Abstract
Using the expression vector λgt11 and immunochemical detection, six cDNA clones that encode the entire precursor polypeptides for spinach thioredoxin m were isolated and characterized. The ca. 1.0 kb cDNA sequence of the largest clone hybridizes to an RNA species of 1.1 kb. In each instance the cDNA sequences display single open reading frames encoding polypeptides of 181 amino acid residues corresponding to a molecular mass of 19.8 kDa. The sequences of the independently selected cDNAs fall into two classes that are indicative of at least two (closely related) genes for this protein. The amino acid sequences deduced from the cDNA sequences differ to some extent from the amino acid sequence published for spinach thioredoxin m. The sequences predict identical mature proteins of 112–114 amino acids corresponding to a polypeptide molecular mass of ca. 12.4–12.6 kDa, and include stroma-targeting N-terminal transit peptides of 67 residues which are removed during or after import into the organelle. Precursor protein was made in vitro from each of the different cDNA clones and imported into isolated intact chloroplasts. Independent of the cDNA clone used, two isoforms were detected in the chloroplasts after import in each instance. They comigrated with authentic thioredoxin mb and mc. These results indicate that the size variants observed for this protein in vivo result from post-translational modification and do not originate in different genes.
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Bartlett SG, Grossmann AR, Chua N-H: In vitro synthesis and uptake of cytoplasmically-synthesized chloroplast proteins. In: Edelman M, Hallick RB, Chua N-H (eds) Methods in Chloroplast Molecular Biology, pp. 000–000. Elsevier, Amsterdam/New York/Oxford (1982).
Birnboim HC, Doly J: A rapid alkaline procedure for screening recombinant plasmid DNA. Nucl Acids Res 7: 1513–1523 (1979).
Clement-Metral D, Holmgren A, Cambillau C, Jörnvall H, Eklund H, Thomas D, Lederer F: Amino acid sequence determination and three-dimensional modelling of thioredoxin from the photosynthetic bacterium Rhodobacter sphaeroides Y. Eur J Biochem 173: 413–419 (1988).
Cline K, Werner-Washburne M, Lubben T, Keegstra K: Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts. Plant Physiol 75: 675–678 (1984).
Cseke C, Buchanan BB: Regulation of the formation and utilization of photosynthate in leaves. Biochim Biophys Acta 853: 43–63 (1986).
Eklund H, Gleason FK, Holmgren A: Structural and functional relations among thioredoxins of different species. Proteins Structure Function Genet 11: 13–28 (1991).
Florencio FJ, Yee BC, Johnson TC, Buchanan BB: An NADP/thioredoxin system in leaves. Purification and characterization of NADP-thioredoxin reductase and thioredoxin h from spinach. Arch Biochem Biophys 266: 496–507 (1988).
Holmes DS, Quigley M: A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 13: 193–197 (1981).
Holmgren A: Thioredoxin and glutaredoxin systems. J Biol Chem 264: 13963–13966 (1989).
Höög J-O, Von Bahr-Lindström H, Josephson S, Wallace BJ, Kushner SR, Jörnvall H, Holmgren A: Nucleotide sequence of the thioredoxin gene from Escherichia coli. Biosci Rep 4: 917–923 (1984).
Kamo M, Tsugita A, Wiessner C, Wedel N, Bartling D, Herrmann RG, Aguilar F, Gardet-Salvi L, Schürmann P: Primary structure of spinach-chloroplast thioredoxin f. Protein sequencing and analysis of complete cDNA clones for spinach-chloroplast thioredoxin f. Eur J Biochem 182: 315–322 (1989).
Kozak M: An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucl Acids Res 15: 8125–8148 (1987).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–684 (1970).
Lautner A, Klein R, Ljungberg U, Reiländer H, Bartling D, Andersson B, Reinke H, Beyreuther K, Herrmann RG: Nucleotide sequence of cDNA clones encoding the complete precursor for the ‘10kDa’ polypeptide of photosystem II from spinach. J Biol Chem 263: 10077–10081 (1988).
Lim CJ, Gleason FK, Fuchs JA: Cloning, expression and characterization of the Anabaena thioredoxin gene in Escherichia coli. J Bact 168: 1258–1264 (1986).
Lüttke HA, Chew KC, Mickel FS, Moss KA, Kern HF, Scheele GA: Selection of AUG initiation codons differs in plants and animals. EMBO J 6: 43–48 (1987).
Maeda K, Tsugita A, Dalzoppo D, Vilbois F, Schürmann P: Further characterization and amino acid sequence of m-type thioredoxins from spinach chloroplasts. Eur J Biochem 154: 197–203 (1986).
Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
Messing J: New M13 vectors for cloning. Meth Enzymol 101: 20–78 (1983).
Muller EGD, Buchanan BB: Thioredoxin is essential for photosynthetic growth. The thioredoxin m gene of Anacystis nidulans. J Biol Chem 264: 4008–4014 (1989).
Sanger F, Nicklen S, Coulsen AR: DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Schägger H, Von Jagow G: Tricine-sodium dodecylsulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368–379 (1987).
Scheibe R: NADP+-malate dehydrogenase in C3-plants: Regulation and role of a light-activated enzyme. Physiol Plant 71: 393–400 (1987).
Schürmann P, Maeda K, Tsugita A: Isomers in thioredoxins of spinach chloroplasts. Eur J Biochem 116: 37–45 (1981).
Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503–517 (1975).
Thomas PS: Hybridization of denatured RNA and small DNA fragments transfered to nitrocellulose. Proc Natl Acad Sci USA 77: 5201–5205 (1980).
Tittgen J, Hermans J, Steppuhn J, Jansen T, Jansson C, Andersson B, Nechushtai R, Nelson N, Herrmann RG: Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins. Mol Gen Genet 204: 258–265 (1986).
Westhoff P, Nelson N, Bünemann H, Herrmann RG: Localization of genes for coupling factor subunits on the spinach plastid chromosome. Curr Genet 4: 109–120 (1981).
Von Heijne G, Steppuhn J, Herrmann RG: Domaine structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180: 535–545 (1989).
Yamamoto KR, Alberts BM, Benzinger R, Lonhorne L, Treiber G: Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 40: 734–744 (1970).
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Wedel, N., Clausmeyer, S., Herrmann, R.G. et al. Nucleotide sequence of cDNAs encoding the entire precursor polypeptide for thioredoxin m from spinach chloroplasts. Plant Mol Biol 18, 527–533 (1992). https://doi.org/10.1007/BF00040668
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DOI: https://doi.org/10.1007/BF00040668