Nucleic Acids Research, 1991, Vol. 19, No. 13 3689-3693
© 1991
MOLECULAR BIOLOGY |
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
Genetic Stock Research Center, National Institute of Genetics and Department of Genetics, The Graduate University for Advanced Studies Mishima, Shizuoka-ken 411, Japan 1DNA Research Center, National Institute of Genetics and Department of Genetics, The Graduate University for Advanced Studies Mishima, Shizuoka-ken 411, Japan
Received February 12, 1991. Revised May 20, 1991. Accepted May 20, 1991.
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'-PyCAA-GGPyCPu-3'. This method may be useful for the determination of a binding sequence of other sequence specific DNA binding factor.
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