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. 1983 Mar;80(5):1318–1321. doi: 10.1073/pnas.80.5.1318

Microinjection of purified ornithine decarboxylase into Xenopus oocytes selectively stimulates ribosomal RNA synthesis.

D H Russell
PMCID: PMC393588  PMID: 6402779

Abstract

This study has utilized stage VI oocytes of Xenopus laevis which have amplified the rDNA gene 1,000-fold to assess whether the microinjection of ornithine decarboxylase (OrnDCase) would stimulate [alpha-32P]guanosine incorporation into 45S and 18S/28S RNA selectively. The injection of purified OrnDCase into individual oocytes resulted in a greater than 2-fold increase in the incorporation of [32P]guanosine into 45S RNA and 18S/28S RNA with no increased incorporation into low molecular weight RNA. Further, an irreversible inhibitor of OrnDCase, alpha-difluoromethylornithine (CHF2-Orn), rapidly inhibited the endogenous activity of OrnDCase when added to the buffered Hepes solution bathing the oocytes and also inhibited the incorporation of [32P]guanosine into rRNA. The inhibitory effect of CHF2-Orn could not be reversed totally by addition of 10 microM putrescine to the oocytes. OrnDCase injected into oocytes in the presence of CHF2-Orn in the media did not stimulate incorporation of [32P]guanosine label into rRNA. However, when CHF2-Orn was removed from the buffered medium at the time of the injection of label and enzyme, a 3-fold increase of 32P incorporation into 18S/28S RNA occurred. Therefore, in an in vivo model in which amplified extrachromosomal rDNA gene copies are present, the microinjection of OrnDCase was capable of specifically stimulating rRNA synthesis. CHF2-Orn, a suicide enzyme inactivator of OrnDCase, was able to inhibit rRNA synthesis and, after washout, there was a more marked stimulation of rRNA synthesis than occurred after only the injection of OrnDCase alone. These data suggest further that OrnDCase is the labile protein that regulates the initiation of RNA synthesis.

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Selected References

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

  1. Atmar V. J., Kuehn G. D. Phosphorylation of ornithine decarboxylase by a polyamine-dependent protein kinase. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5518–5522. doi: 10.1073/pnas.78.9.5518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BROWN D. D., LITTNA E. VARIATIONS IN THE SYNTHESIS OF STABLE RNA'S DURING OOGENESIS AND DEVELOPMENT OF XENOPUS LAEVIS. J Mol Biol. 1964 May;8:688–695. doi: 10.1016/s0022-2836(64)80117-0. [DOI] [PubMed] [Google Scholar]
  3. Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
  4. Brown D. D., Dawid I. B. Specific gene amplification in oocytes. Oocyte nuclei contain extrachromosomal replicas of the genes for ribosomal RNA. Science. 1968 Apr 19;160(3825):272–280. doi: 10.1126/science.160.3825.272. [DOI] [PubMed] [Google Scholar]
  5. Daniels G. R., Atmar V. J., Kuehn G. D. Polyamine-activated protein kinase reaction from nuclei and nucleoli of Physarum polycephalum which phosphorylates a unique Mr 70 000 nonhistone protein. Biochemistry. 1981 Apr 28;20(9):2525–2532. doi: 10.1021/bi00512a025. [DOI] [PubMed] [Google Scholar]
  6. Dumont J. N. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J Morphol. 1972 Feb;136(2):153–179. doi: 10.1002/jmor.1051360203. [DOI] [PubMed] [Google Scholar]
  7. ELSDALE T. R., FISCHBERG M., SMITH S. A mutation that reduces nucleolar number in Xenopus laevis. Exp Cell Res. 1958 Jun;14(3):642–643. doi: 10.1016/0014-4827(58)90175-7. [DOI] [PubMed] [Google Scholar]
  8. Franze-Fernańdez M. T., Fontanive-Sengüesa A. V. Effect of amino acids on the alpha-amanitin-insensitive RNA polymerase activity in the isolated nuclei of Ehrlich ascites cells. Biochim Biophys Acta. 1973 Nov 26;331(1):71–80. doi: 10.1016/0005-2787(73)90420-6. [DOI] [PubMed] [Google Scholar]
  9. Gfeller E., Russell D. H. Autoradiographic distributions of 3 H-putrescine and 3 H-uridine in a Xenopus liver cell line. Z Zellforsch Mikrosk Anat. 1971;120(3):321–331. doi: 10.1007/BF00324895. [DOI] [PubMed] [Google Scholar]
  10. Gross K. J., Pogo A. O. Control mechanism of ribonucleic acid synthesis in eukaryotes. The effect of amino acid and glucose starvation and cycloheximide on yeast deoxyribonucleic acid-dependent ribonucleic acid polymerases. J Biol Chem. 1974 Jan 25;249(2):568–576. [PubMed] [Google Scholar]
  11. Grummt I. Specific transcription of mouse ribosomal DNA in a cell-free system that mimics control in vivo. Proc Natl Acad Sci U S A. 1981 Feb;78(2):727–731. doi: 10.1073/pnas.78.2.727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gurdon J. B. Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal. J Embryol Exp Morphol. 1976 Dec;36(3):523–540. [PubMed] [Google Scholar]
  13. Haddox M. K., Russell D. H. Increased nuclear conjugated polyamines and transglutaminase during liver regeneration. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1712–1716. doi: 10.1073/pnas.78.3.1712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Haddox M. K., Russell D. H. Ornithine decarboxylase from calf liver. Purification and properties. Biochemistry. 1981 Nov 10;20(23):6721–6729. doi: 10.1021/bi00526a030. [DOI] [PubMed] [Google Scholar]
  15. Haddox M. K., Russell D. H. Stimulation in vitro of a heparin-resistant RNA polymerase I transcription complex. Biochem J. 1981 Jul 15;198(1):207–210. doi: 10.1042/bj1980207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hogan B. L., McIlhinney A., Murden S. Effect of growth conditions on the activity of ornithine decarboxylase in cultured hepatoma cells. II. Effect of serum and insulin. J Cell Physiol. 1974 Jun;83(3):353–357. doi: 10.1002/jcp.1040830305. [DOI] [PubMed] [Google Scholar]
  17. James G. T., Yeoman L. C., Matsui S. i., Goldberg A. H., Busch H. Isolation and characterization of nonhistone chromosomal protein C-14 which stimulates RNA synthesis. Biochemistry. 1977 May 31;16(11):2384–2389. doi: 10.1021/bi00630a012. [DOI] [PubMed] [Google Scholar]
  18. Kuehn G. D., Affolter H. U., Atmar V. J., Seebeck T., Gubler U., Braun R. Polyamine-mediated phosphorylation of a nucleolar protein from Physarum polycephalum that stimulates rRNA synthesis. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2541–2545. doi: 10.1073/pnas.76.6.2541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lampert A., Feigelson P. A short lived polypeptide component of one of two discrete functional pools of hepatic nuclear alpha-amanitin resistant RNA polymerases. Biochem Biophys Res Commun. 1974 Jun 18;58(4):1030–1038. doi: 10.1016/s0006-291x(74)80247-0. [DOI] [PubMed] [Google Scholar]
  20. Manen C. A., Russell D. H. Regulation of RNA polymerase I activity by ornithine decarboxylase. Biochem Pharmacol. 1977 Dec 15;26(24):2379–2384. doi: 10.1016/0006-2952(77)90445-2. [DOI] [PubMed] [Google Scholar]
  21. Manen C. A., Russell D. H. Relationship of ornithine decarboxylase to RNA polymerase i activity. Life Sci. 1975 Dec 15;17(12):1769–1775. doi: 10.1016/0024-3205(75)90459-2. [DOI] [PubMed] [Google Scholar]
  22. Manen C., Russel D. H. Ornithine decarboxylase may function as an initiation factor for RNA polymerase I. Science. 1977 Feb 4;195(4277):505–506. doi: 10.1126/science.835013. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Russell D. H., Byus C. V., Manen C. A. Proposed model of major sequential biochemical events of a trophic response. Life Sci. 1976 Nov 1;19(9):1297–1305. [PubMed] [Google Scholar]
  25. Russell D. H., Manen C. A. Posttranslationally modified ornithine decarboxylase may regulate RNA polymerase I activity. Biochem Pharmacol. 1982 Nov 1;31(21):3373–3378. doi: 10.1016/0006-2952(82)90614-1. [DOI] [PubMed] [Google Scholar]
  26. Russell D. H. Ornithine decarboxylase: a key regulatory protein. Med Biol. 1981 Dec;59(5-6):286–295. [PubMed] [Google Scholar]
  27. Russell D. H. Posttranslational modification of ornithine decarboxylase by its product putrescine. Biochem Biophys Res Commun. 1981 Apr 30;99(4):1167–1172. doi: 10.1016/0006-291x(81)90741-5. [DOI] [PubMed] [Google Scholar]
  28. Russell D. H. Putrescine and spermidine biosynthesis in the development of normal and anucleolate mutants of Xenopus laevis. Proc Natl Acad Sci U S A. 1971 Mar;68(3):523–527. doi: 10.1073/pnas.68.3.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Russell D. H., Russell S. D. Relative usefulness of measuring polyamines in serum, plasma, and urine as biochemical markers of cancer. Clin Chem. 1975 Jun;21(7):860–863. [PubMed] [Google Scholar]
  30. Russell D. H., Snyder S. H. Amine synthesis in regenerating rat liver: extremely rapid turnover of ornithine decarboxylase. Mol Pharmacol. 1969 May;5(3):253–262. [PubMed] [Google Scholar]
  31. Russell D. H., Snyder S. H., Medina V. J. Growth hormone induction of ornithine decarboxylase in rat liver. Endocrinology. 1970 Jun;86(6):1414–1419. doi: 10.1210/endo-86-6-1414. [DOI] [PubMed] [Google Scholar]
  32. Russell D., Snyder S. H. Amine synthesis in rapidly growing tissues: ornithine decarboxylase activity in regenerating rat liver, chick embryo, and various tumors. Proc Natl Acad Sci U S A. 1968 Aug;60(4):1420–1427. doi: 10.1073/pnas.60.4.1420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sajdel E. M., Jacob S. T. Mechanism of early effect of hydrocortisone on the transcriptional process: stimulation of the activities of purified rat liver nucleolar RNA polymerases. Biochem Biophys Res Commun. 1971 Nov 5;45(3):707–715. doi: 10.1016/0006-291x(71)90474-8. [DOI] [PubMed] [Google Scholar]
  34. Sjoerdsma A. Suicide enzyme inhibitors as potential drugs. Clin Pharmacol Ther. 1981 Jul;30(1):3–22. doi: 10.1038/clpt.1981.121. [DOI] [PubMed] [Google Scholar]
  35. Tucciarone L. M., Lanclos K. D. Evidence for the involvement of transglutaminase in the uptake of vitellogenin by Xenopus laevis oocytes. Biochem Biophys Res Commun. 1981 Mar 16;99(1):221–227. doi: 10.1016/0006-291x(81)91735-6. [DOI] [PubMed] [Google Scholar]
  36. Yu F. L. Two functional states of the RNA polymerases in the rat hepatic nuclear and nucleolar fractions. Nature. 1974 Sep 27;251(5473):344–346. doi: 10.1038/251344a0. [DOI] [PubMed] [Google Scholar]

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