Skip to main content
SpringerLink
Log in

Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplasts

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

We have examined expression of several insecticidal crystal protein (ICP) genes of Bacillus thuringiensis in transgenic tobacco plants and electroporated carrot protoplasts. We determined that low levels of lepidopteran toxin cryIA(b) ICP gene expression in plants and electroporated carrot cells is due to RNA instability. We used a series of 3′ deleted cryIA(b) constructs directed by the cauliflower mosaic virus 35S promoter to demonstrate that this instability is minimally contained in the first 579 bases of the gene in both systems. This instability may result from 5′ → 3′ as well as 3′ → 5′ RNA metabolism. The coleopteran toxic cryIIIA gene was also examined in electroporated carrot cells, and found to be poorly expressed. A model for improvement of ICP RNA stability in plants is presented.

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. Adang MJ, Firoozabady E, Klein J, DeBoer D, Sekar V, Kemp JD, Murray EE, Rocheleau TA, Rashka K, Staffeld G, Stock C, Sutton D, Merlo DJ: Expression of a Bacillus thuringiensis insecticidal crystal protein gene in tobacco plants. In: Arntzen CJ, Ryan C (eds) Molecular Strategies for Crop Protection. UCLA Symposia on Molecular and Cellular Biology, New Series, Vol 46, pp. 345–353. Alan R. Liss, New York (1987).

    Google Scholar 

  2. Adang MJ, Idler KF, Rocheleau TA: Structural and antigenic relationships among three insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki. In: Maramorosch K (ed) Biotechnology in Invertebrate Pathology and Cell Culture, pp. 85–99. Academic Press, San Diego, CA (1987).

    Google Scholar 

  3. Adang MJ, DeBoer D, Endres J, Firoozabady E, Klein J, Merlo A, Merlo D, Murray EE, Rashka K, Stock C: Manipulation of Bacillus thuringiensis genes for pest insect control. In: Roberts DW, Granados RR (eds) Biotechnology, Biological Pesticides and Novel Plant-Pest Resistance for Insect Pest Management, pp. 31–37. Boyce Thompson Institute for Plant Research, Cornell Univ. Ithaca New York (1989).

    Google Scholar 

  4. Ainley WM, Key JL: Development of a heat shock inducible expression cassette for plants: Characterization of parameters for its use in transient expression assays. Plant Mol Biol 14: 949–967 (1990).

    PubMed  Google Scholar 

  5. Barker RF, Idler KB, Thompson DV, Kemp JD: Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol Biol 2: 335–350 (1983).

    Google Scholar 

  6. Barton KA, Whitely HR, Yang Y-S: Bacillus thuringiensis delta-endotoxin expressed in transgenic Nicotiana tabacum provides resistance to lepidopteran insects. Plant Physiol 85: 1103–1109 (1987).

    Google Scholar 

  7. Baserga SJ, Benz EJJr: Nonsense mutations in the human β-globin gene affect mRNA metabolism. Proc Natl Acad Sci USA 85: 2056–2060 (1988).

    PubMed  Google Scholar 

  8. Baumann B, Potash MJ, Kohler G: Consequences of frameshift mutations at the immunoglobulin heavy chain locus of the mouse. EMBO J 4: 351–359 (1985).

    PubMed  Google Scholar 

  9. Bennetzen JL, Hall BD: Codon selection in yeast. J Biol Chem 257: 3026–3031 (1982).

    PubMed  Google Scholar 

  10. Bernstein P, Peltz SW, Ross J: The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro. Mol Cell Biol 9: 659–670 (1989).

    PubMed  Google Scholar 

  11. Brewer G, Ross J: Regulation of c-myc mRNA stability in vitro by a labile destabilizer with an essential nucleic acid component. Mol Cell Biol 9: 1996–2006.

  12. Daar IO, Maquat LE: Premature translation termination mediates triosephosphate isomerase mRNA degradation. Mol Cell Biol 8: 802–813 (1988).

