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Purification of mouse MEP-1, a nuclear protein which binds to the metal regulatory elements of genes encoding metallothionein. 总被引:1,自引:0,他引:1
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Metal regulatory elements (MREs) shared by metallothionein (MT) gene promoters are essential for metal induction of MT genes. MEP-1, a nuclear protein which binds to these elements has been purified from heavy metal-resistant mouse L cells using footprinting, Southwestern and UV cross-linking techniques to assay its binding activity. The purification scheme, starting from crude nuclear extracts, involved a combination of heparin-Sepharose and MRE-DNA affinity chromatography. The purified protein preparation showed a single polypeptide band of 108 kDa on polyacrylamide gel electrophoresis, and 2D-gel analyses revealed the presence of a protein species migrating as a single population of approximately 110 kDa. MEP-1 does not appear to be glycosylated since it eluted with the flow-through on a Wheat Germ Sepharose column. It was retained by a zinc-Chelating Sepharose column suggesting that amino acid residues (i.e., cysteine, histidine) which have an affinity for zinc ions are exposed on the protein surface. Binding studies with the purified protein indicated that it binds specifically to MRE sequences and that the binding can be abolished by a point mutation in the MRE core consensus sequence or by the addition of the chelating agent 1,10-phenanthroline. Binding activity can be restored by the addition of zinc ions to the chelated protein. These results suggest that MEP-1 is one of the major proteins interacting with MRE sequences. 相似文献
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Functional analyses of promoter elements responsible for the differential expression of the human metallothionein (MT)-IG and MT-IF genes 总被引:4,自引:0,他引:4
The sequences responsible for heavy metal-inducible expression are situated within the proximal 437 and 160 base pairs (bp) of MT-IF and MT-IG 5'-flanking sequence, respectively. Only 105 bp of proximal MT-IG 5'-flanking sequence containing a TATA box, two metal responsive elements (MREs), and three GC motifs and 147 bp of proximal MT-IF 5'-flanking sequence containing a TATCA box, four MREs, and two GC motifs were required for heavy metal-inducible expression. However, the proximal 111 bp of MT-IF 5'-flanking sequences (a TATCA box, two MREs, and two GC motifs) was not responsive to heavy metals and competes less efficiently than the 105-bp MT-IG fragment in a competition transfection analysis. The MT-IF promoter fragment containing MREc and MREd is substantially stronger and a more efficient competitor than the MT-IG promoter fragment containing MREc and MREd. Furthermore, the proximal 160 bp of MT-IG 5'-flanking sequence functions as a strong metal-inducible promoter but not as a metal-inducible enhancer. Mobility shift analysis of MT-IF and MT-IG promoter subregions suggests a correlation between protein binding to MRE sequences and MT gene expression. These data illustrate that the overall structural and functional organization of the MT-IF and MT-IG promoters are very different and that the molecular mechanisms governing differential expression levels of human MT genes are quite complex. 相似文献
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