Abstract
We have isolated a constitutive promoter sequence, tCUP, from tobacco by T-DNA tagging using a promoterless GUS-nos3′ reporter gene construct. The T-DNA integration event produced a translational fusion with the GUS gene that is expressed widely in organs, at both the mRNA and enzyme activity levels. In tobacco transformed with a tCUP-GUS-nos3′ gene, GUS specific activity in leaves was within a range of values similar to those of plants transformed with the widely used constitutive promoter gene fusion, CaMV 35S promoter-GUS-nos3′. Characteristics of the tCUP promoter sequence differ from those of other plant constitutive promoters; for instance, the tCUP sequence lacks a TATA box. Transcription initiates at a single site within the tCUP sequence which is similar to a transcriptional start site consensus sequence determined for plant genes. The tCUP promoter is cryptic as RNA accumulation at the transcriptional start site is not detected in untransformed tobacco. Thus, tCUP is the first example of a cryptic, constitutive promoter isolated from plants. The tCUP-GUS-nos3′ gene fusion produced GUS activity in tissues of all species tested suggesting that tCUP may utilize fundamental transcription mechanisms found in plants.
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An, Y.-Q., McDowell, J.M., Huang,S., McKinney, E.C., Chambliss, S. and Meagher, R.B. 1996. Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J. 10: 107-121.
Callis, J., Raasch, J.A. and Vierstra, R.D. 1990. Ubiquitin extension proteins of Arabidopsis thaliana. J. Biol. Chem. 265: 12486-12493.
Christensen, A.H., Sharrock, R.A. and Quail, P.H. 1992. Maize polyubiquitin genes; structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18: 675-689.
Datla, R.S., Hamerlindl, J.K., Panchuk, B., Pelcher, L.E. and Keller, W. 1992. Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 211: 383-384.
Datla, R.S., Bekkaoui, F., Hammerlindl, J.K., Pilate, G., Dunstan, D.I. and Crosby, W.L. 1993. Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence. Plant Sci. 94: 139-149.
de Pater, B., van der Mark, F., Rueb, S., Katagiri, F., Chua, N.-H., Schilperoort, R.A. and Hensgens, L.A.M. 1992. The promoter of rice gene GOS2 is active in various different monocot tissues and binds rice nuclear factor ASF-1. Plant J. 2: 837-844.
Feldmann, K.A. 1991. T-DNA insertion mutagenesis in Arabidopsis: mutational spectrum. Plant J. 1: 71-82.
Fobert, P.R. 1992. Characterization of chromosomal sites of TDNA integration by activation of a promoterless _-glucuronidase (GUS) gene linked to the T-DNA right border repeat. Ph. D. thesis, Carleton University.
Fobert, P.R., Miki, B.L., Iyer V.N. 1991. Detection of gene regulatory signals in plants revealed by T-DNA-mediated fusions. Plant Mol. Biol. 17: 837-851.
Fobert, P.R., Labbé, H., Cosmopolous, J., Gottlob-McHugh, S., Ouellet, T., Hattori, J., Sunohara, G., Iyer, V.N. and Miki, B.L. 1994. T-DNA tagging of a seed coat-specific cryptic promoter in tobacco. Plant J. 6: 567-577.
Herman, L., Jacobs, A., Van Montagu, M. and Depicker, A. 1990. Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events. Mol. Gen. Genet. 224: 248-256.
Holtorf, S., Apel, K. and Bohlmann, H. 1995. Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana. Plant Mol. Biol. 29: 637-646.
Irniger, S., Egli, C.M., Kuenzler, M. and Braus, G.H. 1992. The yeast actin intron contains a cryptic promoter that can be switched on by preventing transcriptional interference. Nucl. Acids Res. 20: 4733-4739.
Jefferson, R.A. 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5: 387-405.
Kapoun, A.M. and Kaufman, T.C. 1995. A functional analysis of 50, intronic and promoter regions of the homeotic gene proboscipedia in Drosophila melanogater. Development 121: 2127-2141.
