Building a metal-responsive promoter with synthetic regulatory elements.

AUTOR(ES)

Searle, P F

RESUMO

A fusion gene consisting of the promoter region from the mouse metallothionein-I gene joined to the coding region of the herpes simplex virus thymidine kinase gene is efficiently regulated by zinc in a transient assay when transfected into baby hamster kidney cells. Analysis of similar plasmids in which the metallothionein-I promoter region was mutated indicated the presence of multiple metal regulatory elements (MREs) between -176 and -44 base pairs from the cap site. To further investigate the function of MREs, we inserted a synthetic DNA fragment containing the sequence of MRE-a (the element between -55 and -44 base pairs) into the nonresponsive promoter of the thymidine kinase gene in various positions and configurations. Little or no induction by zinc was observed with single insertions of the regulatory sequence, whereas many different constructions having two copies of MRE-a were inducible. The precise position of the two MREs relative to each other or to the thymidine kinase promoter elements had a relatively small effect on the efficiency of induction, but the inducibility could be further increased by the introduction of more MRE-a sequences. MRE-a can function synergistically with the thymidine kinase distal promoter elements, but in the presence of the TATA box alone it functions as a positive, zinc-dependent promoter element.

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