Abstract
Transcriptional and translational fusions between the reading frame of the β-D-glucuronidase gene (gusA) and the 2′ as well as the 1′ promoter of mannopine synthase (mas), a TR locus of Agrobacterium tumefaciens, were made. The expression of these constructs was studied in the transgenic F1 offspring of independent tobacco transformants at the protein level by assaying for GUS activity and western blot analysis of the GUS protein and at the steady-state mRNA level.
In leaves, stems and roots no correlation was found between steady-state levels of GUS mRNA and enzyme activity. In older tissues significantly higher GUS activities were found. This is explained by the stable character of the GUS protein together with an accumulation of protein upon ageing. Three to ten times higher GUS activities were found for in vitro grown plants than for greenhouse-grown plants of the same offspring, despite similar levels of GUS mRNA. Roots from in vitro grown plants display three to ten times higher GUS activities than stems and leaves.
In transgenic plants grown in vitro, containing a translational fusion with two AUGs in phase, the initiation of translation in leaf material occurred at both AUGs. Initiation of translation at the first AUG, however, was ten times more frequent. In contrast, initiation in roots from in vitro grown plants occurred exclusively at the second AUG.
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Barker RF, Idler KB, Thompson DV, Kemp JD: Nucleotide sequence of the Agrobacterium tumefaciens octopine Ti plasmid pTi15855. Plant Mol Biol 2: 335–358 (1983).
Baulcombe DC, Saunders GR, Bevan N, Mayo A, Harrison BD: Expression of biological active viral satellite RNA from the nuclear genome of transformed plants Nature 321: 446–449 (1986).
Benfrey PN, Ren L, Chua N: The CaMV 35 S enhancer contains at least two domains which can confer different developmental and tissue specific expression patterns. EMBO J 8: 2195–2202 (1989).
Bevan M: Binary Agrobacterium vectors for plant transformation. Nucl Acids Res 12: 8711–8721 (1984).
Bradford NM: A rapid and sensitive method for the detection of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 753–760 (1976).
Cavener DR and Ray SC: Eukaryotic start and stop translation sites. Nucl Acids Res 19: 3185–3192 (1991).
Comai L, Moran P and Maslyar D: Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elements. Plant Mol Biol 15: 373–381 (1990).
Deblaere R, Bytebier B, DeGreve H, Deboeck F, Schell J, VanMontagu M, Leemans J: Efficient octopine Ti plasmid-derived vectors for Agrobacterium mediated gene transfer to plants. Nucl Acids Res 13: 4777–4788 (1985).
DeBlock M, Herrera-Estrella L, VanMontagu M, Schell J, Zambryski P: Expression of foreign genes in regenerated plants and their progeny. EMBO J 3: 681–689 (1984).
DiRita VJ, Gelvin SB: Deletion analysis of the mannopine synthase gene promoter in sunflower crown gall tumours and Agrobacterium tumefaciens. Mol Gen Genet 207: 233–241 (1987).
Gallie DR, Lucas WL, Walbot V. Visualizing mRNA expression in plant protoplasts: Factors influencing efficient mRNA uptake and translation. Plant Cell 1: 301–311 (1989).
Gelvin SB, Karcher SJ, Goldsbrough PB: Use of a TR T-DNA promoter to express genes in plants and bacteria. Mol Gen Genet 199: 240–248 (1985).
Gheysen G, Villarroel R, VanMontagu M: Illegitimate recombination in plants: a model for T-DNA integration. Genes Devel 5: 287–297 (1991).
Gritz L, Davies J: Plasmid encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisae. Gene 25: 179–188 (1983).
Hensgens LAM, VanOs-Ruygrok PE: Isolation of RNA and DNA from different rice tissues. Rice Genet Newsl 6: 163–168 (1989).
Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary plant vector strategy based on separation of Vir- and T-region of the Agrobacterium tumefaciens Ti plasmid. Nature 303: 179–180 (1983).
Hoekema A, Hooykaas PJ, Schilperoort RA: Transfer of the Octopine T-DNA segment to plant cells mediated by different types of Agrobacterium tumour- or root-inducing plasmids: generality of virulence systems. J Bact 158: 383–385 (1984).
Hooykaas PJJ, DenDulk-Ras H, Ooms G, Schilperoort RA: Interactions between octopine and nopaline plasmids in Agrobacterium tumefaciens. J Bact 143: 1295–1306 (1980).
Jacks T, Varmus HE: Expression of the rous sarcoma virus pol gene by ribosomal frameshifting. Science 230: 1237–1242 (1985).
Jacks T, Power MD, Masiarz FR, Luciw PA, Barr PJ, Varmus HE: Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature 331: 280–283 (1988).
Jefferson RA, Burgess SM, Hirsch D: Beta-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83: 8447–8451 (1986).
Jefferson RA, Kavanagh TA, Bevan MW: Gus fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).
Jefferson RA: Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 (1987).
Kato T, Shirano Y, Kawazu T, Tada Y, Itoh E, Shibata D: A modified β-glucuronidase gene: Sensitive detection of plant promoter activities in suspension-cultured cells of tobacco and rice. Plant Mol Biol Rep 9: 333–339 (1991).
Kay R, Chan A, Daly M, McPherson J: Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236: 1299–1302 (1987).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227: 680–685 (1970).
Langridge WHR, Fitzgerald KJ, Koncz C, Schell J, Szalay AA: Dual promoter of Agrobacterium tumefaciens mannopine synthase genes is regulated by plant growth hormones. Proc Natl Acad Sci USA 86: 3219–3223 (1989).
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).
