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. 1988 Oct 25;16(20):9597–9609. doi: 10.1093/nar/16.20.9597

Direct measurement of tubulin and bulk message distributions on polysomes of growing, starved and deciliated Tetrahymena using RNA gel blots of sucrose gradients containing acrylamide.

F J Calzone 1, R Callahan 1, M A Gorovsky 1
PMCID: PMC338766  PMID: 3054809

Abstract

A method was developed using sucrose gradients containing acrylamide which greatly simplifies the measurement of the polysomal distribution of messages. After centrifugation, the acrylamide was polymerized, forming a "polysome gel". RNA gel blots of polysome gels were used to determine the polysomal distributions of alpha-tubulin and total polyadenylated mRNA in growing, starved (nongrowing) and starved-deciliated Tetrahymena and the number of messages loaded onto polysomes was calculated. These measurements indicated that the translational efficiencies of alpha-tubulin mRNA and total polyadenylated mRNA are largely unaffected when the rates of tubulin and total protein synthesis vary dramatically. Thus, differential regulation of alpha-tubulin mRNA translation initiation does not contribute to the greater than 100-fold induction of tubulin synthesis observed during cilia regeneration and in growing cells. The major translation-level process regulating tubulin synthesis in Tetrahymena appears to be a change in message loading mediated by a non-specific message recruitment or unmasking factor.

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Selected References

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

  1. Ballinger D. G., Pardue M. L. The control of protein synthesis during heat shock in Drosophila cells involves altered polypeptide elongation rates. Cell. 1983 May;33(1):103–113. doi: 10.1016/0092-8674(83)90339-2. [DOI] [PubMed] [Google Scholar]
  2. Bannon G. A., Bowen J. K., Yao M. C., Gorovsky M. A. Tetrahymena H4 genes: structure, evolution and organization in macro- and micronuclei. Nucleic Acids Res. 1984 Feb 24;12(4):1961–1975. doi: 10.1093/nar/12.4.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bannon G. A., Calzone F. J., Bowen J. K., Allis C. D., Gorovsky M. A. Multiple, independently regulated, polyadenylated messages for histone H3 and H4 in Tetrahymena. Nucleic Acids Res. 1983 Jun 25;11(12):3903–3917. doi: 10.1093/nar/11.12.3903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baum E. Z., Wormington W. M. Coordinate expression of ribosomal protein genes during Xenopus development. Dev Biol. 1985 Oct;111(2):488–498. doi: 10.1016/0012-1606(85)90500-7. [DOI] [PubMed] [Google Scholar]
  5. Bergmann J. E., Lodish H. F. A kinetic model of protein synthesis. Application to hemoglobin synthesis and translational control. J Biol Chem. 1979 Dec 10;254(23):11927–11937. [PubMed] [Google Scholar]
  6. Callahan R. C., Shalke G., Gorovsky M. A. Developmental rearrangements associated with a single type of expressed alpha-tubulin gene in Tetrahymena. Cell. 1984 Feb;36(2):441–445. doi: 10.1016/0092-8674(84)90237-x. [DOI] [PubMed] [Google Scholar]
  7. Calzone F. J., Angerer R. C., Gorovsky M. A. Regulation of protein synthesis in Tetrahymena. Quantitative estimates of the parameters determining the rates of protein synthesis in growing, starved, and starved-deciliated cells. J Biol Chem. 1983 Jun 10;258(11):6887–6898. [PubMed] [Google Scholar]
  8. Calzone F. J., Angerer R. C., Gorovsky M. A. Regulation of protein synthesis in Tetrahymena. Quantitative estimates of the parameters determining the rates of protein synthesis in growing, starved, and starved-deciliated cells. J Biol Chem. 1983 Jun 10;258(11):6887–6898. [PubMed] [Google Scholar]
  9. Calzone F. J., Angerer R. C., Gorovsky M. A. Regulation of protein synthesis in Tetrahymena: isolation and characterization of polysomes by gel filtration and precipitation at pH 5.3. Nucleic Acids Res. 1982 Mar 25;10(6):2145–2161. doi: 10.1093/nar/10.6.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Calzone F. J., Gorovsky M. A. Cilia regeneration in Tetrahymena. A simple reproducible method for producing large numbers of regenerating cells. Exp Cell Res. 1982 Aug;140(2):471–476. doi: 10.1016/0014-4827(82)90144-6. [DOI] [PubMed] [Google Scholar]
  11. DeLeon D. V., Cox K. H., Angerer L. M., Angerer R. C. Most early-variant histone mRNA is contained in the pronucleus of sea urchin eggs. Dev Biol. 1983 Nov;100(1):197–206. doi: 10.1016/0012-1606(83)90211-7. [DOI] [PubMed] [Google Scholar]
  12. Duncan R., McConkey E. H. Rapid alterations in initiation rate and recruitment of inactive RNA are temporally correlated with S6 phosphorylation. Eur J Biochem. 1982 Apr;123(3):539–544. doi: 10.1111/j.1432-1033.1982.tb06565.x. [DOI] [PubMed] [Google Scholar]
  13. Friesen P. D., Rueckert R. R. Early and late functions in a bipartite RNA virus: evidence for translational control by competition between viral mRNAs. J Virol. 1984 Jan;49(1):116–124. doi: 10.1128/jvi.49.1.116-124.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galego L., Barahona I., Rodrigues-Pousada C. Response of Tetrahymena pyriformis to stress induced by starvation. Eur J Biochem. 1984 Feb 15;139(1):163–171. doi: 10.1111/j.1432-1033.1984.tb07990.x. [DOI] [PubMed] [Google Scholar]
  15. Godefroy-Colburn T., Thach R. E. The role of mRNA competition in regulating translation. IV. Kinetic model. J Biol Chem. 1981 Nov 25;256(22):11762–11773. [PubMed] [Google Scholar]
  16. Grainger J. L., Winkler M. M. Fertilization triggers unmasking of maternal mRNAs in sea urchin eggs. Mol Cell Biol. 1987 Nov;7(11):3947–3954. doi: 10.1128/mcb.7.11.3947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hansen L. J., Huang W. I., Jagus R. Inhibitor of translational initiation in sea urchin eggs prevents mRNA utilization. J Biol Chem. 1987 May 5;262(13):6114–6120. [PubMed] [Google Scholar]
  18. Kaempfer R., Konijn A. M. Translational competition by mRNA species encoding albumin, ferritin, haemopexin and globin. Eur J Biochem. 1983 Apr 5;131(3):545–550. doi: 10.1111/j.1432-1033.1983.tb07296.x. [DOI] [PubMed] [Google Scholar]
  19. Katze M. G., DeCorato D., Krug R. M. Cellular mRNA translation is blocked at both initiation and elongation after infection by influenza virus or adenovirus. J Virol. 1986 Dec;60(3):1027–1039. doi: 10.1128/jvi.60.3.1027-1039.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Laskey R. A., Mills A. D., Gurdon J. B., Partington G. A. Protein synthesis in oocytes of Xenopus laevis is not regulated by the supply of messenger RNA. Cell. 1977 Jun;11(2):345–351. doi: 10.1016/0092-8674(77)90051-4. [DOI] [PubMed] [Google Scholar]
  21. Lechner M. C., Sinogas C., Osório-Almeida M. L., Freire M. T., Chaumet-Riffaud P., Frain M., Sala-Trepat J. M. Phenobarbital-mediated modulation of gene expression in rat liver. Analysis of cDNA clones. Eur J Biochem. 1987 Mar 2;163(2):231–238. doi: 10.1111/j.1432-1033.1987.tb10792.x. [DOI] [PubMed] [Google Scholar]
  22. Ledford B. E., Jacobs D. F. Translation kinetics in cultured mouse hepatoma cells. Regulation of albumin synthesis by amino acids. Eur J Biochem. 1985 Nov 4;152(3):611–618. doi: 10.1111/j.1432-1033.1985.tb09239.x. [DOI] [PubMed] [Google Scholar]
  23. Nelson E. M., Winkler M. M. Regulation of mRNA entry into polysomes. Parameters affecting polysome size and the fraction of mRNA in polysomes. J Biol Chem. 1987 Aug 25;262(24):11501–11506. [PubMed] [Google Scholar]
  24. Pierandrei-Amaldi P., Beccari E., Bozzoni I., Amaldi F. Ribosomal protein production in normal and anucleolate Xenopus embryos: regulation at the posttranscriptional and translational levels. Cell. 1985 Aug;42(1):317–323. doi: 10.1016/s0092-8674(85)80127-6. [DOI] [PubMed] [Google Scholar]
  25. Seed B. Diazotizable arylamine cellulose papers for the coupling and hybridization of nucleic acids. Nucleic Acids Res. 1982 Mar 11;10(5):1799–1810. doi: 10.1093/nar/10.5.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shull G. E., Theil E. C. Regulation of ferritin mRNA: a possible gene-sparing phenomenon. Induction of ferritin synthesis by iron in liver as well as red cells combines high translational efficiency with increased utilization of preformed ferritin mRNA. J Biol Chem. 1983 Jul 10;258(13):7921–7923. [PubMed] [Google Scholar]
  27. Sorrentino V., Battistini A., di Francesco P., Curatola A. M., Rossi G. B. Interferon production in L929 cells under impaired translational conditions: comparison of rates of interferon, actin, Newcastle disease and encephalomyocarditis viruses mRNAs initiation of protein synthesis. Arch Virol. 1986;88(3-4):175–187. doi: 10.1007/BF01310873. [DOI] [PubMed] [Google Scholar]
  28. Steel L. F., Jacobson A. Translational control of ribosomal protein synthesis during early Dictyostelium discoideum development. Mol Cell Biol. 1987 Mar;7(3):965–972. doi: 10.1128/mcb.7.3.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Thomas G., Thomas G. Translational control of mRNA expression during the early mitogenic response in Swiss mouse 3T3 cells: identification of specific proteins. J Cell Biol. 1986 Dec;103(6 Pt 1):2137–2144. doi: 10.1083/jcb.103.6.2137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ven Murthy M. R., Bharucha A. D., Charbonneau R. A novel method for sucrose density gradient fractionation of polysomes and mRNA. Nucleic Acids Res. 1986 Aug 11;14(15):6337–6337. doi: 10.1093/nar/14.15.6337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Voorma H. O. Regulatory steps in the initiation of protein synthesis. Horiz Biochem Biophys. 1983;7:139–153. [PubMed] [Google Scholar]
  33. Voss T., Bornstein P. Regulation of type I collagen mRNA levels in fibroblasts. Eur J Biochem. 1986 Jun 2;157(2):433–439. doi: 10.1111/j.1432-1033.1986.tb09686.x. [DOI] [PubMed] [Google Scholar]
  34. Winkler M. M., Nelson E. M., Lashbrook C., Hershey J. W. Multiple levels of regulation of protein synthesis at fertilization in sea urchin eggs. Dev Biol. 1985 Feb;107(2):290–300. doi: 10.1016/0012-1606(85)90312-4. [DOI] [PubMed] [Google Scholar]
  35. Yu S. M., Gorovsky M. A. In situ dot blots: quantitation of mRNA in intact cells. Nucleic Acids Res. 1986 Oct 10;14(19):7597–7615. doi: 10.1093/nar/14.19.7597. [DOI] [PMC free article] [PubMed] [Google Scholar]

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