No abstract

With a view to exploring its use as a metal-binding factor in transgenic plants we prepared the alpha-domain of metallothionein by reconstitution of rabbit apometallothionein and proteolysis of MT-1 and MT-2 with subtilisin. The isolated alpha-domains were characterised by UV and CD spectroscopy Double-Stranded. DNA encoding the alpha-domain (106 bp) of the human MT-IA was constructed from chemically synthesized oligomers by repair synthesis and enzymatic ligation, cloned into pUC19 and sequenced. A expression construct containing the cloned alpha-domain was introduced into tobacco cells on a disarmed Agrobacterium tumefaciens Ti-plasmid. Transformed tobacco cells were selected and regenerated on medium containing cadmium and kanamycin. The growth of roots and shoots of transformants was unaffected by up to 100 microM cadmium, whereas control plants showed severe inhibition of root and shoot growth, and chlorosis of leaves on medium containing only 10 microM cadmium. Southern hybridization confirmed the presence of the transgene in the transformed plant tissues. The concentration of human alpha-domain peptides in transgenic tobacco leaves was determined by the Cd/hemoglobin saturation assay and polarography using the rabbit alpha-domain as standard. The results indicate that the alpha-domain, one of two domains in MT molecules, is not only stable in vitro, but is also expressed efficiently and functions independently in transgenic plant cells.

No abstract

Metallothioneins (MT) are low molecular weight, cysteine-rich, metal-binding proteins. An MT molecule contains two domains which appear to act independently--an alpha-domain, which is characterized by cadmium-binding, and a beta-domain, which binds preferentially to copper. Based on this conception, DNA duplex encoding the alpha-domain (106 bp) of human MT-IA was constructed from a chemically-synthesized oligomer by repair synthesis and enzymatic ligation and cloned into pUC19. The genes cloned were sequenced and found to be in the correct order as designed. Synthetic directional adapters were attached to the terminals of the alpha-domain gene fragment of human MT-IA to establish complete control over fragment orientation during ligation. The use of these directional adapters thereby ensured the production of multiple copies of the alpha-domain in tandem arrays. The successive alpha-domains were linked by a peptide linker consisting of 10 residues. A chimeric gene containing 12 cloned tandemly repeated copies of the 106 bp alpha-domain DNA was introduced into tobacco cells on a disarmed Ti-plasmid of Agrobacterium tumefaciens. A total of 10 different transgenic tobacco plants were generated, of which two showed root and shoot growth unaffected by up to 200 mg/l kanamycin and 100 microM cadmium, whereas root growth of control plants was severely inhibited and leaf chlorosis developed on media containing only 10 microM cadmium.

Zn-metallothioneins (MT-1 and MT-2) were isolated and purified from Wistar rat liver induced by subcutaneous injection with cadmium chloride over a short time. Instead of Sephadex G-50 and DEAE Sephadex A-50, new chromatographic media produced by Pharmacia, Sephacryl S-200, S-100 and DEAE Sepharose Fast Flow were used in the purification of metallothioneins. The time required for purification with the new method was only 1/3 that required with the usual method and had the same purification effect and rate of recovery. The number of mercapto groups measured with modified Ellman's reagent and cysteine as standard is 20 in MT molecules. Zn and Cd concentrations in each fraction were measured by single sweep polarography rather than atomic absorption spectrophotometry. MT-1 and MT-2 contained 6 gram atoms of zinc, but no cadmium. Purified MT-1 and MT-2 were shown by high performance liquid chromatographic analysis to be highly homogeneous and had an amino acid composition similar to that of Cd-MT.