In Vitro Transcription Systems from BY-2 Cells

  • Yasushi Yukawa
  • Masahiro Sugiura
Part of the Biotechnology in Agriculture and Forestry book series (AGRICULTURE, volume 53)


The in vitro transcription system is a powerful method to dissect molecular mechanisms of transcription of genes in question, usually performed in cellular or nuclear extracts containing transcription apparatuses with exogenous DNA templates and four nucleoside triphosphate (NTP) substrates. The first eukaryotic system was reported by Weil et al. (1979). They carried out in vitro transcription using a cytosolic extract and purified Pol II from HeLa cells. Manley et al. (1980) followed with in vitro assays using a whole cell extract from HeLa cells. In 1983, Dignam et al. (1983) established a standard procedure to prepare nuclear extracts from HeLa cells. Based on these efforts, many other in vitro systems have been established from different eukaryotic species, for example, yeast, mouse, and fruit fly. Since then, many significant discoveries have been made with these in vitro transcription systems.


tRNA Gene Transcription System TATA Motif snRNA Gene Primer Extension Assay 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Abelson J, Trotta CR, Li H (1998) tRNA splicing. J Biol Chem 273: 12685–12688Google Scholar
  2. Akama K, Kashihara M (1996) Plant nuclear tRNAMet genes are ubiquitously interrupted by intron. Plant Mol Biol 32: 427–434PubMedCrossRefGoogle Scholar
  3. Akama K, Nass A, Junker V, Beier H (1997) Characterization of nuclear tRNATyr introns: their evolution from red algae to higher plants. FEBS Lett 417: 213–218PubMedCrossRefGoogle Scholar
  4. Akama K, Junker V, Yukawa Y, Sugiura M, Beier H (2000) Splicing of Arabidopsis tRNAMet precursors in tobacco cell and wheat germ extracts. Plant Mol Biol 44: 155–165PubMedCrossRefGoogle Scholar
  5. Arnaud P, Yukawa Y, Lavie L, Pelissier T, Sugiura M, Deragon JM (2001) Analysis of the SINE S1 Pol III promoter from Brassica; impact of methylation and influence of external sequences. Plant J 26: 295–305PubMedCrossRefGoogle Scholar
  6. Beier D, Beier H (1992) Expression of variant nuclear Arabidopsis tRNASer genes and pre-tRNA maturation differ in HeLa, yeast and wheat germ extracts. Mol Gen Genet 233: 201–208Google Scholar
  7. Brown JWS, Echeverria M, Qu LH (2003) Plant snoRNAs: functional evolution and new modes of gene expression. Trends Plant Sci 8: 42–49Google Scholar
  8. Choisne N, Carneiro VTC, Pelletier G, Small I (1997) Implication of 5’-flanking sequence elements in expression of a plant tRNALeu gene. Plant Mol Biol 36: 113–123CrossRefGoogle Scholar
  9. Cloix C, Tutois S, Yukawa Y, Mathieu O, Cuvillier C, Espagnol MC, Picard G, Tourmente S (2002) Analysis of the 5S RNA pool in Arabidopsis thaliana: RNAs are heterogeneous and only two of the genomic 5S loci produce mature 5S RNA. Genome Res 12: 132–144Google Scholar
  10. Deragon JM, Landry BS, Pelissier T, Tutois S, Tourmente S, Picard G (1994) An analysis of retroposition in plants based on a family of SINEs from Brassica napus. J Mol Evol 39: 378–386Google Scholar
  11. Deutscher MP (1995) tRNA processing nucleases. In: Söll D, RajBhandary UL (eds) tRNA: Structure, Biosynthesis, and Function. American Society for Microbiology, Washington, pp 51–65Google Scholar
  12. Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11: 1475–1489PubMedCrossRefGoogle Scholar
  13. Fan H, Sugiura M (1995) A plant basal in vitro system supporting accurate transcription of both RNA polymerase II- and III-dependent genes: supplement of green leaf component(s) drives accurate transcription of a light-responsive rbcS gene. EMBO J 14: 1024–1031PubMedGoogle Scholar
  14. Fan H, Sugiura M (1996) Basal and activated in vitro transcription in plants by RNA polymerase II and III. Methods Enzymol 273: 268–277PubMedCrossRefGoogle Scholar
  15. Fan H, Yakura K, Miyanishi M, Sugita M, Sugiura M (1995) In vitro transcription of plant RNA polymerase I-dependent rRNA genes is species-specific. Plant J 8: 295–298PubMedCrossRefGoogle Scholar
  16. Filipowicz W, Kiss T, Marshallsay C, Waibel F (1990) U-snRNA genes, U-snRNAs and U-snRNPs of higher plants. Mol Biol Rep 14: 125–129Google Scholar
  17. Fuchs T, Beier D, Beier H (1992) The tRNATyr multigene family of Nicotiana rustica: genome organization, sequence analyses and expression in vitro. Plant Mol Biol 20: 869–878PubMedCrossRefGoogle Scholar
  18. Geiduschek EP, Kassavetis GA (1992) RNA polymerase III transcription complexes. In: McKnight SL, Yamamoto KR (eds) Transcription regulation, vol 1, Cold Spring Harbor Laboratory Press, New York, pp 247–280Google Scholar
  19. Goodall GJ, Filipowicz W (1991) Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants. EMBO J 10: 2635–2644PubMedGoogle Scholar
  20. Gopalan V, Vioque A, Altman S (2002) RNase P: variations and uses. J Biol Chem 277: 6759–6762PubMedCrossRefGoogle Scholar
  21. Hasegawa K, Yukawa Y, Sugita M, Sugiura M (2002) Organization and transcription of the gene family encoding chlorophyll a/b-binding proteins in Nicotiana sylvestris. Gene 289: 161–168PubMedCrossRefGoogle Scholar
  22. Hasegawa K, Yukawa Y, Sugiura M (2003a) In vitro analysis of transcription initiation and termination from the Lhcb1 gene family in Nicotiana sylvestris: detection of transcription termination sites. Plant J 33: 1063–1072PubMedCrossRefGoogle Scholar
  23. Hasegawa K, Yukawa Y, Obokata J, Sugiura M (2003b) A tRNALeu-like sequence located immediately upstream of an Arabidopsis clock-regulated gene is transcriptionally active: efficient transcription by an RNA polymerase III-dependent in vitro transcription system. Gene 289: 161–168CrossRefGoogle Scholar
  24. Heard DJ, Filipowicz W, Marques JP, Palme K, Gualberto JM (1995) An upstream U-snRNA gene-like promoter is required for transcription of the Arabidopsis thaliana 7SL RNA gene. Nucleic Acids Res 23: 1970–1976PubMedCrossRefGoogle Scholar
  25. Kassavetis GA, Joazeiro CAP, Pisano M, Geiduschek EP, Colbert T, Hahn S, Blanco JA (1992) The role of the TATA-binding protein in the assembly and function of the multisubunit yeast RNA polymerase III transcription factor, TFIIIB. Cell 71: 1055–1064Google Scholar
  26. Kato A, Nakajima T, Yamashita J, Yakura K, Tanifuji S (1990) The structure of the large spacer region of the rDNA in Vicia faba and Pisum sativum. Plant Mol Biol 14: 983–993PubMedCrossRefGoogle Scholar
  27. Kiss T, Marshallsay C, Filipowicz W (1991) Alteration of the RNA polymerase specificity of U3 snRNA genes during evolution and in vitro. Cell 65: 517–526PubMedCrossRefGoogle Scholar
  28. Kiss T, Marshallsay C, Filipowicz W (1992) 7–2/MRP RNAs in plant and mammalian cells: association with higher order structures in the nucleolus. EMBO J 11: 3737–3746Google Scholar
  29. Manley JL, Fire A, Cano A, Sharp PA, Gefter ML (1980) DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci USA 77: 3855–3859PubMedCrossRefGoogle Scholar
  30. Marshallsay C, Kiss T, Filipowicz W (1990) Nucleotide sequence and expression of a new Arabidopsis U2 snRNA gene. Nucleic Acids Res 18: 5280PubMedCrossRefGoogle Scholar
  31. Mathieu O, Yukawa Y, Sugiura M, Picard G, Tourmente S (2002) 5S rRNA genes expression is not inhibited by DNA methylation in Arabidopsis. Plant J 29: 313–323Google Scholar
  32. Matoušek J, Junker V, Vrba L, Schubert J, Patzak J, Steger G (1999) Molecular characterization and genome organization of 7SL RNA genes from hop (Humulus lupulus L.). Gene 239: 173–183PubMedCrossRefGoogle Scholar
  33. Morl M, Marchfelder A (2001) The final cut. The importance of tRNA 3’-processing. EMBO Rep 2: 17–20PubMedCrossRefGoogle Scholar
  34. Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the “HeLa” cell in the cell biology of higher plants. Int Rev Cytol 132: 1–30Google Scholar
  35. Nemoto Y, Kawano S, Nakamura S, Mita T, Nagata T, Kuroiwa T (1988) Studies on plastid-nuclei (nucleoids) in Nicotiana tabacum L. I. Isolation of proplastid-nuclei from cultured cells and identification of proplastid-nuclear proteins. Plant Cell Physiol 29: 167–177Google Scholar
  36. Ohme-Takagi M, Shinshi H (1990) Structure and expression of a tobacco β-1, 3-glucanase gene. Plant Mol Biol 15: 941–946Google Scholar
  37. Platt T (1986) Transcription termination and the regulation of gene expression. Annu Rev Biochem 55: 339–372PubMedCrossRefGoogle Scholar
  38. Shi P-Y, Maizels N, Weiner AM (1998) CCA addition by tRNA nucleotidyltransferase: polymerization without translocation? EMBO J 17: 3197–3206Google Scholar
