MTT2, a copper-inducible metallothionein gene from Tetrahymena thermophila.
Metallothioneins (MTs) are ubiquitous, cysteine-rich, metal-binding proteins whose transcriptional activation is induced by a variety of stimuli, in particular heavy metals such as cadmium, copper and zinc. Here we describe the sequence and organization of a novel copper-inducible metallothionein gene (MTT2) from Tetrahymena thermophila. Based on its deduced sequence, the gene encodes a protein 108 amino acids, containing 29 cysteine residues (30%) arranged in motifs characteristic of vertebrate and invertebrate MTs. We demonstrate that the 5'-region of the MTT2 gene can act as an efficient promoter to drive the expression of heterologous genes in the Tetrahymena system. In the latter case, a gene for a candidate vaccine antigen against Ichthyophthirius multifiliis, a ubiquitous parasite of freshwater fish, was expressed at high levels in transformed T. thermophila cell lines. Moreover, the protein was properly folded and targeted to the plasma membrane in its correct three-dimensional conformation. This new copper-inducible MT promoter may be an attractive alternative to the cadmium-inducible MTT1 promoter for driving ectopic gene expression in Tetrahymena and could have a great impact on biotechnological perspectives.
Boldrin
Francesco
F
Department of Biology, University of Padova, 35100 Padova, Italy. francesco.boldrin@unipd.it
Santovito
Gianfranco
G
Formigari
Alessia
A
Bisharyan
Yelena
Y
Cassidy-Hanley
Donna
D
Clark
Theodore G
TG
Piccinni
Ester
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019256:Cadmium Chloride / Q000378:metabolism / Q000494:pharmacology; D003001:Cloning, Molecular; D019327:Copper Sulfate / Q000378:metabolism / Q000494:pharmacology*; D005786:Gene Expression Regulation; D008668:Metallothionein / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D030781:Organisms, Genetically Modified; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins; D020539:Sequence Analysis, Protein; D016808:Tetrahymena thermophila / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D012333:RNA, Messenger; D011994:Recombinant Proteins; D008668:Metallothionein; D019256:Cadmium Chloride; D019327:Copper Sulfate