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. 1998 Nov;4(11):1436–1443. doi: 10.1017/s1355838298981043

Overproduction of selenocysteine tRNA in Chinese hamster ovary cells following transfection of the mouse tRNA[Ser]Sec gene.

M E Moustafa 1, M A El-Saadani 1, K M Kandeel 1, D B Mansur 1, B J Lee 1, D L Hatfield 1, A M Diamond 1
PMCID: PMC1369715  PMID: 9814763

Abstract

Selenocysteine insertion during selenoprotein biosynthesis begins with the aminoacylation of selenocysteine tRNA[ser]sec with serine, the conversion of the serine moiety to selenocysteine, and the recognition of specific UGA codons within the mRNA. Selenocysteine tRNA[ser]sec exists as two major forms, differing by methylation of the ribose portion of the nucleotide at the wobble position of the anticodon. The levels and relative distribution of these two forms of the tRNA are influenced by selenium in mammalian cells and tissues. We have generated Chinese hamster ovary cells that exhibit increased levels of tRNA[ser]sec following transfection of the mouse tRNA[ser]sec gene. The levels of selenocysteine tRNA[ser]sec in transfectants increased proportionally to the number of stably integrated copies of the tRNA[ser]sec gene. Although we were able to generate transfectants overproducing tRNA[ser]sec by as much as tenfold, the additional tRNA was principally retained in the unmethylated form. Selenium supplementation could not significantly affect the relative distributions of the two major selenocysteine tRNA[ser]sec isoacceptors. In addition, increased levels of tRNA[ser]sec did not result in measurable alterations in the levels of selenoproteins, including glutathione peroxidase.

