Summary
Ubiquitin DNA sequences were isolated from the higher plant Arabidopsis thaliana L. by screening a lambda-gt11 genomic library with antibodies raised against oat and human ubiquitin. DNA sequence analysis showed that the predicted protein sequence is 100% conserved with that found in oat and barley and differs by only three residues to that found in animals. This gene (UBQ4) encodes a ubiquitin polyprotein with five repeats contiguously linked with no intervening sequences in the coding region and a C-terminal extension of Ser-Phe. Genomic Southern blot analysis showed that ubiquitin sequences comprise a multigene family of approximately 11 members in Arabidopsis. Northern blot analysis identified at least four transcript size classes, which accumulate in sizes ranging from 800 to 1900 bases. A 5′-specific probe for the UBQ4 gene was used to show that after 2 h heat shock stress, the steady state mRNA level decreased significantly in flowers/buds but not in leaves. The UBQ4 transcript accumulates in a differential manner, accumulating to higher levels in germinating tissue, etiolated tissue, and flowers/buds than in mature leaves, roots, or stems.
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Amasino R (1986) Acceleration of nucleic acid hybridization rate by polyethylene glycol. Anal Biochem 152:304–307
Arribas C, Sempredo J, Izquierdo M (1986) The ubiquitin genes in D. melanogaster: transcription and polymorphism. Biochim Biophys Acta 868:119–127
Ball E, Karlik CC, Beall CJ, Saville DL, Sparrow JC, Bullard B, Fyrberg EA (1987) Arthrin, a myofibrillar protein of insect flight muscle, is an actin-ubiquitin conjugate. Cell 52:221–228
Bariaga M, Franco R, Meinkoth J, Ong E, Wahl G (1981) Methods for transfer of DNA, RNA, protein to nitrocellulose diazotized paper solid supports. Schleicher Schuell, Keene, NH
Bond U, Schlesinger M (1985) Ubiquitin is a heat shock protein in chicken embryo fibroblasts. Mol Cell Biol 5:949–956
Bond U, Schlesinger M (1986) The chicken ubiquitin gene contains a heat shock promoter and expresses an unstable mRNA in heat-shocked cells. Mol Cell Biol 6:4602–4610
Craig EA, Jacobsen K (1984) Mutations in cognate genes of Saccharomyces cerevidiae HSP70 result in reduced growth rates at low temperatures. Mol Cell Biol 5:3517–3524
Dworkin-Rastl E, Shrutkowski A, Dworkin MB (1984) Multiple ubiquitin mRNA's during Xenopus laevis development containing tandem repeats of the 76 amino acid coding sequence. Cell 39:321–325
Ecker J, Davis J (1987) Plant defense genes are regulated by ethylene. Proc Natl Acad Sci USA 84 15:5202–5206
Feinberg A, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–14
Finley D, Varshavsky A (1985) The ubiquitin system: functions and mechanisms. Trends Biochem Sci 10:343–347
Finley D, Ciechanover S, Varshavsky A (1984) Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85. Cell 37:43–55
Finley D, Ozkaynak E, Varshavsky A (1987) The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell 48:1035–1045
Gausing K, Barkardottir R (1986) Structure and expression of ubiquitin genes in higher plants. Eur J Biochem 158:57–62
Giorda R, Ennis HL (1987) Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes. Mol Cell Biol 6:2097–2103
Glisin V, Crkvenjakov R, Byus C (1974) Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13:2633–2637
Goldknopf IL, Busch H (1977) Isopeptide linkage between nonhistone and histone 2A polypeptides of chromosomal conjugate-protein A24. Proc Natl Acad Sci USA 74:864–868
Goldknopf IL, Wilson G, Ballal NR, Busch H (1980) Chromatin conjugate protein A24 is cleaved and ubiquitin is lost during chicken erythropoiesis. J Biol Chem 255:10555–10558
Goldstein G, Scheid M, Hammerling U, Boyse EA, Schlesinger DH, Niall HD (1975) Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells. Proc Natl Acad Sci USA 72:11–15
Haas AL, Bright PM (1985) The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates. J Biol Chem 260:12464–12473
Haas AL, Wilkinson KD (1985) The large scale purification of ubiquitin from human erythrocytes. Prep Biochem 15:49–60
Hershko A, Ciechanover A (1986) The ubiquitin pathway for the degradation of intracellular proteins. Prog Nucleic Acid Res Mol Biol 33:19–56
Hough R, Pratt G, Rechsteiner M (1987) Purification of two high molecular weight proteases from rabbit reticulocyte lysate. J Biol Chem 262:8303–8313
Huynh TV, Young RA, Davis RW (1985) Constructing and screening cDNA libraries in lambda gt10 and lambda gt11. In: Glover D (ed) DNA cloning: A practical approach, vol.1. IRL Press, Oxford, p 49–78
Jentsch S, McGrath JP, Varshavsky A (1987) The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature 329:131–134
Kanda F, Sykes DE, Yasuda H, Sandberg AA, Matsui SI (1986) Substrate recognition of isopeptidase: specific cleavage of the e-(a-glycl) lysine linkage in ubiquitin-protein conjugate. Biochim Biophys Acta 870:65–75
Lund PK, Moats-Staats BM, Simmons JG, Hoyt E, D'Ercole AJ, Martin F, Van Wyk JJ (1985) Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveal that ubiquitin is synthesized as a precursor. J Biol Chem 260:7609–7613
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Press, New York
Melton DA, Krieg PA, Robagliatte MR, Maniatis T, Zinn K, Green MR (1984) Effective in vitro synthesis of biologically active RNA and DNA hybridization probes from plasmids containing a bacteriophage sp6 promoter. Nucleic Acids Res 12:7035–7056
Ozkaynak E, Finley D, Varshavsky A (1984) The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein. Nature 312:663–666
Ozkaynak E, Finley D, Solomon MJ, Varshavsky A (1987) The yeast ubiquitin genes: a family of natural gene fusions. EMBO J 6:1429–1439
Pang PP, Meyerowitz EM (1987) Arabidopsis thaliana: a model system for plant molecular biology. Biotechnology 5:1177–1180
Pickart CM, Rose I (1985) Ubiquitin carboxyl-terminal hydrolase acts on ubiquitin carboxyl-terminal amides. J Biol Chem 261:7903–7910
Rechsteiner M (1987) Ubiquitin mediated pathways for intracellular proteolysis. Annu Rev Cell Biol 3:1–30
Sanders PR, Winter JA, Barnason AR, Rogers SJ, Fraley RT (1987) Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants. Nucleic Acids Res 15:1543–1558
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Shanklin J, Jabben M, Vierstra RD (1987) Red light formation of ubiquitin-phytochrome conjugates: identification of possible intermediates of phytochrome degradation. Proc Natl Acad Sci USA 84:359–363
Sharp PM, Li W-H (1987) Ubiquitin genes as a paradigm of concerted evolution of tandem repeats. J Mol Evol 25:58–64
Siegelman M, Bond MW, Gallatin WM, St John T, Smith HT, Fried VA, Weissman IL (1986) Cell surface molecule associated with lymphocyte homing is a ubiquitinated branched chain glycoprotein. Science 231:823–829
Sommerville CR, Ogren WL (1982) Isolation of photorespiration mutants in Arabidopsis thaliana. In: Edelman P (ed) Methods in chloroplast molecular biology. Elsevier Biomedical Press, Amsterdam, p 129–138
St John T, Gallatin WM, Siegelman M, Smith HT, Fried VA, Weissman IL (1986) Expression cloning of a lymphocyte homing receptor cDNA: ubiquitin is the reactive species. Science 231:845–850
Vierstra RD (1987) Demonstration of ATP-dependent ubiquitin conjugating activities in higher plants. Plant Physiol 84:332–336
Vierstra RD, Sullivan ML (1988) Hemin inhibits ubiquitin-dependent proteolysis in a higher plant and yeast. Biochemistry 27:3290–3295
Vierstra RD, Langan S, Haas A (1985) Purification and initial characterization of ubiquitin from the higher plant Avena sativa. J Biol Chem 260:12015–12021
Vierstra RD, Langan SM, Schaller EG (1986) Complete amino acid sequence of ubiquitin from the higher plant Avena sativa. Biochemistry 25:3105–3108
Westphal M, Muller-Taubenberger A, Noegel A, Gerish G (1986) Transcript regulation and carboxyterminal extension of ubiquitin in Dictyostelium discoideum. FEBS Lett 209:92–96
Wiborg O, Pederson MS, Wind A, Berglund LE, Marcker KA, Vuust J (1985) The human ubiquitin multigene family: some genes contain multiple directly repeated ubiquitin coding sequences. EMBO J 4:755–759
Yarden T, Escobedo JA, Kuang WJ, Yang-Feng TL, Daniel TO, Tremble PM, Chen EY, Eo M, Harkins RN, Francke U, Fried VA, Ulrich A, Williams LT (1986) Structure of the receptor for the platelet-derived growth factor helps define a family of closely related growth factor receptors. Nature 323:226–232
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of M13mp18 and pUC vectors. Gene 33:103–119
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Communicated by R.B. Goldberg
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Burke, T.J., Callis, J. & Vierstra, R.D. Characterization of a polyubiquitin gene from Arabidopsis thaliana . Mol Gen Genet 213, 435–443 (1988). https://doi.org/10.1007/BF00339613
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DOI: https://doi.org/10.1007/BF00339613