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To define DNA sequences involved in mouse metallothionein-I (MT-I) gene promoter function and metal regulation, we fused the 5' flanking sequences of the MT-I gene to the coding sequences of a viral thymidine kinase (TK) gene. A series of 5' deletion, 3' deletion, linker-scanning, and internal deletion mutants of the MT-I promoter was constructed and assayed by microinjection into mouse eggs. The results indicate that at least two related promoter elements can confer some metal regulation independently. Those mutations that had the most severe effect on regulation impinge on a 12-base-pair conserved sequence that is repeated several times within the mouse MT-I and other MT promoters. To test the regulatory function of this sequence, it was synthesized as a pair of complementary oligonucleotides and inserted into the promoter of the TK gene. A single insertion of this sequence conferred limited metal regulation onto the TK promoter, whereas a construct with two separate inserts was regulated as efficiently as the MT-I promoter.

A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene.