Characterization of Smubp-2 as a mouse mammary tumor virus promoter-binding protein.
A cDNA encoding a rat Smubp-2 has been cloned from a lambdagt11 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 108 kDa. Northern blot analysis revealed that the gene expression of Smubp-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 Smubp-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 Smubp-2 expression vector with MMTV promoter-containing Luciferase (Luc) reporter plasmids into mouse cells suggested that Smubp-2 is a negative transcription factor. Furthermore, the MMTV promoter activity was suppressed in cells expressing high levels of Smubp-2. Insertion of the 50-bp element upstream of the SV40 promoter negatively responded to the induced expression of Smubp-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.
Uchiumi
Fumiaki
F
Department of Gene Regulation, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
Komuro
Masato
M
Mizuta
Ryushin
R
Tanuma
Sei-ichi
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D047348:Hydrolyzable Tannins / Q000494:pharmacology; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D047348:Hydrolyzable Tannins; C491262:Ighmbp2 protein, rat; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D012697:Serine Endopeptidases