Sequence-specific interaction between ABD-B homeodomain and castor gene in Drosophila
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We have examined the effect of bithorax complex genes on theexpression of castor gene. During the embryonic stages 12-15,both Ultrabithorax and abdominal-A regulated the castor geneexpression negatively, whereas Abdominal-B showed a positivecorrelation with the castor gene expression according toreal-time PCR. To investigate whether ABD-B protein directlyinteracts with the castor gene, electrophoretic mobility shiftassays were performed using the recombinant ABD-Bhomeodomain and oligonucleotides, which are located withinthe region 10 kb upstream of the castor gene. The results showthat ABD-B protein directly binds to the castor genespecifically. ABD-B binds more strongly to oligonucleotidescontaining two 5'-TTAT-3' canonical core motifs than the probecontaining the 5'-TTAC-3' motif. In addition, the sequencesflanking the core motif are also involved in the protein-DNAinteraction. The results demonstrate the importance of HD fordirect binding to target sequences to regulate the expressionlevel of the target genes.
Abstract
We have examined the effect of bithorax complex genes on theexpression of castor gene. During the embryonic stages 12-15,both Ultrabithorax and abdominal-A regulated the castor geneexpression negatively, whereas Abdominal-B showed a positivecorrelation with the castor gene expression according toreal-time PCR. To investigate whether ABD-B protein directlyinteracts with the castor gene, electrophoretic mobility shiftassays were performed using the recombinant ABD-Bhomeodomain and oligonucleotides, which are located withinthe region 10 kb upstream of the castor gene. The results showthat ABD-B protein directly binds to the castor genespecifically. ABD-B binds more strongly to oligonucleotidescontaining two 5'-TTAT-3' canonical core motifs than the probecontaining the 5'-TTAC-3' motif. In addition, the sequencesflanking the core motif are also involved in the protein-DNAinteraction. The results demonstrate the importance of HD fordirect binding to target sequences to regulate the expressionlevel of the target genes.
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