Reverse transcriptase-mediated PCR has been used to isolate two distinct metallothionein (MT) cDNA species from RNA extracted from icefish liver, namely MT-I and MT-II. Northern blot analysis with these cDNA species revealed that significant endogenous levels of MT mRNA were present in liver tissues of normal animals despite the fact that no MT protein could be found accumulating in the same tissue. However, multiple injections of CdCl₂ induced high levels of both MT mRNA and MT protein. Sequence analysis of the cDNA species that were present after cadmium injection revealed the presence of both isoforms. Quantification of the MT-I and MT-II transcripts from normal and heavy-metal-treated fish showed an alteration in the ratio of the MT isoform transcripts. Endogenous transcripts consisted mostly of MT-II, whereas the MT-I transcript was preferentially accumulated only in response to the cadmium salt. The protein encoded by each cDNA isoform was isolated from the heavy-metal-treated fish and the availability of the specific MT mRNA for translation was demonstrated by translation in vitro. These results show that: (1) there is a discrepancy between the significant endogenous levels of MT mRNA and the absence of MT protein; (2) the accumulation of MT in icefish liver can be triggered by heavy metals; (3) genes encoding distinct MT isoforms are differentially regulated by heavy metals.

In the present paper, we examine eight species of Antarctic fish belonging to the suborder Notothenioidei, using reverse-transcriptase polymerase chain reaction, to investigate the presence of mRNAs encoding metallothionein (MT) isoforms. A total of 168 bp from the coding region and the complete (133-165 bp) 3' untranslated region (UTR) was obtained for all species (for three of them, we also sequenced the full-length cDNA, including the 5' UTR). Phylogenetic analyses carried out on the MT-coding region suggest monophyly for Antarctic fish MTs with respect to other teleost MT genes. Analyses also revealed that notothenioid MTs can be divided into at least two groups of paralogy, MT-1 and MT-2. These results indicate that notothenioid MT isoforms arose from at least one gene duplication event occurring in the ancestral lineage of the Notothenioidei. This duplication occurred independent of the one which gave origin to two metallothionein isoforms in the rainbow trout. In addition, an instance of gene conversion was observed between MT-1 and MT-2 genes in Notothenia coriiceps. Analyses of the 5' UTR, combined with quantitative assay of differential expression of MT-1 and MT-2, indicate that only the 3' UTR underwent a gene conversion event in the mentioned species. These findings, together with the observation of a differential pattern of expression for the two MT isoforms, disclose an unexpected complexity in the evolution and function of notothenioid MTs; as in most teleost species examined (apart from the rainbow trout), a single MT form is present.