Fernández-Abalos J M, Fox H, Pitt C, Wells B, Doonan J H
Departamento de Microbiología y Genética, Universidad de Salamanca, Spain.
Mol Microbiol. 1998 Jan;27(1):121-30. doi: 10.1046/j.1365-2958.1998.00664.x.
Green fluorescent protein (GFP) is a useful reporter to follow the in vivo behaviour of proteins, but the wild-type gfp gene does not function in many organisms, including many plants and filamentous fungi. We show that codon-modified forms of gfp, produced for use in plants, function effectively in Aspergillus nidulans both as gene expression reporters and as vital reporters for protein location. To demonstrate the use of these modified gfps as reporter genes we have used fluorescence to follow ethanol-induced GFP expression from the alcA promoter. Translational fusions with the modified gfp were used to follow protein location in living cells; plant ER-retention signals targeted GFP to the endoplasmic reticulum, whereas fusion to the GAL4 DNA-binding domain targeted it to the nucleus. Nuclear-targeted GFP allowed real-time observation of nuclear movement and division. These modified gfp genes should provide useful markers to follow gene expression, organelle behaviour and protein trafficking in real time.
绿色荧光蛋白(GFP)是一种用于追踪蛋白质体内行为的有用报告基因,但野生型gfp基因在许多生物体中不起作用,包括许多植物和丝状真菌。我们表明,为植物使用而产生的密码子修饰形式的gfp,在构巢曲霉中既作为基因表达报告基因,又作为蛋白质定位的重要报告基因发挥有效作用。为了证明这些修饰的gfp作为报告基因的用途,我们利用荧光追踪了来自alcA启动子的乙醇诱导的GFP表达。与修饰的gfp的翻译融合用于追踪活细胞中的蛋白质定位;植物内质网保留信号将GFP靶向内质网,而与GAL4 DNA结合结构域的融合将其靶向细胞核。核靶向GFP允许实时观察核运动和分裂。这些修饰的gfp基因应该为实时追踪基因表达、细胞器行为和蛋白质运输提供有用的标记。