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. 1995 Mar 1;14(5):1024–1031. doi: 10.1002/j.1460-2075.1995.tb07083.x

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.

H Fan 1, M Sugiura 1
PMCID: PMC398174  PMID: 7889933

Abstract

An in vitro transcription initiation system has been developed from nuclei of rapidly growing, non-green tobacco (Nicotiana tabacum) cultured (BY-2) cells. Conditions for nuclear extraction and in vitro transcription reaction have been optimized with a tobacco beta-1,3-glucanase gene, a constitutively expressed gene in BY-2 cells. The in vitro system supports accurate transcription of RNA polymerase II-dependent promoters from not only plant genes (tobacco beta-1,3-glucanase gene, cauliflower mosaic virus 35S promoter) but also animal genes (adenovirus 2 major late promoter, simian virus 40 early major promoter). In addition, this system drives accurate transcription of an RNA polymerase III-dependent Arabidopsis thaliana U6 snRNA gene. As BY-2 cells do not differentiate in response to light or any other stimuli, they would provide a basal transcription system which lacks tissue-specific and light-responsive nuclear signals as well as chloroplast-derived signals. Consequently, the BY-2 cell-free system is unable to transcribe the tomato gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS3C) whose expression is tissue-specific and light-inducible. However, the transcription of rbcS3C was obtained by supplementing the BY-2 system with a nuclear extract of light-grown tomato seedlings. The promoter regions necessary for rbcS transcription was mapped in vitro using a series of 5' deletion mutants. The 351 bp upstream sequence is essential and the further upstream region from -351 to -441 enhances its transcription. The in vitro basal system will be useful to identify specific signals from both the nucleus and chloroplast in green leaves and other organs/tissues.

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Selected References

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

  1. Arias J. A., Dixon R. A., Lamb C. J. Dissection of the functional architecture of a plant defense gene promoter using a homologous in vitro transcription initiation system. Plant Cell. 1993 Apr;5(4):485–496. doi: 10.1105/tpc.5.4.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bustos M. M., Guiltinan M. J., Jordano J., Begum D., Kalkan F. A., Hall T. C. Regulation of beta-glucuronidase expression in transgenic tobacco plants by an A/T-rich, cis-acting sequence found upstream of a French bean beta-phaseolin gene. Plant Cell. 1989 Sep;1(9):839–853. doi: 10.1105/tpc.1.9.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buzby J. S., Yamada T., Tobin E. M. A light-regulated DNA-binding activity interacts with a conserved region of a Lemna gibba rbcS promoter. Plant Cell. 1990 Aug;2(8):805–814. doi: 10.1105/tpc.2.8.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carrasco P., Manzara T., Gruissem W. Developmental and organ-specific changes in DNA-protein interactions in the tomato rbcS3B and rbcS3C promoter regions. Plant Mol Biol. 1993 Jan;21(1):1–15. doi: 10.1007/BF00039613. [DOI] [PubMed] [Google Scholar]
  5. Connelly S., Filipowicz W. Activity of chimeric U small nuclear RNA (snRNA)/mRNA genes in transfected protoplasts of Nicotiana plumbaginifolia: U snRNA 3'-end formation and transcription initiation can occur independently in plants. Mol Cell Biol. 1993 Oct;13(10):6403–6415. doi: 10.1128/mcb.13.10.6403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cooke R., Penon P. In vitro transcription from cauliflower mosaic virus promoters by a cell-free extract from tobacco cells. Plant Mol Biol. 1990 Mar;14(3):391–405. doi: 10.1007/BF00028775. [DOI] [PubMed] [Google Scholar]
  7. Datta N., Cashmore A. R. Binding of a pea nuclear protein to promoters of certain photoregulated genes is modulated by phosphorylation. Plant Cell. 1989 Nov;1(11):1069–1077. doi: 10.1105/tpc.1.11.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Frohnmeyer H., Hahlbrock K., Schäfer E. A light-responsive in vitro transcription system from evacuolated parsley protoplasts. Plant J. 1994 Mar;5(3):437–449. doi: 10.1111/j.1365-313x.1994.00437.x. [DOI] [PubMed] [Google Scholar]
  9. Gorski K., Carneiro M., Schibler U. Tissue-specific in vitro transcription from the mouse albumin promoter. Cell. 1986 Dec 5;47(5):767–776. doi: 10.1016/0092-8674(86)90519-2. [DOI] [PubMed] [Google Scholar]
  10. Green P. J., Kay S. A., Chua N. H. Sequence-specific interactions of a pea nuclear factor with light-responsive elements upstream of the rbcS-3A gene. EMBO J. 1987 Sep;6(9):2543–2549. doi: 10.1002/j.1460-2075.1987.tb02542.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grummt I., Roth E., Paule M. R. Ribosomal RNA transcription in vitro is species specific. Nature. 1982 Mar 11;296(5853):173–174. doi: 10.1038/296173a0. [DOI] [PubMed] [Google Scholar]
  12. Herrera-Estrella L., Van den Broeck G., Maenhaut R., Van Montagu M., Schell J., Timko M., Cashmore A. Light-inducible and chloroplast-associated expression of a chimaeric gene introduced into Nicotiana tabacum using a Ti plasmid vector. Nature. 1984 Jul 12;310(5973):115–120. doi: 10.1038/310115a0. [DOI] [PubMed] [Google Scholar]
  13. Jacobsen K., Laursen N. B., Jensen E. O., Marcker A., Poulsen C., Marcker K. A. HMG I-like proteins from leaf and nodule nuclei interact with different AT motifs in soybean nodulin promoters. Plant Cell. 1990 Jan;2(1):85–94. doi: 10.1105/tpc.2.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jendrisak J., Guilfoyle T. J. Eukaryotic RNA polymerase: comparative subunit structures, immunological properties, and alpha-amanitin sensitivities of the class II enzymes from higher plants. Biochemistry. 1978 Apr 4;17(7):1322–1327. doi: 10.1021/bi00600a029. [DOI] [PubMed] [Google Scholar]
  15. Jensen E. Ø, Marcker K. A., Schell J., Bruijn F. J. Interaction of a nodule specific, trans-acting factor with distinct DNA elements in the soybean leghaemoglobin Ibc(3) 5' upstream region. EMBO J. 1988 May;7(5):1265–1271. doi: 10.1002/j.1460-2075.1988.tb02940.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jofuku K. D., Okamuro J. K., Goldberg R. B. Interaction of an embryo DNA binding protein with a soybean lectin gene upstream region. Nature. 1987 Aug 20;328(6132):734–737. doi: 10.1038/328734a0. [DOI] [PubMed] [Google Scholar]
  17. Jordano J., Almoguera C., Thomas T. L. A sunflower helianthinin gene upstream sequence ensemble contains an enhancer and sites of nuclear protein interaction. Plant Cell. 1989 Sep;1(9):855–866. doi: 10.1105/tpc.1.9.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kadonaga J. T. Assembly and disassembly of the Drosophila RNA polymerase II complex during transcription. J Biol Chem. 1990 Feb 15;265(5):2624–2631. [PubMed] [Google Scholar]
