An embryonic DNA-binding protein specific for a region of the human IFN beta 1 promoter.

Embryonal carcinoma (EC) cells are unable to make interferon in response to inducing agents. This block disappears after differentiation. We have found that nuclear extracts from undifferentiated P19 EC cells contain a DNA-binding activity which specifically recognizes a region within the human interferon-beta 1 promoter. This activity is absent from differentiated cell types, both of EC and non-EC origin. The binding of the factor in undifferentiated EC cells leads to dramatic changes in the overall protein binding pattern of the interferon promoter as compared with differentiated cells, and may be responsible for repression of the endogenous interferon-beta gene prior to differentiation.     

Identification of a novel DNA-binding protein to osmotin promoter

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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.     

Coordinate regulation of bovine prion protein gene promoter activity by two Sp1 binding site polymorphisms

Relationships between insertion/deletion (Ins/Del) polymorphisms of the bovine prion protein gene (PRNP) promoter and bovine spongiform encephalopathy (BSE) susceptibility have been reported. Our previous study has shown that polymorphisms of −6C → T included in the specific protein 1 (Sp1) site in the 5′-flanking region of bovine PRNP influence the promoter activity of bovine PRNP. The present study shows that 12 and 23 bp Ins/Del polymorphisms in the upstream region and an additional polymorphism (−47C → A) in the Sp1 binding site coordinately affect the promoter activity. Reporter gene assays demonstrated that the bovine PRNP promoter containing −47A and 23 bp Del/12 bp Ins or 23 bp Ins/12 bp Ins showed lower promoter activity compared with other haplotypes (23 bp Del/12 bp Ins or 23 bp Ins/12 bp Del with −47C) or the wild-type haplotype (23 bp Del/12 bp Del with −47C). Furthermore, gel shift assays showed that the binding activity of Sp1 to the PRNP promoter was influenced by both polymorphisms with corresponding effects on the promoter activity. The coordinate regulation of the bovine PRNP promoter suggests the two Sp1 binding site polymorphisms control Sp1 binding to the PRNP promoter and its activity.