Isolation and phenotypic characterization ofChlamydomonas reinhardtii mutants defective in chloroplast DNA segregation |
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| Authors: | O. Misumi L. Suzuki Y. Nishimura A. Sakai S. Kawano H. Kuroiwa T. Kuroiwa |
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| Affiliation: | (1) Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, 113-0033 Tokyo, Japan;(2) Present address: Department of Biology, Vanderbilt University, Nashville, Tennessee, USA;(3) Present address: Department of Biological Science, Faculty of Science, Nara Women's University, Nara, Japan;(4) Present address: Department of Integrated Biosciences, Graduate School of Frontier Science, University of Tokyo, Hongo, Tokyo, Japan |
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| Abstract: | Summary Each wild-typeChlamydomonas 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 namedmoc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed thatmoc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in themoc 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 individualmoc 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 themoc 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 ofmoc 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 andmoc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novelmoc 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.Abbreviations DAPI 4 ,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon-counting system |
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| Keywords: | Chlamydomonas reinhardtii Chloroplast DNA Chloroplast nucleus Chloroplast DNA segregation Chloroplast division |
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