    PubMed  Google Scholar 

  13. de boer HA, Kastelein RA: biased codon usage: an exploration of its role in optimization of translation. In: Davis JJ, Reznikiff B, Gold L (eds). From gene to protein: steps dictating the maximal level of gene expression, pp. 225–283. Butterworths, New York.

  14. Fischhoff DA, Bowdish KS, Perlak FJ, Marrone PG, McCormick SM, Niedermeyer JG, Dean DA, Kusano-Kretzmer K, Mayer EJ, Rochester DE, Rogers SG, Fraley RT: Insect tolerant transgenic tomato plants. Bio/technology 5: 807–813 (1987).

    Article  Google Scholar 

  15. Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson TMA: The 5′-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. Nucl Acids Res 15: 3257–3273 (1987).

    PubMed  Google Scholar 

  16. Gay DA, Yen TJ, Lau JTY, Cleveland DW: Sequences that confer β-tubulin autoregulation through modulated mRNA stability reside within exon 1 of a β-tubulin mRNA. Cell 50: 671–679 (1987).

    Article  PubMed  Google Scholar 

  17. Greenberg ME, Hermanowski AL, Ziff EB: Effect of protein synthesis inhibitors on growth factor activation of c-fos, c-myc and actin gene transcription. Mol Cell Biol 6: 1050–1057 (1986).

    PubMed  Google Scholar 

  18. Gubits RM, Fairhurst JL: c-fos mRNA levels are increase by the cellular stressors, heat shock and sodium arsenite. Oncogene 3: 163–168 (1988).

    PubMed  Google Scholar 

  19. Harland R, Misher L: Stability of RNA in Xenopus embryos and identification of a destabilizing sequence in TFIIIA messenger RNA. Development 102: 837–852 (1988).

    PubMed  Google Scholar 

  20. Hoekema A, Kastelein RA, Vasser M, DeBoer HA: Codon 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).

    PubMed  Google Scholar 

  21. Hoffman LM, Donaldson DD, Bookland R, Rashka K, Herman EM: Synthesis and protein body deposition of maize 15-kd zein in transgenic tobacco seeds. EMBO J 6: 3213–3221 (1987).

    Google Scholar 

  22. Hofte H, Whitely HR: Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 2: 242–255 (1989).

    Google Scholar 

  23. Horsch RB, Fry JE, Hoffman NL, Wallroth M, Eichholtz DA, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).

    Google Scholar 

  24. Jobling SA, Gehrke L: Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequence. Nature 325: 622–625 (1987).

    Article  PubMed  Google Scholar 

  25. Jofuku KD, Schipper RD, Goldberg RB: A frameshift mutation prevents Kunitz trypsin inhibitor mRNA accumulation in soybean embryos. Plant Cell 1: 427–425 (1989).

    Article  PubMed  Google Scholar 

  26. Kabnick KS, Housman DE: Determinants that contribute to cytoplasmic stability of human c-fos and β-globin mRNAs are located at several sites in each mRNA. Mol Cell Biol 8: 3244–3250 (1988).

    PubMed  Google Scholar 

  27. Kimple JA, Keys JL: Heat shock in plants. TIBS 10: 353–357 (1985).

    Google Scholar 

  28. Kozak M: Compilation and analysis sequences upstream from the translational start site in eukaryotic mRNAs. Nucl Acids Res 12: 857–872 (1984).

    PubMed  Google Scholar 

  29. Kozak M: Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44: 283–292 (1986).

    Article  PubMed  Google Scholar 

  30. Lim SK, Mullins JJ, Chen C-M, Gross KW, Maquat LE: Novel metabolism of several 1049–1 β-globin mRNAs in the erythroid tissues of transgenic mice. EMBO J 8: 2613–2619 (1989).

    PubMed  Google Scholar 

  31. Losson R, Lacroute F: Interference of nonsense mutations with eukaryotic messenger RNA stability. Proc Natl Acad Sci USA 76: 5134–5137 (1979).

