Two metallothionein genes from mud loach Misgurnus mizolepis (Teleostei; Cypriniformes): Gene structure, genomic organization, and mRNA expression analysis Young Sun Cho, Sang Yoon Lee, Keun-Yong Kim, Yoon Kwon Nam ⁎ Department of Aquaculture, Pukyong National University, Busan 608-737, Republic of Korea abstract article info Article history: Received 23 December 2008 Received in revised form 4 April 2009 Accepted 8 April 2009 Available online 19 April 2009 Keywords: Gene expression Gene structure Heavy metal exposure Tail-to-head array Two MT isoforms Two metallothionein genes, MLMT-IA and MLMT-IB, were isolated and characterized from the mud loach Misgurnus mizolepis (Teleostei; Cypriniformes). For these MTs, we determined a tandem “tail-to-head” genomic organizational pattern, identified conserved genomic features, showed high sequence identities in the coding regions, and examined the closest phylogenetic affiliation, suggesting their divergence by a recent gene duplication event. However, the 5′-flanking upstream regions in MLMT-IA and MLMT-IB exposed large differences in the composition and distribution patterns of various transcription factor binding motifs, especially regarding the organization of the metal response element clusters. Real-time RT-PCR assays showed that mRNA levels of both MLMT-IA and MLMT-IB isoforms were variable among tissues and the ratios between them were also variable across tissues, although the MLMT-IA was always predominant in every adult tissue tested. We also found that the MLMT-IA and MLMT-IB mRNA expression levels were regulated dynamically during embryonic and larval development stages, in which the basal expression level of MLMT- IA was also consistently higher than that of MLMT-IB. Upon acute in vivo metal exposure to cadmium, chromium, copper, iron, manganese, nickel, or zinc at 5 μM for 48 h, the transcriptional modulations of MLMT-IA and MLMT-IB were quite different from each other and the type of response was affected significantly by the kind of metals and tissues. Published by Elsevier Inc. 1. Introduction Metallothionein (MT) is a low molecular weight (6–7 kDa) cysteine (Cys)-rich cytoplasmic protein that plays multifunctional roles in diverse cellular activities, including metal homeostasis, detoxification, and antioxidant protection (Coyle et al., 2002; Haq et al., 2003; Atif et al., 2006). Many previous studies have reported that MT proteins and their gene transcripts can be induced rapidly in fish tissues upon metal exposure. Consequently, these findings have led to the proposed use of fish MTs as potential biomarkers for metal pollution and toxicity in aquatic environments (Langston et al., 2002; Cho et al., 2005; Bourdineaud et al., 2006). Genetic determinants of MTs have been isolated from a number of teleost fishes belonging to a wide array of taxa, and many fish species have been shown to possess at least two MT isoforms in their genomes (Knapen et al., 2005; Scudiero et al., 2005). However, despite numerous literatures on teleost MT genes, the genomic organization of the two isoforms in a given species has not been clearly elucidated yet. More importantly, differential regulation of the two isoforms in response to a given stimulus has not been fully examined. Only a few previous studies have reported the isoform-specific expression of MTs during metal exposures (Carginale et al., 1998; Hermesz et al., 2001; Vergani et al., 2007). The mud loach Misgurnus mizolepis (Teleostei; Cypriniformes), a commercially important freshwater species, is a typical inhabitant in the Korean peninsula, and its natural habitats are widely distributed in most rivers and streams in Korea (Kim et al., 1994). It is benthic species that prefers sandy or muddy bottom. Recently, the mud loach has been suggested as one of potential sentinel organisms for monitoring the pollution-associated risks in freshwater environments of the Korean peninsula. In addition, this species has many attractive merits as a laboratory animal such as fast and transparent embryonic development (hatching in 24 h post fertilization), relatively small adult size (10 cm in total length), short generation time (3–5 months from fertilization), high fecundity (usually more than 10,000 eggs from a single female), year-round multiple spawning under controlled conditions, and relatively well-established chromosome-set manipulation and trans- genesis techniques (Nam et al., 2008). The objective of this study was to characterize the gene structure, genomic organization, and mRNA expression patterns of two distinct MT isoforms, MLMT-IA and MLMT-IB, from the mud loach. For these studies, we isolated the genomic sequences and mRNA transcripts encoding the Comparative Biochemistry and Physiology, Part B 153 (2009) 317–326 ⁎ Corresponding author. Tel.: +82 51 629 5918; fax: +82 51629 5908. E-mail address: yoonknam@pknu.ac.kr (Y.K. Nam). 1096-4959/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.cbpb.2009.04.002 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb