Defining the sequence recognized with BmFTZ-F1, a sequence specific DNA binding factor in the silkworm, Bombyx mori, as revealed by direct sequencing of bound oligonucleotides and gel mobility shift competition analysis.
BmFTZ-F1 is a Bombyx mori homologue of FTZ-F1, a positive regulator of the fushi tarazu gene of Drosophila melanogaster. In order to determine the sequence recognized with this factor, we made three sets of oligonucleotide mixture which contain 4 possible nucleotides at different positions within the previously proposed 12-bp binding consensus sequence. Oligonucleotides which bound to purified BmFTZ-F1 were separated by a gel mobility shift procedure and a binding sequence was determined by direct sequencing through Maxam-Gilbert method. By this analysis, 7 positions showed clear sequence preference and 5 positions showed weak or no sequence preference. The importance of each nucleotide at each position was confirmed by a gel mobility shift competition analysis and results were presented as a quantitative difference in the binding affinity. From these analyses, we conclude that the best binding sequence of BmFTZ-F1 is 5'-PyCAAGGPyCPu-3'. This method may be useful for the determination of a binding sequence of other sequence specific DNA binding factor.
Ueda
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Genetic Stock Research Center, Graduate University for Advanced Studies, Shizuoka-ken, Japan.
Hirose
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D012831:Bombyx / Q000235:genetics*; D016384:Consensus Sequence / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003720:Densitometry; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism*; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D011089:Polydeoxyribonucleotides; D014157:Transcription Factors; C031156:poly d(I-C)