Purification and characterization of chicken ovalbumin gene upstream promoter transcription factor from homologous oviduct cells

Abstract

Previous studies established that the chicken ovalbumin gene upstream promoter (COUP) sequence, which lies between -70 and -90 base pairs upstream from the cap site, is essential for the efficient transcription of the ovalbumin gene. A transcription factor which binds to this sequence has been purified from the homologous chicken oviduct cells. The purification scheme starting from oviduct nuclear extract involved a combination of conventional column and sequence-specific DNA affinity chromatography steps. Using gel retardation and DNase I footprinting techniques to assay COUP-binding activity, we achieved extensive purification of this factor. Binding competition studies with the purified factor indicated that it bound specifically to the COUP sequence and that the binding could be competed for only by the promoter DNA fragments or synthetic oligonucleotides containing the COUP sequence. The purified protein preparation showed multiple polypeptide bands on polyacrylamide gel electrophoresis. Renaturation of separated polypeptides after extraction from the gel matrix was carried out. The majority of renatured polypeptides exhibited specific binding to the COUP sequence.