CONSTRUCTION OF MULTIPLE COPY OF ALPHA-DOMAIN GENE FRAGMENT OF HUMAN LIVER METALLOTHIONEIN-I(A) IN TANDEM ARRAYS AND ITS EXPRESSION IN TRANSGENIC TOBACCO PLANTS

Metallothioneins (MT) are low molecular weight, cysteine-rich, metal-b inding proteins. An MT molecule contains two domains which appear to a ct independently-an alpha-domain, which is characterized by cadmium-bi nding, and a beta-domain, which binds preferentially to copper. Based on this conception, DNA duplex encoding the alpha-domain (106 bp) of h uman MT-IA was constructed from a chemically-synthesized oligomer by r epair synthesis and enzymatic ligation and cloned into pUC19. The gene s cloned were sequenced and found to be in the correct order as design ed. Synthetic directional adapters were attached to the terminals of t he ce-domain gene fragment of human MT-IA to establish complete contro l over fragment orientation during ligation. The use of these directio nal adapters thereby ensured the production of multiple copies of the alpha-domain in tandem arrays. The successive ce-domains were linked b y a peptide linker consisting of 10 residues. A chimeric gene containi ng 12 cloned tandemly repeated copies of the 106 bp alpha-domain DNA w as introduced into tobacco cells on a disarmed Ti-plasmid of Agrobacte rium tumefaciens. A total of 10 different transgenic tobacco plants we re generated, of which two showed root and shoot growth unaffected by up to 200 mg/l kanamycin and 100 muM cadmium, whereas root growth of c ontrol plants was severely inhibited and leaf chlorosis developed on m edia containing only 10 muM cadmium.