Molecular cloning of a cDNA encoding aspartate aminotransferase-P2 from lupin root nodules.
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 lambda ZapII 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.
Reynolds
P H
PH
Department of Scientific and Industrial Research-Fruit and Trees, Palmerston North, New Zealand.
Smith
L A
LA
Dickson
J M
JM
Jones
W T
WT
Jones
S D
SD
Rodber
K A
KA
Carne
A
A
Liddane
C P
CP
D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D007887:Fabaceae / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D011487:Protein Conformation; D012231:Rhizobium / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D013559:Symbiosis
D007527:Isoenzymes; D004247:DNA; D001219:Aspartate Aminotransferases