Cloning and expression of an avian metallothionein-encoding gene

The chicken metallothionein gene (cMT), isolated from a chicken genomic DNA phage lambda library, was found to be approximately 1.5 kb in length and to consist of three exons, separated by two intervening sequences. The number and placement of the introns in the cMT gene is precisely the same as that in the mammalian metallothionein-coding genes. S1 nuclease mapping indicated a prominent transcription start point (tsp) 62 bp 5' to the translation start codon. The promoter region analyzed (623 bp) contained three regions of homology (at -47, -488, and -577 bp relative to the tsp) with the metal regulatory element (MRE) consensus sequence, and three potential Sp1 binding sites. Two of the putative MREs (-47, -577) were 12 to 14-bp palindromes, which suggests that they are binding sites for trans-acting proteins. The intact cMT gene was functional in mammalian cells, and the cMT promoter could confer metal responsiveness on the firefly luciferase cDNA (Luc) in transient expression assays. Deletion mutagenesis established that 107 bp of 5'-flanking sequence, containing the proximal MRE and a putative Sp1-binding site, were sufficient for transient expression and metal induction of the cMT promoter-Luc fusion gene.