Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1970 Apr;65(4):876–883. doi: 10.1073/pnas.65.4.876

Pulse Labeling of RNA of Mammalian Cells*

Giovanni Rovera 1, Stephen Berman 1, Renato Baserga 1
PMCID: PMC282998  PMID: 5266157

Abstract

When cells from a hypotetraploid strain of Ehrlich ascites tumor are exposed to uridine-3H either in vivo or in vitro, the amount of radioactivity incorporated into RNA reaches a maximum within ten minutes, after which any further incorporation stops. 3H-uridine triphosphate disappears from the acid soluble pool within 30 minutes and the findings indicate that the RNA of these cells can be pulse labeled without the use of any antibiotic or the need of a „chase.” The stability of the pulse labeled RNA in the presence of pentobarbital (an inhibitor of RNA synthesis) indicates the virtual absence of RNA breakdown. However, actinomycin D, at a dosage of 250 μg/mouse in vivo and 10 μg/ml in vitro produces breakdown of labeled RNA, thus confirming earlier observations that the drug is not a suitable tool for RNA kinetics determinations. The pulse-labeled RNA leaves the nucleus slowly and some radioactive RNA is still present in the nuclear fraction after 24 hours. Radioactivity begins to appear in cytoplasmic ribosomal RNA after 20 minutes and continues to increase up to six hours.

