ABSTRACT

In this study, the contributions of the two metal-responsive elements (MREs) of the rainbow trout (Salmo gairdnerii) metallothionein (tMT)-B gene promoter (-137 to +5) were analyzed. The effect of MRE mutations on the basal and zinc-induced activities of tMT-B promoter-reporter gene fusions were determined by transfection of a rainbow trout hepatoma (RTH-149) cell line. Together, MREa and MREb cooperate to elicit a significant response to zinc but exhibit differential basal and metal-induced activity. The MREa sequence (-62 to -51) is important for basal promoter activity and can function independently, whereas the more distal MREb (-89 to -100) mainly contributes to metal induction through cooperative interactions with MREa. The degree of basal character of the MREs is partially determined by nucleotide differences at the flexible position N of the MRE consensus TGC(G/A)CNC. In mouse L and HepG2 cells, MREa activity is conserved, but the contributions of the MREb region differ, including reduced cooperativity with MREa. There are also differences in the apparent molecular masses of the rainbow trout and mammalian nuclear factors that bind to the tMT-B promoter and MREa sequence.

FOOTNOTES

*

This study was supported in part by a Medical Research Council of Canada grant (to L. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Supported by a studentship from the Alberta Heritage Foundation for Medical Research.

¶

To whom correspondence should be addressed: Dept. of Biology, University of Calgary, 2500 University Dr. N. W., Calgary, Alberta, Canada T2N 1N4. Tel.: 403-220-5556; Fax: 403-289-9311.

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The abbreviations used are: MT(s), metallothionein(s); MRE, metal-responsive element; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; LUC, firefly luciferase; tMRE, rainbow trout MT-B gene MRE.

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S. L.-A. Samson, unpublished observation.

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S. Scheiman, unpublished observation.

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