Skip to main content
SpringerLink
Log in

cry IA(b) transcript formation in tobacco is inefficient

  • Research Article
  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Chimaeric PCaMV35S cry genes direct in tobacco mesophyll protoplasts mRNA levels of less than one transcript per cell. We provide evidence that this low cytoplasmic cry IA(b) mRNA level is not due to a rapid turnover but rather results from a marginal import flow of cry messenger into the cytoplasm. Run-on assays indicate that the frequency of transcription initiation is not limiting. However, the cry precursor mRNA carries at least three regions that are recognized as introns. The absence of high cytoplasmic levels of spliced cry mRNAs suggests that these mRNAs are unstable and/or not efficiently made. Point mutations in the 5′ splice site of the most distal intron allows high accumulation levels of the full-length mRNA. This implies that the inefficient formation of full-size mRNA is a major cause of the low expression level of chimaeric cry IA(b) genes in tobacco.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Bandyopadhyay R, Coutts M, Krowczynska A, Brawerman G: Nuclease activity associated with mammalian mRNA in its native state: possible basis for selectivity in mRNA decay. Mol Cell Biol 10: 2060–2069 (1990).

    Google Scholar 

  2. Bossé M, Masson L, Brousseau R: Nucleotide sequence of a novel crystal protein gene isolated from Bacillus thuringiensis subspecies kenyae. Nucl Acids Res 18: 7443 (1990).

    Google Scholar 

  3. Brizzard B, Whiteley H: Nucleotide sequence of an additional crystal protein gene cloned from Bacillus thuringiensis subsp. Thuringiensis. Nucl Acids Res 16: 2723 (1988).

    Google Scholar 

  4. Burgess SM, Guthrie C: A mechanism to enhance mRNA splicing fidelity: The RNA-dependent ATPase Prp 16 governs usage of a discard pathway for aberrant lariat intermediates. Cell 73: 1377–1391 (1993).

    Google Scholar 

  5. Celano P, Berchtold C, Casero R: A simplification of the nuclear run-off transcription assay. Bio Techniques 7: 942–943 (1989).

    Google Scholar 

  6. Chen Z, Harless M, Wright D, Kellems R: Identification and characterisation of transcriptional arrest sites in exon 1 of the human adenosine deaminase gene. Mol Cell Biol 10: 4555–4564 (1990).

    Google Scholar 

  7. Cornelissen M: Nuclear and cytoplasmic sites for antisense control. Nucl Acids Res 17: 7203–7209 (1989).

    Google Scholar 

  8. Cornelissen M, Vandewiele M: Both RNA level and translation efficiency are reduced by anti-sense RNA in transgenic tobacco. Nucl Acids Res 17: 833–843 (1989).

    Google Scholar 

  9. Cox S, Goldberg R: Analysis of plant gene expression, In: Shaw C (ed) Plant Molecular Biology: A Practical Approach, pp. 1–35. IRL Press (1989).

  10. Danthinne X, Seurinck J, Meulewaeter F, Van Montagu M, Cornelissen M: The 3′ untranslated region of satellite tobacco necrosis virus RNA stimulates translation in vitro. Mol Cell Biol 13: 3340–3349 (1993).

    Google Scholar 

  11. Denecke J, Gosselé V, Botterman J, Cornelissen M: Quantitative analysis of transiently expressed genes in plant cells. Meth Mol Cell Biol 1: 19–26 (1989).

    Google Scholar 

  12. Foley PF, Leonard MW, Engel JD: Quantitation of RNA using the polymerase chain reaction. Trends Genet 9: 380–385 (1993).

    Google Scholar 

  13. Gallie D: The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Devel 5: 2108–2116 (1991).

    Google Scholar 

  14. Gallie D, Lucas W, Walbot V: Visualising mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation. Plant Cell 1: 301–311 (1989).

    Google Scholar 

  15. Gallie D, Walbot V: RNA pseudoknot domain of tobacco mosaic virus can functionally substitute for a poly(A) tail in plant and animal cells. Genes Devel 4: 1149–1157 (1990).

    Google Scholar 

  16. Green PJ: Control of mRNA stability in higher plants. Plant Physiol 102: 1065–1070 (1993).

    Google Scholar 

  17. Herrick D, Parker R, Jacobson A: Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae. Mol Cell Biol 10: 2269–2284 (1990).

    Google Scholar 

  18. Herschlag D, Johnson F: Synergism in transcriptional activation: a kinetic view. Genes Devel 7: 173–179 (1993).

    Google Scholar 

  19. Hoekema A, Kastelein R, Vasser M, Boer H: Condon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression. Mol Cell Biol 7: 2914–2924 (1987).

    Google Scholar 

  20. Höfte H, Whitely H: Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53: 242–255 (1989).

    Google Scholar 

  21. Honée G, van der Salm T, Visser B: Nucleotide sequence of crystal protein gene isolated from B. thuringiensis subspecies entomocidus 60.5 coding for a toxin highly active against Spodoptera species. Nucl Acids Res 16: 6240 (1988).

    Google Scholar 

  22. Horton R, Hunt H, Ho S, Pullen J, Pease L: Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77: 61–68 (1989).

    Google Scholar 

  23. Joshi C: An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucl Acids Res 15: 6643–6653 (1987).

    Google Scholar 

  24. Kawasaki ES: Amplification of RNA, In: Innes MG, Gelfand D, Sninski J, White T, (eds) PCR protocols: A guide to methods and applications, pp. 21–27. Academic Press (1990).

  25. Kruys V, Marnix O, Shaw G, Deschamps J, Hue G: Translational blockade imposed by cytokine-derived UA-rich sequences. Science 245: 852–855 (1989).

    Google Scholar 

  26. Laird P, Zomerdijk J, de Korte D, Borst P: In vivo labelling of intermediates in the discontinuous synthesis of mRNAs in Trypanosoma brucei. EMBO J 6: 1055–1062 (1987).

    Google Scholar 

  27. Maas C, Laufs J, Grant S, Korfhage C, Werr W: The combination of a novel stimulatory element in the first exon of the maize Shrunken-1 gene with the following intron 1 enhances reporter gene expression up to 1000-fold. Plant Mol Biol 16: 199–207 (1991).

    Google Scholar 

  28. Maliga P, Breznovitz A, Marton L: Streptomycin resistant plants from callus culture of haploid tobacco. Nature 244: 29–30 (1973).

    Google Scholar 

  29. Malter J: Identification of an AUUUA-specific messenger RNA binding protein. Science 246: 664–666 (1989).

    Google Scholar 

  30. Maxam A, Gilbert W: Sequencing end-labeled DNA with base-specific chemical cleavages. Meth Enzymol 65: 499–560 (1980).

    Google Scholar 

  31. Murray E, Lotzer J, Eberle M: Codon usage in plant genes. Nucl Acids Res 17: 477–498 (1989).

    Google Scholar 

  32. Murray E, Rocheleau T, Eberle M, Stock C, Sekar V, Adang M: Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplasts. Plant Mol Biol 16: 1035–1050 (1991).

    Google Scholar 

  33. Neill J, Litts J, Anderson O, Greene F, Stiles J: Expression of a wheat gliadin gene in Saccharomyces cerevisiae. Gene 55: 303–317 (1987).

    Google Scholar 

  34. Perlak F, Deaton R, Armstrong T, Fuchs R, Sims R, Greenplate J, Fischhoff D: Insect resistant cotton. Bio/technology 8: 939–943 (1990).

    Google Scholar 

  35. Perlak F, Fuchs R, Dean D, McPherson S, Fischhoff D: Modification of the coding sequence enhances plant expression of insect control protein genes. Proc Natl Acad Sci USA 88: 3324–3328 (1991).

    Google Scholar 

  36. Sanger F, Nicklen S, Coulson A: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).

    Google Scholar 

  37. Schuler G, Cole M: GM-CSF and oncogene stabilities are independently regulated in trans in a mouse monocytic tumor. Cell 55: 1115–1122 (1988).

    Google Scholar 

  38. Sharp P, Devine K: Codon usage and gene expression in Dictyostelium discodeum: highly expressed genes do ‘prefer’ optimal codons. Nucl Acids Res 17: 5029–5039 (1989).

    Google Scholar 

  39. Shaw G, Kamen R: A conserved AU sequence from the 3′ untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46: 659–667 (1986).

    Google Scholar 

  40. Urlaub G, Mitchell P, Ciudad C, Chasin L: Nonsense mutations in the dihydrofolate reductase gene affect RNA processing. Mol Cell Biol 9: 2868–2880 (1989).

    Google Scholar 

  41. Vaeck M, Reynaerts A, Hofte H: Protein engineering in plants: expression of Bacillus thuringiensis insecticidal protein genes. Cell Cult Somatic Genet Plants 6: 425–439 (1989).

    Google Scholar 

  42. Vaeck M, Reynaerts A, Hofte H, Jansens S, De Beuckeleer M, Dean C, Zabeau M, Van Montagu M, Leemans L: Transgenic plants protected from insect attack. Nature 327: 33–37 (1987).

    Google Scholar 

  43. Yanisch-Perron C, Vieira J, Messing J: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103–119 (1985).

    Google Scholar 

  44. Wilson T, Treisman R: Removal of poly(A) and consequent degradation of c-fos mRNA facilitated by 3′ AU-rich sequences. Nature 336: 396–399 (1988).

    Google Scholar 

  45. Wolin S, Walter P: Ribosome pausing and stacking during translation of a eucaryotic mRNA. EMBO J 7: 3559–3569 (1988).

    Google Scholar 

  46. Zacharias DA, Garamszegi N, Strehler EE: Characterization of persistent artifacts resulting from RT-PCR of alternative spliced mRNAs. Bio Techniques 17: 652–655 (1994).

    Google Scholar 

Download references

Author information

Author notes

  1. Maike Stam

    Present address: Dept. of Genetics, Free University, De Boelelaan 1087, 1081 HV, Amsterdam, Netherlands

  2. Jan Dockx

    Present address: Dept. of Molecular and Cellular Biology, Utrecht University, Padualaan 8, 3584 CH, Utrecht, Netherlands

Authors and Affiliations

  1. Plant Genetic Systems NV, J. Plateaustraat 22, B9000, Gent, Belgium

    Roel van Aarssen, Piet Soetaert, Maike Stam, Jan Dockx, Veronique Gosselé, Jef Seurinck, Arlette Reynaerts & Marc Cornelissen

Authors
  1. Roel van Aarssen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Piet Soetaert
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maike Stam
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jan Dockx
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Veronique Gosselé
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jef Seurinck
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Arlette Reynaerts
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Marc Cornelissen
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

van Aarssen, R., Soetaert, P., Stam, M. et al. cry IA(b) transcript formation in tobacco is inefficient. Plant Mol Biol 28, 513–524 (1995). https://doi.org/10.1007/BF00020398

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00020398

Key words

Search

Navigation