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Purification and characterization of a tRNA nucleotidyltransferase from Lupinus albus and functional complementation of a yeast mutation by the corresponding cDNA

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Abstract

ATP (CTP):tRNA nucleotidyltransferase (EC 2.7.7.25) was purified to apparent homogeneity from a crude extract of Lupinus albus seeds. Purification was accomplised using a multistep protocol including ammonium sulfate fractionation and chromatography on anion-exchange, hydroxylapatite and affinity columns. The lupin enzyme exhibited a pH optimum and salt and ion requirements that were similar to those of tRNA nucleotidyltransferases from other sources. Oligonucleotides, based on partial amino acid sequence of the purified protein, were used to isolate the corresponding cDNA. The cDNA potentially encodes a protein of 560 amino acids with a predicted molecular mass of 64164 Da in good agreement with the apparent molecular mass of the pure protein determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The size and predicted amino acid sequence of the lupin enzyme are more similar to the enzyme from yeast than from Escherichia coli with some blocks of amino acid sequence conserved among all three enzymes. Functionality of the lupin cDNA was shown by complementation of a temperature-sensitive mutation in the yeast tRNA nucleotidyltransferase gene. While the lupin cDNA compensated for the nucleocytoplasmic defect in the yeast mutant it did not enable the mutant strain to grow at the non-permissive temperature on a non-fermentable carbon source.

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Author notes

  1. Kandavel Shanmugam

    Present address: McGill Cancer Centre, McGill University, 3655 Drummond St., H3G 1Y6, Montreal, Quebec

Authors and Affiliations

  1. Departments of Chemistry and Biology, Concordia University, 1455 deMaisonneuve Blvd., West, H3G 1M8, Montreal, Quebec

    Kandavel Shanmugam & Paul B. M. Joyce

  2. Departments of Chemistry and Biochemistry, Concordia University, 1455 deMaisonneuve Blvd., West, H3G 1M8, Montreal, Quebec

    Pamela J. Hanic-Joyce & Paul B. M. Joyce

Authors
  1. Kandavel Shanmugam
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  2. Pamela J. Hanic-Joyce
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  3. Paul B. M. Joyce
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Shanmugam, K., Hanic-Joyce, P.J. & Joyce, P.B.M. Purification and characterization of a tRNA nucleotidyltransferase from Lupinus albus and functional complementation of a yeast mutation by the corresponding cDNA. Plant Mol Biol 30, 281–295 (1996). https://doi.org/10.1007/BF00020114

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  • DOI: https://doi.org/10.1007/BF00020114

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