    PubMed  Google Scholar 

  32. Maquat LE, Kinniburgh AJ, Rachmilewitz EA, Ross J: Cell 27: 543–553 (1981).

    Article  PubMed  Google Scholar 

  33. Marsh JL, Erfle M, Wykes EJ: The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene 32: 481–485 (1984).

    Article  PubMed  Google Scholar 

  34. Müller R, Bravo R, Burckhardt J, Curran T: Induction of c-fos gene and protein by growth factors precedes activation of c-myc. Nature 312: 716–720 (1984).

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  36. Murray EE, Buchholz WG, Bowen B: Direct analysis of RNA transcripts in electroporated carrot cells. Plant Cell Rep 9: 129–132 (1990).

    Article  Google Scholar 

  37. Murray MG, Hoffman LH, Jarvis NP: Improved yield of full-length phaseolin cDNA clones by controlling premature anticomplementary DNA synthesis. Plant Mol Biol 2: 75–84 (1983).

    Google Scholar 

  38. Murray MG: Use of sodium trichloroacetate and mung bean nuclease to increase sensitivity and precision during transcript mapping. Analyt Biochem 158: 165–170 (1985).

    Google Scholar 

  39. Norris DM, Kogan M: Biochemical and morphological bases of resistance. In: Maxwell FG, Jennings PR (eds) Breeding Plants Resistant to Insects, pp. 29–61. John Wiley & Sons, New York (1980).

    Google Scholar 

  40. Odell JT, Nagy F, Chua N-H: Identification of DNA sequences required for the activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810–812 (1985).

    PubMed  Google Scholar 

  41. Parker R, Jacobson A: Translation and a 42-nucleotide segment within the coding region of the mRNA encoded by the matα1 gene are involved in promoting rapid mRNA decay in yeast. Proc Natl Acad Sci USA 87: 2780–27814 (1990).

    PubMed  Google Scholar 

  42. Pedersen S: Escherichia coli ribosomes translate in vivo with variable rate. EMBO J 3: 2895–2898 (1984).

    PubMed  Google Scholar 

  43. Perlak FJ, Deaton RW, Armstrong TA, Fuchs RL, Sims SR, Greenplate JT, Fischhoff DA: Insect resistant cotton plants. Bio/technology 8: 939–943 (1990).

    Article  PubMed  Google Scholar 

  44. Pröls M, Töpfer R, Schell J, Steinbiss H-H: Transient gene expression in tobacco protoplasts: I. Time course of CAt appearance. Plant Cell Rep. 7: 221–224 (1988).

    Google Scholar 

  45. Raghow R: Regulation of messenger RNA turnover in eukaryotes. Trends Biochem. Sci 12: 358–360 (1987).

    Article  Google Scholar 

  46. Ross J: Messenger RNA turnover in eukaryotic cells. Mol Biol Med 5: 1–14 (1988).

    PubMed  Google Scholar 

  47. Scallon BJ, Dickinson CD, Nielsen NC: Characterization of a null-allele for the GY 4 glycinin gene from soybean. Mol Gen Genet 208: 107–113 (1987).

    Article  Google Scholar 

  48. Sekar V, Thompson DV, Maroney MJ, Bookland RG, Adang MJ: Molecular cloning and characterization of the insecticidal crystal protein gene of Bacillus thuringiensis var. tenebionis. Proc Natl Acad Sci USA 84: 7036–7040 (1987).

    Google Scholar 

  49. Shapiro DJ, Blume JE, Nielsen DA: Regulation of messenger RNA stability in eukaryotic cells. Bioessays 6: 221–226 (1987).

    PubMed  Google Scholar 

  50. Sharp PM, Li W-HL: Codon usage in regulatory genes in Escherichia coli does not reflect selection for ’rare‘ codons. Nucl Acids Res. 14: 7737–7749 (1986).

