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

被引:64
作者
UEDA, H
HIROSE, S
机构
[1] GRAD UNIV ADV STUDIES MISHIMA,DEPT GENET,MISHIMA,SHIZUOKA 411,JAPAN
[2] GRAD UNIV ADV STUDIES MISHIMA,NATL INST GENET,DNA RES CTR,MISHIMA,SHIZUOKA 411,JAPAN
关键词
D O I
10.1093/nar/19.13.3689
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:3689 / 3693
页数:5
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