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The expression of a nopaline synthase — human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue

Plant Molecular Biology volume 6, pages 347–357 (1986)Cite this article

Summary

To study whether mammalian RNA processing signals function in plants, we have constructed a chimaeric gene in which the complete human growth hormone (hGH) gene is flanked by DNA fragments containing the promoter and polyadenylation site of the nopaline synthase gene. The hGH gene used contains four introns and an additional 440 bp downstream from the hGH poly(A) addition site. The transcription of this chimaeric gene was studied following its introduction into sunflower and tobacco cells using a Ti plasmid vector. Analysis of poly(A)+ RNA isolated from the transformed tumor tissue demonstrated the following: (1) a single polyadenylated transcript, 2700 bp in length, was transcribed from the chimaeric gene; (2) the transcription was initiated at the published start site of the nopaline synthase gene; (3) the hGH polyadenylation site was not used for processing of the 3′ end; only the poly(A) addition site of the nopaline synthase gene was recognized, (4) no splicing of the hGH introns could be detected. We also demonstrate that the hGH pre-mRNA isolated from plant cells can be spliced in a HeLa cell nuclear extract, indicating that the hGH pre-mRNA was functional. These results show that processing signals of the hGH pre-mRNA are not recognized in these plant cells.

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References

  1. Barton K, Binns A, Matzke AJM, Chilton M-D: Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA, and transmission of T-DNA to R1 progeny. Cell 32: 1033–1043, 1983.

    Google Scholar 

  2. Berget SM: Are U4 small nuclear ribonucleoproteins involved in polyadenylation. Nautere 309: 179–182, 1984.

    Google Scholar 

  3. Bevan MW, Chilton M-D: T-DNA of the Agrobacterium Ti- and Ri-plasmids. Ann Rev Genet 16: 357–384, 1982.

    Google Scholar 

  4. Birnboim HG, Doly J: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7: 1513–1523, 1979.

    Google Scholar 

  5. Breathnach R, Benoist C, O'Hare K, Gannon F, Chambon P: Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries. Proc Natl Acad Sci (USA) 75: 4853–4857, 1978.

    Google Scholar 

  6. Broglie R, Coruzzi G, Fraley RT, Rogers SG, Horsch RB, Niedermeyer JG, Fink CL, Flick JS, Chua N-H: Light-regulated expression of a pea ribulose-1,5-bisphosphate carboxylase small subunit gene in transformed plant cells. Science 224: 838–843.

  7. Caplan A, Herrera-Estrella L, Inze D, VanHaute E, Van Montagu M, Schell J, Zambryski P: Introduction of genetic material into plant cells. Science 222: 815–821, 1983.

    Google Scholar 

  8. Cramer JH, Lea K, Slightom JL: Expression of phaseolin cDNA genes in yeast under control of natural plant DNA sequences. Proc Natl Acad Sci (USA) 82: 334–338, 1985.

    Google Scholar 

  9. DeGreve H, Leemans J, Hernalsteens J-P, Thia-Toong L, DeBeuckeleer M, Willmitzer L, Otten L, VanMontagu M, Schell J: Regeneration of normal and fertile plants that express octopine synthase from tobacco crown galls after deletion of tumor-controlling functions. Nature 300: 752–755, 1982.

    Google Scholar 

  10. Dennis ES, Gerlach WL, Prior AJ, Bennetzen JL, Inglis A, Llewellyn D, Sachs MM, Ferl RJ, Peacock WJ: Molecular analysis of the alcohol dehydrogenase 2 (Adh2) gene of maize. Nucleic Acids Res 12: 3983–4000, 1984.

    Google Scholar 

  11. Dennis ES, Sachs MM, Gerlach WL, Finnegan EJ, Peacock WJ: Molecular analysis of the alcohol dehydrogenase (Adh1) gene of maize. Nucleic Acids Res 13: 727–743, 1985.

