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Structural and functional analysis of the bovine Mx1 promoter.

The bovine Mx1 promoter region was found to contain 4 IFN-stimulated response elements (ISREs), 7 GC boxes, 2 IL-6 responsive elements, 2 NFκB-binding sites and 2 AP-1-binding sites. Among Holstein, Charolai, and Brahman breeds, 5 nucleotide substitutions were detected in the promoter region. After the Mx1 promoter region from Holstein had been constructed with pGL-basic expression vector, the transfected cells showed promoter activity after IFN induction. Several artificial deletion mutants were prepared to determine the important regulatory elements responsible for the promoter activity, and it was found that ISRE has a key function in IFN response. The proximal ISRE1 showed potential induction by IFN. Furthermore, the proximal GC boxes were found to be essential for IFN response in the bovine Mx1 promoter with the deletion mutants. In this case, the 2 GC boxes exhibited a synergistic activation in the IFN response. Mithramycin A, an agent that inhibits gene expression selectively by coating GC boxes, was used, and Mx mRNA expression in MDBK cells was suppressed by this chemical. Therefore, GC boxes were also shown to be essential for IFN response in the bovine Mx1 gene.