Koncz, C., Martini, N., Mayerhofer, R., Koncz-Kalman, Z., Korber, H., Redei, G.P. and Schell, J. 1989. High-frequency T-DNAmediated gene tagging in plants. Proc. Natl. Acad. Sci. USA 86: 8467-8471.
Lo, K. and Smale, S.T. 1996. Generality of a functional initiator consensus sequence. Gene 182: 13-22.
Mandel, T., Fleming, A.J. Krahenbuhl, R. and Kuhlemeier, C. 1995. Definition of constitutive gene expression in plants: the translation initiation factor 4A gene as a model. Plant Mol. Biol. 29: 995-1004.
Mathur, J., Szabados, Z, Schaefer, S., Grunenberg, B., Lassow, A., Jonas-Straube, E., Schell, J., Koncz, C. and Koncz-Kalman, Z. 1998. Gene identification with sequenced T-DNA tags generated by transformation of Arabidopsis cell suspension. Plant J. 13: 707-716.
Miki, B.L.A., McHugh, S.G., Labbé, H., Ouellet, T., Tolman, J.H. and Brandle, J.E. In press. Genetic transformation of tobacco: gene expression and applications. In: Y.P.S. Bajaj (Ed.), Biotechnology in Agriculture and Forestry, Springer-Verlag, Berlin.
Odell, J.T., Nagy, F. and Chua, N.-H. 1985. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810-812.
Ouellet, T., Rutledge, R.G. and Mik, B.L. 1992. Members of the Brassica acetohydroxyacid synthase multigene family of Brassica napus have divergent patterns of expression. Plant J. 2: 321-330.
Palmer, C.N., Hsu, M.-H., Muerhoff, A.S., Griffin, K.J. and Johnson, E.F. 1994. Interaction of the peroxisome proliferatoractivated receptor alpha with the retinoid X receptor alpha unmasks a cryptic peroxisome proliferator response element that overlaps an ARP-1-binding site in the CYP4A6 promoter. J. Biol. Chem. 269: 18083-18089.
Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Sanford, J., Smith, F. and Russell, J. 1993. Optimizing the biolistic process for different biological applications. Meth. Enzymol. 217: 483-509.
Tee, M.K., Babalola, G.O., Aza-Blanc, P., Speek, M., Gitelman, S.E. and Miller, W.L. 1995. A promoter within intron 35 of the human C4A gene initiates abundant adrenal-specific transcription of a 1 kb RNA: location of a cryptic CYP21 promoter element? Hum. Mol. Genet. 4: 2109-2116.
Troyanovsky, S. and Leube, R. 1994. Activation of the silent human cytokeratin 17 pseudogene-promoter region by cryptic enhancer elements of the cytokeratin 17 gene. Eur. J. Biochem. 225: 61-69.
Van Haute, E., Joos, H., Maes, M., Warren, G., Van Montagu, M. and Schell, J. 1983. Intergenic transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J. 2: 411-417.
Walden, R., Hayashi, H. and Schell, J. 1991. T-DNA as a gene tag. Plant J. 1: 281-288.
Wei, W., Twel, D. and Lindsey, K. 1997. A novel nucleic acid helicase gene identified by promoter trapping in Arabidopsis. Plant J. 11: 1307-1314.
Wilmink, A., van de Ven, B. and Dons, J. 1995. Activity of constitutive promoters in various species from the Liliaceae. Plant Mol. Biol. 28: 949-955.
Zhang, W., McElroy, D. and Wu, R. 1991. Analysis of rice Act1 50 region activity in transgenic rice plants. Plant Cell 3: 1155-1165.
Zhu, Q., Dabi, T. and Lamb, C. 1995. TATA box and initiator functions in the accurate transcription of a plant minimal promoter in vivo. Plant Cell 7: 1681-1689.
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Foster, E., Hattori, J., Labbé, H. et al. A tobacco cryptic constitutive promoter, tCUP, revealed by T-DNA tagging. Plant Mol Biol 41, 45–55 (1999). https://doi.org/10.1023/A:1006229501860
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DOI: https://doi.org/10.1023/A:1006229501860