Methylation of chloroplast DNAs in the life cycle of Chlamydomonas

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