Formigari, Alessia (2008) Metallotioneine: Evoluzione molecolare e funzionale nei protozoi ciliati appartenenti al genere Tetrahymena. [Tesi di dottorato]
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The ciliated protozoan, Tetrahymena thermophila, has long served as a model for basic research. At the same time, its unique biological properties make it extremely attractive as practical system for the overexpression of recombinant proteins.
The scientific community has recently taken into great consideration the use of T. thermophila for the production of recombinant proteins for both biotechnological perspectives and as research tools, since no system (e. g., bacteria, fungi, insect and mammalian cell lines) is universally suitable for the expression of foreign genes. The utility of T. thermophila as an experimental model organism has been recently increased by the development of inducible expression vectors based on the MTT1 metallothionein promoter. Metallothioneins (MT) are a group of low molecular weight proteins which bind metals through their numerous cysteine residues and which are present in many phyla. One of the most important properties of these proteins is their ability to be transcriptionally induced by transition metals. The research studies, made during my PhD course, focused on molecular and fuctional evolution of MT in T. thermophila.
In the present study, two new MT (MTT-5 in T. thermophila and MT-2 in T. pyriformis) gene isoforms have been cloned and characterized. The transcriptional activity of MTT-2 and MTT-5 towards heavy metals (cadmium, copper and zinc), was evaluated. Results showed that the three metals induced different MTT-2-mRNA and MTT-5-mRNA levels. Furthermore, using the coding sequence of nrk2-GFP gene as a reporter, I found that the upstream region of MTT5 induced nrk2-GFP protein expression. The fluorescence was present in the basal bodies and at the tips of cilia within transformed cells. These results have substantially contributed to demonstrate the suitability of free-living protozoan in studies of cell tolerance to heavy metals, and they have validated the use of Tetrahymena as a model system to determine ecotoxicological risk. To date, only a limited number of inducible promoters for driving high-level gene expression in T. thermophila has been described. Furthermore, little is known about the sequence elements that regulate gene expression within the upstream regions of ciliate genes. I described a robust new promoter from MTT-5, a T. thermophila cadmium-inducible MT gene of T. thermophila during my research studies. Using, as a reporter, the coding sequence of a gene for a candidate vaccine antigen from a parasitic protist, Ichthyophthirius multifiliis, I found that the upstream region of MTT5 can induce high-level expression of the relevant gene product in its correct 3-dimensional conformation. Expression was sensitive to cadmium, and to lesser extent copper, but not to zinc. Moreover, a direct comparison between this promoter and one from a previously described Cd-inducible MT gene, namely MTT1, shows that MTT5 is considerably stronger.
To begin to define the sequence elements underlying the metal-responsiveness of this promoter, I constructed a series of deletions starting with a 1777 bp fragment immediately upstream of the start codon of MTT5, which is fully functional. Interestingly, a fragment as short as 300 bp upstream of the start codon appears fully functional as well, while a deletion down to 290 bp essentially abolishes activity in response to cadmium. These results have been confirmed by site-specific mutagenesis. This study indicates that the MTT5 promoter can be used as a practical tool for protein manufacture in T. thermophila, and sheds light on potential sequence elements responsible for metal-induced promoter control in this species. The identification of this MRE sequences in Tetrahymena, quite different from those previously identified in mammalians, represents the first step to characterize molecular mechanisms involved in the expression of Tetrahymena MT isoforms.
Phylogenetic analysis performed with all Tetrahymena MT protein and nucleotidic 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 that separated the Tetrahymena borealis group and the Tetrahymena australis group. The mutation rate in the Tetrahymena MTs appears to be heterogeneous, being very low for MT-1 and MTT-1 and higher for other isoforms. The phylogentic analysis indicates a particular evolutionary history independent from other MT isoforms, for MTT-5.
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