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Selected References

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

  1. Amberg R., Urban C., Reuner B., Scharff P., Pomerantz S. C., McCloskey J. A., Gross H. J. Editing does not exist for mammalian selenocysteine tRNAs. Nucleic Acids Res. 1993 Dec 11;21(24):5583–5588. doi: 10.1093/nar/21.24.5583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berggren M., Gallegos A., Gasdaska J., Powis G. Cellular thioredoxin reductase activity is regulated by selenium. Anticancer Res. 1997 Sep-Oct;17(5A):3377–3380. [PubMed] [Google Scholar]
  3. Bermano G., Nicol F., Dyer J. A., Sunde R. A., Beckett G. J., Arthur J. R., Hesketh J. E. Selenoprotein gene expression during selenium-repletion of selenium-deficient rats. Biol Trace Elem Res. 1996 Mar;51(3):211–223. doi: 10.1007/BF02784076. [DOI] [PubMed] [Google Scholar]
  4. Berry M. J., Banu L., Harney J. W., Larsen P. R. Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons. EMBO J. 1993 Aug;12(8):3315–3322. doi: 10.1002/j.1460-2075.1993.tb06001.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bösl M. R., Takaku K., Oshima M., Nishimura S., Taketo M. M. Early embryonic lethality caused by targeted disruption of the mouse selenocysteine tRNA gene (Trsp). Proc Natl Acad Sci U S A. 1997 May 27;94(11):5531–5534. doi: 10.1073/pnas.94.11.5531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carbon P., Krol A. Transcription of the Xenopus laevis selenocysteine tRNA(Ser)Sec gene: a system that combines an internal B box and upstream elements also found in U6 snRNA genes. EMBO J. 1991 Mar;10(3):599–606. doi: 10.1002/j.1460-2075.1991.tb07987.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chittum H. S., Baek H. J., Diamond A. M., Fernandez-Salguero P., Gonzalez F., Ohama T., Hatfield D. L., Kuehn M., Lee B. J. Selenocysteine tRNA[Ser]Sec levels and selenium-dependent glutathione peroxidase activity in mouse embryonic stem cells heterozygous for a targeted mutation in the tRNA[Ser]Sec gene. Biochemistry. 1997 Jul 15;36(28):8634–8639. doi: 10.1021/bi970608t. [DOI] [PubMed] [Google Scholar]
  8. Chittum H. S., Hill K. E., Carlson B. A., Lee B. J., Burk R. F., Hatfield D. L. Replenishment of selenium deficient rats with selenium results in redistribution of the selenocysteine tRNA population in a tissue specific manner. Biochim Biophys Acta. 1997 Oct 30;1359(1):25–34. doi: 10.1016/s0167-4889(97)00092-x. [DOI] [PubMed] [Google Scholar]
  9. Chittum H. S., Lane W. S., Carlson B. A., Roller P. P., Lung F. D., Lee B. J., Hatfield D. L. Rabbit beta-globin is extended beyond its UGA stop codon by multiple suppressions and translational reading gaps. Biochemistry. 1998 Aug 4;37(31):10866–10870. doi: 10.1021/bi981042r. [DOI] [PubMed] [Google Scholar]
  10. Choi I. S., Diamond A. M., Crain P. F., Kolker J. D., McCloskey J. A., Hatfield D. L. Reconstitution of the biosynthetic pathway of selenocysteine tRNAs in Xenopus oocytes. Biochemistry. 1994 Jan 18;33(2):601–605. doi: 10.1021/bi00168a027. [DOI] [PubMed] [Google Scholar]
  11. Diamond A. M., Choi I. S., Crain P. F., Hashizume T., Pomerantz S. C., Cruz R., Steer C. J., Hill K. E., Burk R. F., McCloskey J. A. Dietary selenium affects methylation of the wobble nucleoside in the anticodon of selenocysteine tRNA([Ser]Sec). J Biol Chem. 1993 Jul 5;268(19):14215–14223. [PubMed] [Google Scholar]
  12. Diamond A. M., Dale P., Murray J. L., Grdina D. J. The inhibition of radiation-induced mutagenesis by the combined effects of selenium and the aminothiol WR-1065. Mutat Res. 1996 Sep 23;356(2):147–154. doi: 10.1016/0027-5107(96)00016-4. [DOI] [PubMed] [Google Scholar]
  13. Diamond A. M., Kataoka Y., Murray J., Duan C., Folks T. M., Sandstrom P. A. A T-cell model for the biological role of selenium-dependent glutathione peroxidase. Biomed Environ Sci. 1997 Sep;10(2-3):246–252. [PubMed] [Google Scholar]
  14. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  15. Gladyshev V. N., Jeang K. T., Wootton J. C., Hatfield D. L. A new human selenium-containing protein. Purification, characterization, and cDNA sequence. J Biol Chem. 1998 Apr 10;273(15):8910–8915. doi: 10.1074/jbc.273.15.8910. [DOI] [PubMed] [Google Scholar]
  16. Hatfield D., Lee B. J., Hampton L., Diamond A. M. Selenium induces changes in the selenocysteine tRNA[Ser]Sec population in mammalian cells. Nucleic Acids Res. 1991 Feb 25;19(4):939–943. doi: 10.1093/nar/19.4.939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hatfield D., Matthews C. R., Rice M. Aminoacyl-transfer RNA populations in mammalian cells chromatographic profiles and patterns of codon recognition. Biochim Biophys Acta. 1979 Oct 25;564(3):414–423. doi: 10.1016/0005-2787(79)90032-7. [DOI] [PubMed] [Google Scholar]
  18. Kelmers A. D., Heatherly D. E. Columns for rapid chromatographic separation of small amounts of tracer-labeled transfer ribonucleic acids. Anal Biochem. 1971 Dec;44(2):486–495. doi: 10.1016/0003-2697(71)90236-3. [DOI] [PubMed] [Google Scholar]
  19. Lee B. J., Kang S. G., Hatfield D. Transcription of Xenopus selenocysteine tRNA Ser (formerly designated opal suppressor phosphoserine tRNA) gene is directed by multiple 5'-extragenic regulatory elements. J Biol Chem. 1989 Jun 5;264(16):9696–9702. [PubMed] [Google Scholar]
  20. Lee B. J., Worland P. J., Davis J. N., Stadtman T. C., Hatfield D. L. Identification of a selenocysteyl-tRNA(Ser) in mammalian cells that recognizes the nonsense codon, UGA. J Biol Chem. 1989 Jun 15;264(17):9724–9727. [PubMed] [Google Scholar]
  21. Lee B. J., de la Peña P., Tobian J. A., Zasloff M., Hatfield D. Unique pathway of expression of an opal suppressor phosphoserine tRNA. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6384–6388. doi: 10.1073/pnas.84.18.6384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Low S. C., Berry M. J. Knowing when not to stop: selenocysteine incorporation in eukaryotes. Trends Biochem Sci. 1996 Jun;21(6):203–208. [PubMed] [Google Scholar]
  23. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  24. NIRENBERG M., LEDER P. RNA CODEWORDS AND PROTEIN SYNTHESIS. THE EFFECT OF TRINUCLEOTIDES UPON THE BINDING OF SRNA TO RIBOSOMES. Science. 1964 Sep 25;145(3639):1399–1407. doi: 10.1126/science.145.3639.1399. [DOI] [PubMed] [Google Scholar]
  25. O'Neill V. A., Eden F. C., Pratt K., Hatfield D. L. A human opal suppressor tRNA gene and pseudogene. J Biol Chem. 1985 Feb 25;260(4):2501–2508. [PubMed] [Google Scholar]
  26. Ohama T., Choi I. S., Hatfield D. L., Johnson K. R. Mouse selenocysteine tRNA([Ser]Sec) gene (Trsp) and its localization on chromosome 7. Genomics. 1994 Feb;19(3):595–596. doi: 10.1006/geno.1994.1116. [DOI] [PubMed] [Google Scholar]
  27. Park J. M., Choi I. S., Kang S. G., Lee J. Y., Hatfield D. L., Lee B. J. Upstream promoter elements are sufficient for selenocysteine tRNA[Ser]Sec gene transcription and to determine the transcription start point. Gene. 1995 Aug 30;162(1):13–19. doi: 10.1016/0378-1119(95)00340-c. [DOI] [PubMed] [Google Scholar]
  28. Samuels B. L., Murray J. L., Cohen M. B., Safa A. R., Sinha B. K., Townsend A. J., Beckett M. A., Weichselbaum R. R. Increased glutathione peroxidase activity in a human sarcoma cell line with inherent doxorubicin resistance. Cancer Res. 1991 Jan 15;51(2):521–527. [PubMed] [Google Scholar]
  29. Sandström B. E., Carlsson J., Marklund S. L. Variations among cultured cells in glutathione peroxidase activity in response to selenite supplementation. Biochim Biophys Acta. 1987 Jul 6;929(2):148–153. doi: 10.1016/0167-4889(87)90170-4. [DOI] [PubMed] [Google Scholar]
  30. Schuster C., Myslinski E., Krol A., Carbon P. Staf, a novel zinc finger protein that activates the RNA polymerase III promoter of the selenocysteine tRNA gene. EMBO J. 1995 Aug 1;14(15):3777–3787. doi: 10.1002/j.1460-2075.1995.tb00047.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yeh J. Y., Vendeland S. C., Gu Q., Butler J. A., Ou B. R., Whanger P. D. Dietary selenium increases selenoprotein W levels in rat tissues. J Nutr. 1997 Nov;127(11):2165–2172. doi: 10.1093/jn/127.11.2165. [DOI] [PubMed] [Google Scholar]

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