Isolation and phenotypic characterization of Chlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

Each wild-type Chlamydomonas reinhardtii cell has one large chloroplast con taining 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-fluoresce nce microscopy and microphotometry observations revealed that moc mutant ce lls only contain one cp-nucleus throughout the cell division cycle, and tha t unequal segregation of cpDNA occurred during cell division in the moc mut ant. 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 a lso occurred in the second cell division, producing one cell with a large a mount (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 se veral dozen cpDNA copies 12 h after the completion of cell division, sugges ting that cpDNA synthesis was activated immediately after chloroplast divis ion. In contrast to the cpDNA, the mitochondrial (mt) DNA of the moc mutant s was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-mi croscopic observation of the ultrastructure of moc mutants showed that a lo w-electron-density area, which was identified as the cp-nucleus by immunoel ectron microscopy with anti-DNA antibody, existed near the pyrenoid. Howeve r, there were no other structural differences between the chloroplasts of w ild-type cells and moc mutants. The thylakoid membranes and pyrenoid were i dentical. Therefore, we propose that the novel moc mutants are only defecti ve in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.