Study on morphology and function of yeast mitochondrial nucleids

1.用荧光染料3,3′-二己基氧羰基氰碘[DiOC_<6(>3)]和4′，6-二氨基-2-苯基吲哚（DAPI）对酵母（Saccharomyces cerevisiae）生命周期内不同类型细胞的线粒体和线粒体类核（mt-nucleoids）进行了荧光染色。结果表明，在酵母营养生长、产孢、交配和萌发过程中，线粒体的融合和断裂现象频繁发生。对从酵母中分离的mt -类核进行了分析，以鉴定与mtDNA紧密包装有关的蛋白质成分。分离得到分子量为67 kDa、52 kDa、50 kDa、38 kDa、30 kDa和20 kDa的dna结合蛋白。含有dna结合蛋白的部分能够将mtDNA紧密折叠成核样结构。这些结果表明，至少有六种不同的dna结合蛋白参与了mt-类核的组织。制备了一种针对酿酒酵母mt-nucleoids的67 kda mt-nucleoid蛋白的抗体。从毕赤酵母（Pichia jadinii）和其他四种属毕赤酵母（Pichia willopsis）中分离到了高纯度的mt -类核。sds -聚丙烯酰胺凝胶电泳和免疫印迹分析表明，酵母中存在mt-核样蛋白的多样性。采用dapi染色和罗丹明-phalloidin染色研究mt-类核形态与肌动蛋白分布的关系。在二倍体细胞的孢子形成过程中，mt-类核形成了一个荧光线网络，肌动蛋白点经常重叠在mt-类核上。

1.Mitochondria and mitochondrial nucleoids (mt-nucleoids) of the yeast, Saccharomyces cerevisiae were vitally-stained with two fluorochromes, 3,3'-dihexyloxacarbocyanine iodide [DiOC_<6(>3)] and 4', 6-diamidino-2-phenylindole (DAPI), respectively in the different types of cells during the life cycle. The results showed that the fusion and fragmentation of mitochondria occurred frequently during vegetative growth, sporulation, mating and germination of yeasts.2.Mt-nucleoids isolated from the yeast Saccharomyces cerevisiae were analyzed to indentify the protein components that are involved in the compact packaging of mtDNA.DNA-binding proteins with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa 30 kDa and 20 kDa were separated from isolated mt-nucleoids. The fraction that contained the DNA-binding proteins was able to fold mtDNA compactly into nucleoid-like structures. These results suggested that at least six different DNA-binding proteins were involved in the organization of mt-nucleoids. An antibody was raised against 67-kDa mt-nucleoid protein of S.cerevisiae mt-nucleoids.3.Mt-nucleoids were isolated at high purity from the yeast Pichia jadinii and other four species of yeasts that belong to the genus Pichia, Williopsis. SDS-polyacryl amide gel electrophoresis and immunoblotting suggested that the diversity of mt-nucleoid protein occurred in yeasts.4.Relationships between the morphology of mt-nucleoids and distribution of actin were investigated by DAPI-staining and by the staining with Rhodamine-phalloidin. During sporulation of diploid cells, mt-nucleoids formed a network of fluorescent strings and actin dots were frequently overlapped on the mt-nucleoids.