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. 1974 Dec 1;63(3):831–842. doi: 10.1083/jcb.63.3.831

THE ACTION OF α-AMANITIN ON RNA SYNTHESIS IN CHINESE HAMSTER OVARY CELLS

Ultrastructural and Biochemical Studies

Claude Kedinger 1, Rene Simard 1
PMCID: PMC2109362  PMID: 4474178

Abstract

α-Amanitin acts in vitro as a selective inhibitor of the nucleoplasmic form B RNA polymerases. Treatment of Chinese hamster ovary (CHO) cells with this drug leads principally to a severe fragmentation of the nucleoli. While the ultrastructural lesions induced by α-amanitin in CHO cells and in rat or mouse liver are quite similar, the results diverge concerning the effect on RNA synthesis. It has been shown that in rat or mouse liver α-amanitin blocks both extranucleolar and nucleolar RNA synthesis. Our autoradiographic and biochemical evidence indicates that in CHO cells high molecular weight extranucleolar RNA synthesis (HnRNA) is blocked by the α-amanitin treatment, whereas nucleolar RNA (preribosomal RNA) synthesis remains unaffected even several hours after the inhibition of extranucleolar RNA synthesis. Furthermore, the processing of this RNA as well as its transport to the cytoplasm seem only slightly affected by the treatment. Finally, under these conditions, the synthesis of the low molecular RNA species (4–5S) still occurs, though less actively. The results are interpreted as evidence for a selective impairment of HnRNA synthesis by α-amanitin in CHO cells.

