Methylation of Chloroplast DNAs in the Life Cycle of Chlamydomonas

Methylation patterns of Chlamydomonas chloroplast DNAs (chlDNAs) were examined in the vegetative, gametic, and zygotic stages of the life cycle. Restriction endo-nuclease fragment patterns produced by EcoRI, BamHI, Hpa II, and Msp I were compared; the last two cleave DNA at the sequence C-C-G-G, but Hpa II is blocked by prior methylation of the internal cytidine whereas Msp I is not. chlDNAs from vegetative cells of both mating types showed no evidence of methylation at C-C-G-G. Gametic mt+ chlDNA was heavily methylated at C-C-G-G, whereas the homologous chlDNA from mt- gametes showed very slight methylation at C-C-G-G. Methylation of additional sites in chlDNA from mt+ gametes but not from mt- gametes was shown by blockage of some EcoRI and BamHI sites that were cleaved in the chlDNA from vegetative cells. chlDNA from 6-hr zygotes was much more methylated than gametic mt+ DNA, as shown by its almost total resistance to cleavage by all four restriction enzymes. These findings support and extend previous evidence that chlDNA of mt+ cells is methylated during gametogenesis and that further methylation occurs after gametic fusion in the young zygotes.