Building a metal-responsive promoter with synthetic regulatory elements.

Abstract

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 synergiclly 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.Much interest is centered around understanding the mechanisms of regulation of eucaryotic gene expression. One approach has been to identify cis-acting DNA sequences that modulate gene expression by constructing mutations in cloned genes and examining their function after reintroduction into appropriate eucaryotic cells (6,13,24,29). The metallothionein (MT) genes provide an interesting example because they make up a small multigene family which responds at the transcriptional level to heavy metals such as zinc and cadmium (9), to glucocorticoid hormones (16), to an unidentified factor in the acute-phase response (8), and to interferon (11). In early attempts to localize the metal regulatory region, plasmids containing 5'-flanking DNA from the mouse MT-I gene joined to the coding region of the thymidine kinase (TK) gene from herpes simplex virus (HSV) were assayed by transient expression in microinjected mouse eggs. These experiments indicated that progressive deletion of upstream sequences resulted in a progressive decrease in the overall expression of the fusion gene. However, the minimal sequence capable of mediating regulation by cadmium was localized to between approximately 50 and 90 base pairs (bp) upstream of the site of transcription initiation (2).Our more detailed analysis of a series of mutants of the mouse MT-I promoter region located the proximal metal regulatory element (MRE) to between -59 and approximately -46 bp (35). On the basis of the strong sequence homology in this region between mouse and human MT-I and MT-II genes (33, 35), we ascribed regulatory function to the sequence CCTTTGCGCCCG, which we named MRE-a.However, a deletion between -67 and -42 bp that remove...

Multiple hormone-inducible enhancers as mediators of differential transcription.

“…1), as well as an SV40 enhancer region, the presence of which may have independently affected glucocorticoidresponsive transcription (41 (66). An extension of this view is that differential regulation of separate genes under the same type of biochemical control can be conferred by different numbers of a specific type of enhancer element associated with each gene (13,33,52). We tested this latter possibility in our experimental system.…”

“…An emerging view of enhancer action is that different enhancers may act independently of one another to confer different controls on a gene by two or more distinct biochemical pathways (66). An extension of this view is that differential regulation of transcription for a set of genes under a common control could be conferred by the strength or number of enhancer elements associated with each gene (13,33,52).The regulated transcription of mouse mammary tumor virus (MMTV) provides a relevant biological system in which to test the proposal that differential levels of transcription can be mediated by multiple functionally related enhancer elements of different strength or number. The glucocorticoid-regulated transcription of the proviral genes of MMTV (Fig.…”

“…An emerging view of enhancer action is that different enhancers may act independently of one another to confer different controls on a gene by two or more distinct biochemical pathways (66). An extension of this view is that differential regulation of transcription for a set of genes under a common control could be conferred by the strength or number of enhancer elements associated with each gene (13,33,52).…”

Fluorescence Properties and Functional Roles of Tryptophan Residues 60d, 96, 148, 207, and 215 of Thrombin

“…Cells were transfected with individual pNUT constructs using the calcium phosphate coprecipitation method (25,26). Plasmid DNA (10 g) was precipitated at pH 6.95 and added to 100-mm plates of baby hamster kidney cells at ϳ40% confluence.…”

Cloning and Characterization of the Promoter Region of the Wilson Disease Gene

“…Similar to the MT promoters that contain multiple MRE- like sequence (MLS), the promoter region of the WD gene contained six additional MLSs that only have one or two bases different from that of MRE consensus sequence (TGCRCNC). In the case of metal-regulated genes, the response to heavy metal is generally independent to the positions or orientation of the MRE, and requires at least two copies of the MRE sequence (15). Since this also seems to be the case in the WD gene, the MRE sites in the 5Ј-flanking region of the WD gene may provide as binding sites for metal-dependent transcription factors that play important roles in the control of transcription activity of the WD gene.…”