  19. Kamakaka R. T., Tyree C. M., Kadonaga J. T. Accurate and efficient RNA polymerase II transcription with a soluble nuclear fraction derived from Drosophila embryos. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):1024–1028. doi: 10.1073/pnas.88.3.1024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Laursen N. B., Larsen K., Knudsen J. Y., Hoffmann H. J., Poulsen C., Marcker K. A., Jensen E. O. A protein binding AT-rich sequence in the soybean leghemoglobin c3 promoter is a general cis element that requires proximal DNA elements to stimulate transcription. Plant Cell. 1994 May;6(5):659–668. doi: 10.1105/tpc.6.5.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Li Y., Nagayoshi S., Sugita M., Sugiura M. Structure and expression of the tobacco nuclear gene encoding the 33 kDa chloroplast ribonucleoprotein. Mol Gen Genet. 1993 May;239(1-2):304–309. doi: 10.1007/BF00281632. [DOI] [PubMed] [Google Scholar]
  22. Linard C. G., Mbikay M., Seidah N. G., Chretien M. Primary structure of mouse chromogranin B deduced from cDNA sequence. Nucleic Acids Res. 1990 Mar 11;18(5):1298–1298. doi: 10.1093/nar/18.5.1298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lue N. F., Kornberg R. D. Accurate initiation at RNA polymerase II promoters in extracts from Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8839–8843. doi: 10.1073/pnas.84.24.8839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Manzara T., Carrasco P., Gruissem W. Developmental and organ-specific changes in promoter DNA-protein interactions in the tomato rbcS gene family. Plant Cell. 1991 Dec;3(12):1305–1316. doi: 10.1105/tpc.3.12.1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ohme-Takagi M., Shinshi H. Structure and expression of a tobacco beta-1,3-glucanase gene. Plant Mol Biol. 1990 Dec;15(6):941–946. doi: 10.1007/BF00039434. [DOI] [PubMed] [Google Scholar]
  26. Roberts M. W., Okita T. W. Accurate in vitro transcription of plant promoters with nuclear extracts prepared from cultured plant cells. Plant Mol Biol. 1991 May;16(5):771–786. doi: 10.1007/BF00015070. [DOI] [PubMed] [Google Scholar]
  27. Shapiro D. J., Sharp P. A., Wahli W. W., Keller M. J. A high-efficiency HeLa cell nuclear transcription extract. DNA. 1988 Jan-Feb;7(1):47–55. doi: 10.1089/dna.1988.7.47. [DOI] [PubMed] [Google Scholar]
  28. Sugita M., Gruissem W. Developmental, organ-specific, and light-dependent expression of the tomato ribulose-1,5-bisphosphate carboxylase small subunit gene family. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7104–7108. doi: 10.1073/pnas.84.20.7104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sugita M., Manzara T., Pichersky E., Cashmore A., Gruissem W. Genomic organization, sequence analysis and expression of all five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato. Mol Gen Genet. 1987 Sep;209(2):247–256. doi: 10.1007/BF00329650. [DOI] [PubMed] [Google Scholar]
  30. Sugiura M. The chloroplast genome. Plant Mol Biol. 1992 May;19(1):149–168. doi: 10.1007/BF00015612. [DOI] [PubMed] [Google Scholar]
  31. Sun L., Doxsee R. A., Harel E., Tobin E. M. CA-1, a novel phosphoprotein, interacts with the promoter of the cab140 gene in Arabidopsis and is undetectable in det1 mutant seedlings. Plant Cell. 1993 Jan;5(1):109–121. doi: 10.1105/tpc.5.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Susek R. E., Ausubel F. M., Chory J. Signal transduction mutants of Arabidopsis uncouple nuclear CAB and RBCS gene expression from chloroplast development. Cell. 1993 Sep 10;74(5):787–799. doi: 10.1016/0092-8674(93)90459-4. [DOI] [PubMed] [Google Scholar]
  33. Waibel F., Filipowicz W. RNA-polymerase specificity of transcription of Arabidopsis U snRNA genes determined by promoter element spacing. Nature. 1990 Jul 12;346(6280):199–202. doi: 10.1038/346199a0. [DOI] [PubMed] [Google Scholar]
  34. Waibel F., Filipowicz W. 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. 1990 Jun 25;18(12):3451–3458. doi: 10.1093/nar/18.12.3451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yamazaki K., Katagiri F., Imaseki H., Chua N. H. TGA1a, a tobacco DNA-binding protein, increases the rate of preinitiation complex formation in a plant in vitro transcription system [corrected]. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7035–7039. doi: 10.1073/pnas.87.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yamazaki K, Imamoto F. Selective and accurate initiation of transcription at the T-DNA promoter in a soluble chromatin extract from wheat germ. Mol Gen Genet. 1987 Oct;209(3):445–452. doi: 10.1007/BF00331148. [DOI] [PubMed] [Google Scholar]

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