Construction of multiple copy of α-domain gene fragment of human liver metallothionein I_A in tandem arrays and its expression in transgenic tobacco plants

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.

Structural Adaptation of a Protein to Increased Metal Stress: NMR Structure of a Marine Snail Metallothionein with an Additional Domain

“…Communications MT with at hird domain is expected to provide an increased metal-loading capacity and hence an increased protective effect against harmful intracellular and environmental Cd 2+ concentrations.E xperimental evidence for this assertion comes from studies with tandem-repeat constructs of human MT-1A domains expressed in transgenic Tobacco,w hich confer greatly increased metal resistance on Cd 2+ -exposed plants. [38] Moreover,t he addition of at hird metal-binding domain to an MT may also represent an evolutionary advance,assuggested by studies in yeast cells demonstrating that from an energetic point of view,the expression of alarger protein to improve ag iven function may be slightly less energy-demanding than achieving the same effect through gene duplication. [39] Thus,f rom an evolutionary standpoint, the three-domain structure likely confers on LlMT ah igher functional efficiency without greatly increasing the energy demand for its expression.…”

“…[16] Littorina littorea lives on rocky seashores,where it is intermittently exposed to adverse environmental conditions including anoxia, freezing,desiccation, and stress-dependent metabolic depression. [38] Moreover,t he addition of at hird metal-binding domain to an MT may also represent an evolutionary advance,assuggested by studies in yeast cells demonstrating that from an energetic point of view,the expression of alarger protein to improve ag iven function may be slightly less energy-demanding than achieving the same effect through gene duplication. [38] Moreover,t he addition of at hird metal-binding domain to an MT may also represent an evolutionary advance,assuggested by studies in yeast cells demonstrating that from an energetic point of view,the expression of alarger protein to improve ag iven function may be slightly less energy-demanding than achieving the same effect through gene duplication.…”

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.…”

Strukturanpassung eines Proteins an Metallbelastung: NMR‐Struktur eines marinen Schnecken‐Metallothioneins mit einer zusätzlichen Domäne

“…Die Expression eines MT mit einer zusätzlichen dritten Domäne erhöht daher erwartungsgemäß dessen Metallbindungskapazität und somit seine Schutzfunktion gegen intrazelluläre und umweltbedingte schädliche Cd 2+ ‐Konzentrationen. Ein experimenteller Nachweis für diese Annahme erbrachten Untersuchungen an Tandemkonstrukten mit menschlichen MT‐1A‐Domänen, die in transgenen Tabakpflanzen exprimiert wurden und dabei Cd 2+ ‐exponierten Pflanzen eine um ein Vielfaches gesteigerte Resistenz gegenüber den Schwermetallionen verliehen . Im Übrigen stellt die Vergrößerung eines MT um eine dritte metallbindende Domäne wahrscheinlich auch einen evolutionsbiologischen Fortschritt dar.…”