α-Domain of human metallothionein IA can bind to metals in transgenic tobacco plants

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.

A critical review on the bio-removal of hazardous heavy metals from contaminated soils: Issues, progress, eco-environmental concerns and opportunities

“…Studies on this field are increasing and its mechanism is more and more clear [33][34][35][36][37][38][39][40][41]. Using transgenic technology is a tendency in the future to create an ideal species purposely.…”

Genetic engineering in the improvement of plants for phytoremediation of metal polluted soils

“…Various MT genes Ð mouse MTI, human MTIA (alpha domain), human MTII, Chinese hamster MTII, yeast CUP1, pea PsMTA Ð have been transferred to Nicotiana sp., Brassica sp. or A. thaliana (Lefebvre et al, 1987;Maiti et al, 1988Maiti et al, , 1989Maiti et al, , 1991Misra and Gedamu, 1989;Evans et al, 1992;Yeargan et al, 1992;Brandle et al, 1993;Pan et al, 1993Pan et al, , 1994aElmayan and Tepfer, 1994;Hattori et al, 1994;Hasegawa et al, 1997). As a result, varying degrees of constitutively enhanced Cd tolerance have been achieved, being maximally 20-fold compared with the control.…”

Molecular cloning and characterization of six cDNAs expressed during glucose starvation in excised maize (Zea mays L.) root tips

“…The pZSS5 protein belongs to a family of cysteine proteinases from a wide range of plant and animal species [35,36]. pZSS6 cDNA encodes a metallothionein-like protein which belongs to a family of heavy-metal-binding proteins functioning coordinately as a detoxifying mechanism [39,49]. The nucleotide sequence analysis of pZSS7 cDNA shows that we have isolated the same MPI proteinase inhibitor gene as Cordero et aL [ 12].…”

“…James et al [26] showed that starvationinduced protein degradation was correlated with an overall increase of proteolytic activities; furthermore, the use of inhibitors specific for cysteine proteinases clearly demonstrated that cysteine proteinases participate in protein degradation processes during sugar starvation [26]. The protein breakdown may release metal ions which have to be either stored and/or transported for further processes or sequestered to prevent their toxic effects [9,39,49]. Metallothionein-like proteins are thought to be involved in this detoxification role as recently proposed by BuchananWollaston [9] who isolated a cDNA clone for a senescence-specific metallothionein-like gene from Brassica napus.…”