Abstract
A polyubiquitin clone (ubi7) was isolated from a potato (Solanum tuberosum) genomic library using a copy-specific probe from a stress-induced ubiquitin cDNA. The genomic clone contained a 569-bp intron immediately 5' to the initiation codon for the first ubiquitin-coding unit. Two chimeric beta-glucuronidase (GUS) fusion transgenes were introduced into potato. The first contained GUS fused to a 1156-bp promoter fragment containing only 5' flanking and 5' untranslated sequences from ubi7. The second transgene contained GUS translationally fused to the carboxy terminus of the first ubiquitin-coding unit and thus included the intron present in the 5' untranslated region of the polyubiquitin gene. Both ubi7-GUS transgenes were activated by wounding in tuber tissue and in leaves by application of exogenous methyl jasmonate. They were also expressed constitutively in the potato tuber peel (outer 1-2 mm). Both transgenes were actively expressed in mature leaves. Exceptionally high levels of expression were observed in senescent leaves. Transgenic clones containing the ubi7 intron and the first ubiquitin-coding unit showed GUS expression levels at least 10 times higher than clones containing GUS fused to the intronless promoter.
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- Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
- Bevan M., Barnes W. M., Chilton M. D. Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res. 1983 Jan 25;11(2):369–385. doi: 10.1093/nar/11.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Binet M. N., Weil J. H., Tessier L. H. Structure and expression of sunflower ubiquitin genes. Plant Mol Biol. 1991 Sep;17(3):395–407. doi: 10.1007/BF00040634. [DOI] [PubMed] [Google Scholar]
- Buchman A. R., Berg P. Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol. 1988 Oct;8(10):4395–4405. doi: 10.1128/mcb.8.10.4395. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Butt T. R., Jonnalagadda S., Monia B. P., Sternberg E. J., Marsh J. A., Stadel J. M., Ecker D. J., Crooke S. T. Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2540–2544. doi: 10.1073/pnas.86.8.2540. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Callis J., Fromm M., Walbot V. Introns increase gene expression in cultured maize cells. Genes Dev. 1987 Dec;1(10):1183–1200. doi: 10.1101/gad.1.10.1183. [DOI] [PubMed] [Google Scholar]
- Callis J., Raasch J. A., Vierstra R. D. Ubiquitin extension proteins of Arabidopsis thaliana. Structure, localization, and expression of their promoters in transgenic tobacco. J Biol Chem. 1990 Jul 25;265(21):12486–12493. [PubMed] [Google Scholar]
- Christensen A. H., Sharrock R. A., Quail P. H. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol. 1992 Feb;18(4):675–689. doi: 10.1007/BF00020010. [DOI] [PubMed] [Google Scholar]
- Cornejo M. J., Luth D., Blankenship K. M., Anderson O. D., Blechl A. E. Activity of a maize ubiquitin promoter in transgenic rice. Plant Mol Biol. 1993 Nov;23(3):567–581. doi: 10.1007/BF00019304. [DOI] [PubMed] [Google Scholar]
- Ecker D. J., Stadel J. M., Butt T. R., Marsh J. A., Monia B. P., Powers D. A., Gorman J. A., Clark P. E., Warren F., Shatzman A. Increasing gene expression in yeast by fusion to ubiquitin. J Biol Chem. 1989 May 5;264(13):7715–7719. [PubMed] [Google Scholar]
- Fahrner J., Labruyere W. T., Gaunitz C., Moorman A. F., Gebhardt R., Lamers W. H. Identification and functional characterization of regulatory elements of the glutamine synthetase gene from rat liver. Eur J Biochem. 1993 May 1;213(3):1067–1073. doi: 10.1111/j.1432-1033.1993.tb17854.x. [DOI] [PubMed] [Google Scholar]
- Finley D., Bartel B., Varshavsky A. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature. 1989 Mar 30;338(6214):394–401. doi: 10.1038/338394a0. [DOI] [PubMed] [Google Scholar]
- Foster R., Izawa T., Chua N. H. Plant bZIP proteins gather at ACGT elements. FASEB J. 1994 Feb;8(2):192–200. doi: 10.1096/fasebj.8.2.8119490. [DOI] [PubMed] [Google Scholar]
- Garbarino J. E., Belknap W. R. Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants. Plant Mol Biol. 1994 Jan;24(1):119–127. doi: 10.1007/BF00040579. [DOI] [PubMed] [Google Scholar]
- Genschik P., Marbach J., Uze M., Feuerman M., Plesse B., Fleck J. Structure and promoter activity of a stress and developmentally regulated polyubiquitin-encoding gene of Nicotiana tabacum. Gene. 1994 Oct 21;148(2):195–202. doi: 10.1016/0378-1119(94)90689-0. [DOI] [PubMed] [Google Scholar]
- Genschik P., Parmentier Y., Durr A., Marbach J., Criqui M. C., Jamet E., Fleck J. Ubiquitin genes are differentially regulated in protoplast-derived cultures of Nicotiana sylvestris and in response to various stresses. Plant Mol Biol. 1992 Dec;20(5):897–910. doi: 10.1007/BF00027161. [DOI] [PubMed] [Google Scholar]
- Hershko A. Ubiquitin-mediated protein degradation. J Biol Chem. 1988 Oct 25;263(30):15237–15240. [PubMed] [Google Scholar]
- Jeffers M., Pellicer A. Multiple intragenic elements regulate the expression of the murine N-ras gene. Oncogene. 1992 Nov;7(11):2115–2123. [PubMed] [Google Scholar]
- Jefferson R. A., Kavanagh T. A., Bevan M. W. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987 Dec 20;6(13):3901–3907. doi: 10.1002/j.1460-2075.1987.tb02730.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jonsson J. J., Foresman M. D., Wilson N., McIvor R. S. Intron requirement for expression of the human purine nucleoside phosphorylase gene. Nucleic Acids Res. 1992 Jun 25;20(12):3191–3198. doi: 10.1093/nar/20.12.3191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kim S. R., Choi J. L., Costa M. A., An G. Identification of G-Box Sequence as an Essential Element for Methyl Jasmonate Response of Potato Proteinase Inhibitor II Promoter. Plant Physiol. 1992 Jun;99(2):627–631. doi: 10.1104/pp.99.2.627. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Korb M., Ke Y., Johnson L. F. Stimulation of gene expression by introns: conversion of an inhibitory intron to a stimulatory intron by alteration of the splice donor sequence. Nucleic Acids Res. 1993 Dec 25;21(25):5901–5908. doi: 10.1093/nar/21.25.5901. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
- Luehrsen K. R., Walbot V. Intron enhancement of gene expression and the splicing efficiency of introns in maize cells. Mol Gen Genet. 1991 Jan;225(1):81–93. doi: 10.1007/BF00282645. [DOI] [PubMed] [Google Scholar]
- Mascarenhas D., Mettler I. J., Pierce D. A., Lowe H. W. Intron-mediated enhancement of heterologous gene expression in maize. Plant Mol Biol. 1990 Dec;15(6):913–920. doi: 10.1007/BF00039430. [DOI] [PubMed] [Google Scholar]
- Mason H. S., DeWald D. B., Mullet J. E. Identification of a methyl jasmonate-responsive domain in the soybean vspB promoter. Plant Cell. 1993 Mar;5(3):241–251. doi: 10.1105/tpc.5.3.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mason H. S., Mullet J. E. Expression of two soybean vegetative storage protein genes during development and in response to water deficit, wounding, and jasmonic acid. Plant Cell. 1990 Jun;2(6):569–579. doi: 10.1105/tpc.2.6.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morelli J. K., Shewmaker C. K., Vayda M. E. Biphasic Stimulation of Translational Activity Correlates with Induction of Translation Elongation Factor 1 Subunit [alpha] upon Wounding in Potato Tubers. Plant Physiol. 1994 Nov;106(3):897–903. doi: 10.1104/pp.106.3.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Norris S. R., Meyer S. E., Callis J. The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Mol Biol. 1993 Mar;21(5):895–906. doi: 10.1007/BF00027120. [DOI] [PubMed] [Google Scholar]
- Takimoto I., Christensen A. H., Quail P. H., Uchimiya H., Toki S. Non-systemic expression of a stress-responsive maize polyubiquitin gene (Ubi-1) in transgenic rice plants. Plant Mol Biol. 1994 Nov;26(3):1007–1012. doi: 10.1007/BF00028868. [DOI] [PubMed] [Google Scholar]
- Verwoerd T. C., Dekker B. M., Hoekema A. A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res. 1989 Mar 25;17(6):2362–2362. doi: 10.1093/nar/17.6.2362. [DOI] [PMC free article] [PubMed] [Google Scholar]