scholar]
  25. Macdonald J. S., Goldberg I. H. An effect of pactamycin on the initiation of protein synthesis in reticulocytes. Biochem Biophys Res Commun. 1970 Oct 9;41(1):1–8. doi: 10.1016/0006-291x(70)90460-2. [DOI] [PubMed] [Google Scholar]
  26. Nielsen P. J., McConkey E. H. Evidence for control of protein synthesis in HeLa cells via the elongation rate. J Cell Physiol. 1980 Sep;104(3):269–281. doi: 10.1002/jcp.1041040302. [DOI] [PubMed] [Google Scholar]
  27. Pardee A. B., Dubrow R., Hamlin J. L., Kletzien R. F. Animal cell cycle. Annu Rev Biochem. 1978;47:715–750. doi: 10.1146/annurev.bi.47.070178.003435. [DOI] [PubMed] [Google Scholar]
  28. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  29. Penman S., Smith I., Holtzman E., Greenberg H. RNA metabolism in the HeLa cell nucleus and nucleolus. Natl Cancer Inst Monogr. 1966 Dec;23:489–512. [PubMed] [Google Scholar]
  30. Perry R. P. The nucleolus and the synthesis of ribosomes. Natl Cancer Inst Monogr. 1965 Dec;18:325–340. [PubMed] [Google Scholar]
  31. Rosbash M., Harris P. K., Woolford J. L., Jr, Teem J. L. The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast. Cell. 1981 Jun;24(3):679–686. doi: 10.1016/0092-8674(81)90094-5. [DOI] [PubMed] [Google Scholar]
  32. Rossini M., Baserga S., Huang C. H., Ingles C. J., Baserga R. Changes in RNA polymerase II in a cell cycle-specific temperature-sensitive mutant of hamster cells. J Cell Physiol. 1980 Apr;103(1):97–103. doi: 10.1002/jcp.1041030114. [DOI] [PubMed] [Google Scholar]
  33. Scott M. P., Pardue M. L. Translational control in lysates of Drosophila melanogaster cells. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3353–3357. doi: 10.1073/pnas.78.6.3353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sehgal P. B., Tamm I. Halogenated benzimidazole ribosides, Novel inhibitors of RNA synthesis. Biochem Pharmacol. 1978;27(21):2475–2485. doi: 10.1016/0006-2952(78)90313-1. [DOI] [PubMed] [Google Scholar]
  35. Sheinin R., Lewis P. N. DNA and histone synthesis in mouse cells which exhibit temperature-sensitive DNA synthesis. Somatic Cell Genet. 1980 Mar;6(2):225–239. doi: 10.1007/BF01538798. [DOI] [PubMed] [Google Scholar]
  36. Sheinin R. Preliminary characterization of the temperature-sensitive defect in DNA replication in a mutant mouse L cell. Cell. 1976 Jan;7(1):49–57. doi: 10.1016/0092-8674(76)90254-3. [DOI] [PubMed] [Google Scholar]
  37. Simchen G. Cell cycle mutants. Annu Rev Genet. 1978;12:161–191. doi: 10.1146/annurev.ge.12.120178.001113. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Soderberg K., Nissinen E., Bakay B., Scheffler I. E. The energy charge in wild-type and respiration-deficient Chinese hamster cell mutants. J Cell Physiol. 1980 Apr;103(1):169–172. doi: 10.1002/jcp.1041030121. [DOI] [PubMed] [Google Scholar]
  40. Storti R. V., Scott M. P., Rich A., Pardue M. L. Translational control of protein synthesis in response to heat shock in D. melanogaster cells. Cell. 1980 Dec;22(3):825–834. doi: 10.1016/0092-8674(80)90559-0. [DOI] [PubMed] [Google Scholar]
  41. Tamm I., Sehgal P. B. Halobenzimidazole ribosides and RNA synthesis of cells and viruses. Adv Virus Res. 1978;22:187–258. doi: 10.1016/s0065-3527(08)60775-7. [DOI] [PubMed] [Google Scholar]
  42. Tenner A., Zieg J., Scheffler I. E. Glycoprotein synthesis in a temperature-sensitive Chinese hamster cell cycle mutant. J Cell Physiol. 1977 Feb;90(2):145–160. doi: 10.1002/jcp.1040900202. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Thompson L. H., Lofgren D. J., Adair G. M. CHO cell mutants for arginyl-, asparagyl-, glutaminyl-, histidyl- and methionyl-transfer RNA synthetases: identification and initial characterization. Cell. 1977 May;11(1):157–168. doi: 10.1016/0092-8674(77)90326-9. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Toniolo D., Basilico C. Processing of ribosomal RNA in a temperature sensitive mutant of BHK cells. Biochim Biophys Acta. 1976 Apr 2;425(4):409–418. doi: 10.1016/0005-2787(76)90005-8. [DOI] [PubMed] [Google Scholar]
  47. Toniolo D., Meiss H. K., Basilico C. A temperature-sensitive mutation affecting 28S ribosomal RNA production in mammalian cells. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1273–1277. doi: 10.1073/pnas.70.4.1273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wang R. J. Temperature-sensitive mammalian cell line blocked in mitosis. Nature. 1974 Mar 1;248(5443):76–78. doi: 10.1038/248076a0. [DOI] [PubMed] [Google Scholar]
  49. Weber L. A., Feman E. R., Baglioni C. A cell free system from HeLa cells active in initiation of protein synthesis. Biochemistry. 1975 Dec 2;14(24):5315–5321. doi: 10.1021/bi00695a015. [DOI] [PubMed] [Google Scholar]

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

RESOURCES