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doi:10.1016/0378-1119(89)90349-1 
Copyright © 1989 Published by Elsevier Science B.V.
Cloning and expression of an avian metallothionein-encoding gene
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Received 16 January 1989;
Abstract
The chicken metallothionein gene (cMT), isolated from a chicken genomic DNA phage λ 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 Sp 1-binding site, were sufficient for transient expression and metal induction of the cMT promoter-Luc fusion gene.
Author Keywords: Recombinant DNA; exon; intron; promoter; transfection; luciferase; metal regulatory element; chicken; deletion mutagenesis; transcription start point; Sp1 factor
Abbreviations: bp, base pair(s); cDNA, DNA complementary to mRNA; CEF, chick embryo fibroblast(s); cMT, chicken MT; cMT, gene coding for cMT; cRNA, RNA complementary to mRNA; CV-1, African green monkey kidney cells; DMEM, Dulbecco's modified essential Eagle's medium; FBS, fetal bovine serum; kb, kilobase pair(s) or 1000 bp; Luc, firefly luciferase enzyme; Luc, gene (cDNA) encoding Luc; MRE, metal regulatory element; MT, metallothionein; MT, gene coding for MT; nt, nucleotide(s); SDS, sodium dodecyl sulfate; Spl, eukaryotic transcription factor Spl (Briggs et al., 1986); tsp, transcription start point(s)