Full text

PDF
879

Selected References

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

  1. Attardi G., Huang P. C., Kabat S. Recognition of ribosomal RNA sites in DNA. II. The HeLa cell system. Proc Natl Acad Sci U S A. 1965 Jul;54(1):185–192. doi: 10.1073/pnas.54.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BASERGA R. Mitotic cycle of ascites tumor cells. Arch Pathol. 1963 Feb;75:156–161. [PubMed] [Google Scholar]
  3. Baserga R., Estensen R. D., Petersen R. O. Inhibition of DNA synthesis in Ehrlich ascites cells by actinomycin D. II. The presynthetic block in the cell cycle. Proc Natl Acad Sci U S A. 1965 Oct;54(4):1141–1148. doi: 10.1073/pnas.54.4.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baserga R., Weiss L. Inhibition of deoxyribonucleic acid synthesis by pentobarbital. Biochim Biophys Acta. 1967 Sep 26;145(2):361–367. doi: 10.1016/0005-2787(67)90054-8. [DOI] [PubMed] [Google Scholar]
  5. Bernhardt D., Darnell J. E., Jr tRNA synthesis in HeLa cells: a precursor to tRNA and the effects of methionine starvation on tRNA synthesis. J Mol Biol. 1969 May 28;42(1):43–56. doi: 10.1016/0022-2836(69)90485-9. [DOI] [PubMed] [Google Scholar]
  6. Bishop D. H., Claybrook J. R., Spiegelman S. Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J Mol Biol. 1967 Jun 28;26(3):373–387. doi: 10.1016/0022-2836(67)90310-5. [DOI] [PubMed] [Google Scholar]
  7. Church R. B., Luther S. W., McCarthy B. J. RNA synthesis in Taper hepatoma and mouse-liver cells. Biochim Biophys Acta. 1969 Sep 17;190(1):30–37. doi: 10.1016/0005-2787(69)90151-8. [DOI] [PubMed] [Google Scholar]
  8. Coolsma J. W., Gruber M. RNA synthesis in rooster liver. Biochim Biophys Acta. 1968 Dec 17;169(2):306–315. doi: 10.1016/0005-2787(68)90039-7. [DOI] [PubMed] [Google Scholar]
  9. Cooper H. L. Ribonucleic acid metabolism in lymphocytes stimulated by phytohemagglutinin. II. Rapidly synthesized ribonucleic acid and te production of ribosomal ribonucleic acid. J Biol Chem. 1968 Jan 10;243(1):34–43. [PubMed] [Google Scholar]
  10. FEINENDEGEN L. E., BOND V. P., PAINTER R. B. Studies on the interrelationship of RNA synthesis, DNA synthesis and precursor pool in human tissue culture cells studied with tritiated pyrimidine nucleosides. Exp Cell Res. 1961 Jan;22:381–405. doi: 10.1016/0014-4827(61)90117-3. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. GIRARD M., PENMAN S., DARNELL J. E. THE EFFECT OF ACTINOMYCIN ON RIBOSOME FORMATION IN HELA CELLS. Proc Natl Acad Sci U S A. 1964 Feb;51:205–211. doi: 10.1073/pnas.51.2.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. HARRIS H. Rapidly labelled ribonucleic acid in the cell nucleus. Nature. 1963 Apr 13;198:184–185. doi: 10.1038/198184a0. [DOI] [PubMed] [Google Scholar]
  14. IVES D. H., MORSE P. A., Jr, POTTER V. R. Feedback inhibition of thymodine kinase by thymodine triphosphate. J Biol Chem. 1963 Apr;238:1467–1474. [PubMed] [Google Scholar]
  15. JACQUEZ J. A. Transport and enzymic splitting of pyrimidine nucleosides in Ehrlich cells. Biochim Biophys Acta. 1962 Aug 20;61:265–277. doi: 10.1016/0926-6550(62)90089-0. [DOI] [PubMed] [Google Scholar]
  16. LAUF P., SEEMAYER N., OEHLERT W. [The quantity and mode of RNA synthesis in Ehrlich ascites tumor cells in white mice]. Z Krebsforsch. 1962;64:490–498. [PubMed] [Google Scholar]
  17. Lazarus H. M., Sporin M. B. Purification and properties of a nuclear exoribonuclease from Ehrlich ascites tumor cells. Proc Natl Acad Sci U S A. 1967 May;57(5):1386–1393. doi: 10.1073/pnas.57.5.1386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Loening U. E. The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem J. 1967 Jan;102(1):251–257. doi: 10.1042/bj1020251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Owen M. Uptake of [3H] uridine into precursor pools and RNA in osteogenic cells. J Cell Sci. 1967 Mar;2(1):39–56. doi: 10.1242/jcs.2.1.39. [DOI] [PubMed] [Google Scholar]
  20. Penman S. RNA metabolism in the HeLa cell nucleus. J Mol Biol. 1966 May;17(1):117–130. doi: 10.1016/s0022-2836(66)80098-0. [DOI] [PubMed] [Google Scholar]
  21. Penman S., Vesco C., Penman M. Localization and kinetics of formation of nuclear heterodisperse RNA, cytoplasmic heterodisperse RNA and polyribosome-associated messenger RNA in HeLa cells. J Mol Biol. 1968 May 28;34(1):49–60. doi: 10.1016/0022-2836(68)90234-9. [DOI] [PubMed] [Google Scholar]
  22. Perry R. P., Kelley D. E. Persistent synthesis of 5S RNA when production of 28S and 18S ribosomal RNA is inhibited by low doses of actinomycin D. J Cell Physiol. 1968 Dec;72(3):235–246. doi: 10.1002/jcp.1040720311. [DOI] [PubMed] [Google Scholar]
  23. Perry R. P. The nucleolus and the synthesis of ribosomes. Natl Cancer Inst Monogr. 1965 Dec;18:325–340. [PubMed] [Google Scholar]
  24. SCHERRER K., LATHAM H., DARNELL J. E. Demonstration of an unstable RNA and of a precursor to ribosomal RNA in HeLa cells. Proc Natl Acad Sci U S A. 1963 Feb 15;49:240–248. doi: 10.1073/pnas.49.2.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. SCOTT J. F., FRACCASTORO A. P., TAFT E. B. Studies in histochemistry. I. Determination of nucleic acids in microgram amounts of tissue. J Histochem Cytochem. 1956 Jan;4(1):1–10. doi: 10.1177/4.1.1. [DOI] [PubMed] [Google Scholar]
  26. Stewart G. A., Farber E. The rapid acceleration of hepatic nuclear ribonucleic acid breakdown by actinomycin but not by ethionine. J Biol Chem. 1968 Sep 10;243(17):4479–4485. [PubMed] [Google Scholar]
  27. Tamaoki T., Mueller G. C. The effects of actinomycin D and puromycin on the formation of ribosomes in HeLa cells. Biochim Biophys Acta. 1965 Sep 6;108(1):73–80. doi: 10.1016/0005-2787(65)90109-7. [DOI] [PubMed] [Google Scholar]
  28. Warner J. R., Soeiro R., Birnboim H. C., Girard M., Darnell J. E. Rapidly labeled HeLa cell nuclear RNA. I. Identification by zone sedimentation of a heterogeneous fraction separate from ribosomal precursor RNA. J Mol Biol. 1966 Aug;19(2):349–361. doi: 10.1016/s0022-2836(66)80009-8. [DOI] [PubMed] [Google Scholar]
  29. Weinberg R. A., Loening U., Willems M., Penman S. Acrylamide gel electrophoresis of HeLa cell nucleolar RNA. Proc Natl Acad Sci U S A. 1967 Sep;58(3):1088–1095. doi: 10.1073/pnas.58.3.1088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Weinberg R. A., Penman S. Small molecular weight monodisperse nuclear RNA. J Mol Biol. 1968 Dec;38(3):289–304. doi: 10.1016/0022-2836(68)90387-2. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES