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Molecular cloning of a cDNA encoding aspartate aminotransferase-P2 from lupin root nodules

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Abstract

Two isoenzymic forms of aspartate aminotransferase are present in the plant fraction of developing lupin root nodules. One of these forms, aspartate aminotransferase-P2 (AAT-P2), increases dramatically with the onset of biological nitrogen fixation and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. A day 18 lupin nodule cDNA library in the λZapII vector was immunoscreened with a monoclonal antibody specific for AAT-P2 and yielded two near-full-length 1700 bp clones. These clones were sequenced. Amino acid sequences from three peptides derived from immunopurified AAT-P2 were aligned, and showed 100% homology with the amino acid sequence deduced from the cDNA clones. The DNA sequence showed 50% homology with AAT sequences from a range of animal sources. Conversion of the clones to the phagemid form allowed their expression in Escherichia coli where both exhibited enzyme activity that could be immunoprecipitated with AAT-P2-specific monoclonal antibodies. Western blot analysis revealed protein moieties with molecular masses of 39, 43, 45 and 55 kDa. The 5′ end of the clones coded for a hydrophobic leader sequence of about 50 amino acids indicative of a targeting sequence and consistent with the plastid localisation of nodule AAT-P2.

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Authors and Affiliations

  1. Immunology and Plant Molecular Biology, Department of Scientific and Industrial Research-Fruit and Trees, Private Bag, Palmerston North, New Zealand

    Paul H. S. Reynolds, Laura A. Smith, James M. J. J. Dickson, William T. Jones, Stephen D. Jones, Karen A. Rodber & Cherry P. Liddane

  2. Biochemistry Department, University of Otago, Dunedin, New Zealand

    Alan Carne

Authors
  1. Paul H. S. Reynolds
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  2. Laura A. Smith
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  3. James M. J. J. Dickson
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  4. William T. Jones
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  5. Stephen D. Jones
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  6. Karen A. Rodber
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  7. Alan Carne
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  8. Cherry P. Liddane
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Reynolds, P.H.S., Smith, L.A., Dickson, J.M.J.J. et al. Molecular cloning of a cDNA encoding aspartate aminotransferase-P2 from lupin root nodules. Plant Mol Biol 19, 465–472 (1992). https://doi.org/10.1007/BF00023394

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  • DOI: https://doi.org/10.1007/BF00023394

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