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(W/EH011) Cloning and Sequencing of Bank vole Metallothionein I and II Genes. Savva, Demetris1, Garcia-Aljaro, Cristina2, Lawes, Kathryn P. 1, Swiergosz, Renata 3, 1 2 3 ABSTRACT- Metallothioneins (MTs) are ubiquitous low molecular weight (6000-8000 daltons) proteins that bind heavy metals such as zinc, cadmium, copper and mercury. MT gene expression may be up-regulated as a consequence of high metal contamination of the environment. The aim of this study was to clone and characterise the MT gene from the bank vole (Clethrionomys glareolus) and to use it as a probe to investigate MT gene expression in animals from environments contaminated with heavy metals. DNA was isolated from muscle tissues of bank voles and used in PCR reactions to amplify the MT gene; the primers used for the PCR were based on the consensus sequence of the MT-I gene of other rodents. Analysis of the PCR products showed 3 distinct DNAs (approximately 900, 500 and 200 bp respectively); all of these were ligated individually to plasmid pGEM-T Easy and transformed into Escherichia coli. Recombinant plasmids were isolated from transformed cells and their nucleotide sequences were determined. The results showed that the 200 bp DNA corresponded to the cDNA sequence of MT-I (presumably amplified from traces of RNA contaminating the original DNA). The 900 bp DNA corresponded to the genomic DNA for MT-I; its sequence showed the presence of 2 introns and exon sequences identical to the cDNA for MT-I. The 500 bp DNA was found to have good similarity to genomic sequences of MT-II from other rodents. In both MT-I and MT-II the intron positions and the positions of the 20 cysteines were identical to those in the corresponding genes in other rodents. Key words: metallothionein, bank vole, molecular probe |
