The goal of the present project proposal is to test the hypothesis of metal-specific adaptation of the two
metallothionein (MT) isoforms (Cd-MT and Cu-MT) and their genes from the terrestrial pulmonate mollusc, Helix pomatia at both, the protein expression and the gene regulatory levels. The background of this hypothesis is the fact that the two MT isoforms from this species apparently are differentially induced by and preferentially bind different metals (Cd2+ or Cu+) in vivo and perform metal-specific functions: while one isoform (Cd-MT) serves
detoxification of Cd2+, the second isoform (Cu-MT) seems to be involved in Cu regulation and homaeostasis.
We will achieve our goal by two different approaches: first, metal preference will be tested after recombinant expression of the two MT isoforms by metal replacement reactions of native Zn-thioneins with combined metal solutions (Cd2+ and Cu+). The resulting metal thionein complexes will then be analysed by UV / Vis and CD spectroscopy, as well as mass spectrometry. This part of the study will mainly be carried out in cooperation with the group of Prof. Dra. Silvia Atrian at the institute of Genetics of the University of Barcelona (Spain).
Second, we will test the metal specificity of the two MT isoform genes on the basis of the gene sequence which we have elucidated during the last two years. Specifically, we would like to design reporter gene fusion constructs transfected in Biomphalaria glabrata embryonic cells (Bge), exposed to metal solutions (Cd2+, Zn2+ and Cu+) singly or in combination. Biomphalaria glabrata is a freshwater mollusc, the genomic DNA of which is currently being elucidated. For transfection, we propose to use an array of reporter gene constructs in which functional MT promoters of the two MT isoform genes from Helix pomatia will be fused to a reporter consisting of the mutually exchanged cDNA of the MT counterpart (i.e. Cd-MT promoter with Cu-MT cDNA and vice versa), tagged at its 3'- end by attachment of EGFP (Enhanced Green Fluorescence Protein) cDNA. The promoters will be modified by site-directed mutagenesis to obtain products with changed positions in MREs and in adjacent binding sites for enhancing or silencing nuclear transcription factors. This way, we will be able to study the metal-specific
transcriptional activity of the two genes in dependence of the number and position of Metal Responsive Elements (MREs), and their interaction with other nuclear transcription factors (enhancers and silencers) which could possibly modulate metal-specific transcription. The transcriptional activity of the promoter elements will be detected and quantified by both, real-time detection PCR and laser scanninc microscopy, and the quantified transcriptional activity referred to the number of successfully transfected Bge cells.
In addition to this, the cDNA of MT isoforms and of the Metal Transcription Factor (MTF) from Biomphalaria glabrata will be synthesized.
