A cDNA encoding a rat Smubp-2 has been cloned from a lambdagt1 I 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 Spbp-2 is comparatively high in testis, moderate in brain, and low in other tissues. The recombinant Smubp-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 Spbp-2 protein binds to region 11 (containing the ACTG-motif) in the 50-bp element in the MMTV promoter. A transient transfection assay of the Spbp-2 expression vector with MMTV promoter-containing Luciferase (Luc) reporter plasmids into mouse cells suggested that Spbp-2 is a negative transcription factor. Furthermore, the MMTV promoter activity was suppressed in cells expressing high levels of Spbp-2. Insertion of the 50-bp element upstream of the SV40 promoter negatively responded to the induced expression of Spbp-2. These results suggest that the negative transcriptional effect of Smubp-2 arises from its binding to the 50-bp element located in the MMTV promoter region. (C) 2004 Elsevier Inc. All rights reserved.