Huang H, Mizukami Y, Hu Y, Ma H
Cold Spring Harbor Laboratory, NY 11724-2212.
Nucleic Acids Res. 1993 Oct 11;21(20):4769-76. doi: 10.1093/nar/21.20.4769.
The Arabidopsis floral homeotic gene AGAMOUS (AG) is required for normal flower development. The deduced AG protein contains a region which shares substantial sequence similarity with the DNA-binding domains of known transcription factors, SRF (human) and MCM1 (yeast). Therefore, it is likely that AG is also a DNA-binding protein regulating transcription of floral genes. We describe here several experiments to characterize AG-DNA binding in vitro. We show that AG indeed binds a DNA sequence matching the consensus of SRF targets. Further, we have selected the AG-binding sequences from a pool of random oligonucleotides, and deduced an AG-binding consensus sequence of TT(A/T)CC(A/T)(A/t)2(T/A)NNGG(-G)(A/t)2. We have demonstrated that AG binds to the consensus region of three of the oligonucleotides by footprinting analysis. Finally, we have examined AG's relative binding affinity for different sequences, as compared to SRF, by gel mobility shift analysis. Our results indicate that AG is a sequence-specific DNA-binding protein, and that the AG-binding consensus sequence is similar to those of MCM1 and SRF.
拟南芥花同源异型基因AGAMOUS（AG）是正常花发育所必需的。推导的AG蛋白包含一个与已知转录因子SRF（人类）和MCM1（酵母）的DNA结合域具有大量序列相似性的区域。因此，AG可能也是一种调节花基因转录的DNA结合蛋白。我们在此描述了几个体外鉴定AG-DNA结合的实验。我们表明AG确实结合了与SRF靶标共有序列匹配的DNA序列。此外，我们从随机寡核苷酸库中选择了AG结合序列，并推导了AG结合共有序列为TT(A/T)CC(A/T)(A/t)2(T/A)NNGG(-G)(A/t)2。我们通过足迹分析证明AG与其中三个寡核苷酸的共有区域结合。最后，我们通过凝胶迁移率变动分析检测了与SRF相比AG对不同序列的相对结合亲和力。我们的结果表明AG是一种序列特异性DNA结合蛋白，并且AG结合共有序列与MCM1和SRF的相似。
