Mammary gland-specific nuclear factor is present in lactating rodent and bovine mammary tissue and composed of a single polypeptide of 89 kDa.
Mammary epithelial cells, under the regulation of the lactogenic hormones, produce high amounts of milk proteins during the lactation period. The caseins are the most abundant milk proteins. We have studied the regulation of beta-casein gene expression and found that the lactogenic hormones induce transcription of the beta-casein gene promoter. The hormonal regulation is mediated in part by a mammary gland-specific transcription factor (MGF). MGF is a specific DNA-binding protein which recognizes the sequence 5'-ACTTCT-TGGAATT-3'. This sequence is conserved with slight variations in the alpha- and beta-casein gene promoters of the cow and rodents at position -87 to -99. Bovine MGF and rodent MGF behaved identically when their DNA binding properties and migration in polyacrylamide gels as protein-DNA complexes were compared. MGF was purified to near homogeneity from nuclear extracts of mammary epithelial cells derived from lactating rats. The combination of Bio-Rex 70-, DNA-Sepharose-, and sequence-specific DNA affinity column chromatography yielded a highly purified preparation of MGF. The purification from nuclear extract was more than 2400-fold, and the yield of MGF activity was 11%. A protein of 89 kDa was visualized by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight was confirmed by UV cross-linking of the factor to its cognate DNA binding sequence and subsequent gel electrophoresis. Excision of the 89-kDa band from the gel and renaturation of the protein restored its specific DNA binding ability. This indicates that MGF is composed of a single polypeptide.
Wakao
H
H
Friedrich Miescher Institute, Basel, Switzerland.
Schmitt-Ney
M
M
Groner
B
B
D000818:Animals; D001483:Base Sequence; D002364:Caseins / Q000235:genetics; D002417:Cattle; D002467:Cell Nucleus / Q000502:physiology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004848:Epithelium / Q000502:physiology; D005260:Female; D007774:Lactation / Q000502:physiology*; D008321:Mammary Glands, Animal / Q000502:physiology*; D008894:Milk Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D050799:STAT5 Transcription Factor; D013379:Substrate Specificity; D015534:Trans-Activators
D002364:Caseins; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D009687:Nuclear Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators