Cloning and sequencing of a sheep metallothionein cDNA

Abstract

A partially purified metallothionein mRNA fraction from copper-injected sheep liver was used to synthesize double-stranded cDNA, which was dC-tailed, annealed to dG-tailed pBR322 and used to transform Escherichia coli MC1061. Of the 1500 recombinant clones only one gave a positive signal when screened with a mouse metallothionein 1 probe. This clone (pSMT-1) contained an insert which included the entire coding region of a sheep metallothionein, the whole 3'-untranslated region, part of the poly(A)-tail and 25 bases of the 5'-untranslated region. DNA sequence analysis showed that this sheep metallothionein was very similar to other mammalian metallothioneins except for a threonine to proline change at amino acid 27. The clone also contained a different polyadenylation signal d(A-G-T-A-A-A) from that usually found d(A-A-T-A-A-A). Comparison of the DNA sequence of the sheep metallothionein with those of other species revealed an interesting region of homology close to the poly(A) addition signal in the 3'-untranslated region of the mRNA.