    Google Scholar 

  12. Depicker A, Stachel S, Dhaese P, Zambryski P, Goodman HM: Nopaline synthase: Transcript mapping and DNA sequence. J Molec Appl Genet 1: 561–573, 1982.

    Google Scholar 

  13. Dignam JD, Lebovitz RM, Roeder RG: Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11: 1475–1489, 1983.

    Google Scholar 

  14. Domdey H, Apostol B, Lin RJ, Newman A, Brody E, Abelson J: Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell 39: 611–621, 1984.

    Google Scholar 

  15. Favaloro J, Treisman R, Kamen R: Transcription maps of polyoma specific RNA: analysis by two-dimensional nuclease S1 gel mapping. In: Grossman L, Moldave K (eds) Methods of Enzymology, Vol. 65, 1980, pp 718–749.

  16. Fitzgerald M, Shenk T: The sequences 5′-AAUAAA-3′ forms part of the recognition site for polyadenylation of late SV 40 mRNAs. Cell 24: 251–260, 1981.

    Google Scholar 

  17. Flint SJ: in: Apirion D (ed) Processing of RNA CRC, Boca Raton, FL, 1984, pp 151–179.

  18. Ghosh PK, Reddy VB, Piatak M, Lebovitz P, Weissman SM: Determination of RNA sequences by primer directed synthesis and sequencing of their cDNA transcripts. In: Grossman L, Moldave K (eds) Methods of Enzymology, Vol. 65, 1980, pp 580–595.

  19. Gil A, Proudfoot N: A sequences downstream of AAU-AAA is required for rabbit alpha-globin mRNAs 3′-end formation. Nature 312: 473–474, 1984.

    Google Scholar 

  20. Grabowski PJ, Padgett RA, Sharp PA: Messenger RNA splicing in vitro: An excised intervening sequence and a potential intermediate. Cell 37: 415–427, 1984.

    Google Scholar 

  21. Hall TC: Expression of plant genes in alien cellular environments. Oxford Surveys of Plant Molecular and Cell Biology 2: 329–338, 1985.

    Google Scholar 

  22. Hain R, Stabel P, Czernilofsky AP, Steinbiß HH, Herrera-Estrella L, Schell J: Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts. Mol Gen Genet 199: 161–168, 1985.

    Google Scholar 

  23. Jensen EO, Paludan K, Hyldig-Nielsen JJ, Jorgensen P, Marcker KA: The structure of a chromosomal leghemoglobin gene from soybean. Nature 291: 677–679, 1981.

    Google Scholar 

  24. Keller W: The RNA lariat: A new ring to the splicing of mRNA precursors. Cell 39: 423–425, 1984.

    Google Scholar 

  25. Koncz C, Kreuzaler F, Kalman ZS, Schell J: A simple method to transfer, integrate and study expression of foreign genes, such as chicken ovalbumin and alpha-actin in plant tumors. EMBO J 3: 1029–1037, 1984.

    Google Scholar 

  26. Krämer A, Keller W, Apel B, Lührman R: The 5′-terminus of the RNA moiety of U1 small nuclear ribonucleoprotein particles is required for the splicing of messenger RNA precursors. Cell 38: 299–307, 1984.

    Google Scholar 

  27. Langford CJ, Gallwitz D: Evidence for an intron-contained sequence required for the splicing of yeast RNA polymerase II transcripts. Cell 33: 519–527, 1983.

    Google Scholar 

  28. Langford CJ, Klinz FJ, Donath C, Gallwitz D: Point mutations identify the conserved, intron-contained TACTAAC box as an essential splicing signal sequence in yeast. Cell 36: 645–653, 1984.

    Google Scholar 

  29. Lycett GW, Croy RRD, Shirsat AH, Boulter D: The complete nucleotide sequence of a legumin gene from pea (Pisum sativum L). Nucleic Acids Res 12: 4493–4506, 1984.

