Structural and functional characterizations of mung bean mitochondrial nucleoids
Structural and functional characterizations of mung bean mitochondrial nucleoids
复制标题
DOI：
10.1093/nar/gki783
复制
发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Chiang, KS
中科院分区：
文献类型：
--
作者：
Dai, H;Lo, YS;Chiang, KS
从绿豆幼苗中分离到的线粒体核仁显示出与膜成分相关的染色质样结构。在原位发现了一个类似的结构，在子叶发育过程中经历了离散的变化。分离的类核由基本相同的磷脂组成，包括心磷脂，作为完整的线粒体和起源于线粒体内外膜的蛋白质。肌动蛋白在不同浓度的洗涤剂制备的线粒体类核中一致存在。细胞松弛素B和蛋白酶K处理的线粒体的甲醛交联进一步揭示了肌动蛋白与DNA在类核中的联系。线粒体类核在体外引导DNA合成方面是自给自足的，其模式类似于分离线粒体的mtDNA合成。在脉冲场凝胶电泳中，新合成的线粒体DNA分为两个主要组分，结合良好和快速移动的形式。类核DNA的合成对蚜菌素有抗性，但对N-乙基马来酰亚胺敏感，表明这一活性是由伽马型DNA聚合酶引起的。在体外，线粒体类核能够以非随机的方式自我定向RNA转录。我们的工作与真菌和人类细胞主要在原位完成的结果一致并补充了这一结果，我们的工作有助于建立一个重要的范式，即真核生物中的线粒体类核不仅仅是mtDNA紧凑和分离实体，而是mtDNA维持和表达的中心。
Mitochondrial nucleoids isolated from mung bean seedlings exhibited a chromatin-like structure associated with a membrane component. A similar structure, which underwent discrete changes during cotyledon development, was identified in situ. Isolated nucleoids consisted of essentially the same phospholipids, including cardiolipin, as whole mitochondria and proteins of inner- and outer-mitochondrial-membrane origin. Actin was consistently found with mitochondrial nucleoids prepared with different detergent concentrations. Formaldehyde cross-linking of cytochalasin B- and proteinase K-treated mitochondria further revealed that actin was associated with DNA in nucleoids. Mitochondrial nucleoids were self-sufficient in directing DNA synthesis in vitro in a pattern mimicking mtDNA synthesis in isolated mitochondria. In pulse-field gel electrophoresis, newly synthesized mtDNA separated into two major components, well-bound and fast-moving forms. Nucleoids DNA synthesis was resistant to aphidicolin but sensitive to N-ethylmaleimide, which indicates that a gamma-type DNA polymerase was responsible for this activity. Mitochondrial nucleoids were capable of self-directed RNA transcription in a non-random fashion in vitro. Consistent with and complementary to results from fungi and human cells done mostly in situ, our present work helps to establish the important paradigm that mitochondrial nucleoids in eukaryotes are more than mere mtDNA compaction and segregation entities but are centers of mtDNA maintenance and expression.