Meyer EGM, Schilperoort RA, Rueb S, VanOs-Ruygrok PE, Hensgens LAM: Transgenic rice cell lines and plants: expression of transferred chimeric genes. Plant Mol Biol 16: 807–820 (1991).
Miller WA, Waterhouse PM, Gerlach WL: Sequence and organization of barley yellow dwarf genomic RNA. Nucl Acids Res 16: 6097–6111 (1988).
Morch MD, Drugeon G, Benicourt C: Analysis of the in vitro coding properties of the 3′ region of turnip yellow mosaic virus genomic RNA. Virology 119: 193–198 (1982).
Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497 (1962).
Nagy F, Odell JT, Morelli G, Chua NH: Properties of expression of the 35S promoter from CaMV in transgenic tobacco plants. In: Zaitlin M, Day P, Hollaender A (eds) Biotechnology in Plant Science: Relevance to Agriculture in the Eighties, pp. 227–236, Academic Press, New York (1985).
Odell JT, Nagy F, Chua N-H: Identification of sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810–812 (1985).
Odell JT, Knowlton S, Lin W, Mauvais CJ: Properties of an isolated transcription stimulating sequence derived from the cauliflower mosaic virus 35S promoter. Plant Mol Biol 10: 263–272 (1988).
Offringa IA, Melchers LS, Regensburg-Tuink AJG, Constantino P, Schilperoort RA, Hooykaas PJJ: Complementation of Agrobacterium tumefaciens tumour-inducing aux mutants by genes from the TR-region of the Ri plasmid of Agrobacterium rhizogenes. Proc Natl Acad Sci USA 83: 6935–6939 (1986).
Offringa R, deGroot MJA, Haagsman HJ, Does MP, van denElzen PJM, Hooykaas PJJ: Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation. EMBO J 9: 3077–3084 (1990).
Ooms G, Bakker A, Molendijk L, Wullems GJ, Gordon MP, Nester EW, Schilperoort RA: T-DNA organisation in homogeneous and heterogeneous octopine-type crown gall tissues of Nicotiana tabacum. Cell 30: 589–597 (1982).
Ow DW, Jacobs JD, Howell SH: Functional regions of the cauliflower mosaic virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter of promoter activity. Proc Natl Acad Sci USA 84: 4870–4874 (1987).
Pelham HRB: Leaky UAG termination codon in tobacco mosaic virus RNA. Nature 272: 469–471 (1978).
Pelham HRB: Translation of tobacco rattle virus RNAs in vitro: Four proteins from three RNAs. Virology 97: 256–265 (1979).
Peralta EG, Hellmiss R, Ream W: Overdrive, a T-DNA transmission enhancer on the A. tumefaciens tumour inducing plasmid. EMBO J 5: 1137–1142 (1986).
Rueb S, Hensgens LAM: Improved histochemical staining for β-D-glucuronidase activity in monocotyledonous plants. Rice Genet Newsl 6: 168–169 (1989).
Rothstein SJ, Lahners KN, Lotstein RJ, Carozzi NB, Jayne SM, Rice DA: Promoter cassettes, antibiotic-resistance genes, and vectors for plant transformation. Gene 53: 153–161 (1987).
Sanger M, Daubert S, Goodman RM: Characterization of a strong promoter from figwart mosaic virus, comparison with the analogous 35S promoter from cauliflower mosaic virus and the regulated mannopine synthase promoter. Plant Mol Biol 14: 433–443 (1990).
Schmidhauser TJ, Helinski DR: Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J Bact 164: 446–455 (1985).
Teeri TH, Lehvaslaiho H, Frank M, Uotila J, Heino P, Palva ET, VanMontagu M, Herrera-Estrella L: Gene fusions to lacZ reveal new expression patterns of chimeric genes in transgenic plants. EMBO J 8: 343–350 (1989).
van denElzen PJM, Townsend J, Lee KY, Bedbrook JR: A chimeric hygromycin resistance gene as a selectable marker in plant cells. Plant Mol Biol 5: 2723–2730 (1985).
vanHaaren MJJ, Sedee NJA, deBoer HA, Schilperoort RA, Hooykaas PJJ: Mutational analysis of the conserved domains of a T-region border repeat of Agrobacterium tumefaciens. Plant Mol Biol 13: 523–531 (1989).
Veidt I, Lot H, Leiser H, Scheidecker D, Guilley H, Richards K, Jonard G: Nucleotide sequence of beet western yellows virus RNA. Nucl Acids Res 16: 9917–9932 (1988).
Velten J, Velten L, Hain R, Schell J: Isolation of a dual plant promoter fragment from the Ti plasmid of Agrobacterium tumefaciens. EMBO J 3: 2723–2730 (1984).
Velten J, Schell J: Selection-expression plasmid vectors for use in genetic transformation of higher plants. Nucl Acid Res 13: 6981–6998 (1985).
Williamson JD, Hirsch-Wyncott ME, Larkins BA, Gelvin SB. Differential accumulation of a transcript driven by the CaMV 35S promoter in transgenic tobacco. Plant Physiol 90: 1570–1576 (1989).
Xiong Z, Lommel SA: The complete nucleotide sequence and genome organization of red clover necrotic mosaic virus RNA-1. Virology 171: 543–554 (1989).
Ziegler V, Richards K, Guilley H, Jonard G, Putz C: Cell-free translation of beet necrotic yellow vein virus: Readthrough of the coat protein cistron. J Gen Virol 66: 2079–2087 (1985).
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Hensgens, L.A.M., Fornerod, M.W.J., Rueb, S. et al. Translation controls the expression level of a chimaeric reporter gene. Plant Mol Biol 20, 921–938 (1992). https://doi.org/10.1007/BF00027163
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DOI: https://doi.org/10.1007/BF00027163