    Google Scholar 

  30. Maniatis T, Fritsch E, Sambrook J: Molecular cloning: A Laboratory Mannual. Cold Spring Harbour Laboratory, Cold Spring Harbour, NY, 1982.

    Google Scholar 

  31. Manley JL: Accurate and specific polyadenylation of mRNA precursors in a soluble whole-cell lysate. Cell 33: 595–605, 1983.

    Google Scholar 

  32. Matzke AJM, Chilton M-D: Site specific insertion of genes into T-DNA of the Agrobacterium Ti plasmid: an approach to genetic engineering of higher plant cells. J Molec Appl Genet 1: 39–49, 1981.

    Google Scholar 

  33. Maxam AM, Gilbert W: Sequencing end-labelled DNA with basespecific chemical cleavages. In: Grossman L, Moldave K (eds) Methods in Enzymology, Vol. 65, 1980, pp 499–560.

  34. McDevitt MA, Imperiale MJ, Ali H, Nevins JR: Requirement of a downstream sequence for generation of a poly(A) addition site. Cell 37: 993–999, 1984.

    Google Scholar 

  35. McLauchlan J, Gaffney D, Whitton JL, Clements JB: The consensus sequence YGTGTTYY located downstream from the AATAAA signal is required for efficient formation of mRNA 3′-termini. Nucleic Acids Res 13: 1347–1368, 1985.

    Google Scholar 

  36. Messing J, Geraghty D, Heidecker G, Hu N, Kridl J, Rubenstein T: Plant gene structure. In: Kosuge T, Meredith CP, Hollaender A (eds) Genetic Engineering of Plants. 1983, pp 211–227.

  37. Montell C, Fisher EF, Caruthers MH, Berk AJ: Resolving the functions of overlapping viral genes by site-specific mutagenesis at mRNA splice site. Nature 305: 600–605, 1983.

    Google Scholar 

  38. Moore CL, Sharp PA: Site-specific polyadenylation in a cell-free reaction. Cell 36: 581–591, 1984.

    Google Scholar 

  39. Mount SM: A catalogue of splice junction sequences. Nucleic Acids Res 10: 459–472, 1982.

    Google Scholar 

  40. Mount SM, Petterson I, Hinterberger M, Karmas A, Steitz JA: The U1 small nuclear RNA-protein complex selectively binds a 5′-splice site in vitro. Cell 33: 509–518, 1983.

    Google Scholar 

  41. Murai N, Sutton D, Murray M, Slightom J, Merlo D, Reichert N, Sengupta-Gopalan C, Stock C, Barker R, Kemp J, Hall T: Phaseolin gene from bean is expressed after transfer to sunflower via tumor-inducing plasmid vectors Science (Wash) 222: 476–482, 1983.

    Google Scholar 

  42. Murray HG, Thompson WF: Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8: 4321–4325, 1980.

    Google Scholar 

  43. Otten L, DeGreve H, Hernalsteens JP, VanMontague M, Schieder O, Straub J, Schell J: Mendelian transmission of genes introduced into plants by the Ti plasmid: an approach to genetic engineering of higher plant cells. Mol Gen Genet 183: 209–213, 1981.

    Google Scholar 

  44. Padgett RA, Kornarska MM, Grabowsky PJ, Hardy SF, Sharp PA: Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors. Science (Wash) 205: 898–903, 1984.

    Google Scholar 

  45. Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn B, Portrykus I: Direct gene transfer to plants. EMBO J 3: 2717–2722, 1984.

    Google Scholar 

  46. Pikielny CW, Teem JL, Rosbash M: Evidence for the biochemical role of an internal sequence in yeast nuclear mRNA introns: implications for U1 RNA and metazoan mRNA splicing. Cell 34: 395–403, 1983.

