Plant Physiol. (1 995) 108: 1321-1 322
f h n f Gene Regisfer
Molecular Cloning of the Gene Encoding the i-Asparaginase Gene of Arabidopsis thaliana' Antonio Casado, José L. Caballero, Antonio R. Franco, Jacobo Cárdenas, Murray Juan Muííoz-Blanco*
R. Crant,
and
Departmento de Bioquímica y Biologia Molecular, Facultad de Ciencias, Universidad de Córdoba, Avda de San Alberto Magno s/n, 14071Cordoba, Spain In plants, ammonium is first assimilated into the amino acids L-Gln and L-glutamate and then channeled into L-aspartate and L-Asn. These amino acids serve as nitrogen donors in biosynthetic reactions, as well as compounds for intercellular nitrogen transport. In amide-transporting plants, Asn can act as the sole nitrogen source for the growth of plant tissues. In higher plants the catabolism of Asn is thought to occur mainly through two routes. The first pathway involves the hydrolysis of Asn, releasing ammonia and aspartate by asparaginase activity. The second pathway proceeds via transamination in a reaction catalyzed by the asparaginase:2oxoacid transaminase (Lea et al., 1990). L-Asparaginase is a key enzyme for Asn utilization by plants that plays an important role in the nitrogen metabolism of developing plant tissues (Sieciechowicz et al., 1988). The ammonia released from the hydrolysis of the translocated Asn is utilized in the synthesis of all nitrogen-containing compounds of the cell and in particular in the synthesis of the amino acids needed for protein synthesis. Recently, the genomic clone of asparaginase from Lupinus angustifolius seeds was cloned and characterized (Dickson et al., 1992).The gene consisted of four exons and three introns (Dickson et al., 1992). Also, a cDNA corresponding to the clone that codes for the asparaginase from Lupinus arboreus was isolated (Lough et al., 1992). Southern blot experiments have shown that this gene is a single-copy gene expressed only in seeds (Lough et al., 1992). A genomic clone for L-asparaginase has been isolated from an Arabidopsis tkaliana cv Landsberg erecta AFIX genomic library by heterologous screening with an L. arboreus cDNA probe. The clone, which contains a 14-kb insert, has been studied by restriction and Southern blot analysis (Table I). The L-asparaginase gene was included within two 2.5-kb HindIII fragments. Both fragments were exonuclease I11 deleted, and the corresponding clones were sequenced. Sequences were compared with the genomic sequence of L-asparaginase of L. angustifolius. The results show that both Arabidopsis and Lupinus genes have a similar molecular organization and consist of three introns
Table 1. Characteristic of the L-asparaginase gene of A. thaliana Organism: Arabidopsis thaliana Landsberg erecta and Columbia. Genome Location: Nuclear genome. Function: Encodes L-asparaginase enzyme. Method of Identification: Comparison of the nucleotides and amino acids sequences with those of the L-asparaginase gene of Lupinus. Techniques: Genomic and expression library screening, isolation of phage DNA, restriction fragment subcloning into pBluescript vector, exonuclease 111 deletions, and automatic sequencing using an Applied Biosystems apparatus. Features of Genomic Fragment Containing the L-Asparaginase Sequence: 2200 nucleotides 5' to the putative start of translation; coding region constituted by four exons and three introns; possible TATA box at nucleotide 2214; possible CAAT box at nucleotide at nucleotide 2069. Structural Features of Deduced Protein Sequence: The coding region encodes a polypeptide of 31 5 amino acids, with a calculated molecular mass of 33.03 KDa, a -9 net charge, and a pl of 5.26. Expression Characteristics: Not determined (studies in progress). Anti body: Not available.
and four exons. A high homology between A . thaliana and L. angustifolius genes was found (>71 and 75% for nucleotides and amino acids, respectively). A 2200-bp 5' flanking region was also sequenced, and it was found to contain important putative regulatory sequences. Possible TATA and CAAT boxes are located at nucleotide positions 2214 and 2069, respectively. Our results indicate that the clone studied corresponds to the A. tkaliana L-asparaginase gene. Also, we have isolated and sequenced a full-length L-asparaginase cDNA clone from an A. tkaliana cv Columbia expression library by screening with the first exons of the gene encoding the L-asparaginase from A. tkaliana cv Landsberg erecta. The cDNA clone was 1.2 kb in size and showed a complete open reading frame 948 bp in length, starting at the first ATG codon of the cDNA sequence at
'
This research was supported by grant No. PB93-0719 f r o m Dirección General de Investigación Científica y Tecnológica 3, Spain. A.C. thanks the University of Córdoba for a fellowship. * Corresponding author; e-mail
[email protected]; fax 34-57-
218606. 1321
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Casado et al.
nucleotide position 2214 and terminating with a TGA stop codon (position 3556) of the genomic clone. A leader region of at least 44 bp was observed. Comparison of nucleotide and predicted amino acids sequences between A . tkaliana cv Landsberg erecta and A . tkaliana cv Columbia has shown differences in the third and fourth exons. Moreover, a high percentage of identity between A. thaliana and L. arboreus cDNA clones has been found (>71 and >83% for nucleotides and amino acids, respectively). Received December 23, 1994; accepted January 28, 1995. Copyright Clearance Center: 0032-0889/95/108/1321/02. The GenBank accession number for the sequence reported in this article is 234884.
Plant Physiol. Vol. 108, 1995 LITERATURE ClTED
Dickson JMJJ, Vincze E, Grant MR, Smith LA, Rodber KA, Farnden KJF, Reynolds PHS (1992) Molecular cloning of the gene encoding developing seed L-asparaginase from Lupinus angustifolius. Plant Mo1 Biol 20: 333-336 Lea PJ, Robinson SA, Stewart GR (1990) The enzymology and metabolism of glutamine, glutamate and asparagine. 1n BJ Miflin, PJ Lea, eds, The Biochemistry of Plants, Vol 16. Academic Press, San Diego, CA, pp 147-152 Lough TJ, Reddington BD, Grant MR, Hill DF, Reynolds PHS, Farnden KJF (1992) The isolation and characterisation of a cDNA clone encoding L-asparaginase from developing seeds of lupin (Lupinus nvboreus). Plant Mo1 Biol 19: 391-399 Sieciechowicz KA, Joy KW, Ireland RJ (1988) The metabolism of asparagine in plants. Phytochemistry 27: 663-671
