Isolation and phenotypic characterization of Chlamydomonas reinhardtii mutants defective in chloroplast DNA segregation
Isolation and phenotypic characterization of Chlamydomonas reinhardtii mutants defective in chloroplast DNA segregation
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DOI：
10.1007/bf01453455
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发表时间：
1999-01-01
期刊：
影响因子：
2.9
通讯作者：
Kuroiwa, T
中科院分区：
文献类型：
--
作者：
Misumi, O;Suzuki, L;Kuroiwa, T
每个野生型莱茵衣藻细胞有一个大的叶绿体，其中包含几个核（类核）。我们利用DNA插入诱变技术分离出了含有单个大叶绿体（cp）核的突变衣单胞菌，并将其命名为moc （monkaryotic chloroplast）。dapi荧光显微镜和显微光度观察显示，在整个细胞分裂周期中，moc突变体细胞只含有一个cp-核，并且在细胞分裂过程中发生了cpDNA的不均匀分离。第一次细胞分裂后，产生一个含有大量cpDNA的细胞和一个含有少量cpDNA的细胞。在第二次细胞分裂中也发生不均匀分离，产生一个大量cpDNA（约70拷贝）的细胞和另外三个少量cpDNA（仅2-8拷贝）的细胞，但大多数moc细胞在细胞分裂完成12小时后含有几十个cpDNA拷贝，这表明叶绿体分裂后立即激活了cpDNA合成。与cpDNA相反，moc突变体的线粒体（mt） DNA被观察到分散在整个细胞中的微小颗粒。这些细胞在细胞分裂过程中均匀地分离到每个子细胞。电镜观察moc突变体的超微结构发现，在类pyrenoid附近存在一个低电子密度区，经抗dna抗体免疫电镜鉴定为cp核。然而，野生型细胞和moc突变体的叶绿体在其他结构上没有差异。类囊体膜和类pyrenox膜相同。因此，我们认为新的moc突变体只是在cpDNA的分散和分离上有缺陷。该菌株对阐明cpDNA的分离机制具有重要意义。
Each wild-type Chlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we named moc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed that moc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in the moc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2-8 copies) of cpDNA, However, most individual moc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of the moc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure of moc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells and moc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novel moc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.