    PubMed  Google Scholar 

  51. Sharp PM, Touhy TMF, Mosurski KR: Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes. Nucl Acids Res. 14: 5125–5143 (1986).

    PubMed  Google Scholar 

  52. Shaw G, Kamen R: A conserved AU sequence from the 3′ untranslated region of GM-CSF mediates selective mRNA degradation. Cell 46: 659–667 (1987).

    Article  Google Scholar 

  53. Shyu A-B, Greenberg ME, Belasco JG: The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways. Genes Devel 3: 60–72 (1989).

    PubMed  Google Scholar 

  54. Sleat DE, Gallie DR, Jefferson RA, Bevan MW, Turner PC, Wilson TMA: Characterization of the 5′-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro. Gene 217: 217–225 (1987).

    Article  Google Scholar 

  55. Smith HO: Recovery of DNA from gels. Meth Enzymol 65: 371–380 (1980).

    PubMed  Google Scholar 

  56. Swartout SG, Kinniburgh AJ: c-myc RNA degradation in growing and differentiating cells: possible alternative pathways. Mol Cell Biol 9: 288–295 (1989).

    PubMed  Google Scholar 

  57. Taylor JL, Jones JDG, Sandler S, Mueller GM, Bedbrook J, Dunsmuir P: Optimizing the expression of chimeric genes in plant cells. Mol Gen Genet 210: 572–577 (1987).

    Article  Google Scholar 

  58. Vaeck M, Reynaerts A, Höfte H, Jansens S, DeBeukeleer M, Dean C, Zabeau M, Van Montagu M, Leemans J: Transgenic plants protected from insect attack. Nature 328: 33–37 (1987).

    Article  Google Scholar 

  59. Vancanneyt G, Rosahl S, Willmitzer L: Translatability of a plant-mRNA strongly influences its accumulation in transgenic plants. Nucl Acids Res 18: 2917–2921 (1990).

    PubMed  Google Scholar 

  60. Voelker TA, Moreno J, Chrispeels MJ: Expression analysis of a pseudogene in transgenic tobacco: A frameshift mutation prevents mRNA accumulation. Plant Cell 2: 255–261 (1990).

    Article  PubMed  Google Scholar 

  61. Whiteley HR, Schnepf HR: The molecular biology of parasporal crystal body formation in Bacillus thuringiensis. Annu Rev Microbiol 40: 549–576 (1986).

    Article  PubMed  Google Scholar 

  62. Wong HC, Chang S: Identification of a positive retro-regulator that stabilizes mRNAs in bacteria, Proc Natl Acad Sci USA 83: 3233–3237 (1986).

    PubMed  Google Scholar 

Download references

Author information

Author notes

  1. Elizabeth E. Murray

    Present address: McArdle Laboratory for Cancer Research, University of Wisconsin, 53705, Madison, USA

  2. Thomas Rocheleau

    Present address: Department of Entomology, University of Wisconsin, 53705, Madison, USA

  3. Mary Eberle

    Present address: Blood Center of Southeast Wisconsin, 53233, Milwaukee, WI, USA

  4. Carolyn Stock

    Present address: Biotechnology Center, University of Wisconsin, 53705, Madison, USA

  5. V. Sekar

    Present address: Department of Biotechnology, Madurai Kamaraj University, 625021, Madurai, India

  6. Michael Adang

    Present address: Department of Entomology, University of Georgia, 30605, Athens, GA, USA

Authors and Affiliations

  1. Agrigenetics Advanced Research Corporation, 5649 Buckeye Road, 53716, Madison, WI, USA

    Elizabeth E. Murray, Thomas Rocheleau, Mary Eberle, Carolyn Stock, V. Sekar & Michael Adang

Authors
  1. Elizabeth E. Murray
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas Rocheleau
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mary Eberle
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Carolyn Stock
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. V. Sekar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael Adang
    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

Murray, E.E., Rocheleau, T., Eberle, M. et al. 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). https://doi.org/10.1007/BF00016075

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00016075

Key words

Search

Navigation