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.
Similar content being viewed by others
References
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.
Berget SM: Are U4 small nuclear ribonucleoproteins involved in polyadenylation. Nautere 309: 179–182, 1984.
Bevan MW, Chilton M-D: T-DNA of the Agrobacterium Ti- and Ri-plasmids. Ann Rev Genet 16: 357–384, 1982.
Birnboim HG, Doly J: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7: 1513–1523, 1979.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Flint SJ: in: Apirion D (ed) Processing of RNA CRC, Boca Raton, FL, 1984, pp 151–179.
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.
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.
Grabowski PJ, Padgett RA, Sharp PA: Messenger RNA splicing in vitro: An excised intervening sequence and a potential intermediate. Cell 37: 415–427, 1984.
Hall TC: Expression of plant genes in alien cellular environments. Oxford Surveys of Plant Molecular and Cell Biology 2: 329–338, 1985.
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.
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.
Keller W: The RNA lariat: A new ring to the splicing of mRNA precursors. Cell 39: 423–425, 1984.
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.
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.
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.
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.
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.
Maniatis T, Fritsch E, Sambrook J: Molecular cloning: A Laboratory Mannual. Cold Spring Harbour Laboratory, Cold Spring Harbour, NY, 1982.
Manley JL: Accurate and specific polyadenylation of mRNA precursors in a soluble whole-cell lysate. Cell 33: 595–605, 1983.
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.
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.
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.
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.
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.
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.
Moore CL, Sharp PA: Site-specific polyadenylation in a cell-free reaction. Cell 36: 581–591, 1984.
Mount SM: A catalogue of splice junction sequences. Nucleic Acids Res 10: 459–472, 1982.
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.
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.
Murray HG, Thompson WF: Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8: 4321–4325, 1980.
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.
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.
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.
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.
Proudfoot NJ: The end of the message and beyond. Nature 307: 412–413, 1984.
Rautmann G, Breathnach R: A role for branch points in splicing in vivo. Nature 315: 430–432, 1985.
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.
Seeburg PH: The human growth hormone gene family: nucleotide sequences show recent divergence and predict a new polypeptide hormone. DNA 1: 239–249, 1982.
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.
Shah DM, Hightower RC, Meagher RB: Complete nucleotide sequence of a soybean actin gene. Proc Natl Acad Sci (USA), 79: 1022-S1026, 1982.
Shaw CH: Ti-plasmid-derived plant gene vectors. Oxford Survey of Plant Molecular and Cell Biology 1: 211–216, 1984.
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.
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.
Taylor B, Powell A: Isolation of plant DNA and RNA. Focus 4: 4, 1982.
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.
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.
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.
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.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00034942