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Isolation, characterisation and expression of a cDNA clone encoding plastid aspartate aminotransferase from Arabidopsis thaliana

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Abstract

A clone encoding aspartate aminotransferase (AAT, EC 2.6.1.1) was isolated from an Arabidopsis thaliana leaf cDNA library. This clone contains a 1365 bp open reading frame encoding a polypeptide of 49.8 kDa, designated Ataat1. The clone was shown to contain a chloroplastic isoenzyme as an in organellar protein import assay demonstrated that a radiolabelled transcription/translation product of 49.8 kDa was imported into viable pea chloroplasts and was subsequently processed to yield a mature protein of 45 kDa. The open reading frame corresponding to the predicted mature AAT was manipulated into an expression construct (pEC14). Transformed Escherichia coli cells containing pEC14 expressed up to 16 times more AAT activity than vector only controls, thus demonstrating conclusively that the clone encoded AAT.

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Abbreviations

AAT(1):

aspartate aminotransferase (isoenzyme 1)

Ataat1:

deduced protein sequence for Arabidopsis AAT1 from pcAAT1

pcAAT1:

cDNA clone of Arabidopsis AAT1 in pBluescript SK+

PLP:

pyridoxal phosphate

References

  1. Anderson S, Smith SM: Synthesis of the small subunit of ribulose-bisphosphate carboxylase from genes cloned into plasmids containing an SP6 promoter. Biochem J 240: 709–715 (1986).

    PubMed  Google Scholar 

  2. Brown TA, Ikemura T, McClelland M, Roberts RJ: Bacteria and bacteriophages. In: Brown TA (ed) Molecular Biology Labfax, pp. 1–22. BIOS Scientific Publishers, Oxford (1991).

    Google Scholar 

  3. Christen P, Graf Hausner U, Bossa F, Doonan P: Comparison of covalent structures of the isoenzymes of aspartate aminotransferase. In: Christen P, Metzler DE (eds) Transaminases, pp. 137–184, John Wiley, New York (1985).

    Google Scholar 

  4. Cline K, Werner-Washburne M, Andrews J, Keegstra K: Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts. Plant Physiol 75: 675–678 (1984).

    Google Scholar 

  5. Cline K, Werner-Washburne M, Lubben TH, Keegstra K: Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts. J Biol Chem 260: 3691–3696 (1985).

    PubMed  Google Scholar 

  6. Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation: version 11. Plant Mol Biol Rep 1 (4): 19–21 (1983).

    Google Scholar 

  7. Fotheringham IG, Dacey SA, Taylor PP, Smith TJ, Hunter MG, Finlay ME, Primrose SB, Parker DM, Edwards RM: The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Biochem J 234: 593–604 (1986).

    PubMed  Google Scholar 

  8. Gantt JS, Larson RL, Farnham MW, Pathirana SM, Miller SS, Vance CP: Aspartate aminotransferase in effective and ineffective alfalfa nodules. Plant Physiol 98: 868–878 (1992).

    Google Scholar 

  9. Gavel Y, vonHeijne G: A conserved cleavage site motif in chloroplast transit peptides. FEBS Lett 261: 455–458 (1990).

    PubMed  Google Scholar 

  10. Gelfand DH, Rudo N: Mapping of the aspartate and aromatic amino acid aminotransferase genes of tyrB and aspC. J Bact 130: 441–444 (1977).

    PubMed  Google Scholar 

  11. Harvey MJ, Muehlbauer FJ: Linkages between restriction fragment length, isozyme and morphological markers in lentil. Theor Appl Genet 77: 395–401 (1989).

    Article  Google Scholar 

  12. Jansonius JN, Eichele G, Ford GC, Picot D, Thaller C, Vincent MG: Spatial structure of mitochondrial aspartate aminotransferase. In: Christen P, Metzler DE (eds) Transaminases, pp. 110–137, John Wiley, New York (1985).

    Google Scholar 

  13. Juretic N, Mattes U, Ziak M, Christen P, Jaussi R: Structure of the genes of two homologous intracellularly heterotrophic isoenzymes: cytosolic and mitochondrial aspartate aminotransferase of chicken. Eur J Biochem 192: 119–126 (1990).

    PubMed  Google Scholar 

  14. Kirsch JF, Eicheles G, Ford GC, Vincent MG, Jansonius JN, Gehring H, Christen P: Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure. J Mol Biol 174: 497–525 (1984).

    Article  PubMed  Google Scholar 

  15. Reynolds PHS, Smith LA, Dickson JMJJ, Jones WT, Jones SD, Rodber KA, Carne A, Liddane CP: Molecular cloning of a cDNA encoding aspartate aminotransferase — P2 from lupin root nodules. Plant Mol Biol 19: 465–472 (1992).

    Article  PubMed  Google Scholar 

  16. Robinson DL, Kahn ML, Vance CP: Cellular localisation of nodule-enhanced aspartate aminotransferase in Medicago sativa L. Planta 192: 202–210 (1994).

    Article  Google Scholar 

  17. Schultz CJ, Corruzzi GM: The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localised to three distinct subcellular compartments. Plant J, in press (1994).

  18. Turano FJ, Weisemann JM, Matthews BF: Identification and expression of a cDNA clone encoding aspartate aminotransferase in carrot. Plant Physiol 100: 374–381 (1992).

    Google Scholar 

  19. Udvardi MK, Kahn ML: Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago sativa) aspartate aminotransferase. Mol Gen Genet 231: 97–105 (1991).

    Article  PubMed  Google Scholar 

  20. vonHeijne G, Steppuhn J, Herrmann RG: Domain structure of mitochondrial and chloroplast targetting peptides. Eur J Biochem 180: 535–545 (1989).

    PubMed  Google Scholar 

  21. Wadsworth GJ, Marmaras SM, Matthews BF: Isolation and characterisation of a soybean cDNA clone encoding the plastid form of aspartate aminotransferase. Plant Mol Biol 21: 993–1009 (1993).

    Article  PubMed  Google Scholar 

  22. Yagi T, Kagamiyama, Nozaki M, Soda K: Glutamate-aspartate transaminase from microorganisms. Meth Enzymol 113: 83–89 (1985).

    PubMed  Google Scholar 

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Authors and Affiliations

  1. School of Biological Sciences, Queen Mary and Westfield College, Mile End Road, E1 4NS, London, UK

    Susan E. Wilkie & Martin J. Warren

  2. Department of Plant Sciences, University of Cambridge, Tennis Court Road, CB2 3EA, Cambridge, UK

    Jennifer M. Roper & Alison G. Smith

Authors
  1. Susan E. Wilkie
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  2. Jennifer M. Roper
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  3. Alison G. Smith
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  4. Martin J. Warren
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Wilkie, S.E., Roper, J.M., Smith, A.G. et al. Isolation, characterisation and expression of a cDNA clone encoding plastid aspartate aminotransferase from Arabidopsis thaliana . Plant Mol Biol 27, 1227–1233 (1995). https://doi.org/10.1007/BF00020897

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  • DOI: https://doi.org/10.1007/BF00020897

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