Hyperinducible gene expression from a metallothionein promoter containing additional metal-responsive elements

We describe the development of metallothionein-based vectors with low basal levels of expression that are hyperinducible upon treatment with heavy metals. Vectors were constructed by substituting a region in the hMTIIA promoter (bp -70 to -129) containing an element (BLE) involved in basal level expression with multiple metal responsive elements (MREs). In expression studies utilizing cat as a reporter gene, heavy metal inducibility was examined in both transiently transfected and permanently transformed Chinese hamster ovary (CHO) cells. Our results demonstrate that, within the same promoter structure, inducibility can be increased by altering the ratio of MREs to BLEs. Optimal induction of expression in permanently transformed CHO cells was achieved by exposure to heavy metals for 48 h prior to cell harvest, with an additional boost 12 h before harvest. These vectors have the potential to be used for production of proteins in cultured mammalian cells and in gene expression in transgenic animals.     

Metal-dependent binding of a nuclear factor to the rat metallothionein-I promoter.

Genomic footprinting studies in vivo and experiments using synthetic metal regulatory elements (MREs) in vitro suggest protein binding to the MREs of the mouse and rat metallothionein I (MT-I) genes. Using gel-retardation assays of promoter fragments, we observe a cadmium-dependent binding factor for the rat MT-I promoter in rat hepatoma cells. This factor is present in extracts from both uninduced and cadmium-induced cells, but requires the presence of cadmium to bind to the promoter. The formation of a cadmium-dependent complex is competed by an oligonucleotide containing two MREs. This competition is lost when when one of the MREs is mutated, indicating a requirement for at least two MREs for binding of this factor. The cadmium-dependent factor dissociates more rapidly from the MT-I promoter than does a factor that binds to a consensus Sp1 site present on the same DNA fragment. UV crosslinking analysis using nuclear extracts from cadmium induced cells, in the presence of an oligonucleotide probe containing both 5-bromodeoxyuridine and 32P-deoxycytidine, identifies a 39 kDalton protein associated with the metal inducible complex.     

Building a metal-responsive promoter with synthetic regulatory elements.

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.