ISOLATION, CHARACTERIZATION AND EXPRESSION OF A CDNA CLONE ENCODING PLASTID ASPARTATE-AMINOTRANSFERASE FROM ARABIDOPSIS-THALIANA

A clone encoding aspartate aminotransferase (AAT, EC 2.6.1.1) was isol ated from an Arabidopsis thaliana leaf cDNA library. This clone contai ns a 1365 bp open reading frame encoding a polypeptide of 49.8 kDa, de signated Ataat1. The clone was shown to contain a chloroplastic isoenz yme as an in organellar protein import assay demonstrated that a radio labelled transcription/translation product of 49.8 kDa was imported in to viable pea chloroplasts and was subsequently processed to yield a m ature protein of 45 kDa. The open reading frame corresponding to the p redicted mature AAT was manipulated into an expression construct (pEC1 4). Transformed Escherichia coli cells containing pEC14 expressed up t o 16 times more AAT activity than vector only controls, thus demonstra ting conclusively that the clone encoded AAT.