  39. Solymosy F, Pollák T (1993) Uridylate-rich small nuclear RNAs (U snRNAs), their genes and pseudogenes, and U snRNA in plants: structure and function. A comparative approach. Crit Rev Plant Sci 12: 275–369Google Scholar
  40. Sprague KU (1995) Transcription of eukaryotic tRNA genes. In: Söll D, RajBhandary UL (eds) tRNA: structure, biosynthesis, and function. American Society for Microbiology, Washington, DC, pp 31–50Google Scholar
  41. Stange N, Beier H (1986) A gene for the major cytoplasmic tRNATyr from Nicotiana rustica contains a 13 nucleotides long intron. Nucleic Acids Res 14: 8691PubMedCrossRefGoogle Scholar
  42. Sugiura M (1996) Plant in vitro transcription: the opening of a new era. Trends in Plant Sci 1: 41CrossRefGoogle Scholar
  43. Sugiura M (1997) Plant in vitro transcription systems. Annu Rev Plant Physiol Plant Mol Biol 48: 383–398PubMedCrossRefGoogle Scholar
  44. Vankan P, Filipowicz W (1989) A U-snRNA gene-specific upstream element and a -30 “TATA box” are required for transcription of the U2 snRNA gene of Arabidopsis thaliana. EMBO J 8: 3875–3882PubMedGoogle Scholar
  45. Vankan P, Edoh D, Filipowicz W (1988) Structure and expression of the U5 snRNA gene of Arabidopsis thaliana. Conserved upstream sequence elements in plant U-RNA genes. Nucleic Acids Res 16: 10425–10440Google Scholar
  46. van Tol H, Stange N, Gross HJ, Beier H (1987) A human and a plant intron-containing tRNATyr gene are both transcribed in a HeLa cell extract but spliced along different pathways. EMBO J 6: 35–41Google Scholar
  47. Waibel F, Filipowicz W (1990a) U6 snRNA genes of Arabidopsis are transcribed by RNA polymerase III but contain the same two upstream promoter elements as RNA polymerase II-transcribed U-snRNA genes. Nucleic Acids Res 18: 3451–3458PubMedCrossRefGoogle Scholar
  48. Waibel F, Filipowicz W (1990b) RNA-polymerase specificity of transcription of Arabidopsis U snRNA genes determined by promoter element spacing. Nature 346: 199–202PubMedCrossRefGoogle Scholar
  49. Weil PA, Luse DS, Segall J, Roeder RG (1979) Selective and accurate initiation of transcription at the Ad2 major late promoter in a soluble system dependent on purified RNA polymerase II and DNA. Cell 18: 469–484PubMedCrossRefGoogle Scholar
  50. Westaway SK, Abelson J (1995) Splicing of tRNA precursors. In: Söll D, RajBhandary UL (eds) tRNA: structure, biosynthesis, and function. American Society for Microbiology, Wasington, pp 79–92Google Scholar
  51. White RJ (ed) (1998) RNA Polymerase III transcription. Springer, Berlin Heidelberg New York, and RG Land Company, Georgetown, pp 23–55Google Scholar
  52. Willis IM (1993) RNA polymerase III-Genes, factors and transcriptional specificity. Eur J Biochem 2: 1–11CrossRefGoogle Scholar
  53. Yamashita J, Nakajima T, Tanifuji S, Karo A (1993) Accurate transcription initiation of Vicia faba tDNA in a whole cell extract from embryonic axes. Plant J 3: 187–190CrossRefGoogle Scholar
  54. Yukawa Y, Sugiura M (2002) Plant in vitro transcription systems. Tampaku Kakusan Kouso 47: 583–589Google Scholar
  55. Yukawa Y, Fan H, Akama K, Beier H, Gross HJ, Sugiura M (2001) A tobacco nuclear extract supporting transcription, processing, splicing and modification of plant intron-containing tRNA precursors. Plant J 28: 583–594PubMedCrossRefGoogle Scholar
  56. Yukawa Y, Matoušek J, Grimm M, Vrba L, Steger G, Sugiura M, Beier H (2002) Plant 7SL RNA and tRNATyr genes with inserted antisense sequences are efficiently expressed in an in vitro transcription system from Nicotiana tabacum cells. Plant Mol Biol 50: 713–723PubMedCrossRefGoogle Scholar
  57. Yukawa Y, Sugita M, Choisne N, Small I, Sugiura M (2000) The TATA motif, the CAA motif and the poly(T) transcription termination motif are all important for transcription re-initiation on plant tRNA genes. Plant J 22: 439–447PubMedCrossRefGoogle Scholar
  58. Yukawa Y, Sugita M, Sugiura M (1997) Efficient in vitro transcription of plant nuclear tRNASer genes in a nuclear extract from tobacco cultured cells. Plant J 12: 965–970PubMedCrossRefGoogle Scholar
  59. Zhu Q (1996) RNA polymerase II-dependent plant in vitro transcription systems. Plant J 10:185– 188Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2004

Authors and Affiliations

  • Yasushi Yukawa
    • 1
  • Masahiro Sugiura
    • 1
  1. 1.Graduate School of Natural SciencesNagoya City UniversityNagoyaJapan

Personalised recommendations