    Google Scholar 

  47. Proudfoot NJ: The end of the message and beyond. Nature 307: 412–413, 1984.

    Google Scholar 

  48. Rautmann G, Breathnach R: A role for branch points in splicing in vivo. Nature 315: 430–432, 1985.

    Google Scholar 

  49. Ruskin B, Krainer AR, Maniatis T, Green MR: Excision of an intact intron as a novel lariat structure during pre-mRNA 3′-end formation. Cell 38: 317–331, 1984.

    Google Scholar 

  50. Seeburg PH: The human growth hormone gene family: nucleotide sequences show recent divergence and predict a new polypeptide hormone. DNA 1: 239–249, 1982.

    Google Scholar 

  51. Sengupta-Gopalan C, Reichert N, Barker R, Hall T, Kemp J: Developmentally regulated expression of the bean alphaphaseolin gene in tobacco seed. Proc Natl Acad Sci (USA) 82: 3320–3324, 1985.

    Google Scholar 

  52. Shah DM, Hightower RC, Meagher RB: Complete nucleotide sequence of a soybean actin gene. Proc Natl Acad Sci (USA), 79: 1022-S1026, 1982.

    Google Scholar 

  53. Shaw CH: Ti-plasmid-derived plant gene vectors. Oxford Survey of Plant Molecular and Cell Biology 1: 211–216, 1984.

    Google Scholar 

  54. Simonsen CC, Levinson AD: Analysis of processing and polyadenylation signals of the hepatitis B virus surface antigen gene by using simian virus 40-hepatitis B virus chimeric plasmids. Mol Cell Biol 3: 2250–2258, 1983.

    Google Scholar 

  55. Slightom JL, Sun SM, Hall TC: Complete nucleotide sequence of a French bean storage protein gene: phaseolin. Proc Natl Acad Sci (USA) 80: 1897–1901, 1983.

    Google Scholar 

  56. Taylor B, Powell A: Isolation of plant DNA and RNA. Focus 4: 4, 1982.

    Google Scholar 

  57. VanSanten VL, Spritz RA: mRNA precursor splicing in vivo: sequence requirements determined by deletion analysis of an intervening sequence. Proc Natl Acad Sci (USA) 82: 2885–2889, 1985.

    Google Scholar 

  58. Wickens M, Stephenson P: Role of the conserved AAU-AAA sequence: four AAUAAA point mutants prevent messenger RNA 3′-end, formation. Science (Wash) 226: 1045–1051, 1984.

    Google Scholar 

  59. Wieringa B, Hofer E, Weissmann C: A minimal intron length but no specific internal sequence is required for splicing the large rabbit alpha-globin intron. Cell 37: 915–925, 1984.

    Google Scholar 

  60. Woychick RP, Lyons RH, Prost L, Rottman FM: Requirement for the 3′-flanking region of the bovine growth hormone gene for accurate polyadenylation. Proc Natl Acad Sci (USA) 81: 3944–3948, 1984.

    Google Scholar 

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Affiliations

  1. Institut für Biochemie, Universität Wien, Währingerstraße 17, A-1090, Wien, Austria

    Andrea Barta, Karin Sommergruber, Diana Thompson & Klaus Hartmuth

  2. Institut für Molekularbiologie, Akademie der Wissenschaften, Billrothstraße 11, A-5020, Salzburg, Austria

    Marjori A. Matzke & Antionius J. M. Matzke

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  1. Andrea Barta
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  2. Karin Sommergruber
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  3. Diana Thompson
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  4. Klaus Hartmuth
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  5. Marjori A. Matzke
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  6. Antionius J. M. Matzke
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Barta, A., Sommergruber, K., Thompson, D. et al. The expression of a nopaline synthase — human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue. Plant Mol Biol 6, 347–357 (1986). https://doi.org/10.1007/BF00034942

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  • DOI: https://doi.org/10.1007/BF00034942

Keywords

  • human growth hormone gene
  • plant transformation
  • polyadenylation signal
  • pre-mRNA processing
  • Ti-plasmid