Detection of DNA binding activities of transcription factors with different protein motifs in nuclear extracts of murine brain by using gel-retardation electrophoresis.
DNA binding activities of a variety of transcription factors with different protein motifs were determined in nuclear extracts of mouse brains using radiolabeled double-stranded oligonucleotides containing the respective consensus core elements as probes in gel-retardation electrophoresis. DNA binding activities of the transcription factor with a leucine-zipper motif activator protein 1 (AP1) were markedly modulated by the addition of endogenous monovalent and divalent cations at physiological concentrations. In the presence of both KCl and MgCl2 at maximally effective concentrations, the AP1 binding occurred in a temperature-dependent manner in brain nuclear extracts. Brain nuclear extracts also contained activities to bind probes for seven other transcription factors with leucine-zipper, zinc finger, or helix-turn-helix motifs under the conditions favorable to detecting the AP1 binding. In contrast to brain nuclear extracts, however, both cations and incubation temperature were ineffective in markedly affecting binding of all eight radioprobes tested in hepatic nuclear extracts. Moreover, the addition of hepatic nuclear extracts eliminated the AP1 binding in brain extracts nearly completely, differentially affecting binding of other probes. Size-exclusion chromatography on hepatic nuclear extracts revealed two distinct fractions with different molecular sizes that both have an activity to inhibit the AP1 binding in brain nuclear extracts. These results suggest that monovalent and divalent cations may modulate DNA binding activities of particular transcription factors in brain nuclear extracts but not in hepatic nuclear extracts that could contain high molecular weight materials with an inhibitory potency on brain DNA binding activities.
Ogita
K
K
Department of Pharmacology, Setsunan University, Osaka, Japan.
Yoneda
Y
Y
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004586:Electrophoresis; D018257:Helix-Loop-Helix Motifs; D016350:Leucine Zippers; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers
D015335:Molecular Probes; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D004247:DNA