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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.

nucleic acids research

Defining the sequence recognized with BmFTZ-F1, a sequence specific DNA binding factor in the silkworm,<i>Bombyx mori</i>, as revealed by direct sequencing of bound oligonucleotides and gel mobility shift competition analysis

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