Kanazawa M, Yano M, Namchai C, Yamamoto S, Ohtake A, Takayanagi M, Mori M, Niimi H
Department of Pediatrics, Chiba University, School of Medicine, Japan.
Biochem Biophys Res Commun. 1997 Oct 20;239(2):580-4. doi: 10.1006/bbrc.1997.7448.
cDNAs for green fluorescent protein (GFP) and for a GFP fusion protein containing the presequence of human ornithine transcarbamylase (pOTC-GFP) were transfected into cultured human fibroblasts. GFP cDNA gave diffuse fluorescence throughout the cytoplasm and the nucleus, whereas pOTC-GFP cDNA gave mitochondria-associated fluorescence. Fluorescent mitochondrial structures could be classified into five patterns: thread-like mitochondria, fine thread-like ones, rod-like ones, granular ones, and granular ones with weak cytosolic fluorescence. pOTC-GFP mutants resulted in a loss of mitochondrial fluorescence and an appearance of weak fluorescence throughout the cytoplasm. pOTC-GFP cDNA was transfected into fibroblasts from patients with various mitochondrial diseases. Higher ratios of fibroblasts with granular mitochondria and those with fine thread-like ones were observed in a patient with Reye's syndrome and a patient with Kearns-Sayre syndrome. Weak cytosolic fluorescence was sometimes observed in fibroblasts from these patients. This method will be useful to analyze mitochondrial structural alterations and disorders of mitochondrial protein import.
将绿色荧光蛋白（GFP）以及包含人鸟氨酸转氨甲酰酶前序列的GFP融合蛋白（pOTC-GFP）的互补DNA（cDNA）转染到培养的人成纤维细胞中。GFP cDNA在整个细胞质和细胞核中产生弥漫性荧光，而pOTC-GFP cDNA则产生与线粒体相关的荧光。荧光线粒体结构可分为五种模式：线状线粒体、细线状线粒体、棒状线粒体、颗粒状线粒体以及伴有弱细胞质荧光的颗粒状线粒体。pOTC-GFP突变体导致线粒体荧光丧失，并在整个细胞质中出现弱荧光。将pOTC-GFP cDNA转染到患有各种线粒体疾病患者的成纤维细胞中。在一名瑞氏综合征患者和一名卡恩斯-塞尔综合征患者中，观察到具有颗粒状线粒体的成纤维细胞和具有细线状线粒体的成纤维细胞比例更高。在这些患者的成纤维细胞中有时会观察到弱细胞质荧光。该方法将有助于分析线粒体结构改变和线粒体蛋白导入障碍。