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Selected References

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  1. Aloni Y., Hatlen L. E., Attardi G. Studies of fractionated HeLa cell metaphase chromosomes. II. chromosomal distribution of sites for transfer RNA and 5 s RNA. J Mol Biol. 1971 Mar 28;56(3):555–563. doi: 10.1016/0022-2836(71)90401-3. [DOI] [PubMed] [Google Scholar]
  2. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beermann W. Effect of -amantine on puffing and intranuclear RNA synthesis in Chironomus salivary glands. Chromosoma. 1971;34(2):152–167. doi: 10.1007/BF00285183. [DOI] [PubMed] [Google Scholar]
  4. Bucci S., Nardi I., Mancino G., Fiume L. Incorporation of tritiated uridine in nuclei of Triturus oocytes treated with alpha-amanitin. Exp Cell Res. 1971 Dec;69(2):462–465. doi: 10.1016/0014-4827(71)90255-2. [DOI] [PubMed] [Google Scholar]
  5. CARO L. G., VAN TUBERGEN R. P., KOLB J. A. High-resolution autoradiography. I. Methods. J Cell Biol. 1962 Nov;15:173–188. doi: 10.1083/jcb.15.2.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chesterton C. J., Butterworth P. H. Selective extraction of form I DNA dependent RNA polymerase from rat liver nuclei and its separation into two species. Eur J Biochem. 1971 Mar 11;19(2):232–241. doi: 10.1111/j.1432-1033.1971.tb01309.x. [DOI] [PubMed] [Google Scholar]
  7. Egyházi E., D'Monte B., Edström J. E. Effects of -amanitin on in vitro labeling of RNA from defined nuclear components in salivary gland cells from Chironomus tentans. J Cell Biol. 1972 May;53(2):523–531. doi: 10.1083/jcb.53.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fiume L., Laschi R. Lesioni ultrastrutturali prodotte nelle cellule parenchimali epatiche dalla falloidina e dalla alpha-amanitina. Sperimentale. 1965 Sep-Oct;115(5):288–297. [PubMed] [Google Scholar]
  9. Fiume L., Marinozzi V., Nardi F. The effects of amanitin poisoning on mouse kidney. Br J Exp Pathol. 1969 Jun;50(3):270–276. [PMC free article] [PubMed] [Google Scholar]
  10. Hadjiolov A. A., Dabeva M. D., Mackedonski V. V. The action of alpha-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids. Biochem J. 1974 Mar;138(3):321–334. doi: 10.1042/bj1380321a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hastie N. D., Mahy B. W. Effects of -amanitin in vivo on RNA polymerase activity of cultured chick embryo fibroblast cell nuclei: resistance of ribosomal RNA synthesis to the drug. FEBS Lett. 1973 May 15;32(1):95–99. doi: 10.1016/0014-5793(73)80746-x. [DOI] [PubMed] [Google Scholar]
  12. Jacob S. T. Mammalian RNA polymerases. Prog Nucleic Acid Res Mol Biol. 1973;13:93–126. doi: 10.1016/s0079-6603(08)60101-4. [DOI] [PubMed] [Google Scholar]
  13. Jacob S. T., Sajdel E. M., Munro H. N. Different responses of soluble whole nuclear RNA polymerase and soluble nucleolar RNA polymerase to divalent cations and to inhibition by alpha-amanitin. Biochem Biophys Res Commun. 1970 Feb 20;38(4):765–770. doi: 10.1016/0006-291x(70)90647-9. [DOI] [PubMed] [Google Scholar]
  14. Kedinger C., Gniazdowski M., Mandel J. L., Jr, Gissinger F., Chambon P. Alpha-amanitin: a specific inhibitor of one of two DNA-pendent RNA polymerase activities from calf thymus. Biochem Biophys Res Commun. 1970 Jan 6;38(1):165–171. doi: 10.1016/0006-291x(70)91099-5. [DOI] [PubMed] [Google Scholar]
  15. Lindell T. J., Weinberg F., Morris P. W., Roeder R. G., Rutter W. J. Specific inhibition of nuclear RNA polymerase II by alpha-amanitin. Science. 1970 Oct 23;170(3956):447–449. doi: 10.1126/science.170.3956.447. [DOI] [PubMed] [Google Scholar]
  16. Marinozzi V., Fiume L. Effects of -amanitin on mouse and rat liver cell nuclei. Exp Cell Res. 1971 Aug;67(2):311–322. doi: 10.1016/0014-4827(71)90414-9. [DOI] [PubMed] [Google Scholar]
  17. Montecuccoli G., Novello F., Stirpe F. Inhibition by alpha-amanitin of chromosomal ribonucleic acid synthesis in rat liver. FEBS Lett. 1972 Sep 15;25(2):305–308. doi: 10.1016/0014-5793(72)80510-6. [DOI] [PubMed] [Google Scholar]
  18. Muramatsu M., Shimada N., Higashinakagawa T. Effect of cycloheximide on the nucleolar RNA synthesis in rat liver. J Mol Biol. 1970 Oct 14;53(1):91–106. doi: 10.1016/0022-2836(70)90047-1. [DOI] [PubMed] [Google Scholar]
