Abstract
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.
Highlights
Research Grant CA16672 from the National Cancer Institute
We demonstrate here that the B element-binding factor, or BBF, is a single heat-resistant polypeptide with an apparent molecular weight of approximately 95,000 whose binding to the B elem e n t appears to be modulated by other factors, one of which may be an inhibitor of BBF DNA binding
In previous work we have shown that theB elementof the mouse al(II1) collagen promoter is a positive cis-acting element, that when mutated(5'ATATTXAGA-3t'o
Summary
Preparation of Nuclear Extracts-Nuclear extract from NIH 3T3 fibroblasts was prepared as described previously [7]. Purification of the B Element-binding Factor from NIH 3T3 Fibroblasts and HeLa Cells-Nuclear extracts (prepared from 2.8 g (wet weight) of NIH 3T3 fibroblasts or 12 g (wet weight) of HeLa cells) weredialyzed against 16 volumes of buffer Ccontaining 25 mM HEPES, pH 7.9, 1mM EDTA, 20% glycerol, 0.1% Nonidet P-40, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 pg/ml leupeptin, 10pg/ml pepstatin, and 80 mM NaC1. Renaturation of Proteins from SDS-Polyacrylamide Gels-Protein fractions from DNA affinity chromatography were precipitated with acetone, resuspended in buffer E containing 50 mM HEPES, pH 7.9, 100 mM KC1, 20% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, and 2 pg/ml leupeptin and pepstatin, and mixed with an equal volume of 2 X SDS sample buffer (4% SDS, 5% 2-mercaptoethanol, 18%glycerol, 55 mM Tris-HC1, pH 6.8, and 50 pg/ml bromphenol blue). Since B element-binding activity was present in protein renaturedfrom a gel slice containing proteins of apparent molecular weight of about 68,000 to 110,000, inthe subsequent experiments a smaller region of the separating gel containing proteins of this approximate size was further fractionated into five equal slices
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