Characterization of Vitis vinifera L. glutamine synthetase and molecular cloning of cDNAs for the cytosolic enzyme.
Grapevine (Vitis vinifera L.) glutamine synthetase (GS) was analysed into two distinct classes of isoforms; one of them was present in both leaf and root tissues while the other one showed leaf specificity. Western blot analysis revealed that grapevine GS consists of three types of polypeptides of distinct size and differential tissue specificity. Two structurally distinct cDNA clones, pGS1;1 and pGS1;2, encoding grapevine GS were isolated from a cell suspension library and characterized. Both clones contained open reading frames encoding for polypeptides of 356 amino acids with a predicted molecular mass of about 39 kDa. Although the coding sequences of pGS1;1 and pGS1;2 were 84% similar, their 5'- and 3'-untranslated sequences showed only 40% similarity. The coding sequences of the two clones and the derived amino acid sequences showed higher homology to cytosolic than to chloroplastic GSs of other higher plants indicating that the cDNAs isolated encode for cytosolic isoforms of grapevine GS. Southern blot analysis suggested the existence of more than two GS genes in the grapevine genome. In northern blots both clones were hybridized to mRNAs of about 1.4 kb that are differentially expressed in the various tissues. Supply of nitrate or ammonium in the cell suspension culture medium, as a sole nitrogen source, resulted in differential response of the pGS1;1- and pGS1;2-related genes.
Loulakakis
K A
KA
Department of Biology, University of Crete, Greece.
Roubelakis-Angelakis
K A
KA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003600:Cytosol / Q000201:enzymology*; D018076:DNA, Complementary / Q000235:genetics; D005638:Fruit / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D017343:Genes, Plant; D005974:Glutamate-Ammonia Ligase / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D018749:RNA, Plant / Q000235:genetics; D017422:Sequence Analysis, DNA; D014018:Tissue Distribution
D018076:DNA, Complementary; D007527:Isoenzymes; D018749:RNA, Plant; D005974:Glutamate-Ammonia Ligase