| RHYTHM OF CHLOROPLAST (ROC) genes (ROC15, ROC40, ROC55, ROC66, ROC75, and ROC114) have critical roles in the circadian clock of Chlamydomonas reinhardtii. Insertional mutants of these genes exhibit severe defects in circadian rhythmicity (Matsuo et al., Genes Dev, 2008). To address molecular mechanisms of circadian clockwork in Chlamydomonas, we examined mRNA expression levels of ROC genes in wild-type and roc mutant strains. ROC genes were expressed in circadian manner with different phases in wild-type strain. On the other hand, the circadian patterns were severely disrupted in roc mutants. Especially in roc75 mutant, expression level of ROC40 was markedly elevated. These results indicate that the expressions of ROC genes are themselves circadianly regulated, and there is a strong genetic interaction between ROC75 and ROC40. To address molecular mechanisms further, we generated transgenic strains expressing ROC proteins fused with a variety of tags including the yellow fluorescent protein (YFP), hemagglutinin epitope (HA), and tandem affinity purification tag (TAP). Preliminary results of ongoing studies suggest that ROC55 and ROC75 proteins are localized to the nucleus. |