Construction of multiple copy of alpha-domain gene fragment of human liver metallothionein IA in tandem arrays and its expression in transgenic tobacco plants.
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.
Pan
A
A
National Laboratory of Protein Engineering and Plant Genetic Engineering, Department of Biology, Peking University, Beijing, China.
Tie
F
F
Yang
M
M
Luo
J
J
Wang
Z
Z
Ding
X
X
Li
L
L
Chen
Z
Z
Ru
B
B
D016960:Agrobacterium tumefaciens / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D002104:Cadmium / Q000378:metabolism / Q000494:pharmacology; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D004274:DNA, Recombinant; D015870:Gene Expression; D006801:Humans; D008099:Liver / Q000737:chemistry*; D008668:Metallothionein / Q000235:genetics*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D010957:Plasmids; D015183:Restriction Mapping; D014026:Nicotiana; D014162:Transfection
D004274:DNA, Recombinant; D002104:Cadmium; D004247:DNA; D008668:Metallothionein