Westermann B, Neupert W
Institut für Physiologische Chemie, Ludwig-Maximilians-Universität München, 80336 München, Germany.
Yeast. 2000 Nov;16(15):1421-7. doi: 10.1002/1097-0061(200011)16:15<1421::AID-YEA624>3.0.CO;2-U.
We describe the construction and characterization of a novel set of plasmids for expression of mitochondria-targeted green fluorescent protein (GFP) in Saccharomyces cerevisiae. The vectors include constructs with strong regulatable and constitutive promoters, four different auxotrophic markers for yeast transformation, and a green (S65T) and a blue-shifted (P4-3) variant of GFP. Mitochondria are brightly fluorescent in living yeast cells grown on different carbon sources and at different temperatures, with virtually no background staining. Specific staining of mitochondria is also shown for a respiratory-deficient mutant with abnormal mitochondrial morphology. The plasmids facilitate convenient analysis of mutants defective in mitochondrial morphology or inheritance and, at the same time, are suitable vectors for easy construction of different kinds of GFP fusion proteins to study various aspects of organelle biogenesis in living yeast cells.
我们描述了一组新型质粒的构建和特性，这些质粒用于在酿酒酵母中表达线粒体靶向的绿色荧光蛋白（GFP）。这些载体包括带有强可调控和组成型启动子的构建体、用于酵母转化的四种不同营养缺陷型标记，以及GFP的绿色（S65T）和蓝移（P4-3）变体。在以不同碳源生长且处于不同温度的活酵母细胞中，线粒体发出明亮荧光，几乎没有背景染色。对于线粒体形态异常的呼吸缺陷型突变体，也显示出线粒体的特异性染色。这些质粒便于对线粒体形态或遗传缺陷的突变体进行便捷分析，同时也是用于轻松构建各种GFP融合蛋白以研究活酵母细胞中细胞器生物发生各个方面的合适载体。