  19. Niessing J., Schnieders B., Kunz W., Seifart K. H., Sekeris C. E. Inhibition of RNA synthesis by alpha-amanitin in vivo. Z Naturforsch B. 1970 Oct;25(10):1119–1125. doi: 10.1515/znb-1970-1011. [DOI] [PubMed] [Google Scholar]
  20. Pardue M. L., Brown D. D., Birnstiel M. L. Location of the genes for 5S ribosomal RNA in Xenopus laevis. Chromosoma. 1973;42(2):191–203. doi: 10.1007/BF00320940. [DOI] [PubMed] [Google Scholar]
  21. Peacock A. C., Dingman C. W. Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry. 1968 Feb;7(2):668–674. doi: 10.1021/bi00842a023. [DOI] [PubMed] [Google Scholar]
  22. Perry R. P., Kelley D. E. Inhibition of RNA synthesis by actinomycin D: characteristic dose-response of different RNA species. J Cell Physiol. 1970 Oct;76(2):127–139. doi: 10.1002/jcp.1040760202. [DOI] [PubMed] [Google Scholar]
  23. Petrov P., Sekeris C. E. Early action of alpha-amanitin on extranucleolar ribonucleoproteins, as revealed by electron microscopic observation. Exp Cell Res. 1971 Dec;69(2):393–401. doi: 10.1016/0014-4827(71)90240-0. [DOI] [PubMed] [Google Scholar]
  24. Price R., Penman S. A distinct RNA polymerase activity, synthesizing 5-5 s, 5 s and 4 s RNA in nuclei from adenovirus 2-infected HeLa cells. J Mol Biol. 1972 Oct 14;70(3):435–450. doi: 10.1016/0022-2836(72)90551-7. [DOI] [PubMed] [Google Scholar]
  25. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ritossa F. M., Atwood K. C., Spiegelman S. On the redundancy of DNA complementary to amino acid tranfer RNA and its absence from the nucleolar organizer region of Drosophila melanogaster. Genetics. 1966 Aug;54(2):663–676. doi: 10.1093/genetics/54.2.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. SCHERRER K., DARNELL J. E. Sedimentation characteristics of rapidly labelled RNA from HeLa cells. Biochem Biophys Res Commun. 1962 Jun 4;7:486–490. doi: 10.1016/0006-291x(62)90341-8. [DOI] [PubMed] [Google Scholar]
  28. Schmid W., Sekeris C. E. Possible involvement of nuclear DNA-like RNA in the control of ribosomal RNA synthesis. Biochim Biophys Acta. 1973 Jul 13;312(3):549–554. doi: 10.1016/0005-2787(73)90453-x. [DOI] [PubMed] [Google Scholar]
  29. Sekeris C. E., Schmid W. Action of alpha-Amanitin in vivo and in vitro. FEBS Lett. 1972 Oct 15;27(1):41–45. doi: 10.1016/0014-5793(72)80405-8. [DOI] [PubMed] [Google Scholar]
  30. Serfling E., Wobus U., Panitz R. Effect of alpha-amanitin on chromosomal and nucleolar RNA-synthesis in Chironomus thummi polytene chromosomes. FEBS Lett. 1972 Feb 1;20(2):148–152. doi: 10.1016/0014-5793(72)80780-4. [DOI] [PubMed] [Google Scholar]
  31. Stirpe F., Fiume L. Studies on the pathogenesis of liver necrosis by alpha-amanitin. Effect of alpha-amanitin on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver nuclei. Biochem J. 1967 Nov;105(2):779–782. doi: 10.1042/bj1050779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tata J. R., Hamilton M. J., Shields D. Effects of alpha-amanitin in vivo on RNA polymerase and nuclear RNA synthesis. Nat New Biol. 1972 Aug 9;238(84):161–164. doi: 10.1038/newbio238161a0. [DOI] [PubMed] [Google Scholar]
  33. Tiollais P., Galibert F., Boiron M. Effects of valine deprivation on the biosynthesis of ribosomal and messenger RNA in a mammalian cell line (L 5178 Y). Eur J Biochem. 1971 Jan 1;18(1):35–45. doi: 10.1111/j.1432-1033.1971.tb01211.x. [DOI] [PubMed] [Google Scholar]
  34. Weinmann R., Roeder R. G. Role of DNA-dependent RNA polymerase 3 in the transcription of the tRNA and 5S RNA genes. Proc Natl Acad Sci U S A. 1974 May;71(5):1790–1794. doi: 10.1073/pnas.71.5.1790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wieland T. Poisonous principles of mushrooms of the genus Amanita. Four-carbon amines acting on the central nervous system and cell-destroying cyclic peptides are produced. Science. 1968 Mar 1;159(3818):946–952. doi: 10.1126/science.159.3818.946. [DOI] [PubMed] [Google Scholar]
  36. Zalta J., Zalta J. P., Simard R. Isolation of nucleoli. A method that combines high yield, structural integrity, and biochemical preservation. J Cell Biol. 1971 Nov;51(21):563–568. doi: 10.1083/jcb.51.2.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zylber E. A., Penman S. Products of RNA polymerases in HeLa cell nuclei. Proc Natl Acad Sci U S A. 1971 Nov;68(11):2861–2865. doi: 10.1073/pnas.68.11.2861. [DOI] [PMC free article] [PubMed] [Google Scholar]

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