Cloning and Nucleotide Sequence of a Complementary DNA Encoding Xenopus laevis Metallothionein: mRNA Accumulation in Response to Heavy Metals

A cDNA encoding Xenopus laevis metallothionein (MT) was cloned from a cDNA library constructed using liver poly(A)⁺RNA of X. laevis adult males treated with CdCl₂. The probe used to screen the library was a MT-specific DNA fragment obtained by means of the polymerase chain reaction (PCR) and degenerate oligodeoxynucleotide primers. The cDNA clone encodes a putative protein of 62 amino acids, of which 20 are cysteine residues. The position of all the cysteine residues is conserved with respect to mammalian MT sequences. The amino acid sequence of this X. laevis MT, designated XlMT-A, shares between 60% and 67% identity with various vertebrate MTs. Overall, the structure of XlMT-A is no more similar in sequence to MT-1 than it is to MT-2 isoforms of various vertebrates. Ten different X. laevis MT cDNA isolates were partially sequenced and turned out to be identical, suggesting a single species of MT mRNA. Southern blot analysis of X. laevis DNA reveals that the XlMT-A gene is present in at least two copies. This result is consistent with the suggestion that a genome duplication occurred in a X. laevis ancestor. The in vivo response to increasing doses of Cd²⁺, Zn²⁺, and Cu²⁺ metal salts was tested. In the liver, all three metals proved to be potent inducers, raising MT mRNA levels between 50- and 100-fold. The maximum response to Cd²⁺ was at 12 hr after injection and to Zn²⁺ at 24 hr after injection. High levels of mRNA were maintained for more than 48 hr. Cd²⁺ and Zn²⁺ induced XlMT-A mRNA in all tissues examined (kidney, spleen, heart, intestine, testes, and brain). Dexamethasone did not induce MT mRNA synthesis in the liver.