Use of bimolecular fluorescence complementation to demonstrate transcription factor interaction in nuclei of living cells from the filamentous fungus Acremonium chrysogenum
Use of bimolecular fluorescence complementation to demonstrate transcription factor interaction in nuclei of living cells from the filamentous fungus Acremonium chrysogenum
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DOI：
10.1007/s00294-004-0546-0
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发表时间：
2005-02-01
期刊：
影响因子：
2.5
通讯作者：
Kück, U
中科院分区：
文献类型：
--
作者：
Hoff, B;Kück, U
利用双分子荧光互补实验，我们证实了丝状真菌Acremonium chrysogenum活细胞中转录因子AcFKH1和CPCR1的蛋白-蛋白相互作用。这是通过将增强黄色荧光蛋白（EYFP）的基因分裂成编码N端和c端的两部分来实现的。两个片段被融合到真菌转录因子的不同基因衍生物上。重组质粒用于产生转基因真菌菌株，用于随后的共聚焦激光显微镜。只有当全长转录因子与EYFP片段融合时，由于两种嵌合蛋白的双分子互补，才观察到黄色荧光。用核酸染料TOTO-3对真菌细胞进行染色，验证了蛋白-蛋白相互作用的核定位。
Using bimolecular fluorescence complementation assays, we were able to demonstrate protein-protein interaction of the transcription factors AcFKH1 and CPCR1 in living cells from the filamentous fungus Acremonium chrysogenum. This was accomplished by splitting the gene for the enhanced yellow fluorescent protein (EYFP) into two parts encoding the N- and C-terminus. Both fragments were fused to different gene derivatives of the fungal transcription factors. The recombinant plasmids were used to generate transgenic fungal strains for subsequent confocal laser microscopy. Only when the full-length transcription factors were fused to EYFP fragments yellow fluorescence was observed due to the bimolecular complementation of both chimeric proteins. The nuclear localization of the protein-protein interaction was verified by staining fungal cells with the nucleic acid dye TOTO-3.