Molecular and functional evolution of Tetrahymena metallothioneins: new insights into the gene family of Tetrahymena thermophila.

A new metallothionein (MTT-5) gene isoform has been cloned and characterized from the ciliate Tetrahymena thermophila. Its amino acid sequence shows only limited similarity with other Tetrahymena MTs. To investigate the transcriptional activity of this gene toward heavy metals (Cd, Cu, Zn), mRNA levels were evaluated by real-time quantitative PCR. Results show that the three metals induce different MTT-5-mRNA levels, Cd treatment eliciting the most effective induction in the first 30 min. Phylogenetic analyses of all Tetrahymena MT protein sequences revealed that MTT-5 is closely related to Cd-induced isoforms and quite separate from Cu-induced ones. Our results indicate that Cd and Cu MTs diverged early in evolution, before the speciation event which separated the Tetrahymena borealis group from the Tetrahymena australis group. The mutation rate in the Tetrahymena MT group is heterogeneous, being very low for MT-1 and MTT-1 and higher for the other isoforms, particularly for MTT-5, which shows the maximum divergence among the Cd-induced MTs. This observation, together with the evidence of its inducibility by Zn - a unique condition among T. thermophila MTs - indicates that MTT-5 underwent a particular evolutionary history, independent of other MT isoforms.