Characterization of Sμbp-2 as a mouse mammary tumor virus promoter-binding protein

F Uchiumi, M Komuro, R Mizuta, S Tanuma - Biochemical and biophysical …, 2004 - Elsevier
F Uchiumi, M Komuro, R Mizuta, S Tanuma
Biochemical and biophysical research communications, 2004Elsevier
A cDNA encoding a rat Sμbp-2 has been cloned from a λgt11 library by South-Western blot
screening using a 50-bp tannic acid responsive element [J. Biol. Chem. 273 (1998) 12499]
of the mouse mammary tumor virus (MMTV) promoter region as a probe. The full-length
cDNA encodes a protein with a predicted size of 108kDa. Northern blot analysis revealed
that the gene expression of Sμbp-2 is comparatively high in testis, moderate in brain, and
low in other tissues. The recombinant Sμbp-2 protein was expressed as a GST-or Trx-fusion …
A cDNA encoding a rat Sμbp-2 has been cloned from a λgt11 library by South-Western blot screening using a 50-bp tannic acid responsive element [J. Biol. Chem. 273 (1998) 12499] of the mouse mammary tumor virus (MMTV) promoter region as a probe. The full-length cDNA encodes a protein with a predicted size of 108kDa. Northern blot analysis revealed that the gene expression of Sμbp-2 is comparatively high in testis, moderate in brain, and low in other tissues. The recombinant Sμbp-2 protein was expressed as a GST- or Trx-fusion protein in Escherichia coli and purified by affinity column chromatography. Gel mobility shift competition analysis indicated that the recombinant Sμbp-2 protein binds to region II (containing the ACTG-motif) in the 50-bp element in the MMTV promoter. A transient transfection assay of the Sμbp-2 expression vector with MMTV promoter-containing Luciferase (Luc) reporter plasmids into mouse cells suggested that Sμbp-2 is a negative transcription factor. Furthermore, the MMTV promoter activity was suppressed in cells expressing high levels of Sμbp-2. Insertion of the 50-bp element upstream of the SV40 promoter negatively responded to the induced expression of Sμbp-2. These results suggest that the negative transcriptional effect of Sμbp-2 arises from its binding to the 50-bp element located in the MMTV promoter region.
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