Purification of BBF, a DNA-binding protein recognizing a positive cis-acting element in the mouse alpha 1(III) collagen promoter.
A positive cis-acting element, the B element, located between -83 and -61 in the mouse alpha 1(III) collagen promoter, binds a factor present in nuclear extracts of NIH 3T3 fibroblasts and HeLa cells. We have purified this factor using ion exchange chromatography, sequence-specific DNA affinity chromatography, and sodium dodecyl sulfate-polyacrylamide gel fractionation. The DNA sequence used for the affinity chromatography was a single-base substitution in the B element that increased the stability of the B element-protein complex by 50%. Purification of the B element-binding factor (BBF) by DNA affinity chromatography resulted in the apparent loss of most or all of the DNA-binding activity of this factor. The DNA-binding activity could, however, be reconstituted by combining two chromatographic fractions: the high-salt eluate and the column flow-through. When the partially purified high-salt eluate was size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with subsequent renaturation of gel fractions from guanidine HCl, the purified BBF (apparent molecular weight of about 95,000) bound to the B element with high affinity. These results suggest that during DNA affinity purification of BBF a factor that inhibits BBF DNA binding was co-eluted with BBF. This inhibition of BBF DNA binding was reversed by the addition of the DNA affinity column flow-through. The binding of BBF to the B element of the mouse alpha 1(III) collagen promoter is therefore an apparently complex process involving interactions between BBF and other protein factors.
Ruteshouser
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EC
Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston 77030.
de Crombrugghe
B
B
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003094:Collagen / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D003094:Collagen