Study on morphology and function of yeast mitochondrial nucleids

1.用3，3 '-二己基氧碳菁碘[DiOC_6（>3）]和4'，6-二脒基-2-苯基吲哚（DAPI）荧光染料分别对酿酒酵母（Saccharomycescerevisiae）不同类型细胞的线粒体和线粒体类核（mt-nucleoids）进行了活体染色。2.从酿酒酵母（Saccharomycescerevisiae）中分离得到的线粒体类核蛋白（Mt-nucleoids）的分子量分别为67 kDa、52 kDa、50 kDa、从分离的mt-类核中分离出38 kDa、30 kDa和20 kDa。含有DNA结合蛋白的组分能够将mtDNA复合体折叠成类核结构。这些结果表明，至少有六种不同的DNA结合蛋白参与组织的mt-类核。3.从酵母Pichia jadinii和属于毕赤酵母属的其他四种酵母中分离到高纯度的mt-类核蛋白。SDS-聚丙烯酰胺凝胶电泳和免疫印迹分析表明，线粒体类核蛋白在酵母中存在着多样性。4.通过DAPI染色和罗丹明鬼笔环肽染色，研究了线粒体类核蛋白的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，线粒体类核形成了一个网络的荧光字符串和肌动蛋白点经常重叠的线粒体类核。

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.