Identification and Characterisation of a Copper-inducible Metallothionein Gene from Cockle, Anadara granosa

The cockle, Anadara granosa, was experimentally exposed to low (0.1 mg/L) and sublethal (1.0 mg/L) doses of copper (Cu) for a period of 24 hrs. Significant increase in Cu concentrations in whole tissues and hepatopancreas compared to control animals were observed. In order to study the effect of copper exposure at molecular levels, a subtractive cDNA library was constructed from the hepatopancreas of cockles exposed to 1.0 mg/L Cu. Screening of the subtractive cDNA library using reverse northern analysis resulted in several differentially expressed genes, including one that codes for metallothionein (MT). The complete coding sequence of the MT gene (designated as AnaMT2) reveals an open reading frame of 234 bp in length that encodes a 77 amino acid polypeptide as revealed by the deduced amino acid composition. Although showing similarities with other molluscan MTs, AnaMT2 can be distinguished by its lower glycine and higher asparagine and proline content. Expression analysis of the AnaMT2 by northern analysis indicated higher mRNA level in cockle exposed to 1.0 mg/L Cu and was undetectable in those treated with 0.1 mg/L. This suggests that AnaMT2 represents a primarily inducible MT not highly expressed under basal conditions.