Purification of early-B-cell factor and characterization of its DNA-binding specificity.
Early-B-cell factor (EBF) is a nuclear protein that recognizes a functionally important sequence in the promoter of the mb-1 gene. Like the mb-1 gene, which encodes an immunoglobulin-associated protein, EBF is specifically expressed in the early stages of B-lymphocyte differentiation. We purified EBF by sequence-specific DNA affinity chromatography and examined its biochemical properties and DNA-binding specificity. Crude nuclear extract and affinity-purified EBF generated protein-DNA complexes with the mb-1 promoter that were indistinguishable in electrophoretic mobility shift and DNase I footprint assays. Fractionation of affinity-purified EBF by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation of isolated polypeptides indicated that EBF DNA-binding activity could be reconstituted from polypeptides with molecular masses of 62 to 65 kDa. Gel filtration chromatography suggested that native EBF has a molecular mass of 140 kDa, if a globular shape of the protein is assumed. Thus, EBF appears to be a dimer with subunits of 62 to 65 kDa. To characterize the DNA-binding specificity of purified EBF, we performed two sets of experiments. First, we examined various mutant EBF-binding sites for interaction with purified EBF in an electrophoretic mobility shift assay. Second, we used oligonucleotides containing pairs of randomized bases in a binding-site selection and amplification experiments to determine a preferred sequence for DNA binding by EBF. Taken together, the results of these experiments indicated that EBF recognizes variations on the palindromic sequence 5'-ATTCCCNNGGGAAT, with an optimal spacer of 2 bp between the half-sites.
Howard Hughes Medical Institute, Department of Microbiology, University of California, San Francisco 94143-0414.
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A
Travis
J
J
Hagman
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L
Hwang
R
R
Grosschedl
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D015534:Trans-Activators / Q000302:isolation & purification* / Q000378:metabolism*
D015342:DNA Probes; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D015534:Trans-Activators; D003850:Deoxyribonuclease I