Comparative Biochemistry and Physiology Part C 131 (2002) 217–222 1532-0456/02/$ - see front matter  2002 Elsevier Science Inc. All rights reserved. PII: S1532-0456 Ž 02 . 00008-X Cloning and sequencing of a novel metallothionein gene in Mytilus galloprovincialis Lam  Nadia Ceratto, Francesco Dondero*, Jan-Willem van de Loo, Bruno Burlando, Aldo Viarengo Dipartimento di Scienze e Tecnologie Avanzate, Universita del Piemonte Orientale, C.so Borsalino, 54-15100 Alessandria, Italy ` Received 21 December 2000; received in revised form 29 March 2001; accepted 3 April 2001 Abstract Metallothionein (MT) is a ubiquitous, metal-inducible protein with an important role in the homeostasis and in the detoxification of heavy metals. This work reports the cloning and sequencing of a MT gene encoding a MT isoform (MT20-IIIa) in the mussel Mytilus galloprovincialis Lam, a lamellibranch mollusc known to accumulate and to detoxify large amounts of metal. The MT gene, lacking the 59 promoter region, is 1865 bp long and has a tripartite structure consisting of three exons and two introns. The putative open reading frame (ORF) encodes a polypeptide of 72 amino acids, which corresponds to the MT-I class, type 2 family (http:yywww.unizh.chy;mtpageyclassif.html). The structure of the gene and the putative MT20-III protein have been compared with those of other species. The putative biological significance of the differences at the amino acid level among the different MTs is discussed.  2002 Elsevier Science Inc. All rights reserved. Keywords: Polymerase chain reaction (PCR); Cloning; Metallothionein; Mytilus galloprovincialis; Sequencing; MT20 metallothionein isoform; Genomic DNA 1. Introduction Metallothioneins (MTs) are a class of metallo- proteins showing particular biochemical features: they are low molecular-weight, soluble, sulfhydryl- rich proteins with a characteristic distribution of cysteinyl residues, such as Cys–X–Cys, Cys– XY–Cys, where XY are amino acids different from cysteine (Kagi and Shaffer, 1988). MTs show high affinity for heavy metal cations (Hg )Cu )Cd )Zn ) (Viarengo, 1989), 2q q 2q 2q which at physiologically relevant doses induce the transcription of MT genes. Therefore, MTs can  This paper was originally presented at a symposium dedicated to the memory of Marcel Florkin, held within the ESCPB 21st International Congress, Liege, Belgium, 24–28 ` July 2000. *Corresponding author. Tel.: q39-0131-283842; fax: q39- 0131-254410. E-mail address: fdondero@unipmn.it (F. Dondero). neutralize an excess of heavy metal cations pene- trating into the cell (Viarengo and Nott, 1993), thereby exerting protection of cell structures against non-specific interactions with heavy met- als. Moreover, MTs are implicated in the homeo- stasis of essential heavy metals, such as Cu and Zn, and are supposedly involved in specific cases of gene regulation (i.e. Zn-finger protein activa- tionydeactivation)(Henchel et al., 1994). Finally, MTs may also play a role in the cellular antioxidant system (Sato and Bremner, 1993; Kling and Ols- son, 1997). Due to their inducibility by heavy metal cations, MTs are considered an important, specific biomarker for inorganic pollution (e.g. Cd, Hg, Cu, Zn, etc.). Mussels of the genus Mytilus show widespread geographical distribution and represent an important element of coastal water ecosystems. These molluscs are sessile filter feed- ers that can effectively concentrate seawater pol-